anti h3k4me3  (Millipore)


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    Structured Review

    Millipore anti h3k4me3
    MLL1 knockdown leads to decreased MDR1 expression and trimethylation of H3K4 at the MDR1 promoter A , Western blot analysis of MLL1N , MLL1C , Pgp, and α-tubulin levels (left panel), and RT-PCR analysis of relative MLL1, MDR1, and GAPDH mRNA levels (right panel) in HeLa cells 72 hours following transfection with non-targeting (NT) or MLL1 siRNA. B , MDR1 transcriptional activity was measured as luciferase activity. Cells were transfected with an MDR1 promoter-luciferase construct 72 hours following transfection with 100 nM NT siRNA or MLL1 siRNA. 24 hours post transfection, luciferase activities were measured and normalized to protein concentration and luciferase activity of pGL2B. C , Western blot analysis of MLL1N , MLL1C , NF-YA, Sp1 and α-tubulin levels in untransduced, scrambled shRNA (Scr shRNA) and MLL1 shRNA transduced HeLa cells following puromycin selection for 5 days. D , ChIP analysis of <t>H3K4me3</t> levels at the MDR1, HoxA7 and GAPDH promoters in Scr and MLL1 shRNA transduced HeLa cells 5 days following puromycin selection. The decreased fold of H3K4me3 levels in MLL1 shRNA transduced cells over scrambled shRNA transduced cells was shown at the bottom.
    Anti H3k4me3, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti h3k4me3 - by Bioz Stars, 2019-10
    99/100 stars

    Images

    1) Product Images from "Histone methyltransferase MLL1 regulates MDR1 transcription and chemoresistance"

    Article Title: Histone methyltransferase MLL1 regulates MDR1 transcription and chemoresistance

    Journal:

    doi: 10.1158/0008-5472.CAN-10-0755

    MLL1 knockdown leads to decreased MDR1 expression and trimethylation of H3K4 at the MDR1 promoter A , Western blot analysis of MLL1N , MLL1C , Pgp, and α-tubulin levels (left panel), and RT-PCR analysis of relative MLL1, MDR1, and GAPDH mRNA levels (right panel) in HeLa cells 72 hours following transfection with non-targeting (NT) or MLL1 siRNA. B , MDR1 transcriptional activity was measured as luciferase activity. Cells were transfected with an MDR1 promoter-luciferase construct 72 hours following transfection with 100 nM NT siRNA or MLL1 siRNA. 24 hours post transfection, luciferase activities were measured and normalized to protein concentration and luciferase activity of pGL2B. C , Western blot analysis of MLL1N , MLL1C , NF-YA, Sp1 and α-tubulin levels in untransduced, scrambled shRNA (Scr shRNA) and MLL1 shRNA transduced HeLa cells following puromycin selection for 5 days. D , ChIP analysis of H3K4me3 levels at the MDR1, HoxA7 and GAPDH promoters in Scr and MLL1 shRNA transduced HeLa cells 5 days following puromycin selection. The decreased fold of H3K4me3 levels in MLL1 shRNA transduced cells over scrambled shRNA transduced cells was shown at the bottom.
    Figure Legend Snippet: MLL1 knockdown leads to decreased MDR1 expression and trimethylation of H3K4 at the MDR1 promoter A , Western blot analysis of MLL1N , MLL1C , Pgp, and α-tubulin levels (left panel), and RT-PCR analysis of relative MLL1, MDR1, and GAPDH mRNA levels (right panel) in HeLa cells 72 hours following transfection with non-targeting (NT) or MLL1 siRNA. B , MDR1 transcriptional activity was measured as luciferase activity. Cells were transfected with an MDR1 promoter-luciferase construct 72 hours following transfection with 100 nM NT siRNA or MLL1 siRNA. 24 hours post transfection, luciferase activities were measured and normalized to protein concentration and luciferase activity of pGL2B. C , Western blot analysis of MLL1N , MLL1C , NF-YA, Sp1 and α-tubulin levels in untransduced, scrambled shRNA (Scr shRNA) and MLL1 shRNA transduced HeLa cells following puromycin selection for 5 days. D , ChIP analysis of H3K4me3 levels at the MDR1, HoxA7 and GAPDH promoters in Scr and MLL1 shRNA transduced HeLa cells 5 days following puromycin selection. The decreased fold of H3K4me3 levels in MLL1 shRNA transduced cells over scrambled shRNA transduced cells was shown at the bottom.

    Techniques Used: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Activity Assay, Luciferase, Construct, Protein Concentration, shRNA, Selection, Chromatin Immunoprecipitation

    MLL1 is not involved in TSA induced H3K4 trimethylation at the MDR1 locus Treatment of HeLa cells with 40 or 100 ng/ml TSA for 24h following with A , RT-PCR analysis of relative MDR1 and GAPDH mRNA levels (left panel), and western blot analysis of Pgp and α-tubulin levels (right panel) and B , ChIP analysis of H3K4me3 levels at the MDR1 locus. Immunoprecipitated chromatin was amplified with PCR using MDR1 promoter-specific primers (MDR1-P) and MDR1 coding region-specific primers (MDR1-T). The induction fold of MDR1 mRNA and coding region-associated H3K4me3 levels with TSA treatment over the mock treatment (0) was shown at the bottom. C , ChIP analysis of H3K4me3 levels at the MDR1 coding region in Scr and MLL1 shRNA transduced HeLa cells following treatment with 40 or 100 ng/ml TSA for 24h. The induction fold of H3K4me3 levels with TSA treatment over mock treatment (0) was shown at the bottom.
    Figure Legend Snippet: MLL1 is not involved in TSA induced H3K4 trimethylation at the MDR1 locus Treatment of HeLa cells with 40 or 100 ng/ml TSA for 24h following with A , RT-PCR analysis of relative MDR1 and GAPDH mRNA levels (left panel), and western blot analysis of Pgp and α-tubulin levels (right panel) and B , ChIP analysis of H3K4me3 levels at the MDR1 locus. Immunoprecipitated chromatin was amplified with PCR using MDR1 promoter-specific primers (MDR1-P) and MDR1 coding region-specific primers (MDR1-T). The induction fold of MDR1 mRNA and coding region-associated H3K4me3 levels with TSA treatment over the mock treatment (0) was shown at the bottom. C , ChIP analysis of H3K4me3 levels at the MDR1 coding region in Scr and MLL1 shRNA transduced HeLa cells following treatment with 40 or 100 ng/ml TSA for 24h. The induction fold of H3K4me3 levels with TSA treatment over mock treatment (0) was shown at the bottom.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Polymerase Chain Reaction, shRNA

    MLL1 overexpression induces MDR1 expression and tri-methylation of H3K4 at the MDR1 promoter A , Western blot analysis of MLL1N , MLL1C , and α-tubulin levels and B , RT-PCR analysis of relative MLL1, MDR1, and GAPDH mRNA levels in HeLa cells 24 hours following transfection with f-MLL1 plasmid. C , MDR1 transcriptional activity was measured by luciferase activity, 24h post-cotransfection of a luciferase reporter construct driven by the MDR1 promoter with empty vector, f-MLL1, or f-MLL1-ΔSET. Luciferase activities were measured and normalized to protein concentration and luciferase activities of empty vectors (pGL2B and pCXN2). D , ChIP analysis of H3K4me3 levels at the MDR1 promoter in HeLa cells 24 hours following transfection with f-MLL1 plasmid. Chromatin was immunoprecipitated with anti-H3K4me3. Negative controls include chromatin precipitated with no antibody or rabbit IgG. Immunoprecipitated chromatin was amplified with PCR using MDR1 promoter-specific primers. 1% input DNA was amplified to ensure that equal amounts of DNA were subjected to CHIP analysis.
    Figure Legend Snippet: MLL1 overexpression induces MDR1 expression and tri-methylation of H3K4 at the MDR1 promoter A , Western blot analysis of MLL1N , MLL1C , and α-tubulin levels and B , RT-PCR analysis of relative MLL1, MDR1, and GAPDH mRNA levels in HeLa cells 24 hours following transfection with f-MLL1 plasmid. C , MDR1 transcriptional activity was measured by luciferase activity, 24h post-cotransfection of a luciferase reporter construct driven by the MDR1 promoter with empty vector, f-MLL1, or f-MLL1-ΔSET. Luciferase activities were measured and normalized to protein concentration and luciferase activities of empty vectors (pGL2B and pCXN2). D , ChIP analysis of H3K4me3 levels at the MDR1 promoter in HeLa cells 24 hours following transfection with f-MLL1 plasmid. Chromatin was immunoprecipitated with anti-H3K4me3. Negative controls include chromatin precipitated with no antibody or rabbit IgG. Immunoprecipitated chromatin was amplified with PCR using MDR1 promoter-specific primers. 1% input DNA was amplified to ensure that equal amounts of DNA were subjected to CHIP analysis.

    Techniques Used: Over Expression, Expressing, Methylation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Activity Assay, Luciferase, Cotransfection, Construct, Protein Concentration, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Polymerase Chain Reaction

    2) Product Images from "Analysis of histone modifications at human ribosomal DNA in liver cancer cell"

    Article Title: Analysis of histone modifications at human ribosomal DNA in liver cancer cell

    Journal: Scientific Reports

    doi: 10.1038/srep18100

    Distribution of histone modification markers (H3K4me3, H3K36me3 and H3K27ac) at rDNA in human liver cancer. ( A ) Schematic representation of one human rDNA repeat. ETS: external transcribed spacer; ITS-1/2: internal transcribed spacer-1/2; IGS: intergenic spacer. The coding region, which spans ~0–13.3 kb of the human rDNA repeat, is separated by the non-coding intergenic spacers (IGS). ( B ) Patterns of histone modifications H3K4me3 at rDNA in human liver cancer cell HepG2 were shown below the human rDNA repeat as determined by the analysis of ChIP-seq data. Enrichment P value was calculated using the negative binomial model. ( C ) Patterns of histone modifications H3K36me3 at rDNA in human liver cancer cell HepG2 were shown below the human rDNA repeat as determined by the analysis of ChIP-seq data. Enrichment P value was calculated using the negative binomial model. ( D ) Patterns of histone modifications H3K27me3 at rDNA in human liver cancer cell HepG2 were shown below the human rDNA repeat as determined by the analysis of ChIP-seq data. Enrichment P value was calculated using the negative binomial model. ( E ) Pattern of human liver cancer cell HepG2 at rDNA was shown below the human rDNA repeat as determined by the analysis of ChIP-seq data.
    Figure Legend Snippet: Distribution of histone modification markers (H3K4me3, H3K36me3 and H3K27ac) at rDNA in human liver cancer. ( A ) Schematic representation of one human rDNA repeat. ETS: external transcribed spacer; ITS-1/2: internal transcribed spacer-1/2; IGS: intergenic spacer. The coding region, which spans ~0–13.3 kb of the human rDNA repeat, is separated by the non-coding intergenic spacers (IGS). ( B ) Patterns of histone modifications H3K4me3 at rDNA in human liver cancer cell HepG2 were shown below the human rDNA repeat as determined by the analysis of ChIP-seq data. Enrichment P value was calculated using the negative binomial model. ( C ) Patterns of histone modifications H3K36me3 at rDNA in human liver cancer cell HepG2 were shown below the human rDNA repeat as determined by the analysis of ChIP-seq data. Enrichment P value was calculated using the negative binomial model. ( D ) Patterns of histone modifications H3K27me3 at rDNA in human liver cancer cell HepG2 were shown below the human rDNA repeat as determined by the analysis of ChIP-seq data. Enrichment P value was calculated using the negative binomial model. ( E ) Pattern of human liver cancer cell HepG2 at rDNA was shown below the human rDNA repeat as determined by the analysis of ChIP-seq data.

    Techniques Used: Modification, Chromatin Immunoprecipitation

    ChIP-QPCR for histone modifications (H3K4me3, H3K36me3 and H3K27ac) in human liver cancer cell (LC control) and normal liver cell (CC control). ( A ) Enrichment of H3K4me3 on rDNA analyzed by ChIP-QPCR. Chromatin from HepG2 cells was cross-linked and immunoprecipitated with H3K4me3 antibody, DNA was analyzed by QPCR with different sets of primers indicated in ( A ). The percentage of DNA associated with anti-H3K4me3 antibody was calculated relative to the DNA from ChIP input. Values are represented by means ± SD from three independent ChIP experiments, each experiment was tested by at least three independent QPCR reactions. ( B ) Enrichment of H3K27me3 on rDNA analyzed by ChIP-QPCR. Chromatin from HepG2 cells was cross-linked and immunoprecipitated with H3K27me3 antibody, DNA was analyzed by QPCR with different sets of primers indicated in ( A ). The percentage of DNA associated with anti-H3K27me3 antibody was calculated relative to the DNA from ChIP input. Values are represented by means ± SD from three independent ChIP experiments, each experiment was tested by at least three independent QPCR reactions. Student’s t-test was performed between human liver cancer cell (LC Control) and normal liver cell (CC Control). ( C ) Enrichment of H3K36me3 on rDNA analyzed by ChIP-QPCR. Chromatin from HepG2 cells was cross-linked and immunoprecipitated with H3K27me3 antibody, DNA was analyzed by QPCR with different sets of primers indicated in ( A ). The percentage of DNA associated with anti-H3K36me3 antibody was calculated relative to the DNA from ChIP input. Values are represented by means ± SD derived from three independent ChIP experiments, each experiment was tested by at least three independent QPCR reactions. Student’s t-test was performed between human liver cancer cell (LC Control) and normal liver cell (CC Control).
    Figure Legend Snippet: ChIP-QPCR for histone modifications (H3K4me3, H3K36me3 and H3K27ac) in human liver cancer cell (LC control) and normal liver cell (CC control). ( A ) Enrichment of H3K4me3 on rDNA analyzed by ChIP-QPCR. Chromatin from HepG2 cells was cross-linked and immunoprecipitated with H3K4me3 antibody, DNA was analyzed by QPCR with different sets of primers indicated in ( A ). The percentage of DNA associated with anti-H3K4me3 antibody was calculated relative to the DNA from ChIP input. Values are represented by means ± SD from three independent ChIP experiments, each experiment was tested by at least three independent QPCR reactions. ( B ) Enrichment of H3K27me3 on rDNA analyzed by ChIP-QPCR. Chromatin from HepG2 cells was cross-linked and immunoprecipitated with H3K27me3 antibody, DNA was analyzed by QPCR with different sets of primers indicated in ( A ). The percentage of DNA associated with anti-H3K27me3 antibody was calculated relative to the DNA from ChIP input. Values are represented by means ± SD from three independent ChIP experiments, each experiment was tested by at least three independent QPCR reactions. Student’s t-test was performed between human liver cancer cell (LC Control) and normal liver cell (CC Control). ( C ) Enrichment of H3K36me3 on rDNA analyzed by ChIP-QPCR. Chromatin from HepG2 cells was cross-linked and immunoprecipitated with H3K27me3 antibody, DNA was analyzed by QPCR with different sets of primers indicated in ( A ). The percentage of DNA associated with anti-H3K36me3 antibody was calculated relative to the DNA from ChIP input. Values are represented by means ± SD derived from three independent ChIP experiments, each experiment was tested by at least three independent QPCR reactions. Student’s t-test was performed between human liver cancer cell (LC Control) and normal liver cell (CC Control).

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Liquid Chromatography, Immunoprecipitation, Derivative Assay

    Effect of UBF depletion on the distribution of histone modification marks (H3K4me3, H3K36me3, H3K27me3 and H3K4me1) at rDNA in human liver cancer cell. ( A ) Loss of UBF leads to a decrease of H3K4me3 at H42 region of rDNA. ChIP-QPCR analysis of different regions of rDNA in HepG2 cells transfected with control or UBF siRNA using antibodies against H3K4me3. The ratio of rDNA occupancy between UBF knockdown cells and control cells was calculated for each antibody. Student’s t-test was performed between cells transfected with or without UBF siRNA. ( B–C ) Depletion of UBF does not alter H3K36me3 and H3K4me1 distribution at rDNA. ChIP-QPCR analysis of different regions of rDNA in HepG2 cells transfected with control or UBF siRNA using antibodies against H3K36me3 ( B ), H3K4me1 ( C ). The ratio of rDNA occupancy between UBF knockdown cells and control cells was calculated for each antibody. Student’s t-test was performed between cells transfected with or without UBF siRNA.
    Figure Legend Snippet: Effect of UBF depletion on the distribution of histone modification marks (H3K4me3, H3K36me3, H3K27me3 and H3K4me1) at rDNA in human liver cancer cell. ( A ) Loss of UBF leads to a decrease of H3K4me3 at H42 region of rDNA. ChIP-QPCR analysis of different regions of rDNA in HepG2 cells transfected with control or UBF siRNA using antibodies against H3K4me3. The ratio of rDNA occupancy between UBF knockdown cells and control cells was calculated for each antibody. Student’s t-test was performed between cells transfected with or without UBF siRNA. ( B–C ) Depletion of UBF does not alter H3K36me3 and H3K4me1 distribution at rDNA. ChIP-QPCR analysis of different regions of rDNA in HepG2 cells transfected with control or UBF siRNA using antibodies against H3K36me3 ( B ), H3K4me1 ( C ). The ratio of rDNA occupancy between UBF knockdown cells and control cells was calculated for each antibody. Student’s t-test was performed between cells transfected with or without UBF siRNA.

    Techniques Used: Modification, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Transfection

    3) Product Images from "Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots"

    Article Title: Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1005512

    Expression of PRDM9 in HEK293 cells recapitulates allele-specific hotspot activation in vivo. (A) Western blot showing that expression of PRDM9 in HEK293 cells increases global H3K4me3 levels dependent on the PR/SET domain. (B) and (C) qPCR was performed on enriched DNA from ChIP against H3K4me3 at various intervals across the hotspots indicated by the position of the symbols after expression of PRDM9 protein variants. (B) PRDM9 A -defined recombination hotspots S and F have increased H3K4me3 after expression of PRDM9 A . (C) PRDM9 C -defined recombination hotpots 5A and 22A are enriched for H3K4me3 after expression of PRDM9 C . (D) Expression of PRDM9 in HEK293 cells results in allele-specific H3K4me3 at in vivo DSB hotspots. Heat map of H3K4me3 ChIP-seq (HEK293 cells) and DMC1 SSDS (men) signals for a 2 kb window centered on PRDM9 motifs. DMC1 SSDS data from [ 28 ]. (E) Aggregate plot of H3K4me3 (grey) and DMC1 (purple) signals from A-hotspots after expression of PRDM9 A . PRDM9 A motif derived from the hotspot nucleosome-depleted regions is shown above. (F) Similar to E except showing the result for PRDM9 C .
    Figure Legend Snippet: Expression of PRDM9 in HEK293 cells recapitulates allele-specific hotspot activation in vivo. (A) Western blot showing that expression of PRDM9 in HEK293 cells increases global H3K4me3 levels dependent on the PR/SET domain. (B) and (C) qPCR was performed on enriched DNA from ChIP against H3K4me3 at various intervals across the hotspots indicated by the position of the symbols after expression of PRDM9 protein variants. (B) PRDM9 A -defined recombination hotspots S and F have increased H3K4me3 after expression of PRDM9 A . (C) PRDM9 C -defined recombination hotpots 5A and 22A are enriched for H3K4me3 after expression of PRDM9 C . (D) Expression of PRDM9 in HEK293 cells results in allele-specific H3K4me3 at in vivo DSB hotspots. Heat map of H3K4me3 ChIP-seq (HEK293 cells) and DMC1 SSDS (men) signals for a 2 kb window centered on PRDM9 motifs. DMC1 SSDS data from [ 28 ]. (E) Aggregate plot of H3K4me3 (grey) and DMC1 (purple) signals from A-hotspots after expression of PRDM9 A . PRDM9 A motif derived from the hotspot nucleosome-depleted regions is shown above. (F) Similar to E except showing the result for PRDM9 C .

    Techniques Used: Expressing, Activation Assay, In Vivo, Western Blot, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Derivative Assay

    PRDM9 can form heteromeric complexes at hotspots. (A) PRDM9 protein variants can form homo- and heteromeric complexes. Western blot of various tagged versions of human PRDM9 detected either with anti-FLAG or anti-V5 antibodies (asterisk—non-specific band). V5-PRDM9 C (V5-C) is found in association with FLAG-PRDM9 A (FLAG-A) or FLAG-PRDM9 C (FLAG-C) after FLAG or V5 IP. (B) PRDM9 A is bound to the PRDM9 C -activated hotspot 5A when co-expressed with PRDM9 C . Tagged PRDM9 alleles, indicated on the x-axis, were transfected into HEK293 cells and subsequently subjected to ChIP against FLAG antibody; qPCR was performed on ChIP DNA samples at the center of the 5A hotspot (circles—individual biological replicates, lines—mean). (C) and (D) Similar to B showing ChIP results for PRDM9 C -activated hotspots at Chr 3 114 Mb and Chr 3 4 Mb respectively. (E) PRDM9 A can bind A-hotspots. Similar to B-D showing ChIP results for PRDM9 A -activated hotspot at Chr 3 54 Mb. (F) PRDM9 A can trimethylate C hotspots when in complex with PRDM9 C . Heat map of H3K4me3 ChIP-seq read counts from HEK293 cells for a 2 kb window surrounding PRDM9 C hotspots from Fig 2D .
    Figure Legend Snippet: PRDM9 can form heteromeric complexes at hotspots. (A) PRDM9 protein variants can form homo- and heteromeric complexes. Western blot of various tagged versions of human PRDM9 detected either with anti-FLAG or anti-V5 antibodies (asterisk—non-specific band). V5-PRDM9 C (V5-C) is found in association with FLAG-PRDM9 A (FLAG-A) or FLAG-PRDM9 C (FLAG-C) after FLAG or V5 IP. (B) PRDM9 A is bound to the PRDM9 C -activated hotspot 5A when co-expressed with PRDM9 C . Tagged PRDM9 alleles, indicated on the x-axis, were transfected into HEK293 cells and subsequently subjected to ChIP against FLAG antibody; qPCR was performed on ChIP DNA samples at the center of the 5A hotspot (circles—individual biological replicates, lines—mean). (C) and (D) Similar to B showing ChIP results for PRDM9 C -activated hotspots at Chr 3 114 Mb and Chr 3 4 Mb respectively. (E) PRDM9 A can bind A-hotspots. Similar to B-D showing ChIP results for PRDM9 A -activated hotspot at Chr 3 54 Mb. (F) PRDM9 A can trimethylate C hotspots when in complex with PRDM9 C . Heat map of H3K4me3 ChIP-seq read counts from HEK293 cells for a 2 kb window surrounding PRDM9 C hotspots from Fig 2D .

    Techniques Used: Western Blot, Transfection, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Prdm9 Dom2 is suppressed by competition between alleles. (A) PRDM9 Cst can suppress PRDM9 Dom2 -directed recombination at Ush2a . To detect either parental (top) or recombinant (bottom) molecules, nested PCR was performed on DNA from pooled sperm samples collected from F1 hybrids from the indicated crosses (two representative biological replicates are shown for each genotype). Recombinant molecules are only detected in progeny homozygous or heterozygous null for Prdm9 Dom2 . (B) Proportions of H3K4me3 hotspots in (B6xKI)F1 hybrids due to either PRDM9 Cst or PRDM9 Dom2 . (C) PRDM9 Dom2 hotspots in (B6xKI)F1 hybrids are the same hotspots found in Prdm9 Dom2/- heterozygous mice.
    Figure Legend Snippet: Prdm9 Dom2 is suppressed by competition between alleles. (A) PRDM9 Cst can suppress PRDM9 Dom2 -directed recombination at Ush2a . To detect either parental (top) or recombinant (bottom) molecules, nested PCR was performed on DNA from pooled sperm samples collected from F1 hybrids from the indicated crosses (two representative biological replicates are shown for each genotype). Recombinant molecules are only detected in progeny homozygous or heterozygous null for Prdm9 Dom2 . (B) Proportions of H3K4me3 hotspots in (B6xKI)F1 hybrids due to either PRDM9 Cst or PRDM9 Dom2 . (C) PRDM9 Dom2 hotspots in (B6xKI)F1 hybrids are the same hotspots found in Prdm9 Dom2/- heterozygous mice.

    Techniques Used: Recombinant, Nested PCR, Mouse Assay

    Prdm9 activity is dosage-sensitive for H3K4me3 levels and partially haploinsufficient for male meiotic progress in vivo. (A) Hotspots found in heterozygous null mice (B6- Prdm9 Dom2/- ) correspond to those hotspots with the highest level of H3K4me3 in B6 homozygous mice (B6- Prdm9 Dom2/Dom2 ). Distribution of H3K4me3 level at hotspots in Prdm9 Dom2 homozygous males (white—H3K4me3 level for all B6 hotspots, grey—H3K4me3 level in B6 mice for hotspots found in Prdm9 Dom2/- heterozygous males). (B) H3K4me3 activity at hotspots is reduced in Prdm9 Dom2/- heterozygous mice. MA-plot of H3K4me3 reads from two biological replicates for Prdm9 Dom2/Dom2 and Prdm9 Dom2/- animals (red—PRDM9-defined H3K4me3 hotspots, n = 7,379; blue—H3K4me3 found at promoters, n = 14,602; black—H3K4me3 found at other functional elements, n = 44,463). (C) Examples of pachytene nuclei from B6- Prdm9 Dom2/- males obtained by indirect immunofluorescence labeling. Overlapping SYCP1 and SYCP3 serve as markers for synapsis (yellow). ƴH2AFX is a marker for DSBs and should be restricted to the sex body at pachytene stage. Upper left—no sex body and multiple asynapsed autosomes evident by persistence of ƴH2AFX staining; upper right—asynapsed autosome included in the sex body; lower—normal pachynema. (D) The relative number of aberrant pachynemas increases in male mice heterozygous null for two Prdm9 M . m . domesticus alleles (N—total number of nuclei counted; circles—individual male mice; lines—mean ± standard deviation; p-value—Welsch’s t-test).
    Figure Legend Snippet: Prdm9 activity is dosage-sensitive for H3K4me3 levels and partially haploinsufficient for male meiotic progress in vivo. (A) Hotspots found in heterozygous null mice (B6- Prdm9 Dom2/- ) correspond to those hotspots with the highest level of H3K4me3 in B6 homozygous mice (B6- Prdm9 Dom2/Dom2 ). Distribution of H3K4me3 level at hotspots in Prdm9 Dom2 homozygous males (white—H3K4me3 level for all B6 hotspots, grey—H3K4me3 level in B6 mice for hotspots found in Prdm9 Dom2/- heterozygous males). (B) H3K4me3 activity at hotspots is reduced in Prdm9 Dom2/- heterozygous mice. MA-plot of H3K4me3 reads from two biological replicates for Prdm9 Dom2/Dom2 and Prdm9 Dom2/- animals (red—PRDM9-defined H3K4me3 hotspots, n = 7,379; blue—H3K4me3 found at promoters, n = 14,602; black—H3K4me3 found at other functional elements, n = 44,463). (C) Examples of pachytene nuclei from B6- Prdm9 Dom2/- males obtained by indirect immunofluorescence labeling. Overlapping SYCP1 and SYCP3 serve as markers for synapsis (yellow). ƴH2AFX is a marker for DSBs and should be restricted to the sex body at pachytene stage. Upper left—no sex body and multiple asynapsed autosomes evident by persistence of ƴH2AFX staining; upper right—asynapsed autosome included in the sex body; lower—normal pachynema. (D) The relative number of aberrant pachynemas increases in male mice heterozygous null for two Prdm9 M . m . domesticus alleles (N—total number of nuclei counted; circles—individual male mice; lines—mean ± standard deviation; p-value—Welsch’s t-test).

    Techniques Used: Activity Assay, In Vivo, Mouse Assay, Functional Assay, Immunofluorescence, Labeling, Marker, Staining, Standard Deviation

    4) Product Images from "Characterization and subcellular localization of histone deacetylases and their roles in response to abiotic stresses in soybean"

    Article Title: Characterization and subcellular localization of histone deacetylases and their roles in response to abiotic stresses in soybean

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-018-1454-7

    Levels of histone H3ac, H3K4me2, and H3K4me3 under cold and heat treatments. a Western blot showing the H3ac, H3K4me2, and H3K4me3 status in soybean leaves treated with cold and heat. 10-day-old seedlings were treated under cold (4 °C) and heat (42 °C) conditions for 12 h and the leaves were sampled for total protein extraction. b Quantification of western blot results. Signal intensities were measured using the ImageJ software and normalized to the loaded amount of H3. Values are expressed as fold change over control treatment. Shown is the mean ± SEM of three independent experiments. *, P -value
    Figure Legend Snippet: Levels of histone H3ac, H3K4me2, and H3K4me3 under cold and heat treatments. a Western blot showing the H3ac, H3K4me2, and H3K4me3 status in soybean leaves treated with cold and heat. 10-day-old seedlings were treated under cold (4 °C) and heat (42 °C) conditions for 12 h and the leaves were sampled for total protein extraction. b Quantification of western blot results. Signal intensities were measured using the ImageJ software and normalized to the loaded amount of H3. Values are expressed as fold change over control treatment. Shown is the mean ± SEM of three independent experiments. *, P -value

    Techniques Used: Western Blot, Protein Extraction, Software

    5) Product Images from "Histone demethylase KDM5A is regulated by its reader domain through a positive-feedback mechanism"

    Article Title: Histone demethylase KDM5A is regulated by its reader domain through a positive-feedback mechanism

    Journal:

    doi: 10.1038/ncomms7204

    KDM5A PHD1 recognizes the N-terminal region of the H3 tail and this binding stimulates H3K4me3 demethylation by KDM5A
    Figure Legend Snippet: KDM5A PHD1 recognizes the N-terminal region of the H3 tail and this binding stimulates H3K4me3 demethylation by KDM5A

    Techniques Used: Binding Assay

    6) Product Images from "Female Bias in Rhox6 and 9 Regulation by the Histone Demethylase KDM6A"

    Article Title: Female Bias in Rhox6 and 9 Regulation by the Histone Demethylase KDM6A

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003489

    Rhox6 and 9 are bivalent and preferentially occupied by KDM6A in female ES cells. H3K27me3, H3K4me3 and KDM6A enrichment profiles in undifferentiated female PGK12.1 (pink) and male WD44 ES (blue) cells at representative genes from each Rhox subcluster (α, β, and γ) demonstrate that only Rhox6 and 9 are highly enriched with both histone modifications and are bound by KDM6A (see also Figure S6 ). Rhox3e (α cluster) is enriched in H3K27me3 but not H3K4me3 or KDM6A, and Rhox12 (γ cluster) shows little enrichment for the proteins analyzed. Significant enrichment peaks based on Nimblescan analysis (FDR score
    Figure Legend Snippet: Rhox6 and 9 are bivalent and preferentially occupied by KDM6A in female ES cells. H3K27me3, H3K4me3 and KDM6A enrichment profiles in undifferentiated female PGK12.1 (pink) and male WD44 ES (blue) cells at representative genes from each Rhox subcluster (α, β, and γ) demonstrate that only Rhox6 and 9 are highly enriched with both histone modifications and are bound by KDM6A (see also Figure S6 ). Rhox3e (α cluster) is enriched in H3K27me3 but not H3K4me3 or KDM6A, and Rhox12 (γ cluster) shows little enrichment for the proteins analyzed. Significant enrichment peaks based on Nimblescan analysis (FDR score

    Techniques Used:

    7) Product Images from "Unique molecular events during reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) at naïve state"

    Article Title: Unique molecular events during reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) at naïve state

    Journal: eLife

    doi: 10.7554/eLife.29518

    Dynamics of histone modifications in naïve reprogramming. ( A ) Alluvial plots showing the global dynamics of genes covered by different chromatin states during naïve reprogramming. Each line represents a gene. Red bar represents genes with promoter that covered only by H3K4me2. Yellow bar represents genes with promoter that covered only by H3K4me3. Blue bar represents genes with promoter that covered by both H3K4me2 and H3K4me3. Grey bar represents genes with promoter that covered by neither H3K4me2 nor H3K4me3. ( B ) Enrichment of p-value of representative GO term of the selected clusters in Figure 5C . ( C ) Average H3K9me3 profiles around integrants of SVA family during naïve reprogramming.
    Figure Legend Snippet: Dynamics of histone modifications in naïve reprogramming. ( A ) Alluvial plots showing the global dynamics of genes covered by different chromatin states during naïve reprogramming. Each line represents a gene. Red bar represents genes with promoter that covered only by H3K4me2. Yellow bar represents genes with promoter that covered only by H3K4me3. Blue bar represents genes with promoter that covered by both H3K4me2 and H3K4me3. Grey bar represents genes with promoter that covered by neither H3K4me2 nor H3K4me3. ( B ) Enrichment of p-value of representative GO term of the selected clusters in Figure 5C . ( C ) Average H3K9me3 profiles around integrants of SVA family during naïve reprogramming.

    Techniques Used:

    8) Product Images from "Analysis of Argonaute 4-Associated Long Non-Coding RNA in Arabidopsis thaliana Sheds Novel Insights into Gene Regulation through RNA-Directed DNA Methylation"

    Article Title: Analysis of Argonaute 4-Associated Long Non-Coding RNA in Arabidopsis thaliana Sheds Novel Insights into Gene Regulation through RNA-Directed DNA Methylation

    Journal: Genes

    doi: 10.3390/genes8080198

    Chromatin immunoprecipitation (ChIP) assay and 5’ RACE detect increased H3K4me3 marks and short transcripts inside the gene body of RdDM-activated genes in the AGO4 mutant. Histone H3 Lys 27 (H3K27me3) and Lys 4 (H3K4me3) trimethylation at gene body tiling regions of ( A ) AT5G59820 , ( B ) AT1G76650 and ( C ) AT1G72900. ChIP signals are normalized to the input signals. Error bars are the standard error of the mean from two biological replicates. Green arrows show the start site of 5’ RACE products cloned from ago4-2 tissues. Student’s t -test is used to detect significant differences at the p
    Figure Legend Snippet: Chromatin immunoprecipitation (ChIP) assay and 5’ RACE detect increased H3K4me3 marks and short transcripts inside the gene body of RdDM-activated genes in the AGO4 mutant. Histone H3 Lys 27 (H3K27me3) and Lys 4 (H3K4me3) trimethylation at gene body tiling regions of ( A ) AT5G59820 , ( B ) AT1G76650 and ( C ) AT1G72900. ChIP signals are normalized to the input signals. Error bars are the standard error of the mean from two biological replicates. Green arrows show the start site of 5’ RACE products cloned from ago4-2 tissues. Student’s t -test is used to detect significant differences at the p

    Techniques Used: Chromatin Immunoprecipitation, Mutagenesis, Clone Assay

    9) Product Images from "From transient transcriptome responses to disturbed neurodevelopment: role of histone acetylation and methylation as epigenetic switch between reversible and irreversible drug effects"

    Article Title: From transient transcriptome responses to disturbed neurodevelopment: role of histone acetylation and methylation as epigenetic switch between reversible and irreversible drug effects

    Journal: Archives of Toxicology

    doi: 10.1007/s00204-014-1279-6

    Gene expression and histone methylation patterns of the neuroectodermal marker genes PAX6 and OTX2. a For all experiments, human embryonic stem cells (hESC) were differentiated to neuroepithelial precursor cells (NEP). Marker genes for hESC and NEP at the time points of analysis are indicated in the differentiation scheme. b Samples were taken at the indicated days of differentiation (DoD), and transcript levels of marker genes of neural differentiation were determined by RT-qPCR. Data (gene expression relative to hESC) are mean ± SEM of 3–5 experiments. c The differentiation was performed in the presence of non-cytotoxic concentrations of TSA (10 nM) or VPA (0.6 mM). At the indicated time points, transcript levels were determined by RT-qPCR. Data (expressed relative to control differentiated for the same time in the absence of drug) are mean ± SEM of 3–5 experiments. d Differentiating cells were treated with TSA or VPA for different time periods, and protein acetylation was analyzed by Western blots (WB) with antibodies specific for acetylated histone H3 (H3Ac) or acetylated α-tubulin (tubAc). WB against total histone 3 (H3) or α-tubulin (tub) was performed for normalization. One representative blot for the 6-h time point is displayed. The graphs are based on densitometric analysis of WB from three independent experiments. The levels of acetylated protein are given relative to untreated controls differentiated for the same time period. Data are mean ± SEM of three experiments. e Differentiating cell was treated with TSA (10 nM) for 4 days (day 4), 6 days (day 6), and for 4 days followed by a 2-day washout of the drug (TSA-w, purple bars ). Chromatin immunoprecipitation (ChIP) was performed with antibodies specific for H3K4me3 or H3K27me3. Samples were taken on day 4 of differentiation (day 4) or on day 6 of differentiation (day 6; TSA-w). The figure displays the ratio of the enrichment factors for H3K4me3 and H3K27me3 (individual values are found in supplemental files). A ratio > 1 points to open chromatin (more H3K4me3) and a ratio
    Figure Legend Snippet: Gene expression and histone methylation patterns of the neuroectodermal marker genes PAX6 and OTX2. a For all experiments, human embryonic stem cells (hESC) were differentiated to neuroepithelial precursor cells (NEP). Marker genes for hESC and NEP at the time points of analysis are indicated in the differentiation scheme. b Samples were taken at the indicated days of differentiation (DoD), and transcript levels of marker genes of neural differentiation were determined by RT-qPCR. Data (gene expression relative to hESC) are mean ± SEM of 3–5 experiments. c The differentiation was performed in the presence of non-cytotoxic concentrations of TSA (10 nM) or VPA (0.6 mM). At the indicated time points, transcript levels were determined by RT-qPCR. Data (expressed relative to control differentiated for the same time in the absence of drug) are mean ± SEM of 3–5 experiments. d Differentiating cells were treated with TSA or VPA for different time periods, and protein acetylation was analyzed by Western blots (WB) with antibodies specific for acetylated histone H3 (H3Ac) or acetylated α-tubulin (tubAc). WB against total histone 3 (H3) or α-tubulin (tub) was performed for normalization. One representative blot for the 6-h time point is displayed. The graphs are based on densitometric analysis of WB from three independent experiments. The levels of acetylated protein are given relative to untreated controls differentiated for the same time period. Data are mean ± SEM of three experiments. e Differentiating cell was treated with TSA (10 nM) for 4 days (day 4), 6 days (day 6), and for 4 days followed by a 2-day washout of the drug (TSA-w, purple bars ). Chromatin immunoprecipitation (ChIP) was performed with antibodies specific for H3K4me3 or H3K27me3. Samples were taken on day 4 of differentiation (day 4) or on day 6 of differentiation (day 6; TSA-w). The figure displays the ratio of the enrichment factors for H3K4me3 and H3K27me3 (individual values are found in supplemental files). A ratio > 1 points to open chromatin (more H3K4me3) and a ratio

    Techniques Used: Expressing, Methylation, Marker, Quantitative RT-PCR, Western Blot, Chromatin Immunoprecipitation

    Consequences of different drug washout periods for gene expression and histone methylation patterns. For all experiments, hESC were differentiated to NEP. a Samples for chromatin immunoprecipitation (ChIP) were prepared at the indicated days of differentiation. ChIP was performed with antibodies specific for H3K4me3 or H3K27me3 or control IgG. The enrichment factors of OTX2 and PAX6 promoter sequences are given as % input for H3K4me3 ( dark blue ) and H3K27me3 ( light blue ). Data are mean ± SEM of three independent cell preparations. b Differentiating cells were treated with TSA (10 nM) for the indicated time periods, and ChIP was performed with the same antibodies as described in a . The ratio of enrichment factors of H3K4me3 and H3K27me3 was calculated as measure of chromatin opening. Data are given relative to values of untreated control cells at the same time point ( n = 3). c Scheme of experimental treatment and sampling for the following experiments. Gray bars indicate the period of drug exposure (e.g., P2d: pulsed drug treatment for 2 days) with 10 nM TSA, and white bars indicate medium without TSA. All samples were analyzed on day 6 of differentiation for each treatment scenario. d Protein levels of PAX6 or OTX2 were determined by Western blot, and relative (vs ctr.) protein levels ( n = 3) were quantified. e Transcript levels of PAX6 and OTX2 were determined. They are expressed relative to untreated control on DoD6 (ctr). f ChIP was performed for H3K27me3 ( purple ) or H3K4me3 ( black ) on promoter regions of PAX6 and OTX2, and enrichment factors were calculated relative to ChIP with control IgG. Then, these data were normalized to the values obtained for control cells (ctr). For instance, on day 6 of the differentiation, H3K27me3 was 15-fold higher in cells treated for 1 day with TSA and then left in control medium (P1d), compared to cells that were differentiated under control conditions. Data of d – f are mean ± SEM of 3–5 experiments (color figure online)
    Figure Legend Snippet: Consequences of different drug washout periods for gene expression and histone methylation patterns. For all experiments, hESC were differentiated to NEP. a Samples for chromatin immunoprecipitation (ChIP) were prepared at the indicated days of differentiation. ChIP was performed with antibodies specific for H3K4me3 or H3K27me3 or control IgG. The enrichment factors of OTX2 and PAX6 promoter sequences are given as % input for H3K4me3 ( dark blue ) and H3K27me3 ( light blue ). Data are mean ± SEM of three independent cell preparations. b Differentiating cells were treated with TSA (10 nM) for the indicated time periods, and ChIP was performed with the same antibodies as described in a . The ratio of enrichment factors of H3K4me3 and H3K27me3 was calculated as measure of chromatin opening. Data are given relative to values of untreated control cells at the same time point ( n = 3). c Scheme of experimental treatment and sampling for the following experiments. Gray bars indicate the period of drug exposure (e.g., P2d: pulsed drug treatment for 2 days) with 10 nM TSA, and white bars indicate medium without TSA. All samples were analyzed on day 6 of differentiation for each treatment scenario. d Protein levels of PAX6 or OTX2 were determined by Western blot, and relative (vs ctr.) protein levels ( n = 3) were quantified. e Transcript levels of PAX6 and OTX2 were determined. They are expressed relative to untreated control on DoD6 (ctr). f ChIP was performed for H3K27me3 ( purple ) or H3K4me3 ( black ) on promoter regions of PAX6 and OTX2, and enrichment factors were calculated relative to ChIP with control IgG. Then, these data were normalized to the values obtained for control cells (ctr). For instance, on day 6 of the differentiation, H3K27me3 was 15-fold higher in cells treated for 1 day with TSA and then left in control medium (P1d), compared to cells that were differentiated under control conditions. Data of d – f are mean ± SEM of 3–5 experiments (color figure online)

    Techniques Used: Expressing, Methylation, Chromatin Immunoprecipitation, Sampling, Western Blot

    10) Product Images from "Arabidopsis BREVIPEDICELLUS Interacts with the SWI2/SNF2 Chromatin Remodeling ATPase BRAHMA to Regulate KNAT2 and KNAT6 Expression in Control of Inflorescence Architecture"

    Article Title: Arabidopsis BREVIPEDICELLUS Interacts with the SWI2/SNF2 Chromatin Remodeling ATPase BRAHMA to Regulate KNAT2 and KNAT6 Expression in Control of Inflorescence Architecture

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1005125

    BRM and BP decrease H3K4Me3 levels of KNAT2 and KNAT6 in inflorescences. (A) Schematic diagram of KNAT2 and KNAT6 for ChIP-qPCR analysis. Black boxes indicate the exons. Arrows indicate transcriptional starting sites, whereas black triangles indicate the positions of the TAGC motifs. (B) ChIP-qPCR analysis of relative H3K4me3 levels of KNAT2 chromatin in Col, brm-3 , bp-9 , and brm-3 bp-9 mutants. (C) ChIP-qPCR analysis of relative H3K4me3 levels of KNAT6 chromatin in Col, brm-3 , bp-9 , and brm-3 bp-9 mutants. The amounts of DNA after ChIP were quantified and normalized to TUB2 , the relative enrichment refers to the H3K4me3 enrichment versus the histone H3 occupancy. The values are shown as means±SD. 35-day-old plants were used for analysis. One-way ANOVA (Tukey-Kramer test) analysis was performed, and statistically significant differences (P
    Figure Legend Snippet: BRM and BP decrease H3K4Me3 levels of KNAT2 and KNAT6 in inflorescences. (A) Schematic diagram of KNAT2 and KNAT6 for ChIP-qPCR analysis. Black boxes indicate the exons. Arrows indicate transcriptional starting sites, whereas black triangles indicate the positions of the TAGC motifs. (B) ChIP-qPCR analysis of relative H3K4me3 levels of KNAT2 chromatin in Col, brm-3 , bp-9 , and brm-3 bp-9 mutants. (C) ChIP-qPCR analysis of relative H3K4me3 levels of KNAT6 chromatin in Col, brm-3 , bp-9 , and brm-3 bp-9 mutants. The amounts of DNA after ChIP were quantified and normalized to TUB2 , the relative enrichment refers to the H3K4me3 enrichment versus the histone H3 occupancy. The values are shown as means±SD. 35-day-old plants were used for analysis. One-way ANOVA (Tukey-Kramer test) analysis was performed, and statistically significant differences (P

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    11) Product Images from "Genome-Wide Analysis of the Chromatin Composition of Histone H2A and H3 Variants in Mouse Embryonic Stem Cells"

    Article Title: Genome-Wide Analysis of the Chromatin Composition of Histone H2A and H3 Variants in Mouse Embryonic Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0092689

    H3K4me3 level in H2A.Z- and H3.3-enriched regions. The genome was divided into 150 nucleotide bins, and the bins in which a particular variant was significantly enriched was determined as described in Materials and Methods ( see Figure S4 ). (A) Boxplot of H3K4me3 levels in H2A-and-H3-variant-enriched bins. (B) Boxplot of H3K4me3 levels in H2A.Z- or H3.3-, and both H2A.Z and H3.3 (H2A.Z/H3.3)-enriched bins. The bottom and top of the box are the 25th and 75th percentile, respectively, and the upper and lower whiskers represent 2.5th and 97.5th percentile, respectively. P -values were calculated using Mann–Whitney U-tests; (*) P
    Figure Legend Snippet: H3K4me3 level in H2A.Z- and H3.3-enriched regions. The genome was divided into 150 nucleotide bins, and the bins in which a particular variant was significantly enriched was determined as described in Materials and Methods ( see Figure S4 ). (A) Boxplot of H3K4me3 levels in H2A-and-H3-variant-enriched bins. (B) Boxplot of H3K4me3 levels in H2A.Z- or H3.3-, and both H2A.Z and H3.3 (H2A.Z/H3.3)-enriched bins. The bottom and top of the box are the 25th and 75th percentile, respectively, and the upper and lower whiskers represent 2.5th and 97.5th percentile, respectively. P -values were calculated using Mann–Whitney U-tests; (*) P

    Techniques Used: Variant Assay, MANN-WHITNEY

    12) Product Images from "Eμ and 3′RR IgH enhancers show hierarchic unilateral dependence in mature B-cells"

    Article Title: Eμ and 3′RR IgH enhancers show hierarchic unilateral dependence in mature B-cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-00575-0

    Epigenetic marks in E μ and 3′RR during CSR. ( a ) H3K4me3, H3K9ac and H3K27ac epigenetic marks in the four enhancers (hs3a, hs1,2, hs3b and hs4) of the 3′RR in LPS-stimulated B-cells of E μ -deficient and wt mice. Cells were stimulated with 5 μg/ml LPS for 2 days. Data are the means ± SEM of 3 independent experiments with 2 mice. ( b ) H3K4me3, H3K9ac and H3K27ac epigenetic marks in E μ in LPS-stimulated B-cells of 3′RR-deficient and wt mice. Same experimental protocol as in part ( a ). Data are the means ± SEM of 3 independent experiments with 2 mice. *p
    Figure Legend Snippet: Epigenetic marks in E μ and 3′RR during CSR. ( a ) H3K4me3, H3K9ac and H3K27ac epigenetic marks in the four enhancers (hs3a, hs1,2, hs3b and hs4) of the 3′RR in LPS-stimulated B-cells of E μ -deficient and wt mice. Cells were stimulated with 5 μg/ml LPS for 2 days. Data are the means ± SEM of 3 independent experiments with 2 mice. ( b ) H3K4me3, H3K9ac and H3K27ac epigenetic marks in E μ in LPS-stimulated B-cells of 3′RR-deficient and wt mice. Same experimental protocol as in part ( a ). Data are the means ± SEM of 3 independent experiments with 2 mice. *p

    Techniques Used: Mouse Assay

    13) Product Images from "Trans-tail regulation of MLL4-catalyzed H3K4 methylation by H4R3 symmetric dimethylation is mediated by a tandem PHD of MLL4"

    Article Title: Trans-tail regulation of MLL4-catalyzed H3K4 methylation by H4R3 symmetric dimethylation is mediated by a tandem PHD of MLL4

    Journal:

    doi: 10.1101/gad.203356.112

    PRMT7 knockdown results in increased H3K4me3 and MLL4 levels at MLL4 target genes. ( A – D ) Effect of PRMT7 knockdown on the promoter occupancy of PRMT7, H4R3me2s, MLL4, H3K4me3, and H4R3me2a at the HOXA1 ( A ), HOXA2 ( B ), HOXA3 ( C ), and NESTIN ( D
    Figure Legend Snippet: PRMT7 knockdown results in increased H3K4me3 and MLL4 levels at MLL4 target genes. ( A – D ) Effect of PRMT7 knockdown on the promoter occupancy of PRMT7, H4R3me2s, MLL4, H3K4me3, and H4R3me2a at the HOXA1 ( A ), HOXA2 ( B ), HOXA3 ( C ), and NESTIN ( D

    Techniques Used:

    MLL4-catalyzed H3K4me3 levels inversely correlate with PRMT7-regulated H4R3me2s levels at most HOXA and HOXB cluster genes. ( A , B ) The promoter occupancy of H3K4me3, MLL4, PRMT7, H4R3me2s, PRMT5, and H3 at the HOXA cluster ( A ) and HOXB cluster ( B ) genes
    Figure Legend Snippet: MLL4-catalyzed H3K4me3 levels inversely correlate with PRMT7-regulated H4R3me2s levels at most HOXA and HOXB cluster genes. ( A , B ) The promoter occupancy of H3K4me3, MLL4, PRMT7, H4R3me2s, PRMT5, and H3 at the HOXA cluster ( A ) and HOXB cluster ( B ) genes

    Techniques Used:

    14) Product Images from "Genome-wide positioning of bivalent mononucleosomes"

    Article Title: Genome-wide positioning of bivalent mononucleosomes

    Journal: BMC Medical Genomics

    doi: 10.1186/s12920-016-0221-6

    Distribution of bivalent nucleosomes at gene promoters. a Examples of individual genes showing the patterns of H3K4me3, H3K27me3 and bivalent nucleosomes around the TSS. Direction of gene transcription ( brown arrow ) and CpG islands ( black bar ) shown. b Schematic of distribution of H3K4me3 ( black ) and H3K27me3 ( red ) peaks called within +/−5000 bp from the TSS. Genes previously described as bivalent carry overlapping H3K4me3 and H3K27me3 ( green region ) peaks. c Size distribution of the H3K4me3, H3K27me3 peak calls, and the overlapping regions of these peaks. d – g Distribution of H3K4me3 d H3K27me3 e Sequential ChIP (H3K27me3 → H3K4me3)-seq reads (bivalent nucleosomes, f ), and H2A.Z g around the TSS (x-axis, TSS is 0). Promoters were classified into H3K4me3-exclusive ( black ), H3K27me3-exclusive ( red ), or none (none of the marks detected), promoters based on identification of ChIP-seq enrichment peak calls using SICER in the +/−2500 bp around the TSS. Promoters with overlapping H3K4me3 and H3K27me3 enrichment peak calls in the +/−2500 bp around the TSS were defined as Bivalent promoters ( green ). ChIP-seq reads were binned at 10 bp intervals. Y-axis represents average ChIP-seq reads normalized to corresponding average of the input . h Gene expression ranges of the three different promoter classes
    Figure Legend Snippet: Distribution of bivalent nucleosomes at gene promoters. a Examples of individual genes showing the patterns of H3K4me3, H3K27me3 and bivalent nucleosomes around the TSS. Direction of gene transcription ( brown arrow ) and CpG islands ( black bar ) shown. b Schematic of distribution of H3K4me3 ( black ) and H3K27me3 ( red ) peaks called within +/−5000 bp from the TSS. Genes previously described as bivalent carry overlapping H3K4me3 and H3K27me3 ( green region ) peaks. c Size distribution of the H3K4me3, H3K27me3 peak calls, and the overlapping regions of these peaks. d – g Distribution of H3K4me3 d H3K27me3 e Sequential ChIP (H3K27me3 → H3K4me3)-seq reads (bivalent nucleosomes, f ), and H2A.Z g around the TSS (x-axis, TSS is 0). Promoters were classified into H3K4me3-exclusive ( black ), H3K27me3-exclusive ( red ), or none (none of the marks detected), promoters based on identification of ChIP-seq enrichment peak calls using SICER in the +/−2500 bp around the TSS. Promoters with overlapping H3K4me3 and H3K27me3 enrichment peak calls in the +/−2500 bp around the TSS were defined as Bivalent promoters ( green ). ChIP-seq reads were binned at 10 bp intervals. Y-axis represents average ChIP-seq reads normalized to corresponding average of the input . h Gene expression ranges of the three different promoter classes

    Techniques Used: Chromatin Immunoprecipitation, Expressing

    Distribution of ChIP-seq reads around the TSS for gene promoters that are DNA-hypermethylated or unmethylated. Gene promoters were identified as methylated or unmethylated based on the Infinium methylation array data. H3K4me3 a H3K27me3 b sequential-ChIP c and H2A.Z d ChIP-seq reads were binned at 10 bp intervals and the average ChIP-seq reads normalized to corresponding average of the input plotted (Y-axis)
    Figure Legend Snippet: Distribution of ChIP-seq reads around the TSS for gene promoters that are DNA-hypermethylated or unmethylated. Gene promoters were identified as methylated or unmethylated based on the Infinium methylation array data. H3K4me3 a H3K27me3 b sequential-ChIP c and H2A.Z d ChIP-seq reads were binned at 10 bp intervals and the average ChIP-seq reads normalized to corresponding average of the input plotted (Y-axis)

    Techniques Used: Chromatin Immunoprecipitation, Methylation

    Distribution of ChIP-seq reads around the TSS for gene promoters of different CpG density. Genes were divided into different groups based on the presence or absence of a promoter-associated CpG island. CpG-island genes were further sub-classified by CpG density into quintiles, with the highest CpG density (81–100 percentile, a ), and lowest density (0–20 percentile, b ) CpG-island genes shown. Gene promoters without CpG islands are shown in c . For each plot, genes were additionally characterized for gene expression, with genes belonging to lowest (0–20 percentile) or highest (81–100 percentile) expression depicted in black and red, respectively. ChIP-seq reads were binned at 10 bp intervals. Y-axis represents average ChIP-seq read profiles normalized to corresponding average of the input. 1 st row, H3K4me3 ChIP-seq reads plotted as outlined above. 2 nd row, H3K27me3 ChIP-seq reads. 3 rd row, Sequential ChIP (H3K27me3 → H3K4me3)-seq reads. 4 th row, H2A.Z ChIP-seq reads
    Figure Legend Snippet: Distribution of ChIP-seq reads around the TSS for gene promoters of different CpG density. Genes were divided into different groups based on the presence or absence of a promoter-associated CpG island. CpG-island genes were further sub-classified by CpG density into quintiles, with the highest CpG density (81–100 percentile, a ), and lowest density (0–20 percentile, b ) CpG-island genes shown. Gene promoters without CpG islands are shown in c . For each plot, genes were additionally characterized for gene expression, with genes belonging to lowest (0–20 percentile) or highest (81–100 percentile) expression depicted in black and red, respectively. ChIP-seq reads were binned at 10 bp intervals. Y-axis represents average ChIP-seq read profiles normalized to corresponding average of the input. 1 st row, H3K4me3 ChIP-seq reads plotted as outlined above. 2 nd row, H3K27me3 ChIP-seq reads. 3 rd row, Sequential ChIP (H3K27me3 → H3K4me3)-seq reads. 4 th row, H2A.Z ChIP-seq reads

    Techniques Used: Chromatin Immunoprecipitation, Expressing

    Optimization of mononucleosome isolation and sequential ChIP. a Mononucleosomes were isolated from the indicated cell lines by sucrose gradient (S.G.), and assessed for purity by native PAGE analysis. Dinucleosomal fragments from NCCIT (NCCIT-Di) were included for size reference. b A quick protocol (Q.P.) was developed (see Experimental Procedures), and resulting mononucleosomes from NCCIT cells were assessed for purity by overloading of DNA onto an agarose gel. c Top shows a schematic representation of the TSS region of MLH1 with placement of primer pairs. Below, PCR analysis of mononucleosomes (mono, from Q.P.), dinucleosomes (di, from S.G.) and genomic DNA (gDNA) of NCCIT cells. d Mononucleosomes isolated from RKO and SW480 (using Q.P.) were subjected to ChIP, MLH1 promoter analysis (primer set described above, part C). e Mononucleosomes isolated from RKO and SW480 (using Q.P.) were subjected to sequential ChIP using indicated combinations of anti- H3, H3K4me3 and H3K27me3 immunoprecipitation (IP), and the MLH1 promoter was analyzed by primer set-B described above. f Amplicons depicted in E was quantitated by ImageJ (all data expressed as percentage of input and normalized to IgG background). g Mononucleosomes from NCCIT cells (using Q.P.) were subjected to single or sequential ChIP (precipitation of H3K27me3 followed by H3K4me3). Chromatin patterns of CDO1 , SFRP1 and SOX17 were assessed between treatment groups and compared to MYC as a control. h The schematic depicts isolation of mononucleosomes and individual histone peptides, the latter as depicted by peaks in the HPLC analysis below. i Sequential IP of mononucleosomes (using Q.P.) or purified histone peptide substrates from NCCIT cells were analyzed by dot blot using anti-H3K4me3 antibody (left panel shows sequence of IP)
    Figure Legend Snippet: Optimization of mononucleosome isolation and sequential ChIP. a Mononucleosomes were isolated from the indicated cell lines by sucrose gradient (S.G.), and assessed for purity by native PAGE analysis. Dinucleosomal fragments from NCCIT (NCCIT-Di) were included for size reference. b A quick protocol (Q.P.) was developed (see Experimental Procedures), and resulting mononucleosomes from NCCIT cells were assessed for purity by overloading of DNA onto an agarose gel. c Top shows a schematic representation of the TSS region of MLH1 with placement of primer pairs. Below, PCR analysis of mononucleosomes (mono, from Q.P.), dinucleosomes (di, from S.G.) and genomic DNA (gDNA) of NCCIT cells. d Mononucleosomes isolated from RKO and SW480 (using Q.P.) were subjected to ChIP, MLH1 promoter analysis (primer set described above, part C). e Mononucleosomes isolated from RKO and SW480 (using Q.P.) were subjected to sequential ChIP using indicated combinations of anti- H3, H3K4me3 and H3K27me3 immunoprecipitation (IP), and the MLH1 promoter was analyzed by primer set-B described above. f Amplicons depicted in E was quantitated by ImageJ (all data expressed as percentage of input and normalized to IgG background). g Mononucleosomes from NCCIT cells (using Q.P.) were subjected to single or sequential ChIP (precipitation of H3K27me3 followed by H3K4me3). Chromatin patterns of CDO1 , SFRP1 and SOX17 were assessed between treatment groups and compared to MYC as a control. h The schematic depicts isolation of mononucleosomes and individual histone peptides, the latter as depicted by peaks in the HPLC analysis below. i Sequential IP of mononucleosomes (using Q.P.) or purified histone peptide substrates from NCCIT cells were analyzed by dot blot using anti-H3K4me3 antibody (left panel shows sequence of IP)

    Techniques Used: Isolation, Chromatin Immunoprecipitation, Clear Native PAGE, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Immunoprecipitation, High Performance Liquid Chromatography, Purification, Dot Blot, Sequencing

    15) Product Images from "Epigenetic Modulation of Microglial Inflammatory Gene Loci in Helminth-Induced Immune Suppression"

    Article Title: Epigenetic Modulation of Microglial Inflammatory Gene Loci in Helminth-Induced Immune Suppression

    Journal: ASN NEURO

    doi: 10.1177/1759091415592126

    HSF downregulates positive histone marks. Primary microglia were pulsed with medium alone (NS), HSF at 25 µg/ml, LPS at 10 ng/ml, or HSF at 25 µg/ml prior to the addition of LPS in the medium (HSF + L) and cultured for a total of 24-hr period. Cells were harvested, and ChIP was performed with anti-H3K4Me3, or anti-H3K9/14Ac or isotype IgG or with no antibody. Presence of H3K4Me3 at the promoter regions of CIITA (A1), MHC-II (H2Eβ) (B1), NOS-2 (C1), TNF-α (D1), and IL-6(E1) was assessed by qPCR and expressed as percentage of input. Presence of H3K9/14Ac at the promoter regions of CIITA (A2), MHC-II (H2Eβ) (B2), NOS-2 (C2), TNF-α (D2), and IL-6 (E2) was assessed by qPCR and expressed as percentage of input. The mean ± SE of H3K4Me3 and H3K9/14Ac presence in three independent experiments was determined. Significant differences were measured by Student’s t test (* p
    Figure Legend Snippet: HSF downregulates positive histone marks. Primary microglia were pulsed with medium alone (NS), HSF at 25 µg/ml, LPS at 10 ng/ml, or HSF at 25 µg/ml prior to the addition of LPS in the medium (HSF + L) and cultured for a total of 24-hr period. Cells were harvested, and ChIP was performed with anti-H3K4Me3, or anti-H3K9/14Ac or isotype IgG or with no antibody. Presence of H3K4Me3 at the promoter regions of CIITA (A1), MHC-II (H2Eβ) (B1), NOS-2 (C1), TNF-α (D1), and IL-6(E1) was assessed by qPCR and expressed as percentage of input. Presence of H3K9/14Ac at the promoter regions of CIITA (A2), MHC-II (H2Eβ) (B2), NOS-2 (C2), TNF-α (D2), and IL-6 (E2) was assessed by qPCR and expressed as percentage of input. The mean ± SE of H3K4Me3 and H3K9/14Ac presence in three independent experiments was determined. Significant differences were measured by Student’s t test (* p

    Techniques Used: Cell Culture, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    16) Product Images from "An Arabidopsis E3 ligase HUB2 increases histone H2B monoubiquitination and enhances drought tolerance in transgenic cotton"

    Article Title: An Arabidopsis E3 ligase HUB2 increases histone H2B monoubiquitination and enhances drought tolerance in transgenic cotton

    Journal: Plant Biotechnology Journal

    doi: 10.1111/pbi.12998

    Detection of global histone H2Bub1 and H3K4me3 levels at the Gh DREB chromatin locus. (a) Schematic diagram of the Gh DREB gene. The exons are shown in boxes, and the indicated PCR fragments analysed in the Ch IP assay are indicated with short black lines. P1: 2000 bp upstream of the ATG ; P2: promoter region; P3: transcription start site ( TSS ) region; P4: gene body region. (b) H2Bub1 at the Gh DREB locus in the CCRI 24 and transgenic lines. Immunoprecipitated DNA was analysed via RT ‐ qPCR , and enrichment was determined as a percentage of the input. (c) H3K4me3 at the Gh DREB locus in the CCRI 24 and transgenic lines. (d) The Gh UBI gene was selected as a housekeeping gene that was not differentially expressed in the transgenic lines. (e) Expression of Gh DREB at different time points of air drought treatment in control and At HUB 2 ‐overexpressing lines. Real‐time RT ‐ qPCR quantification was normalized to the expression of Gh UBI . Vertical bars represent SD . (* P
    Figure Legend Snippet: Detection of global histone H2Bub1 and H3K4me3 levels at the Gh DREB chromatin locus. (a) Schematic diagram of the Gh DREB gene. The exons are shown in boxes, and the indicated PCR fragments analysed in the Ch IP assay are indicated with short black lines. P1: 2000 bp upstream of the ATG ; P2: promoter region; P3: transcription start site ( TSS ) region; P4: gene body region. (b) H2Bub1 at the Gh DREB locus in the CCRI 24 and transgenic lines. Immunoprecipitated DNA was analysed via RT ‐ qPCR , and enrichment was determined as a percentage of the input. (c) H3K4me3 at the Gh DREB locus in the CCRI 24 and transgenic lines. (d) The Gh UBI gene was selected as a housekeeping gene that was not differentially expressed in the transgenic lines. (e) Expression of Gh DREB at different time points of air drought treatment in control and At HUB 2 ‐overexpressing lines. Real‐time RT ‐ qPCR quantification was normalized to the expression of Gh UBI . Vertical bars represent SD . (* P

    Techniques Used: Polymerase Chain Reaction, Transgenic Assay, Immunoprecipitation, Quantitative RT-PCR, Expressing

    17) Product Images from "Ago1 Interacts with RNA Polymerase II and Binds to the Promoters of Actively Transcribed Genes in Human Cancer Cells"

    Article Title: Ago1 Interacts with RNA Polymerase II and Binds to the Promoters of Actively Transcribed Genes in Human Cancer Cells

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003821

    Ago1 is preferentially bound to euchromatic regions on chromosomes. ( A ) Shown are bivariate scattergrams with regression analysis between Ago1 peak density (peaks/100 kb) and gene density (genes/100 kb, upper panel), GC% (middle panel), or Repeat% (lower panel). ( B ) Relative density of repetitive and non-repetitive sequences present in the Ago1-bound sequences. The percentages define Ago1-bound sequence composition in comparison to abundance in human genome. Repetitive sequence composition is subdivided in the pie charts to include low complexity (LC), simple repeats (SR), long terminal repeats (LTR), short interspersed elements (SINE), and long interspersed elements (LINE). ( C ) Relative density of Ago1 peaks located in genic (i.e. promoter, 5′UTR, coding exon, intron, 3′UTR, and 3′ flanking region) and intergenic regions. The numbers in the parentheses indicate enrichment ratio relative to the genome with statistical analysis. ( D ) Distribution of Ago1 peaks relative to TSSs of annotated genes and their correlation with H3K4me3 peaks. ( E ) Distribution of H3K4me3 peaks relative to TSSs. ( F ) Genome browser views of Ago1 (red) and H3K4me3 (blue) peaks on 4 representative AbGs including PIK3CA, PRKCH, CDC6 and RRM1. Y-axis is normalized number of reads. All peaks passed the FDR cutoff are shown. Input tracks are included as controls. Major transcription start site (TSS) for each gene is shown by the red arrow. Alternative TSSs are denoted by black arrows. Green bars above gene structures correspond to ChIP amplicons used below. ( G ) Independent ChIP analyses of the representative promoters were performed in PC-3 cells. Ago1 and RNAP II occupancy were determined by qPCR using primer sets encompassing the green bars designated in (F). Results are shown as mean % input ± SD from 3 independent experiments. IgG was used as a negative control. Neg: negative control region.
    Figure Legend Snippet: Ago1 is preferentially bound to euchromatic regions on chromosomes. ( A ) Shown are bivariate scattergrams with regression analysis between Ago1 peak density (peaks/100 kb) and gene density (genes/100 kb, upper panel), GC% (middle panel), or Repeat% (lower panel). ( B ) Relative density of repetitive and non-repetitive sequences present in the Ago1-bound sequences. The percentages define Ago1-bound sequence composition in comparison to abundance in human genome. Repetitive sequence composition is subdivided in the pie charts to include low complexity (LC), simple repeats (SR), long terminal repeats (LTR), short interspersed elements (SINE), and long interspersed elements (LINE). ( C ) Relative density of Ago1 peaks located in genic (i.e. promoter, 5′UTR, coding exon, intron, 3′UTR, and 3′ flanking region) and intergenic regions. The numbers in the parentheses indicate enrichment ratio relative to the genome with statistical analysis. ( D ) Distribution of Ago1 peaks relative to TSSs of annotated genes and their correlation with H3K4me3 peaks. ( E ) Distribution of H3K4me3 peaks relative to TSSs. ( F ) Genome browser views of Ago1 (red) and H3K4me3 (blue) peaks on 4 representative AbGs including PIK3CA, PRKCH, CDC6 and RRM1. Y-axis is normalized number of reads. All peaks passed the FDR cutoff are shown. Input tracks are included as controls. Major transcription start site (TSS) for each gene is shown by the red arrow. Alternative TSSs are denoted by black arrows. Green bars above gene structures correspond to ChIP amplicons used below. ( G ) Independent ChIP analyses of the representative promoters were performed in PC-3 cells. Ago1 and RNAP II occupancy were determined by qPCR using primer sets encompassing the green bars designated in (F). Results are shown as mean % input ± SD from 3 independent experiments. IgG was used as a negative control. Neg: negative control region.

    Techniques Used: Gas Chromatography, Sequencing, Liquid Chromatography, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

    Ago1 knockdown reduces occupancy of Ago1 and RNAP II at gene promoters and downregulates gene expression. ( A ) Genome browser views of Ago1 (red) and H3K4me3 (blue) peaks on 4 representative AbGs including SMC1A, CDC20, SMAD3, and BUB1. Y-axis is normalized number of reads. All peaks passed the FDR cutoff are shown. Input tracks are included as controls. Green bars above gene structures correspond to ChIP amplicons used below. ( B and C ) PC-3 cells were transfected with siControl or siAgo1 for 48 hrs. ChIP analysis was performed to determine Ago1 and RNAP II occupancy at the representative promoters using qPCR in conjunction with the primer sets encompassing the green bars designated in (A). Results are shown as mean fold enrichment relative to negative control region (Neg) ± SD from 3 independent experiments. IgG served as a negative control. ( D ) mRNA expression levels of SMC1A, CDC20, SMAD3 and BUB1 were quantified by qPCR (mean ± SD from 3 independent experiments). Values were normalized to GAPDH.
    Figure Legend Snippet: Ago1 knockdown reduces occupancy of Ago1 and RNAP II at gene promoters and downregulates gene expression. ( A ) Genome browser views of Ago1 (red) and H3K4me3 (blue) peaks on 4 representative AbGs including SMC1A, CDC20, SMAD3, and BUB1. Y-axis is normalized number of reads. All peaks passed the FDR cutoff are shown. Input tracks are included as controls. Green bars above gene structures correspond to ChIP amplicons used below. ( B and C ) PC-3 cells were transfected with siControl or siAgo1 for 48 hrs. ChIP analysis was performed to determine Ago1 and RNAP II occupancy at the representative promoters using qPCR in conjunction with the primer sets encompassing the green bars designated in (A). Results are shown as mean fold enrichment relative to negative control region (Neg) ± SD from 3 independent experiments. IgG served as a negative control. ( D ) mRNA expression levels of SMC1A, CDC20, SMAD3 and BUB1 were quantified by qPCR (mean ± SD from 3 independent experiments). Values were normalized to GAPDH.

    Techniques Used: Expressing, Chromatin Immunoprecipitation, Transfection, Real-time Polymerase Chain Reaction, Negative Control

    18) Product Images from "KDM5A controls bone morphogenic protein 2-induced osteogenic differentiation of bone mesenchymal stem cells during osteoporosis"

    Article Title: KDM5A controls bone morphogenic protein 2-induced osteogenic differentiation of bone mesenchymal stem cells during osteoporosis

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2016.238

    KDM5A knockdown enhanced osteogenic differentiation of MSCs. ( a ) qRT-PCR analysis and ( b ) western blot analysis of Kdm5a in MSCs after infected with lentiviral-Scrsh, lentiviral-Kdm5a-sh1 and lentiviral-Kdm5a-sh2. ( c ) Representative images of ALP staining of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( d ) Quantitative analysis of ALP activity of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( e ) Representative images of Alizarin red staining (including quantitative analysis) of MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 14 days of osteogenic induction. ( f ) qRT-PCR analysis and ( g ) western blot analysis of Col1a1, Ocn and Runx2 expression in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. ( h ) Immunostaining of Runx2 (red) location in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. Scale bar, 20 μ m. ( i ) Representative images of Alizarin red staining of MSCs isolated from OVX mice in Scrsh, Kdm5a-sh1 groups after 14 days of osteogenic induction. ( j ) Western blot analysis of H3K4me3 expression in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor (JIB-04 with 300 nM) treatment. ( k ) qRT-PCR analysis of the expression of Col1a1, Ocn and Runx2 in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor treatment. ( l ) Representative images of Alizarin red staining of MSCs isolated from sham mice and OVX mice with or without Kdm5a inhibitor treatment. All the data were confirmed by three repeated tests. Data were mean±S.D. * P
    Figure Legend Snippet: KDM5A knockdown enhanced osteogenic differentiation of MSCs. ( a ) qRT-PCR analysis and ( b ) western blot analysis of Kdm5a in MSCs after infected with lentiviral-Scrsh, lentiviral-Kdm5a-sh1 and lentiviral-Kdm5a-sh2. ( c ) Representative images of ALP staining of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( d ) Quantitative analysis of ALP activity of MSCs in Scrsh, Kdm5a-sh1, Kdm5a-sh2, Kdm5a-sh1+Kdm5a and Kdm5a-sh2+Kdm5a groups after 7 days of osteogenic induction. ( e ) Representative images of Alizarin red staining (including quantitative analysis) of MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 14 days of osteogenic induction. ( f ) qRT-PCR analysis and ( g ) western blot analysis of Col1a1, Ocn and Runx2 expression in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. ( h ) Immunostaining of Runx2 (red) location in MSCs in Scrsh, Kdm5a-sh1 and Kdm5a-sh1+Kdm5a groups after 7 days of osteogenic induction. Scale bar, 20 μ m. ( i ) Representative images of Alizarin red staining of MSCs isolated from OVX mice in Scrsh, Kdm5a-sh1 groups after 14 days of osteogenic induction. ( j ) Western blot analysis of H3K4me3 expression in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor (JIB-04 with 300 nM) treatment. ( k ) qRT-PCR analysis of the expression of Col1a1, Ocn and Runx2 in MSCs of sham mice and OVX mice with or without Kdm5a inhibitor treatment. ( l ) Representative images of Alizarin red staining of MSCs isolated from sham mice and OVX mice with or without Kdm5a inhibitor treatment. All the data were confirmed by three repeated tests. Data were mean±S.D. * P

    Techniques Used: Quantitative RT-PCR, Western Blot, Infection, ALP Assay, Staining, Activity Assay, Expressing, Immunostaining, Isolation, Mouse Assay

    KDM5A inhibited Runx2 expression in MSC by removal of H3K4me3 marks. ( a ) Western blot analysis of p-Smad1/5/8, Smad1 and Smad4 in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 4 hours of osteogenic induction. ( b ) Quantitative analysis of p-Smad1 expression. Smad1 was used as internal control. ( c ) Immunostaining of p-Smad1/5/8 (green) location in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 4 hours of osteogenic induction. Scale bar, 20 μ m. ( d ) Schematics of Runx2 promoter denoting ChIP-PCR amplified region (−1105 bp to −1065 bp) encompassing the SMAD binding element and the control region 6-kb upstream of the transcription start site (−6173 bp to −6034 bp). ( e ) Western blot analysis of H3K4me3 in MSCs after 0, 3, 7 and 14 days BMP2 treatment. ( f ) Occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment. ( g ) SMAD5 occupancy at the Runx2 promoter after BMP2 treatment. ( h ) Western blot analysis of H3K4me3 in MSCs of sham and OVX mice. ( i ) Occupancy of H3K4me3 at the Runx2 promoter in MSCs of sham and OVX mice following BMP2 treatment. ( j ) Knockdown of Kdm5a increased the occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment in MSCs of sham and OVX mice. ( k ) Overexpression Kdm5a decreased the occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment in MSCs of sham and OVX mice. All the data were confirmed by three repeated tests. Data were mean±S.D. ** P
    Figure Legend Snippet: KDM5A inhibited Runx2 expression in MSC by removal of H3K4me3 marks. ( a ) Western blot analysis of p-Smad1/5/8, Smad1 and Smad4 in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 4 hours of osteogenic induction. ( b ) Quantitative analysis of p-Smad1 expression. Smad1 was used as internal control. ( c ) Immunostaining of p-Smad1/5/8 (green) location in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 4 hours of osteogenic induction. Scale bar, 20 μ m. ( d ) Schematics of Runx2 promoter denoting ChIP-PCR amplified region (−1105 bp to −1065 bp) encompassing the SMAD binding element and the control region 6-kb upstream of the transcription start site (−6173 bp to −6034 bp). ( e ) Western blot analysis of H3K4me3 in MSCs after 0, 3, 7 and 14 days BMP2 treatment. ( f ) Occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment. ( g ) SMAD5 occupancy at the Runx2 promoter after BMP2 treatment. ( h ) Western blot analysis of H3K4me3 in MSCs of sham and OVX mice. ( i ) Occupancy of H3K4me3 at the Runx2 promoter in MSCs of sham and OVX mice following BMP2 treatment. ( j ) Knockdown of Kdm5a increased the occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment in MSCs of sham and OVX mice. ( k ) Overexpression Kdm5a decreased the occupancy of H3K4me3 at the Runx2 promoter following BMP2 treatment in MSCs of sham and OVX mice. All the data were confirmed by three repeated tests. Data were mean±S.D. ** P

    Techniques Used: Expressing, Western Blot, Infection, Plasmid Preparation, Immunostaining, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Binding Assay, Mouse Assay, Over Expression

    KDM5A overexpression impaired osteogenic differentiation of MSCs. ( a ) qRT-PCR analysis and ( b ) western blot analysis of Kdm5a in MSCs after 0, 3, 7 and 14 days osteogenic induction. ( c ) qRT-PCR analysis and ( d ) western blot analysis of Kdm5a in MSCs after infected with lentiviral vector (MSC/V) and lentiviral-Kdm5a (MSC/KDM5A). ( e ) Western blot analysis of H3K4me3, H3K9me3 and H3K27me3 in MSCs with overexpression of Kdm5a. ( f ) ALP activity o f MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction were detected with ALP staining and quantified. ( g ) Mineralized nodules formed by MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 14 days of osteogenic induction were detected with Alizarin red staining and quantified. ( h ) qRT-PCR analysis and ( i ) western blot analysis of Col1a1, Ocn and Runx2 expression in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction. ( j ) Immunostaining of RUNX2 (red) location in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction. Scale bar, 20 μ m. All the data were confirmed by three repeated tests. Data were mean±S.D. ** P
    Figure Legend Snippet: KDM5A overexpression impaired osteogenic differentiation of MSCs. ( a ) qRT-PCR analysis and ( b ) western blot analysis of Kdm5a in MSCs after 0, 3, 7 and 14 days osteogenic induction. ( c ) qRT-PCR analysis and ( d ) western blot analysis of Kdm5a in MSCs after infected with lentiviral vector (MSC/V) and lentiviral-Kdm5a (MSC/KDM5A). ( e ) Western blot analysis of H3K4me3, H3K9me3 and H3K27me3 in MSCs with overexpression of Kdm5a. ( f ) ALP activity o f MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction were detected with ALP staining and quantified. ( g ) Mineralized nodules formed by MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 14 days of osteogenic induction were detected with Alizarin red staining and quantified. ( h ) qRT-PCR analysis and ( i ) western blot analysis of Col1a1, Ocn and Runx2 expression in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction. ( j ) Immunostaining of RUNX2 (red) location in MSCs infected with lentiviral-vector or lentiviral-Kdm5a after 7 days of osteogenic induction. Scale bar, 20 μ m. All the data were confirmed by three repeated tests. Data were mean±S.D. ** P

    Techniques Used: Over Expression, Quantitative RT-PCR, Western Blot, Infection, Plasmid Preparation, ALP Assay, Activity Assay, Staining, Expressing, Immunostaining

    19) Product Images from "Cfp1 integrates both CpG content and gene activity for accurate H3K4me3 deposition in embryonic stem cells"

    Article Title: Cfp1 integrates both CpG content and gene activity for accurate H3K4me3 deposition in embryonic stem cells

    Journal:

    doi: 10.1101/gad.194209.112

    Cfp1 regulates H3K4me3 at many gene promoters. ( A ) Genome browser screenshots representing H3K4me3 signal (as read coverage) in wild-type (wt) and Cfp1 −/− ES cells at selected gene loci ( Sox2 , Actb , and Gapdh ). CGIs are represented in
    Figure Legend Snippet: Cfp1 regulates H3K4me3 at many gene promoters. ( A ) Genome browser screenshots representing H3K4me3 signal (as read coverage) in wild-type (wt) and Cfp1 −/− ES cells at selected gene loci ( Sox2 , Actb , and Gapdh ). CGIs are represented in

    Techniques Used:

    A Cfp1 DNA-binding mutant can restore H3K4me3 at active promoters. ( A ) Screenshots representing H3K4me3 read coverage in wild-type (wt), Cfp1 −/− , wild-type rescue , and C169A rescue ES cells at selected gene loci ( Sox2 , Actb , Gapdh , Pou5f1
    Figure Legend Snippet: A Cfp1 DNA-binding mutant can restore H3K4me3 at active promoters. ( A ) Screenshots representing H3K4me3 read coverage in wild-type (wt), Cfp1 −/− , wild-type rescue , and C169A rescue ES cells at selected gene loci ( Sox2 , Actb , Gapdh , Pou5f1

    Techniques Used: Binding Assay, Mutagenesis

    H3K4me3 accumulates at ectopic sites in the absence of Cfp1 or its DNA-binding activity. ( A ) Screenshots showing H3K4me3 signal read coverage in wild-type (wt), Cfp1 −/− , wild-type rescue , and C169A rescue ES cells at selected genomic regions
    Figure Legend Snippet: H3K4me3 accumulates at ectopic sites in the absence of Cfp1 or its DNA-binding activity. ( A ) Screenshots showing H3K4me3 signal read coverage in wild-type (wt), Cfp1 −/− , wild-type rescue , and C169A rescue ES cells at selected genomic regions

    Techniques Used: Binding Assay, Activity Assay

    Transcription is weakly affected by decreased H3K4me3 at active gene promoters. ( A ) Volcano plots comparing expression values between wild-type and Cfp1 −/− ES cells. Fold change in expression values (wild-type vs. Cfp1 −/−
    Figure Legend Snippet: Transcription is weakly affected by decreased H3K4me3 at active gene promoters. ( A ) Volcano plots comparing expression values between wild-type and Cfp1 −/− ES cells. Fold change in expression values (wild-type vs. Cfp1 −/−

    Techniques Used: Expressing

    Cfp1 deficiency preferentially affects H3K4me3 at highly expressed genes without altering their DNA methylation. ( A ) GRO-seq density distribution in wild-type (wt) ES cells for all Ensembl genes (All genes); for genes whose H3K4me3 is not affected (Nonaffected,
    Figure Legend Snippet: Cfp1 deficiency preferentially affects H3K4me3 at highly expressed genes without altering their DNA methylation. ( A ) GRO-seq density distribution in wild-type (wt) ES cells for all Ensembl genes (All genes); for genes whose H3K4me3 is not affected (Nonaffected,

    Techniques Used: DNA Methylation Assay

    Model for the role of Cfp1 in regulating genome-wide H3K4me3. ( A ) Multiple Cfp1-dependent and Cfp1-independent mechanisms contribute to targeting of the Set1 complex to transcriptionally active CGI promoters in wild-type (wt) mouse ES cells. Cfp1 binding
    Figure Legend Snippet: Model for the role of Cfp1 in regulating genome-wide H3K4me3. ( A ) Multiple Cfp1-dependent and Cfp1-independent mechanisms contribute to targeting of the Set1 complex to transcriptionally active CGI promoters in wild-type (wt) mouse ES cells. Cfp1 binding

    Techniques Used: Genome Wide, Binding Assay

    H3K4me3 ectopic peaks in Cfp1 −/− ES cells coincide with regulatory regions and correlate with enhanced expression of neighboring genes. ( A ) Cluster analysis showing colocalization of ectopic peaks with regions enriched with histone modifications
    Figure Legend Snippet: H3K4me3 ectopic peaks in Cfp1 −/− ES cells coincide with regulatory regions and correlate with enhanced expression of neighboring genes. ( A ) Cluster analysis showing colocalization of ectopic peaks with regions enriched with histone modifications

    Techniques Used: Expressing

    20) Product Images from "The PHD finger protein Spp1 has distinct functions in the Set1 and the meiotic DSB formation complexes"

    Article Title: The PHD finger protein Spp1 has distinct functions in the Set1 and the meiotic DSB formation complexes

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007223

    Illustration of the different functions of Spp1 for H3K4me3 and meiotic DSB formation. 1) in the Set1 complex, Spp1 has a role to allow catalysis of H3K4 trimethylation by Set1, but this function is not essential, since the set1-sid mutant still maintains high levels of H3K4me3. 2) In addition, Spp1 maintains H3K4me3 levels, not by stimulating Set1 catalytic activity, but likely by binding H3K4me3 with its PHD finger. This can take place without interaction with the Set1 complex. We propose this may protect H3K4me3 from active demethylation, by the Jhd2 enzyme. Other possible explanations are described in the text. 3) Finally, the simultaneous binding of Spp1 to H3K4me3 and to the axis-associated Mer2 protein is essential to promote efficient DSB formation by Spo11. It has to be noted that a PHD finger mutant of Spp1 (W45A) is still able to bind Mer2 [ 18 ], so recognition of H3K4me3 by Spp1 PHD finger is not a prerequisite for its subsequent binding to Mer2. NDR: nucleosome-depleted region; Black circle: first nucleosome of genes; H3R2: arginine 2 of histone H3, in its non-asymmetrically methylated form. The blue square represents H3K4me3.
    Figure Legend Snippet: Illustration of the different functions of Spp1 for H3K4me3 and meiotic DSB formation. 1) in the Set1 complex, Spp1 has a role to allow catalysis of H3K4 trimethylation by Set1, but this function is not essential, since the set1-sid mutant still maintains high levels of H3K4me3. 2) In addition, Spp1 maintains H3K4me3 levels, not by stimulating Set1 catalytic activity, but likely by binding H3K4me3 with its PHD finger. This can take place without interaction with the Set1 complex. We propose this may protect H3K4me3 from active demethylation, by the Jhd2 enzyme. Other possible explanations are described in the text. 3) Finally, the simultaneous binding of Spp1 to H3K4me3 and to the axis-associated Mer2 protein is essential to promote efficient DSB formation by Spo11. It has to be noted that a PHD finger mutant of Spp1 (W45A) is still able to bind Mer2 [ 18 ], so recognition of H3K4me3 by Spp1 PHD finger is not a prerequisite for its subsequent binding to Mer2. NDR: nucleosome-depleted region; Black circle: first nucleosome of genes; H3R2: arginine 2 of histone H3, in its non-asymmetrically methylated form. The blue square represents H3K4me3.

    Techniques Used: Mutagenesis, Activity Assay, Binding Assay, Methylation

    In the set1_sid mutant, Spp1 is still important to maintain H3K4me3 levels. (A) Histone H3K4 methylation levels in vegetatively growing cells detected by Western blot. Anti-Spp1, anti H3K4me2, anti-H3K4me3 or anti-Pgk1 antibodies were used, as indicated. A representative experiment is shown. WT: ORT4601; set1∆ : ORT4784; spp1∆ : VBH152; set1_sid : VBH1881; set1_sid spp1∆ : VBH1972; spp1W45A : VBH1419; set1_sid spp1W45A : VBH2021. The bar graph on the right indicates histone modification levels normalized to Pgk1 levels and relative to the WT strain. Values are the mean ± S.E.M. of the normalized relative levels from 3 to 6 replicates for each strain, except for spp1W45A , where only 2 replicates are available and the error bars indicate the range. See also S4 Table . (B) Histone H3K4me3 levels in vegetatively growing cells detected by ChIP at the highly transcribed ADH1 gene. Same strains as in A. Values are expressed as % of input DNA, and are the mean ± S.E.M. of six independent experiments.
    Figure Legend Snippet: In the set1_sid mutant, Spp1 is still important to maintain H3K4me3 levels. (A) Histone H3K4 methylation levels in vegetatively growing cells detected by Western blot. Anti-Spp1, anti H3K4me2, anti-H3K4me3 or anti-Pgk1 antibodies were used, as indicated. A representative experiment is shown. WT: ORT4601; set1∆ : ORT4784; spp1∆ : VBH152; set1_sid : VBH1881; set1_sid spp1∆ : VBH1972; spp1W45A : VBH1419; set1_sid spp1W45A : VBH2021. The bar graph on the right indicates histone modification levels normalized to Pgk1 levels and relative to the WT strain. Values are the mean ± S.E.M. of the normalized relative levels from 3 to 6 replicates for each strain, except for spp1W45A , where only 2 replicates are available and the error bars indicate the range. See also S4 Table . (B) Histone H3K4me3 levels in vegetatively growing cells detected by ChIP at the highly transcribed ADH1 gene. Same strains as in A. Values are expressed as % of input DNA, and are the mean ± S.E.M. of six independent experiments.

    Techniques Used: Mutagenesis, Methylation, Western Blot, Modification, Chromatin Immunoprecipitation

    Related Articles

    Clone Assay:

    Article Title: MeCP2 Reinforces STAT3 Signaling and the Generation of Effector CD4+ T Cells by Promoting miR-124-Mediated Suppression of SOCS5
    Article Snippet: Methylation analysis was quantified by sequencing of genomic DNA after bisulfite conversion with the MethylDetector kit (Active Motif), PCR amplification, and cloning. .. ChIP assays were performed with a standard protocol with anti-H3Ac, anti-H3K4me2, anti-H3K4me3, and anti-H3K27me3 rabbit polyclonal antibodies (Millipore), anti-MeCP2 (D4F3) XP rabbit monoclonal antibody (Cell Signaling), or a nonspecific rabbit anti-mouse immunoglobulin G (IgG, Jackson ImmunoResearch Laboratories).

    Amplification:

    Article Title: MeCP2 Reinforces STAT3 Signaling and the Generation of Effector CD4+ T Cells by Promoting miR-124-Mediated Suppression of SOCS5
    Article Snippet: Methylation analysis was quantified by sequencing of genomic DNA after bisulfite conversion with the MethylDetector kit (Active Motif), PCR amplification, and cloning. .. ChIP assays were performed with a standard protocol with anti-H3Ac, anti-H3K4me2, anti-H3K4me3, and anti-H3K27me3 rabbit polyclonal antibodies (Millipore), anti-MeCP2 (D4F3) XP rabbit monoclonal antibody (Cell Signaling), or a nonspecific rabbit anti-mouse immunoglobulin G (IgG, Jackson ImmunoResearch Laboratories).

    Article Title: Polycomb repressive complex 1 activities determine the columnar organization of motor neurons
    Article Snippet: Hox promoter regions were amplified using SYBR Green PCR master mix (Applied Biosystems) and detected by quantitative PCR. .. The following antibodies were used: rabbit anti-H3K27me3 (Millipore), rabbit anti-H3K4me3 (Millipore), mouse anti-Bmi1 (Millipore), and rabbit anti-mouse IgG (Millipore).

    Mouse Assay:

    Article Title: RXR controlled regulatory networks identified in mouse brain counteract deleterious effects of Aβ oligomers
    Article Snippet: Brains from APOE3 mice treated with bexarotene for 10 days and ES cells treated with bexarotene for 4 days were used for ChIP-qPCR and ChIP-seq. .. For immunoprecipitation, we used a rabbit polyclonal anti-RXR (ΔN 197, #sc-774, Santa Cruz, CA), rabbit polyclonal anti-H3K4me3 (#CS200580, Millipore, CA) and rabbit-polyclonal anti-H3K27me3 (#07-449, Millipore, CA).

    Cytometry:

    Article Title: Neuronal Kmt2a/Mll1 Histone Methyltransferase Is Essential for Prefrontal Synaptic Plasticity and Working Memory
    Article Snippet: Sorting was done on a FACSVantage SE flow cytometer. .. After samples were precleared with protein G agarose slurry, anti H3K4me3 antibody (rabbit polyclonal; Millipore) and ChIP dilution buffer (50 m m EDTA, 200 m m Tris, and 500 m m NaCl) were added and samples incubated for 16 h at 4°C under rotation.

    Immunostaining:

    Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity
    Article Snippet: Immunofluorescent staining was performed as described for tissue sections. .. We used the following primary antibodies for immunostaining: mouse monoclonal anti-Ctbp2 (1:500) (612044; BD Biosciences); anti-S100β (1:2,500) (S-2532; Sigma); anti-FLAG (1:1,000) (M2; Sigma); anti-Pax6 (1:500) (PAX6; Developmental Studies Hybridoma Bank); anti-Ring1B (1:1,000) (D139-3; MBL); rat monoclonal anti-GFP (1:2,000) (GF090R; Nacalai); guinea pig polyclonal anti-Thrβ2 (1:50) (Wako Pure Chemical Industries) ( ); anti-Chx10 (1:500) ( ); goat polyclonal anti–S-opsin (1:500) (sc-14363; Santa Cruz); anti-lamin B (1:500, C-20; Santa Cruz); rabbit polyclonal anti-Samd7 (1:10,000) (generated in this study); anti-rhodopsin (1:2,500) (LB-5597; LSL); anti–M-opsin (1:300) (AB5405; Millipore); anti-Pikachurin (1:500) (Wako Pure Chemical Industries) ( ); anti-Calbindin (1:1,000) (PC253L; Calbiochem); anti-Suz12 (1:500) (D39F6; Cell Signaling Technology); anti-H2AK119ub (1:500) (D27C4; Cell Signaling Technology); anti-H3K27me3 (1:500) (07-449; Millipore); and anti-H3K4me3 (1:500) ( ; Millipore) antibodies. .. Cy3-conjugated secondary antibodies (1:500) (Jackson ImmunoResearch Laboratories) or Alexa Fluor 488-conjugated secondary antibodies (1:500) (Sigma) were used.

    Electrophoresis:

    Article Title: The PHD finger protein Spp1 has distinct functions in the Set1 and the meiotic DSB formation complexes
    Article Snippet: One volume of 2 x SDS protein sample buffer was added and sample were denatured 10 min at 95°C before electrophoresis. .. Antibodies used were as follows: anti-H3K4me3 (MC315, Millipore, 1/5000); anti-H3K4me2 (07–030, Millipore, 1/5000); anti-PGK1 (Invitrogen, 1/20000); anti-HA (Roche, 12CA5, 1/750); anti-Myc (Santa Cruz, 9E10, 1/500); anti-Flag (Sigma, 1/1000); anti-TAP (Invitrogen, 1/2000); anti-Spp1 (rabbit polyclonal, 1/2000).

    Incubation:

    Article Title: Neuronal Kmt2a/Mll1 Histone Methyltransferase Is Essential for Prefrontal Synaptic Plasticity and Working Memory
    Article Snippet: Nuclei were swollen to release chromatin after addition of hypotonization buffer (0.2 m m EDTA, pH 8, containing PMSF, DTT, and benzamidine). .. After samples were precleared with protein G agarose slurry, anti H3K4me3 antibody (rabbit polyclonal; Millipore) and ChIP dilution buffer (50 m m EDTA, 200 m m Tris, and 500 m m NaCl) were added and samples incubated for 16 h at 4°C under rotation. .. Then, the DNA-protein-antibody complexes were captured with protein G agarose slurry under rotation.

    Article Title: Temperature Shift Alters DNA Methylation and Histone Modification Patterns in Gonadal Aromatase (cyp19a1) Gene in Species with Temperature-Dependent Sex Determination
    Article Snippet: Previously-frozen chromatin was thawed on ice, suspended in three volumes of dilution buffer, and pre-cleared with the addition of 20 μL of prepared Dynabeads Protein A and gentle rotation for 2 hrs at 4°C. .. After separating the chromatin from the beads, they were incubated overnight at 4°C with 2–4 μg of one of the following antibodies: rabbit polyclonal H3K4me3 (Millipore, 07–473), rabbit polyclonal H3K27me3 (Millipore, 07–449), mouse monoclonal RNA Polymerase II CTD repeat (Abcam, ab817), or rabbit polyclonal anti-mouse IgG (Abcam, ab46540) as a negative control. .. These antibodies were previously shown to be compatible with ChIP or ChIP-seq experiments [ – ] and the amount of antibodies required was optimized earlier using chromatin collected from turtle embryonic gonads.

    Article Title: CHD4 Has Oncogenic Functions in Initiating and Maintaining Epigenetic Suppression of Multiple Tumor Suppressor Genes
    Article Snippet: The recovered supernatants were incubated with specific antibodies or an isotype control IgG for 2 hr in the presence of herring sperm DNA and Protein A/G Magnetic beads (Thermo Fisher). .. These antibodies are anti-8OHdG (Millipore, MAB3560), anti-γH2AX (Millipore, 05-636) anti-CHD4 (Sigma, WH0001108M1), anti-DNMT1 (Sigma, D4692), anti-DNMT3A (Novusbio, NB120-13888), anti-DNMT3B (A rabbit polyclonal antibody was raised against a fusion protein containing residues 376–390 (ENKTRRRTADDSATS)), anti-EZH2 (Active Motif, 39875), anti-G9a (Perseus Proteomics Inc, PP-A8620A-00), anti-5mc (Diagenode, C15200081), anti-H3 (Millipore, 17-10046), anti-H3K27me3 (Millipore, 17-622), anti-H3K9me2 (Millipore, 05-1249), anti-H3K4me3 (Millipore, 17-614), and anti-H4K16ac (Millipore, 17-10101).

    Article Title: Recruitment of RAG1 and RAG2 to Chromatinized DNA during V(D)J Recombination
    Article Snippet: The pellets were then resuspended in radioimmunoprecipitation assay (RIPA) buffer containing 0.8 M NaCl and sonicated. .. The chromatin was incubated with anti-RAG1 or anti-RAG2 monoclonal antibody , anti-H3K4me3 antibody (Millipore), or rabbit IgG (Millipore), and immune complexes were isolated with protein A-agarose beads (Millipore). .. After reversal of cross-links and purification of the DNA (DNA Clean concentrator; Zymo), input and immunoprecipitated DNAs were quantitated by duplicate trials of TaqMan qPCR Qiagen HotStart Taq with a 3000 XP thermocycler (Stratagene).

    Article Title: Epigenetics and Sex Differences in the Brain: A Genome-Wide Comparison of Histone-3 Lysine-4 Trimethylation (H3K4me3) in Male and Female Mice
    Article Snippet: Nuclei were lysed after the addition of hypotonisation buffer (0.2 mM EDTA, 0.1mM Benzamidin, 0.1mM PMSF, and 1 mM DTT). .. Samples were pre-cleared with protein G agarose, and incubated with anti-H3K4me3 antibody (rabbit polyclonal, Millipore, #07–473) and ChIP dilution buffer (50 mM EDTA, 200 mM Tris and 500 mM NaCl) for 16 h at 4 °C under rotation. .. The antibody-DNA complexes were bound to protein G agarose beads, and the beads were washed with low salt, high salt, lithium chloride, and TE buffers.

    Article Title: Unique roles for histone H3K9me states in RNAi and heritable silencing of transcription
    Article Snippet: For H3K9me2, H3K9me3, H3K4me3, H3K36me3, H3K14ac, H4K16ac, RNA pol II, Chp1, and Flag-Ago1 ChIP, cells were fixed with 1% formaldehyde for 15 min. For Swi6 and other Flag-protein ChIP, we used a modified dual-crosslinking protocol in which cells were first incubated at 18 °C for 2 hr, resuspended in 5 ml of room temperature PBS, and fixed with 1.5 mM ethylene glycol bis-succinimidyl succinate (EGS, Thermo Scientific 21565) for 30 min, followed by addition of formaldehyde to 1% final concentration for another 30 min . .. For each IP, ~ 2 μg of antibody was pre-incubated with 30 μl of Invitrogen Dynabeads Protein A or Protein G: anti-H3K9me2 (Abcam 1220) with Protein A, anti-H3K4me3 (Millipore 04–745) with protein G, anti-H3K36me3 (Abcam 9050) with protein A, anti-H3K14ac (Millipore 07–353) with protein-A, anti-H4K16ac (Active Motif 39167) with protein A, anti-Chp1 (Abcam 18191) with protein A, anti-Flag (Sigma F1804) with protein G, anti-Swi6 (rabbit polyclonal) with protein A, anti-RNA pol II 8WG16 (BioLegend MMS-126R-500) with protein A.

    Article Title: The PHD finger protein Spp1 has distinct functions in the Set1 and the meiotic DSB formation complexes
    Article Snippet: The beads were heated at 95 °C for 10 min. For Mer2-Flag IP, Sigma Anti-FLAG M2 magnetic beads were added to the lysate and incubated overnight at 4°C. .. Antibodies used were as follows: anti-H3K4me3 (MC315, Millipore, 1/5000); anti-H3K4me2 (07–030, Millipore, 1/5000); anti-PGK1 (Invitrogen, 1/20000); anti-HA (Roche, 12CA5, 1/750); anti-Myc (Santa Cruz, 9E10, 1/500); anti-Flag (Sigma, 1/1000); anti-TAP (Invitrogen, 1/2000); anti-Spp1 (rabbit polyclonal, 1/2000).

    Modification:

    Article Title: Unique roles for histone H3K9me states in RNAi and heritable silencing of transcription
    Article Snippet: For H3K9me2, H3K9me3, H3K4me3, H3K36me3, H3K14ac, H4K16ac, RNA pol II, Chp1, and Flag-Ago1 ChIP, cells were fixed with 1% formaldehyde for 15 min. For Swi6 and other Flag-protein ChIP, we used a modified dual-crosslinking protocol in which cells were first incubated at 18 °C for 2 hr, resuspended in 5 ml of room temperature PBS, and fixed with 1.5 mM ethylene glycol bis-succinimidyl succinate (EGS, Thermo Scientific 21565) for 30 min, followed by addition of formaldehyde to 1% final concentration for another 30 min . .. For each IP, ~ 2 μg of antibody was pre-incubated with 30 μl of Invitrogen Dynabeads Protein A or Protein G: anti-H3K9me2 (Abcam 1220) with Protein A, anti-H3K4me3 (Millipore 04–745) with protein G, anti-H3K36me3 (Abcam 9050) with protein A, anti-H3K14ac (Millipore 07–353) with protein-A, anti-H4K16ac (Active Motif 39167) with protein A, anti-Chp1 (Abcam 18191) with protein A, anti-Flag (Sigma F1804) with protein G, anti-Swi6 (rabbit polyclonal) with protein A, anti-RNA pol II 8WG16 (BioLegend MMS-126R-500) with protein A.

    Western Blot:

    Article Title: The PHD finger protein Spp1 has distinct functions in the Set1 and the meiotic DSB formation complexes
    Article Snippet: Paragraph title: Co-immunoprecipitation and western blot ... Antibodies used were as follows: anti-H3K4me3 (MC315, Millipore, 1/5000); anti-H3K4me2 (07–030, Millipore, 1/5000); anti-PGK1 (Invitrogen, 1/20000); anti-HA (Roche, 12CA5, 1/750); anti-Myc (Santa Cruz, 9E10, 1/500); anti-Flag (Sigma, 1/1000); anti-TAP (Invitrogen, 1/2000); anti-Spp1 (rabbit polyclonal, 1/2000).

    Transfection:

    Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity
    Article Snippet: After 48 h of transfection, cells were fixed in 4% PFA/PBS for 10 min at room temperature. .. We used the following primary antibodies for immunostaining: mouse monoclonal anti-Ctbp2 (1:500) (612044; BD Biosciences); anti-S100β (1:2,500) (S-2532; Sigma); anti-FLAG (1:1,000) (M2; Sigma); anti-Pax6 (1:500) (PAX6; Developmental Studies Hybridoma Bank); anti-Ring1B (1:1,000) (D139-3; MBL); rat monoclonal anti-GFP (1:2,000) (GF090R; Nacalai); guinea pig polyclonal anti-Thrβ2 (1:50) (Wako Pure Chemical Industries) ( ); anti-Chx10 (1:500) ( ); goat polyclonal anti–S-opsin (1:500) (sc-14363; Santa Cruz); anti-lamin B (1:500, C-20; Santa Cruz); rabbit polyclonal anti-Samd7 (1:10,000) (generated in this study); anti-rhodopsin (1:2,500) (LB-5597; LSL); anti–M-opsin (1:300) (AB5405; Millipore); anti-Pikachurin (1:500) (Wako Pure Chemical Industries) ( ); anti-Calbindin (1:1,000) (PC253L; Calbiochem); anti-Suz12 (1:500) (D39F6; Cell Signaling Technology); anti-H2AK119ub (1:500) (D27C4; Cell Signaling Technology); anti-H3K27me3 (1:500) (07-449; Millipore); and anti-H3K4me3 (1:500) ( ; Millipore) antibodies.

    Sequencing:

    Article Title: RXR controlled regulatory networks identified in mouse brain counteract deleterious effects of Aβ oligomers
    Article Snippet: Paragraph title: Chromatin immunoprecipitation and sequencing (ChIP-seq) ... For immunoprecipitation, we used a rabbit polyclonal anti-RXR (ΔN 197, #sc-774, Santa Cruz, CA), rabbit polyclonal anti-H3K4me3 (#CS200580, Millipore, CA) and rabbit-polyclonal anti-H3K27me3 (#07-449, Millipore, CA).

    Article Title: MeCP2 Reinforces STAT3 Signaling and the Generation of Effector CD4+ T Cells by Promoting miR-124-Mediated Suppression of SOCS5
    Article Snippet: Methylation analysis was quantified by sequencing of genomic DNA after bisulfite conversion with the MethylDetector kit (Active Motif), PCR amplification, and cloning. .. ChIP assays were performed with a standard protocol with anti-H3Ac, anti-H3K4me2, anti-H3K4me3, and anti-H3K27me3 rabbit polyclonal antibodies (Millipore), anti-MeCP2 (D4F3) XP rabbit monoclonal antibody (Cell Signaling), or a nonspecific rabbit anti-mouse immunoglobulin G (IgG, Jackson ImmunoResearch Laboratories).

    Article Title: RAG Represents a Widespread Threat to the Lymphocyte Genome
    Article Snippet: Instead, we propose that an evolutionary response unfolded through gradual selection for mutations that deactivated dangerous ectopic cRSSs, though it is clear that this purifying process is not complete ( ). .. ChIP experiments (assayed either by qPCR or sequencing) were performed as described previously ( ) using custom monoclonal rabbit antibodies for RAG1 (mAb #23) and RAG2 (mAb #39) ( ) or a polyclonal rabbit antibody for H3K4me3 (Millipore, 07-475). .. Taqman qPCR primer and probe sequences can be found in or in .

    Article Title: Epigenetics and Sex Differences in the Brain: A Genome-Wide Comparison of Histone-3 Lysine-4 Trimethylation (H3K4me3) in Male and Female Mice
    Article Snippet: Paragraph title: Chromatin Immunoprecipitation and Deep Sequencing (ChIP-seq) ... Samples were pre-cleared with protein G agarose, and incubated with anti-H3K4me3 antibody (rabbit polyclonal, Millipore, #07–473) and ChIP dilution buffer (50 mM EDTA, 200 mM Tris and 500 mM NaCl) for 16 h at 4 °C under rotation.

    Article Title: The gene signature in CCAAT-enhancer-binding protein ? dysfunctional acute myeloid leukemia predicts responsiveness to histone deacetylase inhibitors
    Article Snippet: Paragraph title: Chromatin immunoprecipitation followed by sequencing or quantitative reverse transcriptase polymerase chain reaction ... Immunoprecipitation was performed using Protein G Dynabeads (Invitrogen), and 20 μg rabbit polyclonal IgG anti-ERα (Santa Cruz sc543X), 20 μg normal rabbit IgG control, 2 μg H3K4me3 (Millipore, 17-678), or 2 μg normal mouse IgG control.

    Ligation:

    Article Title: Extended Self-Renewal and Accelerated Reprogramming in the Absence of Kdm5b
    Article Snippet: The polyclonal H3K4me3 antibody ( ) was obtained from Millipore. .. Sonicated cell extracts were used for ChIP assays.

    Protease Inhibitor:

    Article Title: CHD4 Has Oncogenic Functions in Initiating and Maintaining Epigenetic Suppression of Multiple Tumor Suppressor Genes
    Article Snippet: These antibodies are anti-8OHdG (Millipore, MAB3560), anti-γH2AX (Millipore, 05-636) anti-CHD4 (Sigma, WH0001108M1), anti-DNMT1 (Sigma, D4692), anti-DNMT3A (Novusbio, NB120-13888), anti-DNMT3B (A rabbit polyclonal antibody was raised against a fusion protein containing residues 376–390 (ENKTRRRTADDSATS)), anti-EZH2 (Active Motif, 39875), anti-G9a (Perseus Proteomics Inc, PP-A8620A-00), anti-5mc (Diagenode, C15200081), anti-H3 (Millipore, 17-10046), anti-H3K27me3 (Millipore, 17-622), anti-H3K9me2 (Millipore, 05-1249), anti-H3K4me3 (Millipore, 17-614), and anti-H4K16ac (Millipore, 17-10101). .. These antibodies are anti-8OHdG (Millipore, MAB3560), anti-γH2AX (Millipore, 05-636) anti-CHD4 (Sigma, WH0001108M1), anti-DNMT1 (Sigma, D4692), anti-DNMT3A (Novusbio, NB120-13888), anti-DNMT3B (A rabbit polyclonal antibody was raised against a fusion protein containing residues 376–390 (ENKTRRRTADDSATS)), anti-EZH2 (Active Motif, 39875), anti-G9a (Perseus Proteomics Inc, PP-A8620A-00), anti-5mc (Diagenode, C15200081), anti-H3 (Millipore, 17-10046), anti-H3K27me3 (Millipore, 17-622), anti-H3K9me2 (Millipore, 05-1249), anti-H3K4me3 (Millipore, 17-614), and anti-H4K16ac (Millipore, 17-10101).

    SYBR Green Assay:

    Article Title: Polycomb repressive complex 1 activities determine the columnar organization of motor neurons
    Article Snippet: Hox promoter regions were amplified using SYBR Green PCR master mix (Applied Biosystems) and detected by quantitative PCR. .. The following antibodies were used: rabbit anti-H3K27me3 (Millipore), rabbit anti-H3K4me3 (Millipore), mouse anti-Bmi1 (Millipore), and rabbit anti-mouse IgG (Millipore).

    Magnetic Beads:

    Article Title: CHD4 Has Oncogenic Functions in Initiating and Maintaining Epigenetic Suppression of Multiple Tumor Suppressor Genes
    Article Snippet: The recovered supernatants were incubated with specific antibodies or an isotype control IgG for 2 hr in the presence of herring sperm DNA and Protein A/G Magnetic beads (Thermo Fisher). .. These antibodies are anti-8OHdG (Millipore, MAB3560), anti-γH2AX (Millipore, 05-636) anti-CHD4 (Sigma, WH0001108M1), anti-DNMT1 (Sigma, D4692), anti-DNMT3A (Novusbio, NB120-13888), anti-DNMT3B (A rabbit polyclonal antibody was raised against a fusion protein containing residues 376–390 (ENKTRRRTADDSATS)), anti-EZH2 (Active Motif, 39875), anti-G9a (Perseus Proteomics Inc, PP-A8620A-00), anti-5mc (Diagenode, C15200081), anti-H3 (Millipore, 17-10046), anti-H3K27me3 (Millipore, 17-622), anti-H3K9me2 (Millipore, 05-1249), anti-H3K4me3 (Millipore, 17-614), and anti-H4K16ac (Millipore, 17-10101).

    Article Title: The PHD finger protein Spp1 has distinct functions in the Set1 and the meiotic DSB formation complexes
    Article Snippet: The beads were heated at 95 °C for 10 min. For Mer2-Flag IP, Sigma Anti-FLAG M2 magnetic beads were added to the lysate and incubated overnight at 4°C. .. Antibodies used were as follows: anti-H3K4me3 (MC315, Millipore, 1/5000); anti-H3K4me2 (07–030, Millipore, 1/5000); anti-PGK1 (Invitrogen, 1/20000); anti-HA (Roche, 12CA5, 1/750); anti-Myc (Santa Cruz, 9E10, 1/500); anti-Flag (Sigma, 1/1000); anti-TAP (Invitrogen, 1/2000); anti-Spp1 (rabbit polyclonal, 1/2000).

    Cell Culture:

    Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity
    Article Snippet: HEK293 and U2OS cells were cultured in DMEM (Sigma) with 10% FCS. .. We used the following primary antibodies for immunostaining: mouse monoclonal anti-Ctbp2 (1:500) (612044; BD Biosciences); anti-S100β (1:2,500) (S-2532; Sigma); anti-FLAG (1:1,000) (M2; Sigma); anti-Pax6 (1:500) (PAX6; Developmental Studies Hybridoma Bank); anti-Ring1B (1:1,000) (D139-3; MBL); rat monoclonal anti-GFP (1:2,000) (GF090R; Nacalai); guinea pig polyclonal anti-Thrβ2 (1:50) (Wako Pure Chemical Industries) ( ); anti-Chx10 (1:500) ( ); goat polyclonal anti–S-opsin (1:500) (sc-14363; Santa Cruz); anti-lamin B (1:500, C-20; Santa Cruz); rabbit polyclonal anti-Samd7 (1:10,000) (generated in this study); anti-rhodopsin (1:2,500) (LB-5597; LSL); anti–M-opsin (1:300) (AB5405; Millipore); anti-Pikachurin (1:500) (Wako Pure Chemical Industries) ( ); anti-Calbindin (1:1,000) (PC253L; Calbiochem); anti-Suz12 (1:500) (D39F6; Cell Signaling Technology); anti-H2AK119ub (1:500) (D27C4; Cell Signaling Technology); anti-H3K27me3 (1:500) (07-449; Millipore); and anti-H3K4me3 (1:500) ( ; Millipore) antibodies.

    Generated:

    Article Title: RXR controlled regulatory networks identified in mouse brain counteract deleterious effects of Aβ oligomers
    Article Snippet: For immunoprecipitation, we used a rabbit polyclonal anti-RXR (ΔN 197, #sc-774, Santa Cruz, CA), rabbit polyclonal anti-H3K4me3 (#CS200580, Millipore, CA) and rabbit-polyclonal anti-H3K27me3 (#07-449, Millipore, CA). .. For ChIP validation we performed ChIP-qPCR with mouse Glucagon (Gcg ) as a negative control and determined Fold enrichment normalized to % input of negative control (target gene % input/negative control % input).

    Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity
    Article Snippet: Immunofluorescent staining was performed as described for tissue sections. .. We used the following primary antibodies for immunostaining: mouse monoclonal anti-Ctbp2 (1:500) (612044; BD Biosciences); anti-S100β (1:2,500) (S-2532; Sigma); anti-FLAG (1:1,000) (M2; Sigma); anti-Pax6 (1:500) (PAX6; Developmental Studies Hybridoma Bank); anti-Ring1B (1:1,000) (D139-3; MBL); rat monoclonal anti-GFP (1:2,000) (GF090R; Nacalai); guinea pig polyclonal anti-Thrβ2 (1:50) (Wako Pure Chemical Industries) ( ); anti-Chx10 (1:500) ( ); goat polyclonal anti–S-opsin (1:500) (sc-14363; Santa Cruz); anti-lamin B (1:500, C-20; Santa Cruz); rabbit polyclonal anti-Samd7 (1:10,000) (generated in this study); anti-rhodopsin (1:2,500) (LB-5597; LSL); anti–M-opsin (1:300) (AB5405; Millipore); anti-Pikachurin (1:500) (Wako Pure Chemical Industries) ( ); anti-Calbindin (1:1,000) (PC253L; Calbiochem); anti-Suz12 (1:500) (D39F6; Cell Signaling Technology); anti-H2AK119ub (1:500) (D27C4; Cell Signaling Technology); anti-H3K27me3 (1:500) (07-449; Millipore); and anti-H3K4me3 (1:500) ( ; Millipore) antibodies. .. Cy3-conjugated secondary antibodies (1:500) (Jackson ImmunoResearch Laboratories) or Alexa Fluor 488-conjugated secondary antibodies (1:500) (Sigma) were used.

    other:

    Article Title: Chd5 Requires PHD-mediated Histone 3 Binding for Tumor Suppression
    Article Snippet: Anti-Chd5 (Santa-cruz, M-182: sc-68389, and in-house raised polyclonal antibody Chd5–232), anti-H3K4me3 (Millipore, 07–473), anti-H3K9me3 (Millipore, 07–442), normal rabbit IgG (Cell signaling, 2729), anti-Histidine (Clontech, 631212), anti-β-actin (Sigma, A5441), and anti-MAP2 (Abcam, ab11267) were used.

    Polymerase Chain Reaction:

    Article Title: CHD4 Has Oncogenic Functions in Initiating and Maintaining Epigenetic Suppression of Multiple Tumor Suppressor Genes
    Article Snippet: These antibodies are anti-8OHdG (Millipore, MAB3560), anti-γH2AX (Millipore, 05-636) anti-CHD4 (Sigma, WH0001108M1), anti-DNMT1 (Sigma, D4692), anti-DNMT3A (Novusbio, NB120-13888), anti-DNMT3B (A rabbit polyclonal antibody was raised against a fusion protein containing residues 376–390 (ENKTRRRTADDSATS)), anti-EZH2 (Active Motif, 39875), anti-G9a (Perseus Proteomics Inc, PP-A8620A-00), anti-5mc (Diagenode, C15200081), anti-H3 (Millipore, 17-10046), anti-H3K27me3 (Millipore, 17-622), anti-H3K9me2 (Millipore, 05-1249), anti-H3K4me3 (Millipore, 17-614), and anti-H4K16ac (Millipore, 17-10101). .. The immunoprecipitated DNA was retrieved from the beads with 1% SDS and a 1.1MNaHCO3 solution at 65 °C for 6 hr.

    Article Title: Recruitment of RAG1 and RAG2 to Chromatinized DNA during V(D)J Recombination
    Article Snippet: The chromatin was incubated with anti-RAG1 or anti-RAG2 monoclonal antibody , anti-H3K4me3 antibody (Millipore), or rabbit IgG (Millipore), and immune complexes were isolated with protein A-agarose beads (Millipore). .. The chromatin was incubated with anti-RAG1 or anti-RAG2 monoclonal antibody , anti-H3K4me3 antibody (Millipore), or rabbit IgG (Millipore), and immune complexes were isolated with protein A-agarose beads (Millipore).

    Article Title: MeCP2 Reinforces STAT3 Signaling and the Generation of Effector CD4+ T Cells by Promoting miR-124-Mediated Suppression of SOCS5
    Article Snippet: Methylation analysis was quantified by sequencing of genomic DNA after bisulfite conversion with the MethylDetector kit (Active Motif), PCR amplification, and cloning. .. ChIP assays were performed with a standard protocol with anti-H3Ac, anti-H3K4me2, anti-H3K4me3, and anti-H3K27me3 rabbit polyclonal antibodies (Millipore), anti-MeCP2 (D4F3) XP rabbit monoclonal antibody (Cell Signaling), or a nonspecific rabbit anti-mouse immunoglobulin G (IgG, Jackson ImmunoResearch Laboratories).

    Article Title: Polycomb repressive complex 1 activities determine the columnar organization of motor neurons
    Article Snippet: Hox promoter regions were amplified using SYBR Green PCR master mix (Applied Biosystems) and detected by quantitative PCR. .. The following antibodies were used: rabbit anti-H3K27me3 (Millipore), rabbit anti-H3K4me3 (Millipore), mouse anti-Bmi1 (Millipore), and rabbit anti-mouse IgG (Millipore).

    Article Title: The gene signature in CCAAT-enhancer-binding protein ? dysfunctional acute myeloid leukemia predicts responsiveness to histone deacetylase inhibitors
    Article Snippet: Paragraph title: Chromatin immunoprecipitation followed by sequencing or quantitative reverse transcriptase polymerase chain reaction ... Immunoprecipitation was performed using Protein G Dynabeads (Invitrogen), and 20 μg rabbit polyclonal IgG anti-ERα (Santa Cruz sc543X), 20 μg normal rabbit IgG control, 2 μg H3K4me3 (Millipore, 17-678), or 2 μg normal mouse IgG control.

    Article Title: Histone methyltransferase MLL1 regulates MDR1 transcription and chemoresistance
    Article Snippet: Sheared chromatin was immunoprecipitated with anti-H3K4me3, rabbit IgG (Millipore) or no antibody overnight at 4°C with rotation. .. Purified DNA was subjected to PCR using MDR1 promoter-specific primers P (forward: 5’-ACCTGTTTCGCAGTTTCTCG-3’; reverse: 5’-CCTCTGCTTCTTTGAGCTTG-3’), MDR1 5’ coding region-specific primers T (forward: 5’-AACTCTGCCTTCGTGGAGAT-3’; reverse: 5’-ATCCATTCCGACCTGAAGAG-3’), GAPDH promoter-specific primers (provided in the kit), and HoxA7 promoter-specific primers (forward: 5’-GAGCCTCCAGGTCTTTTTCC-3’; reverse: 5’-ACACCCCCAGATTTACACCA-3’).

    Sonication:

    Article Title: RXR controlled regulatory networks identified in mouse brain counteract deleterious effects of Aβ oligomers
    Article Snippet: Brain lysates were sonicated with 3 pulses of 15 sec at amplitude 30, a 120 sec pause and 3 pulses of 15 sec at amplitude 40 using a Model 705 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA), to obtain fragments of 200–600 bp. .. For immunoprecipitation, we used a rabbit polyclonal anti-RXR (ΔN 197, #sc-774, Santa Cruz, CA), rabbit polyclonal anti-H3K4me3 (#CS200580, Millipore, CA) and rabbit-polyclonal anti-H3K27me3 (#07-449, Millipore, CA).

    Article Title: CHD4 Has Oncogenic Functions in Initiating and Maintaining Epigenetic Suppression of Multiple Tumor Suppressor Genes
    Article Snippet: Sonicated chromatin was diluted to a final SDS concentration of 0.1% and aliquots were rotated with antibody O/N at 4 °C. .. These antibodies are anti-8OHdG (Millipore, MAB3560), anti-γH2AX (Millipore, 05-636) anti-CHD4 (Sigma, WH0001108M1), anti-DNMT1 (Sigma, D4692), anti-DNMT3A (Novusbio, NB120-13888), anti-DNMT3B (A rabbit polyclonal antibody was raised against a fusion protein containing residues 376–390 (ENKTRRRTADDSATS)), anti-EZH2 (Active Motif, 39875), anti-G9a (Perseus Proteomics Inc, PP-A8620A-00), anti-5mc (Diagenode, C15200081), anti-H3 (Millipore, 17-10046), anti-H3K27me3 (Millipore, 17-622), anti-H3K9me2 (Millipore, 05-1249), anti-H3K4me3 (Millipore, 17-614), and anti-H4K16ac (Millipore, 17-10101).

    Article Title: Recruitment of RAG1 and RAG2 to Chromatinized DNA during V(D)J Recombination
    Article Snippet: The pellets were then resuspended in radioimmunoprecipitation assay (RIPA) buffer containing 0.8 M NaCl and sonicated. .. The chromatin was incubated with anti-RAG1 or anti-RAG2 monoclonal antibody , anti-H3K4me3 antibody (Millipore), or rabbit IgG (Millipore), and immune complexes were isolated with protein A-agarose beads (Millipore).

    Article Title: Extended Self-Renewal and Accelerated Reprogramming in the Absence of Kdm5b
    Article Snippet: The polyclonal H3K4me3 antibody ( ) was obtained from Millipore. .. The polyclonal H3K4me3 antibody ( ) was obtained from Millipore.

    Immunofluorescence:

    Article Title: Characterization of DXZ4 conservation in primates implies important functional roles for CTCF binding, array expression and tandem repeat organization on the X chromosome
    Article Snippet: Paragraph title: Immunofluorescence on interphase cells and metaphase chromosomes ... Rabbit polyclonal anti-H3K4me2 (07-030), anti-H3K4me3 (05-745), anti-CTCF (07-729), anti-H3K36me2 (07-274) and anti-acetyl-lysine (06-933) were all obtained from Millipore (Billerica, MA, USA).

    ChIP-sequencing:

    Article Title: Neuronal Kmt2a/Mll1 Histone Methyltransferase Is Essential for Prefrontal Synaptic Plasticity and Working Memory
    Article Snippet: Paragraph title: Nuclei sorting and ChIP-seq. ... After samples were precleared with protein G agarose slurry, anti H3K4me3 antibody (rabbit polyclonal; Millipore) and ChIP dilution buffer (50 m m EDTA, 200 m m Tris, and 500 m m NaCl) were added and samples incubated for 16 h at 4°C under rotation.

    Article Title: RXR controlled regulatory networks identified in mouse brain counteract deleterious effects of Aβ oligomers
    Article Snippet: Paragraph title: Chromatin immunoprecipitation and sequencing (ChIP-seq) ... For immunoprecipitation, we used a rabbit polyclonal anti-RXR (ΔN 197, #sc-774, Santa Cruz, CA), rabbit polyclonal anti-H3K4me3 (#CS200580, Millipore, CA) and rabbit-polyclonal anti-H3K27me3 (#07-449, Millipore, CA).

    Article Title: RAG Represents a Widespread Threat to the Lymphocyte Genome
    Article Snippet: Paragraph title: ChIP and ChIP-seq ... ChIP experiments (assayed either by qPCR or sequencing) were performed as described previously ( ) using custom monoclonal rabbit antibodies for RAG1 (mAb #23) and RAG2 (mAb #39) ( ) or a polyclonal rabbit antibody for H3K4me3 (Millipore, 07-475).

    Article Title: Epigenetics and Sex Differences in the Brain: A Genome-Wide Comparison of Histone-3 Lysine-4 Trimethylation (H3K4me3) in Male and Female Mice
    Article Snippet: Paragraph title: Chromatin Immunoprecipitation and Deep Sequencing (ChIP-seq) ... Samples were pre-cleared with protein G agarose, and incubated with anti-H3K4me3 antibody (rabbit polyclonal, Millipore, #07–473) and ChIP dilution buffer (50 mM EDTA, 200 mM Tris and 500 mM NaCl) for 16 h at 4 °C under rotation.

    Article Title: Unique roles for histone H3K9me states in RNAi and heritable silencing of transcription
    Article Snippet: For each IP, ~ 2 μg of antibody was pre-incubated with 30 μl of Invitrogen Dynabeads Protein A or Protein G: anti-H3K9me2 (Abcam 1220) with Protein A, anti-H3K4me3 (Millipore 04–745) with protein G, anti-H3K36me3 (Abcam 9050) with protein A, anti-H3K14ac (Millipore 07–353) with protein-A, anti-H4K16ac (Active Motif 39167) with protein A, anti-Chp1 (Abcam 18191) with protein A, anti-Flag (Sigma F1804) with protein G, anti-Swi6 (rabbit polyclonal) with protein A, anti-RNA pol II 8WG16 (BioLegend MMS-126R-500) with protein A. .. For IP with H3K9me3 antibody, Dynabeads M-280 Streptavidin beads were first incubated with 1 μg of anti-H3K9me3 (Diagenode C15500003), followed by blocking with 5 μM biotin.

    Article Title: The gene signature in CCAAT-enhancer-binding protein ? dysfunctional acute myeloid leukemia predicts responsiveness to histone deacetylase inhibitors
    Article Snippet: Chromatin immunoprecipitation followed by sequencing (ChIP-seq analysis) ( ) was performed in K562 C/EBPα-ER-expressing cells treated with 1 μM β-estradiol or a control ethanol vehicle, and in K562 ER control-expressing cells treated with 1 μM β-estradiol (100×106 cells). .. Immunoprecipitation was performed using Protein G Dynabeads (Invitrogen), and 20 μg rabbit polyclonal IgG anti-ERα (Santa Cruz sc543X), 20 μg normal rabbit IgG control, 2 μg H3K4me3 (Millipore, 17-678), or 2 μg normal mouse IgG control.

    Article Title: Extended Self-Renewal and Accelerated Reprogramming in the Absence of Kdm5b
    Article Snippet: Paragraph title: ChIP-Seq. ... The polyclonal H3K4me3 antibody ( ) was obtained from Millipore.

    Methylation:

    Article Title: MeCP2 Reinforces STAT3 Signaling and the Generation of Effector CD4+ T Cells by Promoting miR-124-Mediated Suppression of SOCS5
    Article Snippet: Methylation analysis was quantified by sequencing of genomic DNA after bisulfite conversion with the MethylDetector kit (Active Motif), PCR amplification, and cloning. .. ChIP assays were performed with a standard protocol with anti-H3Ac, anti-H3K4me2, anti-H3K4me3, and anti-H3K27me3 rabbit polyclonal antibodies (Millipore), anti-MeCP2 (D4F3) XP rabbit monoclonal antibody (Cell Signaling), or a nonspecific rabbit anti-mouse immunoglobulin G (IgG, Jackson ImmunoResearch Laboratories).

    Isolation:

    Article Title: Recruitment of RAG1 and RAG2 to Chromatinized DNA during V(D)J Recombination
    Article Snippet: The pellets were then resuspended in radioimmunoprecipitation assay (RIPA) buffer containing 0.8 M NaCl and sonicated. .. The chromatin was incubated with anti-RAG1 or anti-RAG2 monoclonal antibody , anti-H3K4me3 antibody (Millipore), or rabbit IgG (Millipore), and immune complexes were isolated with protein A-agarose beads (Millipore). .. After reversal of cross-links and purification of the DNA (DNA Clean concentrator; Zymo), input and immunoprecipitated DNAs were quantitated by duplicate trials of TaqMan qPCR Qiagen HotStart Taq with a 3000 XP thermocycler (Stratagene).

    Flow Cytometry:

    Article Title: Neuronal Kmt2a/Mll1 Histone Methyltransferase Is Essential for Prefrontal Synaptic Plasticity and Working Memory
    Article Snippet: Sorting was done on a FACSVantage SE flow cytometer. .. After samples were precleared with protein G agarose slurry, anti H3K4me3 antibody (rabbit polyclonal; Millipore) and ChIP dilution buffer (50 m m EDTA, 200 m m Tris, and 500 m m NaCl) were added and samples incubated for 16 h at 4°C under rotation.

    Size-exclusion Chromatography:

    Article Title: RXR controlled regulatory networks identified in mouse brain counteract deleterious effects of Aβ oligomers
    Article Snippet: Brain lysates were sonicated with 3 pulses of 15 sec at amplitude 30, a 120 sec pause and 3 pulses of 15 sec at amplitude 40 using a Model 705 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA), to obtain fragments of 200–600 bp. .. For immunoprecipitation, we used a rabbit polyclonal anti-RXR (ΔN 197, #sc-774, Santa Cruz, CA), rabbit polyclonal anti-H3K4me3 (#CS200580, Millipore, CA) and rabbit-polyclonal anti-H3K27me3 (#07-449, Millipore, CA).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: The gene signature in CCAAT-enhancer-binding protein ? dysfunctional acute myeloid leukemia predicts responsiveness to histone deacetylase inhibitors
    Article Snippet: ChIP followed by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was performed in K562 cells treated with trichostatin A (TSA) or ethanol control (10×106 cells). .. Immunoprecipitation was performed using Protein G Dynabeads (Invitrogen), and 20 μg rabbit polyclonal IgG anti-ERα (Santa Cruz sc543X), 20 μg normal rabbit IgG control, 2 μg H3K4me3 (Millipore, 17-678), or 2 μg normal mouse IgG control.

    Blocking Assay:

    Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity
    Article Snippet: We used the following primary antibodies for immunostaining: mouse monoclonal anti-Ctbp2 (1:500) (612044; BD Biosciences); anti-S100β (1:2,500) (S-2532; Sigma); anti-FLAG (1:1,000) (M2; Sigma); anti-Pax6 (1:500) (PAX6; Developmental Studies Hybridoma Bank); anti-Ring1B (1:1,000) (D139-3; MBL); rat monoclonal anti-GFP (1:2,000) (GF090R; Nacalai); guinea pig polyclonal anti-Thrβ2 (1:50) (Wako Pure Chemical Industries) ( ); anti-Chx10 (1:500) ( ); goat polyclonal anti–S-opsin (1:500) (sc-14363; Santa Cruz); anti-lamin B (1:500, C-20; Santa Cruz); rabbit polyclonal anti-Samd7 (1:10,000) (generated in this study); anti-rhodopsin (1:2,500) (LB-5597; LSL); anti–M-opsin (1:300) (AB5405; Millipore); anti-Pikachurin (1:500) (Wako Pure Chemical Industries) ( ); anti-Calbindin (1:1,000) (PC253L; Calbiochem); anti-Suz12 (1:500) (D39F6; Cell Signaling Technology); anti-H2AK119ub (1:500) (D27C4; Cell Signaling Technology); anti-H3K27me3 (1:500) (07-449; Millipore); and anti-H3K4me3 (1:500) ( ; Millipore) antibodies. .. We used the following primary antibodies for immunostaining: mouse monoclonal anti-Ctbp2 (1:500) (612044; BD Biosciences); anti-S100β (1:2,500) (S-2532; Sigma); anti-FLAG (1:1,000) (M2; Sigma); anti-Pax6 (1:500) (PAX6; Developmental Studies Hybridoma Bank); anti-Ring1B (1:1,000) (D139-3; MBL); rat monoclonal anti-GFP (1:2,000) (GF090R; Nacalai); guinea pig polyclonal anti-Thrβ2 (1:50) (Wako Pure Chemical Industries) ( ); anti-Chx10 (1:500) ( ); goat polyclonal anti–S-opsin (1:500) (sc-14363; Santa Cruz); anti-lamin B (1:500, C-20; Santa Cruz); rabbit polyclonal anti-Samd7 (1:10,000) (generated in this study); anti-rhodopsin (1:2,500) (LB-5597; LSL); anti–M-opsin (1:300) (AB5405; Millipore); anti-Pikachurin (1:500) (Wako Pure Chemical Industries) ( ); anti-Calbindin (1:1,000) (PC253L; Calbiochem); anti-Suz12 (1:500) (D39F6; Cell Signaling Technology); anti-H2AK119ub (1:500) (D27C4; Cell Signaling Technology); anti-H3K27me3 (1:500) (07-449; Millipore); and anti-H3K4me3 (1:500) ( ; Millipore) antibodies.

    Lysis:

    Article Title: CHD4 Has Oncogenic Functions in Initiating and Maintaining Epigenetic Suppression of Multiple Tumor Suppressor Genes
    Article Snippet: Cells were cross-linked in 1% formaldehyde at 37 °C for 10 min. After washing with PBS, the cells were resuspended in 300 μl of lysis buffer. .. These antibodies are anti-8OHdG (Millipore, MAB3560), anti-γH2AX (Millipore, 05-636) anti-CHD4 (Sigma, WH0001108M1), anti-DNMT1 (Sigma, D4692), anti-DNMT3A (Novusbio, NB120-13888), anti-DNMT3B (A rabbit polyclonal antibody was raised against a fusion protein containing residues 376–390 (ENKTRRRTADDSATS)), anti-EZH2 (Active Motif, 39875), anti-G9a (Perseus Proteomics Inc, PP-A8620A-00), anti-5mc (Diagenode, C15200081), anti-H3 (Millipore, 17-10046), anti-H3K27me3 (Millipore, 17-622), anti-H3K9me2 (Millipore, 05-1249), anti-H3K4me3 (Millipore, 17-614), and anti-H4K16ac (Millipore, 17-10101).

    Article Title: The PHD finger protein Spp1 has distinct functions in the Set1 and the meiotic DSB formation complexes
    Article Snippet: Beads were washed twice with lysis buffer, and eluted for 2 hrs at 4°C with 12.5 μg of Flag peptide in elution buffer (20 mM Tris pH8; 150 mM NaCl; 0.1% Tween; 10% Glycerol; 5mM MgCl2; 0.5 mM EDTA). .. Antibodies used were as follows: anti-H3K4me3 (MC315, Millipore, 1/5000); anti-H3K4me2 (07–030, Millipore, 1/5000); anti-PGK1 (Invitrogen, 1/20000); anti-HA (Roche, 12CA5, 1/750); anti-Myc (Santa Cruz, 9E10, 1/500); anti-Flag (Sigma, 1/1000); anti-TAP (Invitrogen, 1/2000); anti-Spp1 (rabbit polyclonal, 1/2000).

    Concentration Assay:

    Article Title: CHD4 Has Oncogenic Functions in Initiating and Maintaining Epigenetic Suppression of Multiple Tumor Suppressor Genes
    Article Snippet: Sonicated chromatin was diluted to a final SDS concentration of 0.1% and aliquots were rotated with antibody O/N at 4 °C. .. These antibodies are anti-8OHdG (Millipore, MAB3560), anti-γH2AX (Millipore, 05-636) anti-CHD4 (Sigma, WH0001108M1), anti-DNMT1 (Sigma, D4692), anti-DNMT3A (Novusbio, NB120-13888), anti-DNMT3B (A rabbit polyclonal antibody was raised against a fusion protein containing residues 376–390 (ENKTRRRTADDSATS)), anti-EZH2 (Active Motif, 39875), anti-G9a (Perseus Proteomics Inc, PP-A8620A-00), anti-5mc (Diagenode, C15200081), anti-H3 (Millipore, 17-10046), anti-H3K27me3 (Millipore, 17-622), anti-H3K9me2 (Millipore, 05-1249), anti-H3K4me3 (Millipore, 17-614), and anti-H4K16ac (Millipore, 17-10101).

    Article Title: Unique roles for histone H3K9me states in RNAi and heritable silencing of transcription
    Article Snippet: For H3K9me2, H3K9me3, H3K4me3, H3K36me3, H3K14ac, H4K16ac, RNA pol II, Chp1, and Flag-Ago1 ChIP, cells were fixed with 1% formaldehyde for 15 min. For Swi6 and other Flag-protein ChIP, we used a modified dual-crosslinking protocol in which cells were first incubated at 18 °C for 2 hr, resuspended in 5 ml of room temperature PBS, and fixed with 1.5 mM ethylene glycol bis-succinimidyl succinate (EGS, Thermo Scientific 21565) for 30 min, followed by addition of formaldehyde to 1% final concentration for another 30 min . .. For each IP, ~ 2 μg of antibody was pre-incubated with 30 μl of Invitrogen Dynabeads Protein A or Protein G: anti-H3K9me2 (Abcam 1220) with Protein A, anti-H3K4me3 (Millipore 04–745) with protein G, anti-H3K36me3 (Abcam 9050) with protein A, anti-H3K14ac (Millipore 07–353) with protein-A, anti-H4K16ac (Active Motif 39167) with protein A, anti-Chp1 (Abcam 18191) with protein A, anti-Flag (Sigma F1804) with protein G, anti-Swi6 (rabbit polyclonal) with protein A, anti-RNA pol II 8WG16 (BioLegend MMS-126R-500) with protein A.

    Agarose Gel Electrophoresis:

    Article Title: RXR controlled regulatory networks identified in mouse brain counteract deleterious effects of Aβ oligomers
    Article Snippet: For immunoprecipitation, we used a rabbit polyclonal anti-RXR (ΔN 197, #sc-774, Santa Cruz, CA), rabbit polyclonal anti-H3K4me3 (#CS200580, Millipore, CA) and rabbit-polyclonal anti-H3K27me3 (#07-449, Millipore, CA). .. For each steps, samples were purified by AMPure XP beads (Beckman Coulter, Brea, CA).

    Article Title: Histone methyltransferase MLL1 regulates MDR1 transcription and chemoresistance
    Article Snippet: Sheared chromatin was immunoprecipitated with anti-H3K4me3, rabbit IgG (Millipore) or no antibody overnight at 4°C with rotation. .. Purified DNA was subjected to PCR using MDR1 promoter-specific primers P (forward: 5’-ACCTGTTTCGCAGTTTCTCG-3’; reverse: 5’-CCTCTGCTTCTTTGAGCTTG-3’), MDR1 5’ coding region-specific primers T (forward: 5’-AACTCTGCCTTCGTGGAGAT-3’; reverse: 5’-ATCCATTCCGACCTGAAGAG-3’), GAPDH promoter-specific primers (provided in the kit), and HoxA7 promoter-specific primers (forward: 5’-GAGCCTCCAGGTCTTTTTCC-3’; reverse: 5’-ACACCCCCAGATTTACACCA-3’).

    Purification:

    Article Title: Neuronal Kmt2a/Mll1 Histone Methyltransferase Is Essential for Prefrontal Synaptic Plasticity and Working Memory
    Article Snippet: After samples were precleared with protein G agarose slurry, anti H3K4me3 antibody (rabbit polyclonal; Millipore) and ChIP dilution buffer (50 m m EDTA, 200 m m Tris, and 500 m m NaCl) were added and samples incubated for 16 h at 4°C under rotation. .. After samples were precleared with protein G agarose slurry, anti H3K4me3 antibody (rabbit polyclonal; Millipore) and ChIP dilution buffer (50 m m EDTA, 200 m m Tris, and 500 m m NaCl) were added and samples incubated for 16 h at 4°C under rotation.

    Article Title: RXR controlled regulatory networks identified in mouse brain counteract deleterious effects of Aβ oligomers
    Article Snippet: For immunoprecipitation, we used a rabbit polyclonal anti-RXR (ΔN 197, #sc-774, Santa Cruz, CA), rabbit polyclonal anti-H3K4me3 (#CS200580, Millipore, CA) and rabbit-polyclonal anti-H3K27me3 (#07-449, Millipore, CA). .. ChIP libraries were generated using TruSeq ChIP Sample Prep Kit (Illumina, San Diego, CA) following manufacturer’s protocols.

    Article Title: CHD4 Has Oncogenic Functions in Initiating and Maintaining Epigenetic Suppression of Multiple Tumor Suppressor Genes
    Article Snippet: These antibodies are anti-8OHdG (Millipore, MAB3560), anti-γH2AX (Millipore, 05-636) anti-CHD4 (Sigma, WH0001108M1), anti-DNMT1 (Sigma, D4692), anti-DNMT3A (Novusbio, NB120-13888), anti-DNMT3B (A rabbit polyclonal antibody was raised against a fusion protein containing residues 376–390 (ENKTRRRTADDSATS)), anti-EZH2 (Active Motif, 39875), anti-G9a (Perseus Proteomics Inc, PP-A8620A-00), anti-5mc (Diagenode, C15200081), anti-H3 (Millipore, 17-10046), anti-H3K27me3 (Millipore, 17-622), anti-H3K9me2 (Millipore, 05-1249), anti-H3K4me3 (Millipore, 17-614), and anti-H4K16ac (Millipore, 17-10101). .. The immunoprecipitated DNA was retrieved from the beads with 1% SDS and a 1.1MNaHCO3 solution at 65 °C for 6 hr.

    Article Title: MeCP2 Reinforces STAT3 Signaling and the Generation of Effector CD4+ T Cells by Promoting miR-124-Mediated Suppression of SOCS5
    Article Snippet: Genomic DNA was purified with GenElute Mammalian Genomic DNA Miniprep Kit (Sigma, Cat. G1N79). .. ChIP assays were performed with a standard protocol with anti-H3Ac, anti-H3K4me2, anti-H3K4me3, and anti-H3K27me3 rabbit polyclonal antibodies (Millipore), anti-MeCP2 (D4F3) XP rabbit monoclonal antibody (Cell Signaling), or a nonspecific rabbit anti-mouse immunoglobulin G (IgG, Jackson ImmunoResearch Laboratories).

    Article Title: Epigenetics and Sex Differences in the Brain: A Genome-Wide Comparison of Histone-3 Lysine-4 Trimethylation (H3K4me3) in Male and Female Mice
    Article Snippet: Samples were pre-cleared with protein G agarose, and incubated with anti-H3K4me3 antibody (rabbit polyclonal, Millipore, #07–473) and ChIP dilution buffer (50 mM EDTA, 200 mM Tris and 500 mM NaCl) for 16 h at 4 °C under rotation. .. The antibody-DNA complexes were eluted from the beads, and incubated with Proteinase K for protein digestion.

    Article Title: Polycomb repressive complex 1 activities determine the columnar organization of motor neurons
    Article Snippet: DNA was purified via QIAprep spin columns (Qiagen). .. The following antibodies were used: rabbit anti-H3K27me3 (Millipore), rabbit anti-H3K4me3 (Millipore), mouse anti-Bmi1 (Millipore), and rabbit anti-mouse IgG (Millipore).

    Chromatin Immunoprecipitation:

    Article Title: Histone H3K27me3 demethylases KDM6A and KDM6B modulate definitive endoderm differentiation from human ESCs by regulating WNT signaling pathway
    Article Snippet: Relative expression levels were normalized to GAPDH andcalculated using the 2−ΔCt method. .. For ChIP analysis, undifferentiated human ESCs or differentiated cells from knockdownor control were used for H3K27me3 or H3K4me3 or H3 ChIP using Imprint® Chromatin Immunoprecipitation Kit (Sigma) according to the manufacturer's instructions.The antibodies used are all polyclonal rabbit IgG and included anti-H3K27me3(Millipore), anti-H3K4me3 (Millipore) and anti-H3 (Abcam). .. Quantitative PCR was carriedout as above.

    Article Title: Neuronal Kmt2a/Mll1 Histone Methyltransferase Is Essential for Prefrontal Synaptic Plasticity and Working Memory
    Article Snippet: Nuclei were swollen to release chromatin after addition of hypotonization buffer (0.2 m m EDTA, pH 8, containing PMSF, DTT, and benzamidine). .. After samples were precleared with protein G agarose slurry, anti H3K4me3 antibody (rabbit polyclonal; Millipore) and ChIP dilution buffer (50 m m EDTA, 200 m m Tris, and 500 m m NaCl) were added and samples incubated for 16 h at 4°C under rotation. .. Then, the DNA-protein-antibody complexes were captured with protein G agarose slurry under rotation.

    Article Title: RXR controlled regulatory networks identified in mouse brain counteract deleterious effects of Aβ oligomers
    Article Snippet: Paragraph title: Chromatin immunoprecipitation and sequencing (ChIP-seq) ... For immunoprecipitation, we used a rabbit polyclonal anti-RXR (ΔN 197, #sc-774, Santa Cruz, CA), rabbit polyclonal anti-H3K4me3 (#CS200580, Millipore, CA) and rabbit-polyclonal anti-H3K27me3 (#07-449, Millipore, CA).

    Article Title: Temperature Shift Alters DNA Methylation and Histone Modification Patterns in Gonadal Aromatase (cyp19a1) Gene in Species with Temperature-Dependent Sex Determination
    Article Snippet: Paragraph title: Chromatin immunoprecipitation (ChIP) assay ... After separating the chromatin from the beads, they were incubated overnight at 4°C with 2–4 μg of one of the following antibodies: rabbit polyclonal H3K4me3 (Millipore, 07–473), rabbit polyclonal H3K27me3 (Millipore, 07–449), mouse monoclonal RNA Polymerase II CTD repeat (Abcam, ab817), or rabbit polyclonal anti-mouse IgG (Abcam, ab46540) as a negative control.

    Article Title: CHD4 Has Oncogenic Functions in Initiating and Maintaining Epigenetic Suppression of Multiple Tumor Suppressor Genes
    Article Snippet: Paragraph title: Chromatin Immunoprecipitation Assay (ChIP) ... These antibodies are anti-8OHdG (Millipore, MAB3560), anti-γH2AX (Millipore, 05-636) anti-CHD4 (Sigma, WH0001108M1), anti-DNMT1 (Sigma, D4692), anti-DNMT3A (Novusbio, NB120-13888), anti-DNMT3B (A rabbit polyclonal antibody was raised against a fusion protein containing residues 376–390 (ENKTRRRTADDSATS)), anti-EZH2 (Active Motif, 39875), anti-G9a (Perseus Proteomics Inc, PP-A8620A-00), anti-5mc (Diagenode, C15200081), anti-H3 (Millipore, 17-10046), anti-H3K27me3 (Millipore, 17-622), anti-H3K9me2 (Millipore, 05-1249), anti-H3K4me3 (Millipore, 17-614), and anti-H4K16ac (Millipore, 17-10101).

    Article Title: Recruitment of RAG1 and RAG2 to Chromatinized DNA during V(D)J Recombination
    Article Snippet: Paragraph title: ChIP. ... The chromatin was incubated with anti-RAG1 or anti-RAG2 monoclonal antibody , anti-H3K4me3 antibody (Millipore), or rabbit IgG (Millipore), and immune complexes were isolated with protein A-agarose beads (Millipore).

    Article Title: MeCP2 Reinforces STAT3 Signaling and the Generation of Effector CD4+ T Cells by Promoting miR-124-Mediated Suppression of SOCS5
    Article Snippet: Methylation analysis was quantified by sequencing of genomic DNA after bisulfite conversion with the MethylDetector kit (Active Motif), PCR amplification, and cloning. .. ChIP assays were performed with a standard protocol with anti-H3Ac, anti-H3K4me2, anti-H3K4me3, and anti-H3K27me3 rabbit polyclonal antibodies (Millipore), anti-MeCP2 (D4F3) XP rabbit monoclonal antibody (Cell Signaling), or a nonspecific rabbit anti-mouse immunoglobulin G (IgG, Jackson ImmunoResearch Laboratories). .. The amount of DNA immunoprecipitated by antibodies was quantified by quantitative PCR with primers specific for the indicated gene regulatory regions and normalized to the amount of input DNA before immunoprecipitation.

    Article Title: RAG Represents a Widespread Threat to the Lymphocyte Genome
    Article Snippet: Instead, we propose that an evolutionary response unfolded through gradual selection for mutations that deactivated dangerous ectopic cRSSs, though it is clear that this purifying process is not complete ( ). .. ChIP experiments (assayed either by qPCR or sequencing) were performed as described previously ( ) using custom monoclonal rabbit antibodies for RAG1 (mAb #23) and RAG2 (mAb #39) ( ) or a polyclonal rabbit antibody for H3K4me3 (Millipore, 07-475). .. Taqman qPCR primer and probe sequences can be found in or in .

    Article Title: Epigenetics and Sex Differences in the Brain: A Genome-Wide Comparison of Histone-3 Lysine-4 Trimethylation (H3K4me3) in Male and Female Mice
    Article Snippet: Nuclei were lysed after the addition of hypotonisation buffer (0.2 mM EDTA, 0.1mM Benzamidin, 0.1mM PMSF, and 1 mM DTT). .. Samples were pre-cleared with protein G agarose, and incubated with anti-H3K4me3 antibody (rabbit polyclonal, Millipore, #07–473) and ChIP dilution buffer (50 mM EDTA, 200 mM Tris and 500 mM NaCl) for 16 h at 4 °C under rotation. .. The antibody-DNA complexes were bound to protein G agarose beads, and the beads were washed with low salt, high salt, lithium chloride, and TE buffers.

    Article Title: Unique roles for histone H3K9me states in RNAi and heritable silencing of transcription
    Article Snippet: Paragraph title: Chromatin Immunoprecipitation (ChIP) ... For each IP, ~ 2 μg of antibody was pre-incubated with 30 μl of Invitrogen Dynabeads Protein A or Protein G: anti-H3K9me2 (Abcam 1220) with Protein A, anti-H3K4me3 (Millipore 04–745) with protein G, anti-H3K36me3 (Abcam 9050) with protein A, anti-H3K14ac (Millipore 07–353) with protein-A, anti-H4K16ac (Active Motif 39167) with protein A, anti-Chp1 (Abcam 18191) with protein A, anti-Flag (Sigma F1804) with protein G, anti-Swi6 (rabbit polyclonal) with protein A, anti-RNA pol II 8WG16 (BioLegend MMS-126R-500) with protein A.

    Article Title: Polycomb repressive complex 1 activities determine the columnar organization of motor neurons
    Article Snippet: Paragraph title: ChIP assays ... The following antibodies were used: rabbit anti-H3K27me3 (Millipore), rabbit anti-H3K4me3 (Millipore), mouse anti-Bmi1 (Millipore), and rabbit anti-mouse IgG (Millipore).

    Article Title: The gene signature in CCAAT-enhancer-binding protein ? dysfunctional acute myeloid leukemia predicts responsiveness to histone deacetylase inhibitors
    Article Snippet: Paragraph title: Chromatin immunoprecipitation followed by sequencing or quantitative reverse transcriptase polymerase chain reaction ... Immunoprecipitation was performed using Protein G Dynabeads (Invitrogen), and 20 μg rabbit polyclonal IgG anti-ERα (Santa Cruz sc543X), 20 μg normal rabbit IgG control, 2 μg H3K4me3 (Millipore, 17-678), or 2 μg normal mouse IgG control.

    Article Title: Histone methyltransferase MLL1 regulates MDR1 transcription and chemoresistance
    Article Snippet: Paragraph title: Chromatin Immunoprecipitation (ChIP) ... Sheared chromatin was immunoprecipitated with anti-H3K4me3, rabbit IgG (Millipore) or no antibody overnight at 4°C with rotation.

    Article Title: NF-κB regulates PD-1 expression in macrophages
    Article Snippet: Paragraph title: Chromatin Immunoprecipitation ... 5 µg of chromatin was immunoprecipitated with Protein A beads using 0.5µg of antibodies for control IgG (rabbit polyclonal antibody, EMD Millipore, Billerica, MA), NF-κB p65 (clone sc-372, Santa Cruz Biotechnology Inc., Santa Cruz, CA), H3K4me1 (rabbit polyclonal antibody, EMD Millipore, Billerica, MA), H3K4me3 (rabbit polyclonal antibody, EMD Millipore, Billerica, MA), or H3K27ac (rabbit polyclonal antibody, EMD Millipore, Billerica, MA).

    Article Title: Extended Self-Renewal and Accelerated Reprogramming in the Absence of Kdm5b
    Article Snippet: The polyclonal H3K4me3 antibody ( ) was obtained from Millipore. .. The polyclonal H3K4me3 antibody ( ) was obtained from Millipore.

    Plasmid Preparation:

    Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity
    Article Snippet: We used the following primary antibodies for immunostaining: mouse monoclonal anti-Ctbp2 (1:500) (612044; BD Biosciences); anti-S100β (1:2,500) (S-2532; Sigma); anti-FLAG (1:1,000) (M2; Sigma); anti-Pax6 (1:500) (PAX6; Developmental Studies Hybridoma Bank); anti-Ring1B (1:1,000) (D139-3; MBL); rat monoclonal anti-GFP (1:2,000) (GF090R; Nacalai); guinea pig polyclonal anti-Thrβ2 (1:50) (Wako Pure Chemical Industries) ( ); anti-Chx10 (1:500) ( ); goat polyclonal anti–S-opsin (1:500) (sc-14363; Santa Cruz); anti-lamin B (1:500, C-20; Santa Cruz); rabbit polyclonal anti-Samd7 (1:10,000) (generated in this study); anti-rhodopsin (1:2,500) (LB-5597; LSL); anti–M-opsin (1:300) (AB5405; Millipore); anti-Pikachurin (1:500) (Wako Pure Chemical Industries) ( ); anti-Calbindin (1:1,000) (PC253L; Calbiochem); anti-Suz12 (1:500) (D39F6; Cell Signaling Technology); anti-H2AK119ub (1:500) (D27C4; Cell Signaling Technology); anti-H3K27me3 (1:500) (07-449; Millipore); and anti-H3K4me3 (1:500) ( ; Millipore) antibodies. .. Cy3-conjugated secondary antibodies (1:500) (Jackson ImmunoResearch Laboratories) or Alexa Fluor 488-conjugated secondary antibodies (1:500) (Sigma) were used.

    Software:

    Article Title: Characterization of DXZ4 conservation in primates implies important functional roles for CTCF binding, array expression and tandem repeat organization on the X chromosome
    Article Snippet: Rabbit polyclonal anti-H3K4me2 (07-030), anti-H3K4me3 (05-745), anti-CTCF (07-729), anti-H3K36me2 (07-274) and anti-acetyl-lysine (06-933) were all obtained from Millipore (Billerica, MA, USA). .. Cell staining and preparation of metaphase chromosomes was performed essentially as described [ ].

    Article Title: The PHD finger protein Spp1 has distinct functions in the Set1 and the meiotic DSB formation complexes
    Article Snippet: Antibodies used were as follows: anti-H3K4me3 (MC315, Millipore, 1/5000); anti-H3K4me2 (07–030, Millipore, 1/5000); anti-PGK1 (Invitrogen, 1/20000); anti-HA (Roche, 12CA5, 1/750); anti-Myc (Santa Cruz, 9E10, 1/500); anti-Flag (Sigma, 1/1000); anti-TAP (Invitrogen, 1/2000); anti-Spp1 (rabbit polyclonal, 1/2000). .. Antibodies used were as follows: anti-H3K4me3 (MC315, Millipore, 1/5000); anti-H3K4me2 (07–030, Millipore, 1/5000); anti-PGK1 (Invitrogen, 1/20000); anti-HA (Roche, 12CA5, 1/750); anti-Myc (Santa Cruz, 9E10, 1/500); anti-Flag (Sigma, 1/1000); anti-TAP (Invitrogen, 1/2000); anti-Spp1 (rabbit polyclonal, 1/2000).

    Real-time Polymerase Chain Reaction:

    Article Title: RAG Represents a Widespread Threat to the Lymphocyte Genome
    Article Snippet: Instead, we propose that an evolutionary response unfolded through gradual selection for mutations that deactivated dangerous ectopic cRSSs, though it is clear that this purifying process is not complete ( ). .. ChIP experiments (assayed either by qPCR or sequencing) were performed as described previously ( ) using custom monoclonal rabbit antibodies for RAG1 (mAb #23) and RAG2 (mAb #39) ( ) or a polyclonal rabbit antibody for H3K4me3 (Millipore, 07-475). .. Taqman qPCR primer and probe sequences can be found in or in .

    Article Title: Unique roles for histone H3K9me states in RNAi and heritable silencing of transcription
    Article Snippet: For each IP, ~ 2 μg of antibody was pre-incubated with 30 μl of Invitrogen Dynabeads Protein A or Protein G: anti-H3K9me2 (Abcam 1220) with Protein A, anti-H3K4me3 (Millipore 04–745) with protein G, anti-H3K36me3 (Abcam 9050) with protein A, anti-H3K14ac (Millipore 07–353) with protein-A, anti-H4K16ac (Active Motif 39167) with protein A, anti-Chp1 (Abcam 18191) with protein A, anti-Flag (Sigma F1804) with protein G, anti-Swi6 (rabbit polyclonal) with protein A, anti-RNA pol II 8WG16 (BioLegend MMS-126R-500) with protein A. .. All (500 μl for ChIP-seq) or one fifth (100 μl for ChIP-qPCR) of sheared chromatin lysate was added to the antibody-bead mixture and incubated for 2 hr at 4 °C on a rotating device.

    Article Title: Polycomb repressive complex 1 activities determine the columnar organization of motor neurons
    Article Snippet: Hox promoter regions were amplified using SYBR Green PCR master mix (Applied Biosystems) and detected by quantitative PCR. .. The following antibodies were used: rabbit anti-H3K27me3 (Millipore), rabbit anti-H3K4me3 (Millipore), mouse anti-Bmi1 (Millipore), and rabbit anti-mouse IgG (Millipore).

    Negative Control:

    Article Title: Temperature Shift Alters DNA Methylation and Histone Modification Patterns in Gonadal Aromatase (cyp19a1) Gene in Species with Temperature-Dependent Sex Determination
    Article Snippet: Previously-frozen chromatin was thawed on ice, suspended in three volumes of dilution buffer, and pre-cleared with the addition of 20 μL of prepared Dynabeads Protein A and gentle rotation for 2 hrs at 4°C. .. After separating the chromatin from the beads, they were incubated overnight at 4°C with 2–4 μg of one of the following antibodies: rabbit polyclonal H3K4me3 (Millipore, 07–473), rabbit polyclonal H3K27me3 (Millipore, 07–449), mouse monoclonal RNA Polymerase II CTD repeat (Abcam, ab817), or rabbit polyclonal anti-mouse IgG (Abcam, ab46540) as a negative control. .. These antibodies were previously shown to be compatible with ChIP or ChIP-seq experiments [ – ] and the amount of antibodies required was optimized earlier using chromatin collected from turtle embryonic gonads.

    Sample Prep:

    Article Title: RXR controlled regulatory networks identified in mouse brain counteract deleterious effects of Aβ oligomers
    Article Snippet: For immunoprecipitation, we used a rabbit polyclonal anti-RXR (ΔN 197, #sc-774, Santa Cruz, CA), rabbit polyclonal anti-H3K4me3 (#CS200580, Millipore, CA) and rabbit-polyclonal anti-H3K27me3 (#07-449, Millipore, CA). .. For ChIP validation we performed ChIP-qPCR with mouse Glucagon (Gcg ) as a negative control and determined Fold enrichment normalized to % input of negative control (target gene % input/negative control % input).

    Radio Immunoprecipitation:

    Article Title: Recruitment of RAG1 and RAG2 to Chromatinized DNA during V(D)J Recombination
    Article Snippet: The pellets were then resuspended in radioimmunoprecipitation assay (RIPA) buffer containing 0.8 M NaCl and sonicated. .. The chromatin was incubated with anti-RAG1 or anti-RAG2 monoclonal antibody , anti-H3K4me3 antibody (Millipore), or rabbit IgG (Millipore), and immune complexes were isolated with protein A-agarose beads (Millipore).

    DNA Methylation Assay:

    Article Title: MeCP2 Reinforces STAT3 Signaling and the Generation of Effector CD4+ T Cells by Promoting miR-124-Mediated Suppression of SOCS5
    Article Snippet: Paragraph title: ChIP assay and DNA methylation analysis ... ChIP assays were performed with a standard protocol with anti-H3Ac, anti-H3K4me2, anti-H3K4me3, and anti-H3K27me3 rabbit polyclonal antibodies (Millipore), anti-MeCP2 (D4F3) XP rabbit monoclonal antibody (Cell Signaling), or a nonspecific rabbit anti-mouse immunoglobulin G (IgG, Jackson ImmunoResearch Laboratories).

    Produced:

    Article Title: Viral Reprogramming of the Daxx Histone H3.3 Chaperone during Early Epstein-Barr Virus Infection
    Article Snippet: Rabbit anti-histones H3.3 (catalog no. 09-838), pan-H3 (catalog no. 07-690), and H3K4me3 (catalog no. 07-473) were purchased from Millipore. .. Rabbit anti-histones H3.3 (catalog no. 09-838), pan-H3 (catalog no. 07-690), and H3K4me3 (catalog no. 07-473) were purchased from Millipore.

    Immunoprecipitation:

    Article Title: RXR controlled regulatory networks identified in mouse brain counteract deleterious effects of Aβ oligomers
    Article Snippet: Brain lysates were sonicated with 3 pulses of 15 sec at amplitude 30, a 120 sec pause and 3 pulses of 15 sec at amplitude 40 using a Model 705 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA), to obtain fragments of 200–600 bp. .. For immunoprecipitation, we used a rabbit polyclonal anti-RXR (ΔN 197, #sc-774, Santa Cruz, CA), rabbit polyclonal anti-H3K4me3 (#CS200580, Millipore, CA) and rabbit-polyclonal anti-H3K27me3 (#07-449, Millipore, CA). .. For ChIP validation we performed ChIP-qPCR with mouse Glucagon (Gcg ) as a negative control and determined Fold enrichment normalized to % input of negative control (target gene % input/negative control % input).

    Article Title: CHD4 Has Oncogenic Functions in Initiating and Maintaining Epigenetic Suppression of Multiple Tumor Suppressor Genes
    Article Snippet: These antibodies are anti-8OHdG (Millipore, MAB3560), anti-γH2AX (Millipore, 05-636) anti-CHD4 (Sigma, WH0001108M1), anti-DNMT1 (Sigma, D4692), anti-DNMT3A (Novusbio, NB120-13888), anti-DNMT3B (A rabbit polyclonal antibody was raised against a fusion protein containing residues 376–390 (ENKTRRRTADDSATS)), anti-EZH2 (Active Motif, 39875), anti-G9a (Perseus Proteomics Inc, PP-A8620A-00), anti-5mc (Diagenode, C15200081), anti-H3 (Millipore, 17-10046), anti-H3K27me3 (Millipore, 17-622), anti-H3K9me2 (Millipore, 05-1249), anti-H3K4me3 (Millipore, 17-614), and anti-H4K16ac (Millipore, 17-10101). .. These antibodies are anti-8OHdG (Millipore, MAB3560), anti-γH2AX (Millipore, 05-636) anti-CHD4 (Sigma, WH0001108M1), anti-DNMT1 (Sigma, D4692), anti-DNMT3A (Novusbio, NB120-13888), anti-DNMT3B (A rabbit polyclonal antibody was raised against a fusion protein containing residues 376–390 (ENKTRRRTADDSATS)), anti-EZH2 (Active Motif, 39875), anti-G9a (Perseus Proteomics Inc, PP-A8620A-00), anti-5mc (Diagenode, C15200081), anti-H3 (Millipore, 17-10046), anti-H3K27me3 (Millipore, 17-622), anti-H3K9me2 (Millipore, 05-1249), anti-H3K4me3 (Millipore, 17-614), and anti-H4K16ac (Millipore, 17-10101).

    Article Title: Recruitment of RAG1 and RAG2 to Chromatinized DNA during V(D)J Recombination
    Article Snippet: The chromatin was incubated with anti-RAG1 or anti-RAG2 monoclonal antibody , anti-H3K4me3 antibody (Millipore), or rabbit IgG (Millipore), and immune complexes were isolated with protein A-agarose beads (Millipore). .. After reversal of cross-links and purification of the DNA (DNA Clean concentrator; Zymo), input and immunoprecipitated DNAs were quantitated by duplicate trials of TaqMan qPCR Qiagen HotStart Taq with a 3000 XP thermocycler (Stratagene).

    Article Title: MeCP2 Reinforces STAT3 Signaling and the Generation of Effector CD4+ T Cells by Promoting miR-124-Mediated Suppression of SOCS5
    Article Snippet: ChIP assays were performed with a standard protocol with anti-H3Ac, anti-H3K4me2, anti-H3K4me3, and anti-H3K27me3 rabbit polyclonal antibodies (Millipore), anti-MeCP2 (D4F3) XP rabbit monoclonal antibody (Cell Signaling), or a nonspecific rabbit anti-mouse immunoglobulin G (IgG, Jackson ImmunoResearch Laboratories). .. The amount of DNA immunoprecipitated by antibodies was quantified by quantitative PCR with primers specific for the indicated gene regulatory regions and normalized to the amount of input DNA before immunoprecipitation.

    Article Title: Unique roles for histone H3K9me states in RNAi and heritable silencing of transcription
    Article Snippet: For each IP, ~ 2 μg of antibody was pre-incubated with 30 μl of Invitrogen Dynabeads Protein A or Protein G: anti-H3K9me2 (Abcam 1220) with Protein A, anti-H3K4me3 (Millipore 04–745) with protein G, anti-H3K36me3 (Abcam 9050) with protein A, anti-H3K14ac (Millipore 07–353) with protein-A, anti-H4K16ac (Active Motif 39167) with protein A, anti-Chp1 (Abcam 18191) with protein A, anti-Flag (Sigma F1804) with protein G, anti-Swi6 (rabbit polyclonal) with protein A, anti-RNA pol II 8WG16 (BioLegend MMS-126R-500) with protein A. .. For each IP, ~ 2 μg of antibody was pre-incubated with 30 μl of Invitrogen Dynabeads Protein A or Protein G: anti-H3K9me2 (Abcam 1220) with Protein A, anti-H3K4me3 (Millipore 04–745) with protein G, anti-H3K36me3 (Abcam 9050) with protein A, anti-H3K14ac (Millipore 07–353) with protein-A, anti-H4K16ac (Active Motif 39167) with protein A, anti-Chp1 (Abcam 18191) with protein A, anti-Flag (Sigma F1804) with protein G, anti-Swi6 (rabbit polyclonal) with protein A, anti-RNA pol II 8WG16 (BioLegend MMS-126R-500) with protein A.

    Article Title: The gene signature in CCAAT-enhancer-binding protein ? dysfunctional acute myeloid leukemia predicts responsiveness to histone deacetylase inhibitors
    Article Snippet: Cells were treated for 6 h prior to formaldehyde fixation. .. Immunoprecipitation was performed using Protein G Dynabeads (Invitrogen), and 20 μg rabbit polyclonal IgG anti-ERα (Santa Cruz sc543X), 20 μg normal rabbit IgG control, 2 μg H3K4me3 (Millipore, 17-678), or 2 μg normal mouse IgG control. .. For ChIP-seq the purified ChIP DNA was used to construct ChIP-seq libraries with the Illumina ChIP-seq sample preparation kit (Illumina), as indicated by manufacturer.

    Article Title: Histone methyltransferase MLL1 regulates MDR1 transcription and chemoresistance
    Article Snippet: Basically, chromatin crosslinked with 0.5% formaldehyde was sheared to an average length of 500 bp by sonicating 10 times with 10-sec pulse followed by 60-sec recovery period at a setting of 5 in a Fisher Scientific 550 Sonic Dismembrator. .. Sheared chromatin was immunoprecipitated with anti-H3K4me3, rabbit IgG (Millipore) or no antibody overnight at 4°C with rotation. .. Purified DNA was subjected to PCR using MDR1 promoter-specific primers P (forward: 5’-ACCTGTTTCGCAGTTTCTCG-3’; reverse: 5’-CCTCTGCTTCTTTGAGCTTG-3’), MDR1 5’ coding region-specific primers T (forward: 5’-AACTCTGCCTTCGTGGAGAT-3’; reverse: 5’-ATCCATTCCGACCTGAAGAG-3’), GAPDH promoter-specific primers (provided in the kit), and HoxA7 promoter-specific primers (forward: 5’-GAGCCTCCAGGTCTTTTTCC-3’; reverse: 5’-ACACCCCCAGATTTACACCA-3’).

    Article Title: NF-κB regulates PD-1 expression in macrophages
    Article Snippet: Chromatin was prepared from RAW 264.7 cells treated with LPS for 4 hours and crosslinked in 1% formaldehyde for 10 minutes. .. 5 µg of chromatin was immunoprecipitated with Protein A beads using 0.5µg of antibodies for control IgG (rabbit polyclonal antibody, EMD Millipore, Billerica, MA), NF-κB p65 (clone sc-372, Santa Cruz Biotechnology Inc., Santa Cruz, CA), H3K4me1 (rabbit polyclonal antibody, EMD Millipore, Billerica, MA), H3K4me3 (rabbit polyclonal antibody, EMD Millipore, Billerica, MA), or H3K27ac (rabbit polyclonal antibody, EMD Millipore, Billerica, MA). .. Immunoprecipitates were then quantitated by quantitative real-time PCR and calculated as a percent of input.

    Staining:

    Article Title: Samd7 is a cell type-specific PRC1 component essential for establishing retinal rod photoreceptor identity
    Article Snippet: We used the following primary antibodies for immunostaining: mouse monoclonal anti-Ctbp2 (1:500) (612044; BD Biosciences); anti-S100β (1:2,500) (S-2532; Sigma); anti-FLAG (1:1,000) (M2; Sigma); anti-Pax6 (1:500) (PAX6; Developmental Studies Hybridoma Bank); anti-Ring1B (1:1,000) (D139-3; MBL); rat monoclonal anti-GFP (1:2,000) (GF090R; Nacalai); guinea pig polyclonal anti-Thrβ2 (1:50) (Wako Pure Chemical Industries) ( ); anti-Chx10 (1:500) ( ); goat polyclonal anti–S-opsin (1:500) (sc-14363; Santa Cruz); anti-lamin B (1:500, C-20; Santa Cruz); rabbit polyclonal anti-Samd7 (1:10,000) (generated in this study); anti-rhodopsin (1:2,500) (LB-5597; LSL); anti–M-opsin (1:300) (AB5405; Millipore); anti-Pikachurin (1:500) (Wako Pure Chemical Industries) ( ); anti-Calbindin (1:1,000) (PC253L; Calbiochem); anti-Suz12 (1:500) (D39F6; Cell Signaling Technology); anti-H2AK119ub (1:500) (D27C4; Cell Signaling Technology); anti-H3K27me3 (1:500) (07-449; Millipore); and anti-H3K4me3 (1:500) ( ; Millipore) antibodies. .. We used the following primary antibodies for immunostaining: mouse monoclonal anti-Ctbp2 (1:500) (612044; BD Biosciences); anti-S100β (1:2,500) (S-2532; Sigma); anti-FLAG (1:1,000) (M2; Sigma); anti-Pax6 (1:500) (PAX6; Developmental Studies Hybridoma Bank); anti-Ring1B (1:1,000) (D139-3; MBL); rat monoclonal anti-GFP (1:2,000) (GF090R; Nacalai); guinea pig polyclonal anti-Thrβ2 (1:50) (Wako Pure Chemical Industries) ( ); anti-Chx10 (1:500) ( ); goat polyclonal anti–S-opsin (1:500) (sc-14363; Santa Cruz); anti-lamin B (1:500, C-20; Santa Cruz); rabbit polyclonal anti-Samd7 (1:10,000) (generated in this study); anti-rhodopsin (1:2,500) (LB-5597; LSL); anti–M-opsin (1:300) (AB5405; Millipore); anti-Pikachurin (1:500) (Wako Pure Chemical Industries) ( ); anti-Calbindin (1:1,000) (PC253L; Calbiochem); anti-Suz12 (1:500) (D39F6; Cell Signaling Technology); anti-H2AK119ub (1:500) (D27C4; Cell Signaling Technology); anti-H3K27me3 (1:500) (07-449; Millipore); and anti-H3K4me3 (1:500) ( ; Millipore) antibodies.

    Article Title: Characterization of DXZ4 conservation in primates implies important functional roles for CTCF binding, array expression and tandem repeat organization on the X chromosome
    Article Snippet: Rabbit polyclonal anti-H3K4me2 (07-030), anti-H3K4me3 (05-745), anti-CTCF (07-729), anti-H3K36me2 (07-274) and anti-acetyl-lysine (06-933) were all obtained from Millipore (Billerica, MA, USA). .. Rabbit polyclonal anti-H3K4me2 (07-030), anti-H3K4me3 (05-745), anti-CTCF (07-729), anti-H3K36me2 (07-274) and anti-acetyl-lysine (06-933) were all obtained from Millipore (Billerica, MA, USA).

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    Millipore monoclonal h3k4me3 antibody
    KDM5B controls enhancer activity by regulating H3K4 methylation. (A) KDM5B binding at p300 enhancer sites sorted by enrichment level of acetylated H3K27 (H3K27ac). (B) Heat map density profiles of <t>H3K4me3/2/1</t> around intergenic p300 binding sites, sorted by H3K27ac levels. (C) Top: average profile of H3K4me3/2/1 density at intergenic p300 sites in shLuc and shKdm5b ES cells. Bottom: log2 fold change normalized tag density ratios (shKdm5b/shLuc) of H3K4me3, H3K4me2, and H3K4me1 at p300 enhancers. RPBM, reads per base pair per million reads. (D) Average profiles of H3K27ac tag densities around H3K27ac peaks in shLuc and shKdm5b ES cells. (E) Average profiles of H3K27ac tag densities of H3K27ac around p300 binding sites (proxies of enhancers) in shLuc and shKdm5b ES cells. (F) Scatter plot of H3K27ac tag densities in shLuc and shKdm5b ES cells. (G) Heat map density profiles of H3K27ac peaks, sorted by H3K27ac levels. (H) Spreading of H3K4 methylation into enhancer shores in Kdm5b knockdown ES cells is associated with a decrease in H3K27ac levels. Shown are empirical cumulative distributions for the change in H3K27ac (shKdm5b/shLuc) across two groups of enhancers sorted by changes in spreading indices, calculated independently for H3K4me3 (left panel), H3K4me2 (middle panel) or H3K4me1 (right panel). Y-axis shows the percentage of enhancers that exhibit a change in H3K27ac density less than the value specified by the x-axis. A line shifted to the left means a systematic decrease in H3K27ac levels. P -value for all
    Monoclonal H3k4me3 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 81/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    KDM5B controls enhancer activity by regulating H3K4 methylation. (A) KDM5B binding at p300 enhancer sites sorted by enrichment level of acetylated H3K27 (H3K27ac). (B) Heat map density profiles of H3K4me3/2/1 around intergenic p300 binding sites, sorted by H3K27ac levels. (C) Top: average profile of H3K4me3/2/1 density at intergenic p300 sites in shLuc and shKdm5b ES cells. Bottom: log2 fold change normalized tag density ratios (shKdm5b/shLuc) of H3K4me3, H3K4me2, and H3K4me1 at p300 enhancers. RPBM, reads per base pair per million reads. (D) Average profiles of H3K27ac tag densities around H3K27ac peaks in shLuc and shKdm5b ES cells. (E) Average profiles of H3K27ac tag densities of H3K27ac around p300 binding sites (proxies of enhancers) in shLuc and shKdm5b ES cells. (F) Scatter plot of H3K27ac tag densities in shLuc and shKdm5b ES cells. (G) Heat map density profiles of H3K27ac peaks, sorted by H3K27ac levels. (H) Spreading of H3K4 methylation into enhancer shores in Kdm5b knockdown ES cells is associated with a decrease in H3K27ac levels. Shown are empirical cumulative distributions for the change in H3K27ac (shKdm5b/shLuc) across two groups of enhancers sorted by changes in spreading indices, calculated independently for H3K4me3 (left panel), H3K4me2 (middle panel) or H3K4me1 (right panel). Y-axis shows the percentage of enhancers that exhibit a change in H3K27ac density less than the value specified by the x-axis. A line shifted to the left means a systematic decrease in H3K27ac levels. P -value for all

    Journal: Genome Biology

    Article Title: KDM5B focuses H3K4 methylation near promoters and enhancers during embryonic stem cell self-renewal and differentiation

    doi: 10.1186/gb-2014-15-2-r32

    Figure Lengend Snippet: KDM5B controls enhancer activity by regulating H3K4 methylation. (A) KDM5B binding at p300 enhancer sites sorted by enrichment level of acetylated H3K27 (H3K27ac). (B) Heat map density profiles of H3K4me3/2/1 around intergenic p300 binding sites, sorted by H3K27ac levels. (C) Top: average profile of H3K4me3/2/1 density at intergenic p300 sites in shLuc and shKdm5b ES cells. Bottom: log2 fold change normalized tag density ratios (shKdm5b/shLuc) of H3K4me3, H3K4me2, and H3K4me1 at p300 enhancers. RPBM, reads per base pair per million reads. (D) Average profiles of H3K27ac tag densities around H3K27ac peaks in shLuc and shKdm5b ES cells. (E) Average profiles of H3K27ac tag densities of H3K27ac around p300 binding sites (proxies of enhancers) in shLuc and shKdm5b ES cells. (F) Scatter plot of H3K27ac tag densities in shLuc and shKdm5b ES cells. (G) Heat map density profiles of H3K27ac peaks, sorted by H3K27ac levels. (H) Spreading of H3K4 methylation into enhancer shores in Kdm5b knockdown ES cells is associated with a decrease in H3K27ac levels. Shown are empirical cumulative distributions for the change in H3K27ac (shKdm5b/shLuc) across two groups of enhancers sorted by changes in spreading indices, calculated independently for H3K4me3 (left panel), H3K4me2 (middle panel) or H3K4me1 (right panel). Y-axis shows the percentage of enhancers that exhibit a change in H3K27ac density less than the value specified by the x-axis. A line shifted to the left means a systematic decrease in H3K27ac levels. P -value for all

    Article Snippet: The monoclonal H3K4me3 antibody (CS200580) was obtained from EMD Millipore.

    Techniques: Activity Assay, Methylation, Binding Assay

    Spreading of H3K4me3 to gene bodies leads to defects in gene expression during ES cell differentiation. Scatter plot of gene expression measured by RPKM for shLuc and shKdm5b ES cells differentiated in the absence of LIF for (A) 48 h (day 2) and (B) 72 h (day 3). Log2-adjusted differentially expressed genes ( > 1.5-fold; FDR

    Journal: Genome Biology

    Article Title: KDM5B focuses H3K4 methylation near promoters and enhancers during embryonic stem cell self-renewal and differentiation

    doi: 10.1186/gb-2014-15-2-r32

    Figure Lengend Snippet: Spreading of H3K4me3 to gene bodies leads to defects in gene expression during ES cell differentiation. Scatter plot of gene expression measured by RPKM for shLuc and shKdm5b ES cells differentiated in the absence of LIF for (A) 48 h (day 2) and (B) 72 h (day 3). Log2-adjusted differentially expressed genes ( > 1.5-fold; FDR

    Article Snippet: The monoclonal H3K4me3 antibody (CS200580) was obtained from EMD Millipore.

    Techniques: Expressing, Cell Differentiation

    KDM5B and LSD1 regulate H3K4 methylation at promoters and enhancers in ES cells. (A) Average profile of H3K4me3/2/1 density at p300 enhancer regions in LSD1i-treated ES cells (red line) relative to shLuc ES cells (black line). RPBM, reads per base pair per million reads. (B) Relationship between changes in promoter and gene body H3K4me3 density and the percentage of genes with bivalent marks (red, increased H3K4me3; black, no-change in H3K4me3; green, decreased H3K4me3). (C) Genome browser view of H3K4me3 levels at HoxA cluster bivalent genes upon inhibition of LSD1 in ES cells.

    Journal: Genome Biology

    Article Title: KDM5B focuses H3K4 methylation near promoters and enhancers during embryonic stem cell self-renewal and differentiation

    doi: 10.1186/gb-2014-15-2-r32

    Figure Lengend Snippet: KDM5B and LSD1 regulate H3K4 methylation at promoters and enhancers in ES cells. (A) Average profile of H3K4me3/2/1 density at p300 enhancer regions in LSD1i-treated ES cells (red line) relative to shLuc ES cells (black line). RPBM, reads per base pair per million reads. (B) Relationship between changes in promoter and gene body H3K4me3 density and the percentage of genes with bivalent marks (red, increased H3K4me3; black, no-change in H3K4me3; green, decreased H3K4me3). (C) Genome browser view of H3K4me3 levels at HoxA cluster bivalent genes upon inhibition of LSD1 in ES cells.

    Article Snippet: The monoclonal H3K4me3 antibody (CS200580) was obtained from EMD Millipore.

    Techniques: Methylation, Inhibition

    KDM5B regulates H3K4 methylation at bivalent genes during differentiation without LIF and Oct4. (A) Relationship between changes in promoter H3K4me3 density during differentiation and the percentage of genes with bivalent marks (H3K4me3/H3K27me3), using a sliding window of 500 genes (red, increased H3K4me3; black, no-change in H3K4me3; green, decreased H3K4me3). (B) Average profile of H3K4me3 density at genes with increased H3K4me3 during shKdm5b differentiation. RPBM, reads per base pair per million reads. (C) Oct4-regulatable ES cells (ZHBTc4) infected with shLuc or shKdm5b lentiviral particles were (D) cultured in the presence of doxycycline to downregulate OCT4 levels and induce differentiation. Relationship between changes in promoter H3K4me3 density during differentiation in the absence of OCT4 and KDM5B and the percentage of genes with bivalent marks (red, increased H3K4me3; black, no-change in H3K4me3; green, decreased H3K4me3). (E) Browser view of H3K4me3 density following differentiation of shKdm5b ZHBTc4 ES cells. (F) Relationship between changes in gene body H3K4me3 density during differentiation and the percentage of genes with bivalent marks.

    Journal: Genome Biology

    Article Title: KDM5B focuses H3K4 methylation near promoters and enhancers during embryonic stem cell self-renewal and differentiation

    doi: 10.1186/gb-2014-15-2-r32

    Figure Lengend Snippet: KDM5B regulates H3K4 methylation at bivalent genes during differentiation without LIF and Oct4. (A) Relationship between changes in promoter H3K4me3 density during differentiation and the percentage of genes with bivalent marks (H3K4me3/H3K27me3), using a sliding window of 500 genes (red, increased H3K4me3; black, no-change in H3K4me3; green, decreased H3K4me3). (B) Average profile of H3K4me3 density at genes with increased H3K4me3 during shKdm5b differentiation. RPBM, reads per base pair per million reads. (C) Oct4-regulatable ES cells (ZHBTc4) infected with shLuc or shKdm5b lentiviral particles were (D) cultured in the presence of doxycycline to downregulate OCT4 levels and induce differentiation. Relationship between changes in promoter H3K4me3 density during differentiation in the absence of OCT4 and KDM5B and the percentage of genes with bivalent marks (red, increased H3K4me3; black, no-change in H3K4me3; green, decreased H3K4me3). (E) Browser view of H3K4me3 density following differentiation of shKdm5b ZHBTc4 ES cells. (F) Relationship between changes in gene body H3K4me3 density during differentiation and the percentage of genes with bivalent marks.

    Article Snippet: The monoclonal H3K4me3 antibody (CS200580) was obtained from EMD Millipore.

    Techniques: Methylation, Infection, Cell Culture

    KDM5B and LSD1 co-regulate H3K4 methylation in ES cells. (A) Venn diagram showing overlap of KDM5B- and LSD1-bound genes in ES cells. (B) Bright field microscopy of shLuc and shKdm5b ES cells cultured in the presence of 2 μM of the LSD1 inhibitor tranylcypromine/parnate (LSD1i) for 4 days. (C) Correlation between changes in histone methylation and expression level. Fold change normalized tag density ratios of H3K4me3, H3K4me2, and H3K4me1 at refseq genes were sorted into four groups based on their absolute expression level. Note that the lowest expressed genes have increased H3K4me3/2/1 levels in LSD1i-treated ES cells (red line, highest 25% expressed; green line, lowest 25% expressed).

    Journal: Genome Biology

    Article Title: KDM5B focuses H3K4 methylation near promoters and enhancers during embryonic stem cell self-renewal and differentiation

    doi: 10.1186/gb-2014-15-2-r32

    Figure Lengend Snippet: KDM5B and LSD1 co-regulate H3K4 methylation in ES cells. (A) Venn diagram showing overlap of KDM5B- and LSD1-bound genes in ES cells. (B) Bright field microscopy of shLuc and shKdm5b ES cells cultured in the presence of 2 μM of the LSD1 inhibitor tranylcypromine/parnate (LSD1i) for 4 days. (C) Correlation between changes in histone methylation and expression level. Fold change normalized tag density ratios of H3K4me3, H3K4me2, and H3K4me1 at refseq genes were sorted into four groups based on their absolute expression level. Note that the lowest expressed genes have increased H3K4me3/2/1 levels in LSD1i-treated ES cells (red line, highest 25% expressed; green line, lowest 25% expressed).

    Article Snippet: The monoclonal H3K4me3 antibody (CS200580) was obtained from EMD Millipore.

    Techniques: Methylation, Microscopy, Cell Culture, Expressing

    KDM5B regulates H3K4 methylation at gene body regions. Knockdown of Kdm5b transcripts results in altered H3K4 methylation profiles at promoters and enhancers and within gene body regions. (A) Heat map density of ChIP-Seq data at refseq genes sorted according to their absolute expression in ES cells. shKdm5b ES cells exhibit increased H3K4me3/2 methylation in gene body regions and decreased H3K4me3/2/1 methylation at promoter regions. (B) Correlation between changes in gene body histone methylation and expression level. Log2 fold change normalized tag density ratios (shKdm5b/shLuc) of H3K4me3, H3K4me2, and H3K4me1 at all refseq genes, which were sorted into four groups based on their absolute expression level in ES cells (red line, highest 25% expressed; green line, lowest 25% expressed). (C) Scatter plot of gene body densities of H3K4me3/2/1 in shLuc and shKdm5b ES cells. (D) UCSC genome browser examples of altered profiles of H3K4me3/2 within gene bodies of pluripotency-related genes such as Lsd1 and Nr5a2 in shKdm5b ES cells.

    Journal: Genome Biology

    Article Title: KDM5B focuses H3K4 methylation near promoters and enhancers during embryonic stem cell self-renewal and differentiation

    doi: 10.1186/gb-2014-15-2-r32

    Figure Lengend Snippet: KDM5B regulates H3K4 methylation at gene body regions. Knockdown of Kdm5b transcripts results in altered H3K4 methylation profiles at promoters and enhancers and within gene body regions. (A) Heat map density of ChIP-Seq data at refseq genes sorted according to their absolute expression in ES cells. shKdm5b ES cells exhibit increased H3K4me3/2 methylation in gene body regions and decreased H3K4me3/2/1 methylation at promoter regions. (B) Correlation between changes in gene body histone methylation and expression level. Log2 fold change normalized tag density ratios (shKdm5b/shLuc) of H3K4me3, H3K4me2, and H3K4me1 at all refseq genes, which were sorted into four groups based on their absolute expression level in ES cells (red line, highest 25% expressed; green line, lowest 25% expressed). (C) Scatter plot of gene body densities of H3K4me3/2/1 in shLuc and shKdm5b ES cells. (D) UCSC genome browser examples of altered profiles of H3K4me3/2 within gene bodies of pluripotency-related genes such as Lsd1 and Nr5a2 in shKdm5b ES cells.

    Article Snippet: The monoclonal H3K4me3 antibody (CS200580) was obtained from EMD Millipore.

    Techniques: Methylation, Chromatin Immunoprecipitation, Expressing

    KDM5B regulates H3K4 methylation at promoters. (A) Log2 normalized tag density ratios (reads per base pair per million reads (RPBM)) of H3K4me3, H3K4me2, and H3K4me1 at TSSs of all refseq genes. (B) Browser view of altered profiles of H3K4me3/2/1 at promoters of genes such as Nanog , Pou5f1 , and Tbx3 . (C) Scatter plot of ChIP-Seq tag densities of H3K4me3/2/1 peaks in promoter regions in shLuc and shKdm5b ES cells.

    Journal: Genome Biology

    Article Title: KDM5B focuses H3K4 methylation near promoters and enhancers during embryonic stem cell self-renewal and differentiation

    doi: 10.1186/gb-2014-15-2-r32

    Figure Lengend Snippet: KDM5B regulates H3K4 methylation at promoters. (A) Log2 normalized tag density ratios (reads per base pair per million reads (RPBM)) of H3K4me3, H3K4me2, and H3K4me1 at TSSs of all refseq genes. (B) Browser view of altered profiles of H3K4me3/2/1 at promoters of genes such as Nanog , Pou5f1 , and Tbx3 . (C) Scatter plot of ChIP-Seq tag densities of H3K4me3/2/1 peaks in promoter regions in shLuc and shKdm5b ES cells.

    Article Snippet: The monoclonal H3K4me3 antibody (CS200580) was obtained from EMD Millipore.

    Techniques: Methylation, Chromatin Immunoprecipitation

    Spreading of H3K4me3 to gene bodies leads to defects in gene expression in ES cells. (A) Schematic representation describing the calculation used to determine the SI of genes marked by H3K4 methylation. The promoter bin is defined as a 1 kb, 1.5 kb, or 3 kb window around the TSS of genes marked by H3K4me3, H3K4me2, and H3K4me1, respectively, while the transcribed region (gene body) is defined as the region extending to the TES. The SI is calculated from the ratio of the density of H3K4 methylation in the gene body bin to the density of H3K4 methylation in the promoter bin. (B) Empirical cumulative distribution for the SI of H3K4me3 (top panel), H3K4me2 (middle panel), and H3K4me1 (bottom panel) across all genes for shLuc (black) and shKdm5b (red) ES cells. Y-axis shows the percentage of genes that exhibit a SI less than the value specified by the x-axis. A line shifted to the right means a systematic increase in the spreading index. P -value for all

    Journal: Genome Biology

    Article Title: KDM5B focuses H3K4 methylation near promoters and enhancers during embryonic stem cell self-renewal and differentiation

    doi: 10.1186/gb-2014-15-2-r32

    Figure Lengend Snippet: Spreading of H3K4me3 to gene bodies leads to defects in gene expression in ES cells. (A) Schematic representation describing the calculation used to determine the SI of genes marked by H3K4 methylation. The promoter bin is defined as a 1 kb, 1.5 kb, or 3 kb window around the TSS of genes marked by H3K4me3, H3K4me2, and H3K4me1, respectively, while the transcribed region (gene body) is defined as the region extending to the TES. The SI is calculated from the ratio of the density of H3K4 methylation in the gene body bin to the density of H3K4 methylation in the promoter bin. (B) Empirical cumulative distribution for the SI of H3K4me3 (top panel), H3K4me2 (middle panel), and H3K4me1 (bottom panel) across all genes for shLuc (black) and shKdm5b (red) ES cells. Y-axis shows the percentage of genes that exhibit a SI less than the value specified by the x-axis. A line shifted to the right means a systematic increase in the spreading index. P -value for all

    Article Snippet: The monoclonal H3K4me3 antibody (CS200580) was obtained from EMD Millipore.

    Techniques: Expressing, Methylation

    KDM5B regulates H3K4 methylation during differentiation. (A) ES cells cultured in the absence of LIF for 3 to 4 days to induce differentiation. Note the loss of normal three-dimensional colony morphology of shLuc ES cells at day 3 of differentiation, while shKdm5b ES cells maintained their normal three-dimensional colony morphology in the absence of LIF. (B) Correlation between changes in histone methylation and expression level. Fold change normalized tag density ratios (shKdm5b/shLuc) of H3K4me3, H3K4me2, and H3K4me1 at all refseq genes, which were sorted into three groups based on their expression level (red line, highest expressed; green line, lowest expressed). (C) UCSC genome browser view of H3K4me3/2/1 marks at the self-renewal gene Tbx3 during differentiation. shKdm5b ES cells have altered H3K4me3/2/1 levels relative to shLuc ES cells. (D) Average profile of H3K4me3/2/1 densities at p300 enhancer regions following 4 days of shLuc and shKdm5b ES cell differentiation. RPBM, reads per base pair per million reads. (E) Promoter density of H3K4me3 in ES cells and day 3 differentiated ES cells. (F) UCSC browser view of H3K4me3 density at HoxA cluster genes during differentiation. Note the delayed decrease of H3K4me3 levels during shKdm5b ES cell differentiation relative to shLuc ES cells.

    Journal: Genome Biology

    Article Title: KDM5B focuses H3K4 methylation near promoters and enhancers during embryonic stem cell self-renewal and differentiation

    doi: 10.1186/gb-2014-15-2-r32

    Figure Lengend Snippet: KDM5B regulates H3K4 methylation during differentiation. (A) ES cells cultured in the absence of LIF for 3 to 4 days to induce differentiation. Note the loss of normal three-dimensional colony morphology of shLuc ES cells at day 3 of differentiation, while shKdm5b ES cells maintained their normal three-dimensional colony morphology in the absence of LIF. (B) Correlation between changes in histone methylation and expression level. Fold change normalized tag density ratios (shKdm5b/shLuc) of H3K4me3, H3K4me2, and H3K4me1 at all refseq genes, which were sorted into three groups based on their expression level (red line, highest expressed; green line, lowest expressed). (C) UCSC genome browser view of H3K4me3/2/1 marks at the self-renewal gene Tbx3 during differentiation. shKdm5b ES cells have altered H3K4me3/2/1 levels relative to shLuc ES cells. (D) Average profile of H3K4me3/2/1 densities at p300 enhancer regions following 4 days of shLuc and shKdm5b ES cell differentiation. RPBM, reads per base pair per million reads. (E) Promoter density of H3K4me3 in ES cells and day 3 differentiated ES cells. (F) UCSC browser view of H3K4me3 density at HoxA cluster genes during differentiation. Note the delayed decrease of H3K4me3 levels during shKdm5b ES cell differentiation relative to shLuc ES cells.

    Article Snippet: The monoclonal H3K4me3 antibody (CS200580) was obtained from EMD Millipore.

    Techniques: Methylation, Cell Culture, Expressing, Cell Differentiation

    KDM5B occupies active genes, pluripotency regulators, and bivalent genes in ES cells. KDM5B is associated with transcriptional start sites (TSSs) and gene body regions of highly expressed genes in ES cells. (A) ChIP-Seq tag density of KDM5B binding at TSS normalized by input (log2 scale) of all refseq genes sorted into quartiles based on their mRNA expression level in ES cells. (B) ChIP-Seq tag densities of KDM5B and H3K4me3 around TSSs in ES cells. KDM5B binding profiles are similar to H3K4me3 marks near TSS regions, while KDM5B occupancy is enriched more in gene body regions relative to H3K4me3. (C) Scatter plot of the ratio of relative tag densities of KDM5B and H3K4me3 in promoter versus gene body regions. (D) RNA polymerase II and MLL4 are also enriched at TSS regions. (E) KDM5B occupies promoters of pluripotency-related genes in ES cells (Pou5f1/Oct4, Sox2, and Nanog). ChIP-Seq binding profiles of KDM5B, H3K4me3, RNA polymerase II, and Mll4 at core pluripotency genes. (F) Venn diagrams showing the co-occupancy of KDM5B and H3K4me3 (left panel), H3K27me3 (middle panel), and both modifications (right panel) at promoter regions. (G) Example of KDM5B binding at promoters marked with H3K4me3 and H3K27me3 (for example, HoxA cluster). (H) Correlation matrix of KDM5B binding with an assortment of TFs and epigenetic modifiers that are highly expressed in ES cells. Hierarchical clustering heat map generated by evaluating pair-wise affinities at promoters between ChIP-Seq datasets generated from this study (KDM5B, H3K4me3, RNAPII) and from published datasets [ 3 , 35 - 39 ]. AutoSOME [ 40 ] was used to generate pair-wise affinity values. (I) Venn diagrams showing co-occupancy of KDM5B, OCT4, SOX2, and NANOG at promoter regions.

    Journal: Genome Biology

    Article Title: KDM5B focuses H3K4 methylation near promoters and enhancers during embryonic stem cell self-renewal and differentiation

    doi: 10.1186/gb-2014-15-2-r32

    Figure Lengend Snippet: KDM5B occupies active genes, pluripotency regulators, and bivalent genes in ES cells. KDM5B is associated with transcriptional start sites (TSSs) and gene body regions of highly expressed genes in ES cells. (A) ChIP-Seq tag density of KDM5B binding at TSS normalized by input (log2 scale) of all refseq genes sorted into quartiles based on their mRNA expression level in ES cells. (B) ChIP-Seq tag densities of KDM5B and H3K4me3 around TSSs in ES cells. KDM5B binding profiles are similar to H3K4me3 marks near TSS regions, while KDM5B occupancy is enriched more in gene body regions relative to H3K4me3. (C) Scatter plot of the ratio of relative tag densities of KDM5B and H3K4me3 in promoter versus gene body regions. (D) RNA polymerase II and MLL4 are also enriched at TSS regions. (E) KDM5B occupies promoters of pluripotency-related genes in ES cells (Pou5f1/Oct4, Sox2, and Nanog). ChIP-Seq binding profiles of KDM5B, H3K4me3, RNA polymerase II, and Mll4 at core pluripotency genes. (F) Venn diagrams showing the co-occupancy of KDM5B and H3K4me3 (left panel), H3K27me3 (middle panel), and both modifications (right panel) at promoter regions. (G) Example of KDM5B binding at promoters marked with H3K4me3 and H3K27me3 (for example, HoxA cluster). (H) Correlation matrix of KDM5B binding with an assortment of TFs and epigenetic modifiers that are highly expressed in ES cells. Hierarchical clustering heat map generated by evaluating pair-wise affinities at promoters between ChIP-Seq datasets generated from this study (KDM5B, H3K4me3, RNAPII) and from published datasets [ 3 , 35 - 39 ]. AutoSOME [ 40 ] was used to generate pair-wise affinity values. (I) Venn diagrams showing co-occupancy of KDM5B, OCT4, SOX2, and NANOG at promoter regions.

    Article Snippet: The monoclonal H3K4me3 antibody (CS200580) was obtained from EMD Millipore.

    Techniques: Chromatin Immunoprecipitation, Binding Assay, Expressing, Generated

    H3K4 methylation profiles in KDM5B-depleted and LSD1-inhibited ES cells. Browser views of H3K4me3/2/1 densities at (A) olfactory receptor and (B) pluripotency genes ( Nanog , Pou5f1 ). (C) Fold change densities of H3K4me3/2/1 peaks in shLuc?+?LSD1i and shKdm5b?+?LSD1i ES cells relative to shLuc ES cells. RPBM, reads per base pair per million reads.

    Journal: Genome Biology

    Article Title: KDM5B focuses H3K4 methylation near promoters and enhancers during embryonic stem cell self-renewal and differentiation

    doi: 10.1186/gb-2014-15-2-r32

    Figure Lengend Snippet: H3K4 methylation profiles in KDM5B-depleted and LSD1-inhibited ES cells. Browser views of H3K4me3/2/1 densities at (A) olfactory receptor and (B) pluripotency genes ( Nanog , Pou5f1 ). (C) Fold change densities of H3K4me3/2/1 peaks in shLuc?+?LSD1i and shKdm5b?+?LSD1i ES cells relative to shLuc ES cells. RPBM, reads per base pair per million reads.

    Article Snippet: The monoclonal H3K4me3 antibody (CS200580) was obtained from EMD Millipore.

    Techniques: Methylation

    Conserved broadest H3K4me3 domains are associated with synaptic signaling and neuronal functions. ( a ) Number of H3K4me3 peaks, total and top 5% broadest in prefrontal neurons of chimpanzee, macaque and adult mouse cerebral cortex, before and after liftover into HG19, as indicated. Brain coronal sections reproduced with permission from http://www.brains.rad.msu.edu , and http://brainmuseum.org , supported by the US National Science Foundation. Venn diagram shows that 131/525 top 5% broadest human NeuN+ H3K4me3 peaks are conserved across the four species. ( b ) Representative examples for conserved broadest peaks. ( c ) Circosplot highlighting top 10 DAVID Gene Ontology categories, comprised of 55 genes (shown in d ) with FDR (Benjamin Hochberg) P

    Journal: Translational Psychiatry

    Article Title: Deciphering H3K4me3 broad domains associated with gene-regulatory networks and conserved epigenomic landscapes in the human brain

    doi: 10.1038/tp.2015.169

    Figure Lengend Snippet: Conserved broadest H3K4me3 domains are associated with synaptic signaling and neuronal functions. ( a ) Number of H3K4me3 peaks, total and top 5% broadest in prefrontal neurons of chimpanzee, macaque and adult mouse cerebral cortex, before and after liftover into HG19, as indicated. Brain coronal sections reproduced with permission from http://www.brains.rad.msu.edu , and http://brainmuseum.org , supported by the US National Science Foundation. Venn diagram shows that 131/525 top 5% broadest human NeuN+ H3K4me3 peaks are conserved across the four species. ( b ) Representative examples for conserved broadest peaks. ( c ) Circosplot highlighting top 10 DAVID Gene Ontology categories, comprised of 55 genes (shown in d ) with FDR (Benjamin Hochberg) P

    Article Snippet: Purified mononucleosomal DNA were pulled downed using anti-H3K4me3 antibody (Upstate/Millipore 07473) with chromatin immunoprecipitation assay and then purified.

    Techniques:

    Cell-type-specific transcriptomics and epigenomics. ( a ) Browser tracks for eight genes subject to cell-type expression, showing (top to bottom) RNAseq tracks (orange/yellow) for PFC gray (G) and white matter (W) from subjects C6 and C10, as indicated. H3K4me3 ChIP-seq tracks from PFC NeuN+ (purple) and NeuN- (pink) nuclei of subjects and blood (green), from additional subjects. Note that robust expression in PFC gray matter (G) corresponds to strong H3K4me3 peaks in neuronal (NeuN+) chromatin. H3K4me3 domain length for each gene is listed in kb. ( b ) Lower-right panel shows examples of GO categories enriched in specific cell types (FDR-adjusted P

    Journal: Translational Psychiatry

    Article Title: Deciphering H3K4me3 broad domains associated with gene-regulatory networks and conserved epigenomic landscapes in the human brain

    doi: 10.1038/tp.2015.169

    Figure Lengend Snippet: Cell-type-specific transcriptomics and epigenomics. ( a ) Browser tracks for eight genes subject to cell-type expression, showing (top to bottom) RNAseq tracks (orange/yellow) for PFC gray (G) and white matter (W) from subjects C6 and C10, as indicated. H3K4me3 ChIP-seq tracks from PFC NeuN+ (purple) and NeuN- (pink) nuclei of subjects and blood (green), from additional subjects. Note that robust expression in PFC gray matter (G) corresponds to strong H3K4me3 peaks in neuronal (NeuN+) chromatin. H3K4me3 domain length for each gene is listed in kb. ( b ) Lower-right panel shows examples of GO categories enriched in specific cell types (FDR-adjusted P

    Article Snippet: Purified mononucleosomal DNA were pulled downed using anti-H3K4me3 antibody (Upstate/Millipore 07473) with chromatin immunoprecipitation assay and then purified.

    Techniques: Expressing, Chromatin Immunoprecipitation

    Features associated with top 5% broadest NeuN+ neuron-specific H3K4me3 peaks. ( a ) Pie charts express proportions of 523 (top 5%) broadest NeuN+-specific H3K4me3 peaks in relation to Refseq database, including ‘promoter TSS', intron, exon, intergenic, 5′UTR, 3′UTR and TTSs, as indicated. Pie charts are defined from left to right by increasing the window size of ‘promoter TSS'. Note that overwhelming proportion of peaks ( > 85%) are found within 4 kb of annotated Refseq TSSs. ( b ) Top sequence motif enriched in broadest domain (top 5%) neuronal H3K4me3 peaks. ( c ) Clustered H3K4me3 profiles within a ±4-kb window for the 523 (top 5%) broadest NeuN+-specific H3K4me3 peaks. The strength of the H3K4me3 signal is shown on a color scale of yellow (strong) to red (intermediate) to blue (weak) signal. Notice highly consistent enrichments across all subjects' PFC NeuN+ samples in cohorts 1 and 2, in comparison with non-neuronal (NeuN−) cells and blood. ( d ) Bean plot showing distribution of 523 NeuN+ broad peaks ordered by breadth of H3K4me3 signal, with broadest peak ( LINC00966 ) extending across ~13 kb. Purple area, estimated density of peak distribution. Tall vertical line, average bp length of top 5% broadest peaks (~5.4 kb). H3K4me3, H3-trimethyl lysine 4; TTS, transcription termination site; UTR, untranslated region.

    Journal: Translational Psychiatry

    Article Title: Deciphering H3K4me3 broad domains associated with gene-regulatory networks and conserved epigenomic landscapes in the human brain

    doi: 10.1038/tp.2015.169

    Figure Lengend Snippet: Features associated with top 5% broadest NeuN+ neuron-specific H3K4me3 peaks. ( a ) Pie charts express proportions of 523 (top 5%) broadest NeuN+-specific H3K4me3 peaks in relation to Refseq database, including ‘promoter TSS', intron, exon, intergenic, 5′UTR, 3′UTR and TTSs, as indicated. Pie charts are defined from left to right by increasing the window size of ‘promoter TSS'. Note that overwhelming proportion of peaks ( > 85%) are found within 4 kb of annotated Refseq TSSs. ( b ) Top sequence motif enriched in broadest domain (top 5%) neuronal H3K4me3 peaks. ( c ) Clustered H3K4me3 profiles within a ±4-kb window for the 523 (top 5%) broadest NeuN+-specific H3K4me3 peaks. The strength of the H3K4me3 signal is shown on a color scale of yellow (strong) to red (intermediate) to blue (weak) signal. Notice highly consistent enrichments across all subjects' PFC NeuN+ samples in cohorts 1 and 2, in comparison with non-neuronal (NeuN−) cells and blood. ( d ) Bean plot showing distribution of 523 NeuN+ broad peaks ordered by breadth of H3K4me3 signal, with broadest peak ( LINC00966 ) extending across ~13 kb. Purple area, estimated density of peak distribution. Tall vertical line, average bp length of top 5% broadest peaks (~5.4 kb). H3K4me3, H3-trimethyl lysine 4; TTS, transcription termination site; UTR, untranslated region.

    Article Snippet: Purified mononucleosomal DNA were pulled downed using anti-H3K4me3 antibody (Upstate/Millipore 07473) with chromatin immunoprecipitation assay and then purified.

    Techniques: Sequencing

    Broadest H3K4me3 peaks in neurons are developmentally regulated and enriched for super-enhancer and other cis -regulatory sequences. ( a ) Polar plot showing enrichment of top 5% broadest H3K4me3 peaks in PFC neurons for regulatory elements, including enhancers and super-enhancers, 14 , 36 , 37 and published data sets on developmental and autism-associated H3K4me3 peak profiles in PFC neurons. 16 , 18 ( b ) Browser window showing in PFC neurons from controls C1–C4 and autism subjects A1–A4, 16 representative examples for autism-associated broadest H3K4me3 peak, including SCRT1 transcriptional repressor (and PLEC encoding a cytoskleleton-associated protein. SCRT1 up- and PLEC downregulated in PFC neurons of diseased subjects, as indicated. ( c ) Heatmap showing gene-to-gene correlations of subset of 44 transcripts associated with 45 top 5% broadest H3K4me3 peaks that show age-dependent regulation across the postnatal lifespan of adult human cerebral cortex (transcript levels from BrainSpan database 78 and H3K4me3 peaks from Shulha et al. 18 ). Notice large number of strong (dark blue) gene-to-gene correlations, suggesting that subset top 5% broadest H3K4me3-peak-associated transcripts are co-regulated from postnatal to old age. H3K4me3, H3-trimethyl lysine 4; miRNA, microRNA; ncRNA, noncoding RNA; PFC, prefrontal cortex.

    Journal: Translational Psychiatry

    Article Title: Deciphering H3K4me3 broad domains associated with gene-regulatory networks and conserved epigenomic landscapes in the human brain

    doi: 10.1038/tp.2015.169

    Figure Lengend Snippet: Broadest H3K4me3 peaks in neurons are developmentally regulated and enriched for super-enhancer and other cis -regulatory sequences. ( a ) Polar plot showing enrichment of top 5% broadest H3K4me3 peaks in PFC neurons for regulatory elements, including enhancers and super-enhancers, 14 , 36 , 37 and published data sets on developmental and autism-associated H3K4me3 peak profiles in PFC neurons. 16 , 18 ( b ) Browser window showing in PFC neurons from controls C1–C4 and autism subjects A1–A4, 16 representative examples for autism-associated broadest H3K4me3 peak, including SCRT1 transcriptional repressor (and PLEC encoding a cytoskleleton-associated protein. SCRT1 up- and PLEC downregulated in PFC neurons of diseased subjects, as indicated. ( c ) Heatmap showing gene-to-gene correlations of subset of 44 transcripts associated with 45 top 5% broadest H3K4me3 peaks that show age-dependent regulation across the postnatal lifespan of adult human cerebral cortex (transcript levels from BrainSpan database 78 and H3K4me3 peaks from Shulha et al. 18 ). Notice large number of strong (dark blue) gene-to-gene correlations, suggesting that subset top 5% broadest H3K4me3-peak-associated transcripts are co-regulated from postnatal to old age. H3K4me3, H3-trimethyl lysine 4; miRNA, microRNA; ncRNA, noncoding RNA; PFC, prefrontal cortex.

    Article Snippet: Purified mononucleosomal DNA were pulled downed using anti-H3K4me3 antibody (Upstate/Millipore 07473) with chromatin immunoprecipitation assay and then purified.

    Techniques:

    Transcripts associated with top 5% broadest NeuN+ and NeuN−-specific H3K4me3 peaks. ( a ) Box plots comparing for 36 representative gene transcripts the gray (G) and white matter (W) RNA-seq signal from PFC tissue blocks of six subjects, showing much higher FKPM/expression in G compared with W. All transcripts are within 4 kb of a top 5% broadest H3K4me3 peak specific to PFC neurons. ( b ) Box plot comparison of PFC gray (G) and (W) white matter expression for 25 representative transcripts associated with broad non-neuronal H3K4me3 peaks ( n =6 (G) gray and n =6 (W) white matter samples). FKPM, fragments per kilobase per million; H3K4me3, H3-trimethyl lysine 4; PFC, prefrontal cortex.

    Journal: Translational Psychiatry

    Article Title: Deciphering H3K4me3 broad domains associated with gene-regulatory networks and conserved epigenomic landscapes in the human brain

    doi: 10.1038/tp.2015.169

    Figure Lengend Snippet: Transcripts associated with top 5% broadest NeuN+ and NeuN−-specific H3K4me3 peaks. ( a ) Box plots comparing for 36 representative gene transcripts the gray (G) and white matter (W) RNA-seq signal from PFC tissue blocks of six subjects, showing much higher FKPM/expression in G compared with W. All transcripts are within 4 kb of a top 5% broadest H3K4me3 peak specific to PFC neurons. ( b ) Box plot comparison of PFC gray (G) and (W) white matter expression for 25 representative transcripts associated with broad non-neuronal H3K4me3 peaks ( n =6 (G) gray and n =6 (W) white matter samples). FKPM, fragments per kilobase per million; H3K4me3, H3-trimethyl lysine 4; PFC, prefrontal cortex.

    Article Snippet: Purified mononucleosomal DNA were pulled downed using anti-H3K4me3 antibody (Upstate/Millipore 07473) with chromatin immunoprecipitation assay and then purified.

    Techniques: RNA Sequencing Assay, Expressing

    Cell-type-specific histone methylation profiling in PFC. ( a ) Graphical outline of experiment starting with postmortem cerebral cortex (PFC) to generate cell-type-specific H3K4me3 maps. ( b , c ) Heatmaps for Spearman's rank correlation coefficients comparing H3K4me3 profiles for three peripheral mononuclear blood cells (blood), two sorted NeuN− PFC cells, compared with ( b ) 11 PFC NeuN+ samples (cohort 1) and ( c ) 14 PFC NeuN+ samples (cohort 2) each from a different individual, showing much higher correlations between samples from the same cell type as compared with sample correlations across cell types and tissues. Principal component analyses showing complete separation of NeuN+ samples from other cell types with the first two principal components. ( d , e ) Venn diagrams showing absolute number of peaks for NeuN+, NeuN− and blood, confirming for ( d ) cohort 1 and ( e ) cohort 2 the enrichment cell-type-specific peaks among the top 5% broadest H3K4me3 peaks, as compared with the total set of peaks. ( f ) Venn diagram confirming that large majority of neuron-specific peaks from cohort 1 are confirmed in replication sample (cohort 2). ChIP-seq, chromatin immunoprecipitation and next-generation sequencing; H3K4me3, H3-trimethyl lysine 4; PFC, prefrontal cortex.

    Journal: Translational Psychiatry

    Article Title: Deciphering H3K4me3 broad domains associated with gene-regulatory networks and conserved epigenomic landscapes in the human brain

    doi: 10.1038/tp.2015.169

    Figure Lengend Snippet: Cell-type-specific histone methylation profiling in PFC. ( a ) Graphical outline of experiment starting with postmortem cerebral cortex (PFC) to generate cell-type-specific H3K4me3 maps. ( b , c ) Heatmaps for Spearman's rank correlation coefficients comparing H3K4me3 profiles for three peripheral mononuclear blood cells (blood), two sorted NeuN− PFC cells, compared with ( b ) 11 PFC NeuN+ samples (cohort 1) and ( c ) 14 PFC NeuN+ samples (cohort 2) each from a different individual, showing much higher correlations between samples from the same cell type as compared with sample correlations across cell types and tissues. Principal component analyses showing complete separation of NeuN+ samples from other cell types with the first two principal components. ( d , e ) Venn diagrams showing absolute number of peaks for NeuN+, NeuN− and blood, confirming for ( d ) cohort 1 and ( e ) cohort 2 the enrichment cell-type-specific peaks among the top 5% broadest H3K4me3 peaks, as compared with the total set of peaks. ( f ) Venn diagram confirming that large majority of neuron-specific peaks from cohort 1 are confirmed in replication sample (cohort 2). ChIP-seq, chromatin immunoprecipitation and next-generation sequencing; H3K4me3, H3-trimethyl lysine 4; PFC, prefrontal cortex.

    Article Snippet: Purified mononucleosomal DNA were pulled downed using anti-H3K4me3 antibody (Upstate/Millipore 07473) with chromatin immunoprecipitation assay and then purified.

    Techniques: Methylation, Chromatin Immunoprecipitation, Next-Generation Sequencing

    Integrative network-based analyses identify and prioritize key drivers associated with top 5% broadest neuronal H3K4me3 peaks. ( a ) Mapping 475 genes associated with top 5% broadest neuronal H3K4me3 peaks in the constructed Bayesian network of human prefrontal cortex revealed that only 158 genes (out of 475 genes) fell in the center of the network composed of about 7000 genes and 8000 edges. On the basis of the figure, there is not strong concentration in the center of the network; however, statistics show that the 158 neuron-specific genes do have significant higher degree and significant smaller average path lengths than the other genes in the network. Network statistics comparison suggesting that these genes actually tend to be located in the middle of the network ( Supplementary Tables 17 ). The purple nodes are representing genes that annotated to 4 kb TSS −4 kb. Blue nodes representing a subset of genes, including the synaptic vesicle gene SV2B and the NMDA receptor subunit GRIN2A were among the top three genes in multiple network categories (see Supplementary Figure 18 ; SV2B in ‘average shortest pathway', SV2B and GRIN2A in ‘between centrality', SV2B in ‘degree' and SV2B and GRIN2A in ‘stress'. ( b ) TOM plots of a weighted gene co-expression network constructed from PFC of normal control subjects (see the Materials and Methods section). In this symmetric TOM heatmap, the rows and the columns represent genes and color intensity (green: no connection and blue: strong connection) represents network connection strength between any pair of nodes (genes) based on topological overlap between genes. The brown and purple modules, highlighted with a brown and a purple box, respectively, overlap most significantly with the H3K4me3 peaks genes and are enriched for the neuronal activities and synaptic transmission signature. H3K4me3, H3-trimethyl lysine 4; PFC, prefrontal cortex; TOM, topological overlap matrix; TSS, transcription start site.

    Journal: Translational Psychiatry

    Article Title: Deciphering H3K4me3 broad domains associated with gene-regulatory networks and conserved epigenomic landscapes in the human brain

    doi: 10.1038/tp.2015.169

    Figure Lengend Snippet: Integrative network-based analyses identify and prioritize key drivers associated with top 5% broadest neuronal H3K4me3 peaks. ( a ) Mapping 475 genes associated with top 5% broadest neuronal H3K4me3 peaks in the constructed Bayesian network of human prefrontal cortex revealed that only 158 genes (out of 475 genes) fell in the center of the network composed of about 7000 genes and 8000 edges. On the basis of the figure, there is not strong concentration in the center of the network; however, statistics show that the 158 neuron-specific genes do have significant higher degree and significant smaller average path lengths than the other genes in the network. Network statistics comparison suggesting that these genes actually tend to be located in the middle of the network ( Supplementary Tables 17 ). The purple nodes are representing genes that annotated to 4 kb TSS −4 kb. Blue nodes representing a subset of genes, including the synaptic vesicle gene SV2B and the NMDA receptor subunit GRIN2A were among the top three genes in multiple network categories (see Supplementary Figure 18 ; SV2B in ‘average shortest pathway', SV2B and GRIN2A in ‘between centrality', SV2B in ‘degree' and SV2B and GRIN2A in ‘stress'. ( b ) TOM plots of a weighted gene co-expression network constructed from PFC of normal control subjects (see the Materials and Methods section). In this symmetric TOM heatmap, the rows and the columns represent genes and color intensity (green: no connection and blue: strong connection) represents network connection strength between any pair of nodes (genes) based on topological overlap between genes. The brown and purple modules, highlighted with a brown and a purple box, respectively, overlap most significantly with the H3K4me3 peaks genes and are enriched for the neuronal activities and synaptic transmission signature. H3K4me3, H3-trimethyl lysine 4; PFC, prefrontal cortex; TOM, topological overlap matrix; TSS, transcription start site.

    Article Snippet: Purified mononucleosomal DNA were pulled downed using anti-H3K4me3 antibody (Upstate/Millipore 07473) with chromatin immunoprecipitation assay and then purified.

    Techniques: Construct, Concentration Assay, Expressing, Transmission Assay

    Cfp1 regulates H3K4me3 at many gene promoters. ( A ) Genome browser screenshots representing H3K4me3 signal (as read coverage) in wild-type (wt) and Cfp1 −/− ES cells at selected gene loci ( Sox2 , Actb , and Gapdh ). CGIs are represented in

    Journal:

    Article Title: Cfp1 integrates both CpG content and gene activity for accurate H3K4me3 deposition in embryonic stem cells

    doi: 10.1101/gad.194209.112

    Figure Lengend Snippet: Cfp1 regulates H3K4me3 at many gene promoters. ( A ) Genome browser screenshots representing H3K4me3 signal (as read coverage) in wild-type (wt) and Cfp1 −/− ES cells at selected gene loci ( Sox2 , Actb , and Gapdh ). CGIs are represented in

    Article Snippet: Anti-H3K4me3 was obtained from Millipore (07-473).

    Techniques:

    A Cfp1 DNA-binding mutant can restore H3K4me3 at active promoters. ( A ) Screenshots representing H3K4me3 read coverage in wild-type (wt), Cfp1 −/− , wild-type rescue , and C169A rescue ES cells at selected gene loci ( Sox2 , Actb , Gapdh , Pou5f1

    Journal:

    Article Title: Cfp1 integrates both CpG content and gene activity for accurate H3K4me3 deposition in embryonic stem cells

    doi: 10.1101/gad.194209.112

    Figure Lengend Snippet: A Cfp1 DNA-binding mutant can restore H3K4me3 at active promoters. ( A ) Screenshots representing H3K4me3 read coverage in wild-type (wt), Cfp1 −/− , wild-type rescue , and C169A rescue ES cells at selected gene loci ( Sox2 , Actb , Gapdh , Pou5f1

    Article Snippet: Anti-H3K4me3 was obtained from Millipore (07-473).

    Techniques: Binding Assay, Mutagenesis

    H3K4me3 accumulates at ectopic sites in the absence of Cfp1 or its DNA-binding activity. ( A ) Screenshots showing H3K4me3 signal read coverage in wild-type (wt), Cfp1 −/− , wild-type rescue , and C169A rescue ES cells at selected genomic regions

    Journal:

    Article Title: Cfp1 integrates both CpG content and gene activity for accurate H3K4me3 deposition in embryonic stem cells

    doi: 10.1101/gad.194209.112

    Figure Lengend Snippet: H3K4me3 accumulates at ectopic sites in the absence of Cfp1 or its DNA-binding activity. ( A ) Screenshots showing H3K4me3 signal read coverage in wild-type (wt), Cfp1 −/− , wild-type rescue , and C169A rescue ES cells at selected genomic regions

    Article Snippet: Anti-H3K4me3 was obtained from Millipore (07-473).

    Techniques: Binding Assay, Activity Assay

    Transcription is weakly affected by decreased H3K4me3 at active gene promoters. ( A ) Volcano plots comparing expression values between wild-type and Cfp1 −/− ES cells. Fold change in expression values (wild-type vs. Cfp1 −/−

    Journal:

    Article Title: Cfp1 integrates both CpG content and gene activity for accurate H3K4me3 deposition in embryonic stem cells

    doi: 10.1101/gad.194209.112

    Figure Lengend Snippet: Transcription is weakly affected by decreased H3K4me3 at active gene promoters. ( A ) Volcano plots comparing expression values between wild-type and Cfp1 −/− ES cells. Fold change in expression values (wild-type vs. Cfp1 −/−

    Article Snippet: Anti-H3K4me3 was obtained from Millipore (07-473).

    Techniques: Expressing

    Cfp1 deficiency preferentially affects H3K4me3 at highly expressed genes without altering their DNA methylation. ( A ) GRO-seq density distribution in wild-type (wt) ES cells for all Ensembl genes (All genes); for genes whose H3K4me3 is not affected (Nonaffected,

    Journal:

    Article Title: Cfp1 integrates both CpG content and gene activity for accurate H3K4me3 deposition in embryonic stem cells

    doi: 10.1101/gad.194209.112

    Figure Lengend Snippet: Cfp1 deficiency preferentially affects H3K4me3 at highly expressed genes without altering their DNA methylation. ( A ) GRO-seq density distribution in wild-type (wt) ES cells for all Ensembl genes (All genes); for genes whose H3K4me3 is not affected (Nonaffected,

    Article Snippet: Anti-H3K4me3 was obtained from Millipore (07-473).

    Techniques: DNA Methylation Assay

    Model for the role of Cfp1 in regulating genome-wide H3K4me3. ( A ) Multiple Cfp1-dependent and Cfp1-independent mechanisms contribute to targeting of the Set1 complex to transcriptionally active CGI promoters in wild-type (wt) mouse ES cells. Cfp1 binding

    Journal:

    Article Title: Cfp1 integrates both CpG content and gene activity for accurate H3K4me3 deposition in embryonic stem cells

    doi: 10.1101/gad.194209.112

    Figure Lengend Snippet: Model for the role of Cfp1 in regulating genome-wide H3K4me3. ( A ) Multiple Cfp1-dependent and Cfp1-independent mechanisms contribute to targeting of the Set1 complex to transcriptionally active CGI promoters in wild-type (wt) mouse ES cells. Cfp1 binding

    Article Snippet: Anti-H3K4me3 was obtained from Millipore (07-473).

    Techniques: Genome Wide, Binding Assay

    H3K4me3 ectopic peaks in Cfp1 −/− ES cells coincide with regulatory regions and correlate with enhanced expression of neighboring genes. ( A ) Cluster analysis showing colocalization of ectopic peaks with regions enriched with histone modifications

    Journal:

    Article Title: Cfp1 integrates both CpG content and gene activity for accurate H3K4me3 deposition in embryonic stem cells

    doi: 10.1101/gad.194209.112

    Figure Lengend Snippet: H3K4me3 ectopic peaks in Cfp1 −/− ES cells coincide with regulatory regions and correlate with enhanced expression of neighboring genes. ( A ) Cluster analysis showing colocalization of ectopic peaks with regions enriched with histone modifications

    Article Snippet: Anti-H3K4me3 was obtained from Millipore (07-473).

    Techniques: Expressing

    NURF remodeling and core promoter architecture. (A) TSSs were categorized based on associated core promoter motifs and averaged nucleosome probability plots flanking the TSS of each category generated for both wild type (WT) and Nurf301/E(bx) mutants. +1 nucleosome dyad position is labeled. +1 nucleosome shifts were detected at promoters containing DREs and Ohler box 1 and 7 consensi (TRF2/DREF targets). (B) Heatmaps of NURF, DREF and H3K4me3 signals relative to the TSS for genes ordered according to the H3K4me3 signal strength. Colocalization of NURF (green) and DREF (red) signals is indicated by yellow merge. (C) NURF immunoprecipitated from S2 soluble nuclear fraction associates with the DREF/TRF2 subunit Wash. Reciprocal co-immunoprecipitation from S2 soluble nuclear fraction using anti-Wash and anti-PAR antibodies indicates that Wash is PARylated. An abundant PARylated protein partially overlaps with Wash in Input but is not immunoprecipitated by anti-Wash antibodies. Wash is indicated by arrowheads. 5% Input is loaded as control in all experiments.

    Journal: PLoS Genetics

    Article Title: Genome-Wide Mapping Targets of the Metazoan Chromatin Remodeling Factor NURF Reveals Nucleosome Remodeling at Enhancers, Core Promoters and Gene Insulators

    doi: 10.1371/journal.pgen.1005969

    Figure Lengend Snippet: NURF remodeling and core promoter architecture. (A) TSSs were categorized based on associated core promoter motifs and averaged nucleosome probability plots flanking the TSS of each category generated for both wild type (WT) and Nurf301/E(bx) mutants. +1 nucleosome dyad position is labeled. +1 nucleosome shifts were detected at promoters containing DREs and Ohler box 1 and 7 consensi (TRF2/DREF targets). (B) Heatmaps of NURF, DREF and H3K4me3 signals relative to the TSS for genes ordered according to the H3K4me3 signal strength. Colocalization of NURF (green) and DREF (red) signals is indicated by yellow merge. (C) NURF immunoprecipitated from S2 soluble nuclear fraction associates with the DREF/TRF2 subunit Wash. Reciprocal co-immunoprecipitation from S2 soluble nuclear fraction using anti-Wash and anti-PAR antibodies indicates that Wash is PARylated. An abundant PARylated protein partially overlaps with Wash in Input but is not immunoprecipitated by anti-Wash antibodies. Wash is indicated by arrowheads. 5% Input is loaded as control in all experiments.

    Article Snippet: The following antibodies were used: rabbit anti-NURF301, rabbit anti-H3K4me3 (17–614, Millipore), rabbit anti-CP190 [ ].

    Techniques: Generated, Labeling, Immunoprecipitation

    NURF interacts with insulator components at promoters. (A) Heatmaps of NURF, CP190 and H3K4me3 signals relative to the TSS for genes ordered according to the H3K4me3 signal strength. Colocalization of NURF (red) and CP190 (green) signals is indicated by yellow merge. (B) Normalized NURF and CP190 ChIP tag density relative to TSSs. TSSs are categorized as active (+H3K4me3) or inactive (-H3K4me3) based on H3K4me3 signal. (C) Transcript levels of NURF targets in CP190 mutant hemocytes, determined by real-time PCR relative to WT and normalized to rp49 . Transcripts are ordered according to associated H3K4me3. (D) NURF, CP190 and DREF signals are well correlated at TSSs.

    Journal: PLoS Genetics

    Article Title: Genome-Wide Mapping Targets of the Metazoan Chromatin Remodeling Factor NURF Reveals Nucleosome Remodeling at Enhancers, Core Promoters and Gene Insulators

    doi: 10.1371/journal.pgen.1005969

    Figure Lengend Snippet: NURF interacts with insulator components at promoters. (A) Heatmaps of NURF, CP190 and H3K4me3 signals relative to the TSS for genes ordered according to the H3K4me3 signal strength. Colocalization of NURF (red) and CP190 (green) signals is indicated by yellow merge. (B) Normalized NURF and CP190 ChIP tag density relative to TSSs. TSSs are categorized as active (+H3K4me3) or inactive (-H3K4me3) based on H3K4me3 signal. (C) Transcript levels of NURF targets in CP190 mutant hemocytes, determined by real-time PCR relative to WT and normalized to rp49 . Transcripts are ordered according to associated H3K4me3. (D) NURF, CP190 and DREF signals are well correlated at TSSs.

    Article Snippet: The following antibodies were used: rabbit anti-NURF301, rabbit anti-H3K4me3 (17–614, Millipore), rabbit anti-CP190 [ ].

    Techniques: Chromatin Immunoprecipitation, Mutagenesis, Real-time Polymerase Chain Reaction

    NURF nucleosome remodeling at insulator sites requires DREF. (A) Heatmap of nucleosomes and Nurf301/E(bx) at Su(Hw) and CTCF sites that also contain DREF ordered according to DREF signal. (B) Averaged nucleosome probability at all Su(Hw) and CTCF sites that either contain (+DREF) or lack (-DREF) DREF reveals that insulator nucleosome-depleted domain requires associated DREF (threshold of 50 reads at peak co-ordinate). (C) Anti-CP190 antibodies immunoprecipitate Wash. 5% Input is loaded as control in all experiments. (D) The enhancer blocking action of the ct 6 gyspy transposon, reduces mechanosensory bristle number on the anterior wing margin. Simultaneous removal of one copy of Nurf301 and CP190 partially restores mechanosensory bristles. Box-plots indicate at least 100 independent determinations. (E) Anterior wing margin showing mechanosensory bristles (arrowheads). (F) The H3K27me3-containing eve domain is flanked by NURF and CP190 peaks that separate it from surrounding H3K4me3-containing active genes. (G) Genome-wide profile of NURF, CP190, H3K4me3 and H3K27me3 signals at the boundaries of H3K27me3-enriched domains reveals NURF and CP190 flank H3K27me3 domains.

    Journal: PLoS Genetics

    Article Title: Genome-Wide Mapping Targets of the Metazoan Chromatin Remodeling Factor NURF Reveals Nucleosome Remodeling at Enhancers, Core Promoters and Gene Insulators

    doi: 10.1371/journal.pgen.1005969

    Figure Lengend Snippet: NURF nucleosome remodeling at insulator sites requires DREF. (A) Heatmap of nucleosomes and Nurf301/E(bx) at Su(Hw) and CTCF sites that also contain DREF ordered according to DREF signal. (B) Averaged nucleosome probability at all Su(Hw) and CTCF sites that either contain (+DREF) or lack (-DREF) DREF reveals that insulator nucleosome-depleted domain requires associated DREF (threshold of 50 reads at peak co-ordinate). (C) Anti-CP190 antibodies immunoprecipitate Wash. 5% Input is loaded as control in all experiments. (D) The enhancer blocking action of the ct 6 gyspy transposon, reduces mechanosensory bristle number on the anterior wing margin. Simultaneous removal of one copy of Nurf301 and CP190 partially restores mechanosensory bristles. Box-plots indicate at least 100 independent determinations. (E) Anterior wing margin showing mechanosensory bristles (arrowheads). (F) The H3K27me3-containing eve domain is flanked by NURF and CP190 peaks that separate it from surrounding H3K4me3-containing active genes. (G) Genome-wide profile of NURF, CP190, H3K4me3 and H3K27me3 signals at the boundaries of H3K27me3-enriched domains reveals NURF and CP190 flank H3K27me3 domains.

    Article Snippet: The following antibodies were used: rabbit anti-NURF301, rabbit anti-H3K4me3 (17–614, Millipore), rabbit anti-CP190 [ ].

    Techniques: Blocking Assay, Genome Wide

    NURF maintains nucleosome spacing on active genes. (A) Averaged nucleosome probability relative to all TSSs, and active (+H3K4me3) or inactive (-H3K4me3) TSSs. +1 nucleosome dyad position is indicated. (B) Heatmap of H3K4me3 in hemocytes. (C) Nucleosome probability trace at CG10699 in wild type (WT) and Nurf301/E(bx) mutant hemocytes corroborates NURF-dependent nucleosome positioning of six nucleosomes downstream of the TSS. (D) Genes were binned according to expression quintile from highest (1st) to lowest (5th) and averaged nucleosome probability relative to the TSS determined. (E) Transcript levels of genes containing H3K4me3 were increased in Nurf301/E(bx) mutants. Levels determined by real-time PCR relative to WT normalized using rp49 . (F) Shifts in +1 nucleosome dyad position (labeled) only occur for genes with increased expression in Nurf301/E(bx) mutants.

    Journal: PLoS Genetics

    Article Title: Genome-Wide Mapping Targets of the Metazoan Chromatin Remodeling Factor NURF Reveals Nucleosome Remodeling at Enhancers, Core Promoters and Gene Insulators

    doi: 10.1371/journal.pgen.1005969

    Figure Lengend Snippet: NURF maintains nucleosome spacing on active genes. (A) Averaged nucleosome probability relative to all TSSs, and active (+H3K4me3) or inactive (-H3K4me3) TSSs. +1 nucleosome dyad position is indicated. (B) Heatmap of H3K4me3 in hemocytes. (C) Nucleosome probability trace at CG10699 in wild type (WT) and Nurf301/E(bx) mutant hemocytes corroborates NURF-dependent nucleosome positioning of six nucleosomes downstream of the TSS. (D) Genes were binned according to expression quintile from highest (1st) to lowest (5th) and averaged nucleosome probability relative to the TSS determined. (E) Transcript levels of genes containing H3K4me3 were increased in Nurf301/E(bx) mutants. Levels determined by real-time PCR relative to WT normalized using rp49 . (F) Shifts in +1 nucleosome dyad position (labeled) only occur for genes with increased expression in Nurf301/E(bx) mutants.

    Article Snippet: The following antibodies were used: rabbit anti-NURF301, rabbit anti-H3K4me3 (17–614, Millipore), rabbit anti-CP190 [ ].

    Techniques: Mutagenesis, Expressing, Real-time Polymerase Chain Reaction, Labeling

    NURF organizes nucleosomes around insulator sites. (A) Genome browser view showing NURF, CP190, DREF and H3K4me3 distribution at the bithorax complex. Predicted (cdBEST) [ 21 ] and known insulator sites (Mcp and Fab8) are indicated. (B) NURF, CP190 and DREF signals are well correlated at predicted insulator sites (cdBEST). (C) Heatmap of nucleosomes and Nurf301/E(bx) at Su(Hw) and CTCF sites that also contain CP190, ordered according to CP190 signal (threshold of at least 50 reads at peak co-ordinate). Averaged nucleosome probability at all Su(Hw) and CTCF sites that either contain (+CP190) or lack (-CP190) CP190 reveals that insulator nucleosome-depleted domain requires associated CP190. (D) Reciprocal co-immunoprecipitation from S2 soluble nuclear fraction using anti-Nurf301 and anti-CP190 antibodies indicates proteins physically associate. As negative controls, anti-Enhancer of zeste (E(z)) and anti-Groucho (Gro) antibodies do not immunoprecipitate CP190, and anti-Nurf301 pull-down from Nurf301 -RNAi knockdown cells fails to immunoprecipitate CP190. (E) Anti-Nurf301 antibodies immunoprecipitate CP190 from MNase-treated soluble chromatin. This association is lost following Benzonase or RNase A treatment. 5% Input is loaded as control in all experiments.

    Journal: PLoS Genetics

    Article Title: Genome-Wide Mapping Targets of the Metazoan Chromatin Remodeling Factor NURF Reveals Nucleosome Remodeling at Enhancers, Core Promoters and Gene Insulators

    doi: 10.1371/journal.pgen.1005969

    Figure Lengend Snippet: NURF organizes nucleosomes around insulator sites. (A) Genome browser view showing NURF, CP190, DREF and H3K4me3 distribution at the bithorax complex. Predicted (cdBEST) [ 21 ] and known insulator sites (Mcp and Fab8) are indicated. (B) NURF, CP190 and DREF signals are well correlated at predicted insulator sites (cdBEST). (C) Heatmap of nucleosomes and Nurf301/E(bx) at Su(Hw) and CTCF sites that also contain CP190, ordered according to CP190 signal (threshold of at least 50 reads at peak co-ordinate). Averaged nucleosome probability at all Su(Hw) and CTCF sites that either contain (+CP190) or lack (-CP190) CP190 reveals that insulator nucleosome-depleted domain requires associated CP190. (D) Reciprocal co-immunoprecipitation from S2 soluble nuclear fraction using anti-Nurf301 and anti-CP190 antibodies indicates proteins physically associate. As negative controls, anti-Enhancer of zeste (E(z)) and anti-Groucho (Gro) antibodies do not immunoprecipitate CP190, and anti-Nurf301 pull-down from Nurf301 -RNAi knockdown cells fails to immunoprecipitate CP190. (E) Anti-Nurf301 antibodies immunoprecipitate CP190 from MNase-treated soluble chromatin. This association is lost following Benzonase or RNase A treatment. 5% Input is loaded as control in all experiments.

    Article Snippet: The following antibodies were used: rabbit anti-NURF301, rabbit anti-H3K4me3 (17–614, Millipore), rabbit anti-CP190 [ ].

    Techniques: Immunoprecipitation