Structured Review

Abcam histone h3
Partially closed chromatin structure and increased H3K methylation in the ATM promoter in Vav-IDH1-KI cells (A) ATAC-qPCR to analyze the indicated genes in LSK cells from Vav-IDH1-KI and WT mice (n=3). Data are the mean ± SD. (B) Immunoblot to detect the indicated methylated forms of <t>histone</t> H3 in Lin − cells from Vav-IDH1-KI and WT mice. (C) ChIP-qPCR analysis of the Atm promoter in LSK cells from Vav-IDH1-KI and WT mice (n=3) using anti-histone H3-K9me3 Ab. Data are the mean ± SD. (D) Immunoblot to detect the indicated proteins in LSK cells isolated from Vav-IDH1-KI and WT mice and cultured with/without UNC1999 (1 µM) or UNC0642(0.25 or 1 µM). UT, untreated. (E) qRT-PCR determination of Atm .
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Images

1) Product Images from "Mutant IDH1 downregulates ATM and alters DNA repair and sensitivity to DNA damage independent of TET2"

Article Title: Mutant IDH1 downregulates ATM and alters DNA repair and sensitivity to DNA damage independent of TET2

Journal: Cancer cell

doi: 10.1016/j.ccell.2016.05.018

Partially closed chromatin structure and increased H3K methylation in the ATM promoter in Vav-IDH1-KI cells (A) ATAC-qPCR to analyze the indicated genes in LSK cells from Vav-IDH1-KI and WT mice (n=3). Data are the mean ± SD. (B) Immunoblot to detect the indicated methylated forms of histone H3 in Lin − cells from Vav-IDH1-KI and WT mice. (C) ChIP-qPCR analysis of the Atm promoter in LSK cells from Vav-IDH1-KI and WT mice (n=3) using anti-histone H3-K9me3 Ab. Data are the mean ± SD. (D) Immunoblot to detect the indicated proteins in LSK cells isolated from Vav-IDH1-KI and WT mice and cultured with/without UNC1999 (1 µM) or UNC0642(0.25 or 1 µM). UT, untreated. (E) qRT-PCR determination of Atm .
Figure Legend Snippet: Partially closed chromatin structure and increased H3K methylation in the ATM promoter in Vav-IDH1-KI cells (A) ATAC-qPCR to analyze the indicated genes in LSK cells from Vav-IDH1-KI and WT mice (n=3). Data are the mean ± SD. (B) Immunoblot to detect the indicated methylated forms of histone H3 in Lin − cells from Vav-IDH1-KI and WT mice. (C) ChIP-qPCR analysis of the Atm promoter in LSK cells from Vav-IDH1-KI and WT mice (n=3) using anti-histone H3-K9me3 Ab. Data are the mean ± SD. (D) Immunoblot to detect the indicated proteins in LSK cells isolated from Vav-IDH1-KI and WT mice and cultured with/without UNC1999 (1 µM) or UNC0642(0.25 or 1 µM). UT, untreated. (E) qRT-PCR determination of Atm .

Techniques Used: Methylation, Real-time Polymerase Chain Reaction, Mouse Assay, Chromatin Immunoprecipitation, Isolation, Cell Culture, Quantitative RT-PCR

2) Product Images from "Transcription Factor Sp3 Represses Expression of p21CIP1 via Inhibition of Productive Elongation by RNA Polymerase II"

Article Title: Transcription Factor Sp3 Represses Expression of p21CIP1 via Inhibition of Productive Elongation by RNA Polymerase II

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00323-12

Sp3 inhibits H3S10 phosphorylation at the promoter region of the p21 gene. (A) ChIP analysis of multiple positions of the p21 locus using a specific antibody against histone H3 phosphorylated at Ser10 (H3S10ph) demonstrated that phosphorylation of Ser
Figure Legend Snippet: Sp3 inhibits H3S10 phosphorylation at the promoter region of the p21 gene. (A) ChIP analysis of multiple positions of the p21 locus using a specific antibody against histone H3 phosphorylated at Ser10 (H3S10ph) demonstrated that phosphorylation of Ser

Techniques Used: Chromatin Immunoprecipitation

3) Product Images from "Chromatin condensation via the condensin II complex is required for peripheral T-cell quiescence"

Article Title: Chromatin condensation via the condensin II complex is required for peripheral T-cell quiescence

Journal: The EMBO Journal

doi: 10.1038/emboj.2010.314

Standard immunofluorescence techniques fail to detect histones in naive peripheral T cells. ( A ) Naive T cells were left untreated, stimulated for 20 h with anti-CD3 antibodies or growing in culture for 5 days. DAPI was used to mark the nucleus. Antibodies
Figure Legend Snippet: Standard immunofluorescence techniques fail to detect histones in naive peripheral T cells. ( A ) Naive T cells were left untreated, stimulated for 20 h with anti-CD3 antibodies or growing in culture for 5 days. DAPI was used to mark the nucleus. Antibodies

Techniques Used: Immunofluorescence

Detection of histones in peripheral T lymphocytes by western blot. ( A ) Purified naive T cells were left untreated or stimulated with anti-CD3 antibodies for 1, 3, 6, 12 or 24 h. Half of each sample was either acid extracted overnight or lysed using NP40
Figure Legend Snippet: Detection of histones in peripheral T lymphocytes by western blot. ( A ) Purified naive T cells were left untreated or stimulated with anti-CD3 antibodies for 1, 3, 6, 12 or 24 h. Half of each sample was either acid extracted overnight or lysed using NP40

Techniques Used: Western Blot, Purification

Chromatin condenses rapidly during thymocyte development. ( A ) Thymocytes were isolated and stained with DAPI and with antibodies to mono-, di- and tri-methylated histone H3K4, tri-methylated histone H3K9/27 and acetylated histone H3 to detect modification
Figure Legend Snippet: Chromatin condenses rapidly during thymocyte development. ( A ) Thymocytes were isolated and stained with DAPI and with antibodies to mono-, di- and tri-methylated histone H3K4, tri-methylated histone H3K9/27 and acetylated histone H3 to detect modification

Techniques Used: Isolation, Staining, Methylation, Modification

4) Product Images from "Free DNA in Cystic Fibrosis Airway Fluids Correlates with Airflow Obstruction"

Article Title: Free DNA in Cystic Fibrosis Airway Fluids Correlates with Airflow Obstruction

Journal: Mediators of Inflammation

doi: 10.1155/2015/408935

Free NET-like DNA structures in CF lung disease. (a) Immunological characterization of free DNA structures in CF airway fluids by CLSM. Upper two panel rows: NET-like DNA structures in induced CF sputum. Blue: DAPI stains DNA-NET backbone. Red: elastase. Lower two panel rows: Blue: DAPI, red: citrullinated histones. Scale bar: 20 μ m. (b) Ultrastructure of free NET-like DNA structures. Upper and middle panels: SEM images of CF airway fluids. Arrow marks bacteria entrapped in DNA-NET-like structures. Scale bar: 2 μ m; Middle right panel: TEM staining of citrullinated histones in CF airway fluids (sputa). Lower panel: Ultrathin sections of CF airway DNA-NET-like structures. The NETs and the bacterial extracellular polysaccharides are visualized by the ruthenium-red-osmium-tetroxide technique. Bacteria embedded in a dense wickerwork of NETs. Arrowheads mark NETs; asterisk: bacterial extracellular polysaccharide. (c) Upper panel: free DNA structures in CF lung tissue. Red: MPO (as characteristic NET component). Blue: DAPI (DNA). Inlays mark characteristic NET-areas. Lower panel: CF airway NETs in CF BAL fluids. Red: elastase (as characteristic NET component). Blue: DAPI (DNA). (d) Costainings of DAPI, citrullinated histones, and F-actin. Scale bar: 20 μ m. (e) Dead/live staining of CF airway fluids (induced sputum). Free DNA and dead bacteria appear red; vital bacteria appear green.
Figure Legend Snippet: Free NET-like DNA structures in CF lung disease. (a) Immunological characterization of free DNA structures in CF airway fluids by CLSM. Upper two panel rows: NET-like DNA structures in induced CF sputum. Blue: DAPI stains DNA-NET backbone. Red: elastase. Lower two panel rows: Blue: DAPI, red: citrullinated histones. Scale bar: 20 μ m. (b) Ultrastructure of free NET-like DNA structures. Upper and middle panels: SEM images of CF airway fluids. Arrow marks bacteria entrapped in DNA-NET-like structures. Scale bar: 2 μ m; Middle right panel: TEM staining of citrullinated histones in CF airway fluids (sputa). Lower panel: Ultrathin sections of CF airway DNA-NET-like structures. The NETs and the bacterial extracellular polysaccharides are visualized by the ruthenium-red-osmium-tetroxide technique. Bacteria embedded in a dense wickerwork of NETs. Arrowheads mark NETs; asterisk: bacterial extracellular polysaccharide. (c) Upper panel: free DNA structures in CF lung tissue. Red: MPO (as characteristic NET component). Blue: DAPI (DNA). Inlays mark characteristic NET-areas. Lower panel: CF airway NETs in CF BAL fluids. Red: elastase (as characteristic NET component). Blue: DAPI (DNA). (d) Costainings of DAPI, citrullinated histones, and F-actin. Scale bar: 20 μ m. (e) Dead/live staining of CF airway fluids (induced sputum). Free DNA and dead bacteria appear red; vital bacteria appear green.

Techniques Used: Confocal Laser Scanning Microscopy, Transmission Electron Microscopy, Staining

5) Product Images from "Nonsense suppression induced readthrough of a novel PAX6 mutation in patient‐derived cells of congenital aniridia. Nonsense suppression induced readthrough of a novel PAX6 mutation in patient‐derived cells of congenital aniridia"

Article Title: Nonsense suppression induced readthrough of a novel PAX6 mutation in patient‐derived cells of congenital aniridia. Nonsense suppression induced readthrough of a novel PAX6 mutation in patient‐derived cells of congenital aniridia

Journal: Molecular Genetics & Genomic Medicine

doi: 10.1002/mgg3.1198

Readthrough efficiency of nonsense suppression drugs. The readthrough efficiency was calculated as a percentage of increased full‐length PAX6 over the solvent‐treated control (−) using lymphocytes of patient III3 by Western blot analysis. Histone H3 was used as an internal control. (a) The readthrough in response to varying doses of ataluren (PTC124) and geneticin (G418). Cells were harvested at four days of treatment for analysis. (b) The readthrough in response to varied treatment times, with optimized doses of 10 μg/ml PTC124 and 500 μM G418. * p
Figure Legend Snippet: Readthrough efficiency of nonsense suppression drugs. The readthrough efficiency was calculated as a percentage of increased full‐length PAX6 over the solvent‐treated control (−) using lymphocytes of patient III3 by Western blot analysis. Histone H3 was used as an internal control. (a) The readthrough in response to varying doses of ataluren (PTC124) and geneticin (G418). Cells were harvested at four days of treatment for analysis. (b) The readthrough in response to varied treatment times, with optimized doses of 10 μg/ml PTC124 and 500 μM G418. * p

Techniques Used: Western Blot

Recovered expression of PAX6 by nonsense suppression therapy. Patients II2 and III3 were compared with the age‐matched II4 and III2 healthy controls, respectively (taken as “1”). (a) Both ataluren (PTC124) and geneticin (G418) restored the PAX6 protein to about 65%–70% of a healthy level by Western blot. Histone H3 was used as an internal control. (b) The PAX6 mRNA expression was restored to about 80% of the healthy level in G418 but not PTC124 treatment by real‐time PCR analysis. The β‐actin mRNA was used as an internal control. * p
Figure Legend Snippet: Recovered expression of PAX6 by nonsense suppression therapy. Patients II2 and III3 were compared with the age‐matched II4 and III2 healthy controls, respectively (taken as “1”). (a) Both ataluren (PTC124) and geneticin (G418) restored the PAX6 protein to about 65%–70% of a healthy level by Western blot. Histone H3 was used as an internal control. (b) The PAX6 mRNA expression was restored to about 80% of the healthy level in G418 but not PTC124 treatment by real‐time PCR analysis. The β‐actin mRNA was used as an internal control. * p

Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction

6) Product Images from "Deacetylation of Histone H4 Accompanying Cardiomyogenesis is Weakened in HDAC1-Depleted ES Cells"

Article Title: Deacetylation of Histone H4 Accompanying Cardiomyogenesis is Weakened in HDAC1-Depleted ES Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19082425

Distribution pattern and levels of histone H4 acetylation (green) and α-actinin (red) in ( Aa ) wt mESCs and ( Ab ) HDAC1 dn mESCs differentiated at dd20 and dd25. Panel ( Ba ) shows non-treated wt mESCs and HDCA1 dn mESCs at dd25, and panels ( Bb – d ) show wt mESCs and HDCA1 dn mESCs treated by HDACi at dd25: ( Bb ) TSA treatment, ( Bc ) SAHA treatment, and ( Bd ) VPA treatment. DAPI (blue) was used as a counterstain of the cell nuclei.
Figure Legend Snippet: Distribution pattern and levels of histone H4 acetylation (green) and α-actinin (red) in ( Aa ) wt mESCs and ( Ab ) HDAC1 dn mESCs differentiated at dd20 and dd25. Panel ( Ba ) shows non-treated wt mESCs and HDCA1 dn mESCs at dd25, and panels ( Bb – d ) show wt mESCs and HDCA1 dn mESCs treated by HDACi at dd25: ( Bb ) TSA treatment, ( Bc ) SAHA treatment, and ( Bd ) VPA treatment. DAPI (blue) was used as a counterstain of the cell nuclei.

Techniques Used:

Histone acetylation and methylation in HDAC1 wt and HDAC1 dn mESCs induced into cardiomyocytes and treated with HDACi. The level of H3K9ac, H3K9me3, H4ac, H4K20ac, pan-acetylated lysines (K-ac), and α-actinin in ( A ) HDAC1 wt mESCs and ( B ) HDAC1 dn mESCs. In three biological replicates, Western blots were performed on one gel. For the data presented in panel A or B, the gel was separated by Photoshop to show samples that were compared in one relevant subset. Data on histone levels were normalized to the level of histone H3 and non-histone proteins were normalized and quantified to the level of GAPDH ( C ). In wt and HDAC1 dn non-treated cells and in TSA-, SAHA-, or VPA-treated mESCs, panel ( Ca ) shows the levels of H4ac, ( Cb ) shows H4K20ac, and ( Cc ) shows the levels of α-actinin. The total protein levels were measured using a µQuant spectrophotometer for each sample, and an identical protein amount was loaded on the gels. In panel ( A , B ), the levels of histone markers are also shown for embryonic hearts (e15). Quantification of the protein levels in panel ( C ) was performed using ImageJ software (NIH, freeware). Statistical analyses were performed using Student’s t -test; asterisks (*) in panel ( Ca – c ) show statistically significant differences at p ≤ 0.05. Note that the y -axis-scale in panel ( Ca ) is different (red frames) for the wt and HDAC1 dn cells for technical purposes. In panel ( Ca ), the level of H4ac is significantly less in the wt mESCs when compared with the HDAC1 dn cells ( Cb ).
Figure Legend Snippet: Histone acetylation and methylation in HDAC1 wt and HDAC1 dn mESCs induced into cardiomyocytes and treated with HDACi. The level of H3K9ac, H3K9me3, H4ac, H4K20ac, pan-acetylated lysines (K-ac), and α-actinin in ( A ) HDAC1 wt mESCs and ( B ) HDAC1 dn mESCs. In three biological replicates, Western blots were performed on one gel. For the data presented in panel A or B, the gel was separated by Photoshop to show samples that were compared in one relevant subset. Data on histone levels were normalized to the level of histone H3 and non-histone proteins were normalized and quantified to the level of GAPDH ( C ). In wt and HDAC1 dn non-treated cells and in TSA-, SAHA-, or VPA-treated mESCs, panel ( Ca ) shows the levels of H4ac, ( Cb ) shows H4K20ac, and ( Cc ) shows the levels of α-actinin. The total protein levels were measured using a µQuant spectrophotometer for each sample, and an identical protein amount was loaded on the gels. In panel ( A , B ), the levels of histone markers are also shown for embryonic hearts (e15). Quantification of the protein levels in panel ( C ) was performed using ImageJ software (NIH, freeware). Statistical analyses were performed using Student’s t -test; asterisks (*) in panel ( Ca – c ) show statistically significant differences at p ≤ 0.05. Note that the y -axis-scale in panel ( Ca ) is different (red frames) for the wt and HDAC1 dn cells for technical purposes. In panel ( Ca ), the level of H4ac is significantly less in the wt mESCs when compared with the HDAC1 dn cells ( Cb ).

Techniques Used: Methylation, Western Blot, Spectrophotometry, Software

Histone post-translational modifications studied in mouse embryonic hearts (e15) treated with HDACi. ( A ) Western blots showed changes in H3K9ac, H3K9me3, H4ac, H4K20ac, pan-acetylated lysines (K-ac), and α-actinin in e15 embryonic hearts treated with HDACi (TSA, SAHA, and VPA). Data on histone levels were normalized to the level of histone H3 and non-histone proteins were normalized to the level of GAPDH. An identical protein amount for each experimental event was loaded on the gel. ( B ) Data from panel ( A ) were normalized to the relevant reference protein GAPDH, and the density of Western blot fragments was statistically analyzed using Student’s t -test; asterisks show statistically significant differences at p ≤ 0.05. GAPDH was used for data normalization, and α-actinin was used as a marker of cardiomyocytes. ( C ) The distribution pattern of H3K9ac (red) in the e15 mouse embryonic hearts is shown. DAPI (blue) was used as a counterstain of the cell nuclei. Arrows show the accumulation of H3K9ac in ventricular portions.
Figure Legend Snippet: Histone post-translational modifications studied in mouse embryonic hearts (e15) treated with HDACi. ( A ) Western blots showed changes in H3K9ac, H3K9me3, H4ac, H4K20ac, pan-acetylated lysines (K-ac), and α-actinin in e15 embryonic hearts treated with HDACi (TSA, SAHA, and VPA). Data on histone levels were normalized to the level of histone H3 and non-histone proteins were normalized to the level of GAPDH. An identical protein amount for each experimental event was loaded on the gel. ( B ) Data from panel ( A ) were normalized to the relevant reference protein GAPDH, and the density of Western blot fragments was statistically analyzed using Student’s t -test; asterisks show statistically significant differences at p ≤ 0.05. GAPDH was used for data normalization, and α-actinin was used as a marker of cardiomyocytes. ( C ) The distribution pattern of H3K9ac (red) in the e15 mouse embryonic hearts is shown. DAPI (blue) was used as a counterstain of the cell nuclei. Arrows show the accumulation of H3K9ac in ventricular portions.

Techniques Used: Western Blot, Marker

7) Product Images from "MDC1 methylation mediated by lysine methyltransferases EHMT1 and EHMT2 regulates active ATM accumulation flanking DNA damage sites"

Article Title: MDC1 methylation mediated by lysine methyltransferases EHMT1 and EHMT2 regulates active ATM accumulation flanking DNA damage sites

Journal: Scientific Reports

doi: 10.1038/s41598-018-29239-3

EHMT2 promote methylation of MDC1 at lysine 45. ( a ) Schematic illustration of the full-length (FL) and deletion mutant (N1) of MDC1. Asterisk indicates the position of lysine 45 (wild type; WT or the alanine-substituted mutant; K45A). FHA, forkhead-associated domain; SDT, Ser-Asp-Thr repeats; TQXF, Thr-Gln-X-Phe repeats; PST, Pro-Ser-Thr repeats; BRCT, tandem BRCT domain. ( b–d ) Immunoblotting analysis of whole-cell extracts from U2OS cells transfected with ( b ) HA-tagged MDC1 (WT or K45A) and control or MDC1 -targeting siRNAs, ( c ) HA-tagged MDC1-N1 (WT or K45A), and ( d ) HA-tagged MDC1-N1 (WT) and control, two EHMT1 -targeting siRNAs ( EHMT1 siRNA-1 and siRNA-2) or three EHMT2 -targeting siRNAs ( EHMT1 siRNA-1 siRNA-2 and siRNA-3; see methods), using the indicated antibodies. Quantification of methylation levels in histone H3 and MDC1 shown in right panels. Relative density was calculated based on signal intensities normalized against levels of total histone H3 (upper panel) and total MDC1-N1 (lower panel). Error bars, SD from triplicate measurements. ( e ) Immunofluorescence analysis of U2OS cells transfected with HA-tagged MDC1 (WT or K45A) and MDC1 -targeting siRNA, and co-immunostained with indicated antibodies at 2 h after exposure to neocarzinostatin (NCS, 50 ng/ml for 15 min). A representative image of each treated or control cells is shown, as indicated. Scale bar, 10 μm.
Figure Legend Snippet: EHMT2 promote methylation of MDC1 at lysine 45. ( a ) Schematic illustration of the full-length (FL) and deletion mutant (N1) of MDC1. Asterisk indicates the position of lysine 45 (wild type; WT or the alanine-substituted mutant; K45A). FHA, forkhead-associated domain; SDT, Ser-Asp-Thr repeats; TQXF, Thr-Gln-X-Phe repeats; PST, Pro-Ser-Thr repeats; BRCT, tandem BRCT domain. ( b–d ) Immunoblotting analysis of whole-cell extracts from U2OS cells transfected with ( b ) HA-tagged MDC1 (WT or K45A) and control or MDC1 -targeting siRNAs, ( c ) HA-tagged MDC1-N1 (WT or K45A), and ( d ) HA-tagged MDC1-N1 (WT) and control, two EHMT1 -targeting siRNAs ( EHMT1 siRNA-1 and siRNA-2) or three EHMT2 -targeting siRNAs ( EHMT1 siRNA-1 siRNA-2 and siRNA-3; see methods), using the indicated antibodies. Quantification of methylation levels in histone H3 and MDC1 shown in right panels. Relative density was calculated based on signal intensities normalized against levels of total histone H3 (upper panel) and total MDC1-N1 (lower panel). Error bars, SD from triplicate measurements. ( e ) Immunofluorescence analysis of U2OS cells transfected with HA-tagged MDC1 (WT or K45A) and MDC1 -targeting siRNA, and co-immunostained with indicated antibodies at 2 h after exposure to neocarzinostatin (NCS, 50 ng/ml for 15 min). A representative image of each treated or control cells is shown, as indicated. Scale bar, 10 μm.

Techniques Used: Methylation, Mutagenesis, Transfection, Immunofluorescence

8) Product Images from "Regulation of the DNA Damage Response and Gene Expression by the Dot1L Histone Methyltransferase and the 53Bp1 Tumour Suppressor"

Article Title: Regulation of the DNA Damage Response and Gene Expression by the Dot1L Histone Methyltransferase and the 53Bp1 Tumour Suppressor

Journal: PLoS ONE

doi: 10.1371/journal.pone.0014714

Histone H3 Lysine 79 is not methylated in Dot1L −/− cells. A. Western blot showing the loss of H3K79 dimethylation in Dot1L −/− cells. The membrane was stripped and reprobed with an antibody against total H3 to demonstrate equivalent loading. B. Collision-induced fragmentation spectrum of precursor ion m/z 706.37 2+ (shown in C). The spectrum shows a nearly complete y -ion series. The mass difference between the y 4 and y6-ion confirms the presence of a single propionyl and methyl group on H3K79 (pr1+me1). C. Doubly charged precursor ion with the measured m/z ratio expected for peptide EIAQDFK 79(pr1+me1) TDLR. The monoisotopic peak containing only 12 C atoms (arrow) and those containing a single 13 C (*) or two naturally occurring 13 C atoms (**) are shown. D. Extracted ion chromatograms (XIC) showing the abundance of the doubly charged precursor peptide containing H3K79me1 (pr1+me1) in WT and Dot1L −/− cells. E. Relative abundance of the various H3K79 methylation states determined by MRM mass spectrometry in total histones isolated from WT and Dot1L −/− cells. ND: not detected. F. Western blot showing that the absence of H3K79me2 in Dot1L −/− cells can be complemented by transient transfection of plasmid containing the human Dot1L cDNA.
Figure Legend Snippet: Histone H3 Lysine 79 is not methylated in Dot1L −/− cells. A. Western blot showing the loss of H3K79 dimethylation in Dot1L −/− cells. The membrane was stripped and reprobed with an antibody against total H3 to demonstrate equivalent loading. B. Collision-induced fragmentation spectrum of precursor ion m/z 706.37 2+ (shown in C). The spectrum shows a nearly complete y -ion series. The mass difference between the y 4 and y6-ion confirms the presence of a single propionyl and methyl group on H3K79 (pr1+me1). C. Doubly charged precursor ion with the measured m/z ratio expected for peptide EIAQDFK 79(pr1+me1) TDLR. The monoisotopic peak containing only 12 C atoms (arrow) and those containing a single 13 C (*) or two naturally occurring 13 C atoms (**) are shown. D. Extracted ion chromatograms (XIC) showing the abundance of the doubly charged precursor peptide containing H3K79me1 (pr1+me1) in WT and Dot1L −/− cells. E. Relative abundance of the various H3K79 methylation states determined by MRM mass spectrometry in total histones isolated from WT and Dot1L −/− cells. ND: not detected. F. Western blot showing that the absence of H3K79me2 in Dot1L −/− cells can be complemented by transient transfection of plasmid containing the human Dot1L cDNA.

Techniques Used: Methylation, Western Blot, Mass Spectrometry, Isolation, Transfection, Plasmid Preparation

9) Product Images from "Alpha Radiation as a Way to Target Heterochromatic and Gamma Radiation-Exposed Breast Cancer Cells"

Article Title: Alpha Radiation as a Way to Target Heterochromatic and Gamma Radiation-Exposed Breast Cancer Cells

Journal: Cells

doi: 10.3390/cells9051165

( A ) Two parallel cell replicates (denoted 1 and 2) were exposed to 6 × 2 Gy fractions of gamma radiation, 2 days per week, aiming to establish gamma radiation-exposed (RE) MDA-MB-231 cells. Analysis was performed at early (2 replicates) and late time points (from replicate 1, three independent experiments). ( B ) Cell growth was assayed and presented as number of doublings during the first 9 days, with the 2 Gy fractions indicated as arrows. The mRNA levels of cancer/normal stem cell markers CD44, CD133, Sox2, Oct4, and Nanog were analysed by real-time PCR at 24 h after 2 Gy or in total 6 Gy, i.e., in samples collected on day 2 and 9 ( C ) or in samples 4–6 weeks after 12 Gy ( D ). Trimethylated lysine 9 of histone H3 (H3K9me3) levels, normalised to GAPDH, were assayed using Western blot ( E ), and clonogenic survival was analysed in RE cells versus control cells in response to gamma and alpha radiation in samples 4–6 weeks after 12 Gy ( F ). * p
Figure Legend Snippet: ( A ) Two parallel cell replicates (denoted 1 and 2) were exposed to 6 × 2 Gy fractions of gamma radiation, 2 days per week, aiming to establish gamma radiation-exposed (RE) MDA-MB-231 cells. Analysis was performed at early (2 replicates) and late time points (from replicate 1, three independent experiments). ( B ) Cell growth was assayed and presented as number of doublings during the first 9 days, with the 2 Gy fractions indicated as arrows. The mRNA levels of cancer/normal stem cell markers CD44, CD133, Sox2, Oct4, and Nanog were analysed by real-time PCR at 24 h after 2 Gy or in total 6 Gy, i.e., in samples collected on day 2 and 9 ( C ) or in samples 4–6 weeks after 12 Gy ( D ). Trimethylated lysine 9 of histone H3 (H3K9me3) levels, normalised to GAPDH, were assayed using Western blot ( E ), and clonogenic survival was analysed in RE cells versus control cells in response to gamma and alpha radiation in samples 4–6 weeks after 12 Gy ( F ). * p

Techniques Used: Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Western Blot

( A ) Levels of acetylated lysine 8 of histone H4 (H4K8ac) were analysed in MDA-MB-231 cells using Western blot after treatment with 0.25–1 µM trichostatin A (TSA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. ( B ) Effects of TSA alone on clonogenic survival using 0.5 and 1 µM TSA relative to untreated control cells. * p
Figure Legend Snippet: ( A ) Levels of acetylated lysine 8 of histone H4 (H4K8ac) were analysed in MDA-MB-231 cells using Western blot after treatment with 0.25–1 µM trichostatin A (TSA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. ( B ) Effects of TSA alone on clonogenic survival using 0.5 and 1 µM TSA relative to untreated control cells. * p

Techniques Used: Multiple Displacement Amplification, Western Blot

( A ) Basal levels of H3K9me3 and total protein levels of histone H3 were analysed in MDA-MB-231 and MCF7 cells using Western blot. GAPDH was used as a loading control. ( B ) γH2AX foci numbers are presented at 30 min and 24 h postexposure to 6 Gy of gamma or 2 Gy of alpha radiation in MDA-MB-231 and MCF7 cells. * p
Figure Legend Snippet: ( A ) Basal levels of H3K9me3 and total protein levels of histone H3 were analysed in MDA-MB-231 and MCF7 cells using Western blot. GAPDH was used as a loading control. ( B ) γH2AX foci numbers are presented at 30 min and 24 h postexposure to 6 Gy of gamma or 2 Gy of alpha radiation in MDA-MB-231 and MCF7 cells. * p

Techniques Used: Multiple Displacement Amplification, Western Blot

10) Product Images from "The Histone Deacetylase Inhibitor, MS-275 (Entinostat), Downregulates c-FLIP, Sensitizes Osteosarcoma Cells to FasL, and Induces the Regression of Osteosarcoma Lung Metastases"

Article Title: The Histone Deacetylase Inhibitor, MS-275 (Entinostat), Downregulates c-FLIP, Sensitizes Osteosarcoma Cells to FasL, and Induces the Regression of Osteosarcoma Lung Metastases

Journal: Current cancer drug targets

doi:

MS-275 induces accumulation of acetylated histone H3
Figure Legend Snippet: MS-275 induces accumulation of acetylated histone H3

Techniques Used: Mass Spectrometry

11) Product Images from "Specialized compartments of cardiac nuclei exhibit distinct proteomic anatomy *"

Article Title: Specialized compartments of cardiac nuclei exhibit distinct proteomic anatomy *

Journal: Molecular & Cellular Proteomics : MCP

doi: 10.1074/mcp.M110.000703

Features of cardiac histone proteins.
Figure Legend Snippet: Features of cardiac histone proteins.

Techniques Used:

12) Product Images from "Histone H3K4 and K36 Methylation, Chd1 and Rpd3S Oppose the Functions of Saccharomyces cerevisiae Spt4-Spt5 in Transcription"

Article Title: Histone H3K4 and K36 Methylation, Chd1 and Rpd3S Oppose the Functions of Saccharomyces cerevisiae Spt4-Spt5 in Transcription

Journal: Genetics

doi: 10.1534/genetics.109.111526

Mutations that interfere with histone acetylation do not suppress spt5-242 . (A) Loss of the SAGA subunit Spt8 decreases the viability of spt5-242 mutants. A strain containing spt5-242 was crossed to a strain lacking SPT8 . The resulting diploid was transformed
Figure Legend Snippet: Mutations that interfere with histone acetylation do not suppress spt5-242 . (A) Loss of the SAGA subunit Spt8 decreases the viability of spt5-242 mutants. A strain containing spt5-242 was crossed to a strain lacking SPT8 . The resulting diploid was transformed

Techniques Used: Transformation Assay

13) Product Images from "A Nucleosome Surface Formed by Histone H4, H2A, and H3 Residues Is Needed for Proper Histone H3 Lys36 Methylation, Histone Acetylation, and Repression of Cryptic Transcription *"

Article Title: A Nucleosome Surface Formed by Histone H4, H2A, and H3 Residues Is Needed for Proper Histone H3 Lys36 Methylation, Histone Acetylation, and Repression of Cryptic Transcription *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.085043

Histone H4 Lys 44 and the histone H4 interaction motif of Set2 are needed to maintain the repression of intragenic cryptic transcription. A , Northern blot analysis of total RNA isolated from WT or the indicated strains. For detection of cryptic transcripts,
Figure Legend Snippet: Histone H4 Lys 44 and the histone H4 interaction motif of Set2 are needed to maintain the repression of intragenic cryptic transcription. A , Northern blot analysis of total RNA isolated from WT or the indicated strains. For detection of cryptic transcripts,

Techniques Used: Northern Blot, Isolation

Both histone H4 Lys 44  and H2A Leu 116  and Leu 117  are needed for  trans -histone H3 Lys 36  di- and trimethylation  in vivo  and  in vitro . A , yeast whole cell extracts generated from cells expressing WT or the indicated histone mutants, including histone H4 single
Figure Legend Snippet: Both histone H4 Lys 44 and H2A Leu 116 and Leu 117 are needed for trans -histone H3 Lys 36 di- and trimethylation in vivo and in vitro . A , yeast whole cell extracts generated from cells expressing WT or the indicated histone mutants, including histone H4 single

Techniques Used: In Vivo, In Vitro, Generated, Expressing

Both H4 Lys 44  and H2A Leu 116  and Leu 117  are needed to mediate  trans -histone H3 Lys 36  di- and trimethylation at gene-specific loci. A  and  B , ChIP assays from the indicated strains are performed using antibodies specific to H3 Lys 36  dimethyl ( A ) and trimethyl
Figure Legend Snippet: Both H4 Lys 44 and H2A Leu 116 and Leu 117 are needed to mediate trans -histone H3 Lys 36 di- and trimethylation at gene-specific loci. A and B , ChIP assays from the indicated strains are performed using antibodies specific to H3 Lys 36 dimethyl ( A ) and trimethyl

Techniques Used: Chromatin Immunoprecipitation

X-ray crystal structure of the nucleosome core particle indicating the positions of histone H4 Lys 44 and H2A Leu 116 and Leu 117 . A , the structure of the human nucleosome core particle is shown (H3, cyan ; H4, green; H2A, gray ; H2B, yellow ). Histone H3 Lys
Figure Legend Snippet: X-ray crystal structure of the nucleosome core particle indicating the positions of histone H4 Lys 44 and H2A Leu 116 and Leu 117 . A , the structure of the human nucleosome core particle is shown (H3, cyan ; H4, green; H2A, gray ; H2B, yellow ). Histone H3 Lys

Techniques Used:

Multiple histone H2A and H3 residues located near histone H4 Lys 44 are needed for proper H3 Lys 36 di- and trimethylation. A , a zoomed in view of the nucleosome is shown indicating multiple residues in the close proximity within 5 angstroms of histone
Figure Legend Snippet: Multiple histone H2A and H3 residues located near histone H4 Lys 44 are needed for proper H3 Lys 36 di- and trimethylation. A , a zoomed in view of the nucleosome is shown indicating multiple residues in the close proximity within 5 angstroms of histone

Techniques Used:

The histone H4 and H2A mutants showing  trans -histone H3 Lys 36  di- and trimethylation defects exhibit increased histone H4 acetylation and intragenic cryptic transcription. A  and  B , ChIP assays from the indicated strains are performed using antibodies
Figure Legend Snippet: The histone H4 and H2A mutants showing trans -histone H3 Lys 36 di- and trimethylation defects exhibit increased histone H4 acetylation and intragenic cryptic transcription. A and B , ChIP assays from the indicated strains are performed using antibodies

Techniques Used: Chromatin Immunoprecipitation

Histone H2A Leu 116  and Leu 117  are needed for  trans -histone H3 Lys 36  di- and trimethylation  in vivo  and for appropriate 6-AU sensitivity. A , whole cell extracts prepared from cells expressing WT histones or the indicated histone mutants are immunoblotted
Figure Legend Snippet: Histone H2A Leu 116 and Leu 117 are needed for trans -histone H3 Lys 36 di- and trimethylation in vivo and for appropriate 6-AU sensitivity. A , whole cell extracts prepared from cells expressing WT histones or the indicated histone mutants are immunoblotted

Techniques Used: In Vivo, Expressing

14) Product Images from "Counteracting H3K4 methylation modulators Set1 and Jhd2 co-regulate chromatin dynamics and gene transcription"

Article Title: Counteracting H3K4 methylation modulators Set1 and Jhd2 co-regulate chromatin dynamics and gene transcription

Journal: Nature Communications

doi: 10.1038/ncomms11949

Transcriptional co-regulation is dependent on Set1 and Jhd2 catalytic activities and H3K4 methylation. ( a ) Western blots for H3K4me1, H3K4me2 and H3K4me3 in WT, deletion mutants ( set1Δ , jhd2Δ and set1Δ jhd2Δ ), catalytic-dead mutants ( set1-C1019A and jhd2-H427A ) and Jhd2 overexpressing strain ( ADH1pro-JHD2 ) are shown. Histone H3 serves as loading control. Twofold serial dilutions of cell extracts from the indicated strains were applied in lanes 5–16. Graph shows quantitation of western blots using densitometry. Fold-change in a H3K4 methyl mark level normalized to the total H3 level in a mutant is shown relative to that in control WT (set as 1). ( b ) Fold-change in PHO89 and LCP5 sense transcript levels in the indicated mutants relative to WT are shown. For ( b , c ), error bars denote ±s.e.m. from four independent experiments ( n =4). Statistical significance calculated using the Student's t -test. PHO89 : * P -value
Figure Legend Snippet: Transcriptional co-regulation is dependent on Set1 and Jhd2 catalytic activities and H3K4 methylation. ( a ) Western blots for H3K4me1, H3K4me2 and H3K4me3 in WT, deletion mutants ( set1Δ , jhd2Δ and set1Δ jhd2Δ ), catalytic-dead mutants ( set1-C1019A and jhd2-H427A ) and Jhd2 overexpressing strain ( ADH1pro-JHD2 ) are shown. Histone H3 serves as loading control. Twofold serial dilutions of cell extracts from the indicated strains were applied in lanes 5–16. Graph shows quantitation of western blots using densitometry. Fold-change in a H3K4 methyl mark level normalized to the total H3 level in a mutant is shown relative to that in control WT (set as 1). ( b ) Fold-change in PHO89 and LCP5 sense transcript levels in the indicated mutants relative to WT are shown. For ( b , c ), error bars denote ±s.e.m. from four independent experiments ( n =4). Statistical significance calculated using the Student's t -test. PHO89 : * P -value

Techniques Used: Methylation, Western Blot, Quantitation Assay, Mutagenesis

Co-regulation of nucleosomal occupancy and transcription by Set1 and Jhd2 at candidate target genes. ( a – c ) Changes to H3K4 methylation levels, nucleosomal occupancy and transcripts at SER3 , CHA1 and PHO5 genes in jhd2Δ or set1Δ are shown. The normal nucleosomal, H3K4 methylation, Jhd2 and Set1 occupancies at target genes are also shown: reads for mononucleosomal DNA in WT shows native chromatin organization (brown). Reads for H3K4 monomethyl (me1, magenta), H3K4 dimethyl (me2, cyan) and H3K4 trimethyl (me3, green) marks in WT shows their distribution over candidate target genes. Enrichment values (qpois) for Jhd2 (black) and Set1 (pink) in WT show their occupancy at the indicated genes. Fold-change in the levels of H3K4me1, H3K4me2 and H3K4me3 marks in jhd2Δ or set1Δ relative to that in WT are shown. Fold-change in H3K4 methylation in set1Δ relative to WT was calculated using published datasets 42 . Increase or decrease in a given H3K4 methyl mark in the mutant is shown in red or blue, respectively. Mononucleosomal DNA from WT were subtracted from those obtained from jhd2Δ or set1Δ and visualized using a genome browser. Gain or loss in nucleosome occupancy in the mutant is represented in red or blue, respectively. RNA-seq reads showing transcript levels for the three target genes in WT , jhd2Δ or set1Δ strains are shown (orange). Teal arrow indicates SRG1 , a gene coding for regulatory non-coding RNA involved in SER3 regulation 34 . In c , schematic diagram at top shows the nucleosome organization at PHO5 . Circles, well-positioned promoter nucleosomes; black arrow, TSS; and pink box, the Pho4 binding site, determined using published datasets 69 70 . Grape coloured arrow and dotted line indicate decrease in nucleosomal occupancy at the Pho4 binding site. ( d ) The average mean profile for histone H3 turnover across 800 bp region upstream (−) or downstream (+) of the TSSs (0) at all 236 yeast ribosome biogenesis or Ribi genes in WT , set1Δ and jhd2Δ are shown.
Figure Legend Snippet: Co-regulation of nucleosomal occupancy and transcription by Set1 and Jhd2 at candidate target genes. ( a – c ) Changes to H3K4 methylation levels, nucleosomal occupancy and transcripts at SER3 , CHA1 and PHO5 genes in jhd2Δ or set1Δ are shown. The normal nucleosomal, H3K4 methylation, Jhd2 and Set1 occupancies at target genes are also shown: reads for mononucleosomal DNA in WT shows native chromatin organization (brown). Reads for H3K4 monomethyl (me1, magenta), H3K4 dimethyl (me2, cyan) and H3K4 trimethyl (me3, green) marks in WT shows their distribution over candidate target genes. Enrichment values (qpois) for Jhd2 (black) and Set1 (pink) in WT show their occupancy at the indicated genes. Fold-change in the levels of H3K4me1, H3K4me2 and H3K4me3 marks in jhd2Δ or set1Δ relative to that in WT are shown. Fold-change in H3K4 methylation in set1Δ relative to WT was calculated using published datasets 42 . Increase or decrease in a given H3K4 methyl mark in the mutant is shown in red or blue, respectively. Mononucleosomal DNA from WT were subtracted from those obtained from jhd2Δ or set1Δ and visualized using a genome browser. Gain or loss in nucleosome occupancy in the mutant is represented in red or blue, respectively. RNA-seq reads showing transcript levels for the three target genes in WT , jhd2Δ or set1Δ strains are shown (orange). Teal arrow indicates SRG1 , a gene coding for regulatory non-coding RNA involved in SER3 regulation 34 . In c , schematic diagram at top shows the nucleosome organization at PHO5 . Circles, well-positioned promoter nucleosomes; black arrow, TSS; and pink box, the Pho4 binding site, determined using published datasets 69 70 . Grape coloured arrow and dotted line indicate decrease in nucleosomal occupancy at the Pho4 binding site. ( d ) The average mean profile for histone H3 turnover across 800 bp region upstream (−) or downstream (+) of the TSSs (0) at all 236 yeast ribosome biogenesis or Ribi genes in WT , set1Δ and jhd2Δ are shown.

Techniques Used: Methylation, Mutagenesis, RNA Sequencing Assay, Binding Assay

Set1 and Jhd2 co-regulate nucleosomal occupancy and histone turnover at their shared target genes. ( a , b ) Heat maps for nucleosome occupancy ratios (Δnucleosome occupancy) between jhd2Δ and WT or between set1Δ and WT at genes either co-repressed ( a ) or co-activated ( b ) by Set1 and Jhd2. Red, gain in nucleosome occupancy; blue, loss in nucleosome occupancy. Genes in rows are organized into six groups using k -means clustering. Columns represent 50 bp windows over ±800 bp regions relative to the TSS (0). Windows overlapping neighbouring genes were excluded. Total reads for mononucleosomal DNA in WT show the native state of nucleosome occupancy (leftmost panel). Asterisk, NDR; black arrow, the well-positioned +1 nucleosome. ( c ) Western blots showing RITE. WT , set1Δ and jhd2Δ strains expressing histone H3 tagged with V5- LoxP -HygMX- LoxP -2Flag cassette were grown overnight in YPD medium and then grown to saturation followed by addition of β-estradiol to induce tag switch. Saturated cultures were re-inoculated into fresh medium to release cells into the cell cycle in the presence of β-estradiol. Cells were arrested in G1 using α-factor. Cells before addition of β-estradiol (pre) and β-estradiol-treated G1 cells (post) were collected for whole-cell lysate preparation and detection of old (V5) and new (Flag) histone H3. The levels of H3K4me1, H3K4me2, H3K4me3 and total histone H3 in the indicated strains are shown. 2Flag , two copies of Flag tag; asterisk, truncated H3. See Supplementary Fig. 22 for full- length image of blots. ( d ) The average mean profile for histone H3 turnover across 800 bp region upstream (−) or downstream (+) of the TSSs (0) at all yeast genes. ( e , f ) Mean profiles for histone H3 turnover across regions (as described for d ) at genes co-repressed ( e ) or co-activated ( f ) by Set1 and Jhd2 in WT, set1Δ and jhd2Δ strains.
Figure Legend Snippet: Set1 and Jhd2 co-regulate nucleosomal occupancy and histone turnover at their shared target genes. ( a , b ) Heat maps for nucleosome occupancy ratios (Δnucleosome occupancy) between jhd2Δ and WT or between set1Δ and WT at genes either co-repressed ( a ) or co-activated ( b ) by Set1 and Jhd2. Red, gain in nucleosome occupancy; blue, loss in nucleosome occupancy. Genes in rows are organized into six groups using k -means clustering. Columns represent 50 bp windows over ±800 bp regions relative to the TSS (0). Windows overlapping neighbouring genes were excluded. Total reads for mononucleosomal DNA in WT show the native state of nucleosome occupancy (leftmost panel). Asterisk, NDR; black arrow, the well-positioned +1 nucleosome. ( c ) Western blots showing RITE. WT , set1Δ and jhd2Δ strains expressing histone H3 tagged with V5- LoxP -HygMX- LoxP -2Flag cassette were grown overnight in YPD medium and then grown to saturation followed by addition of β-estradiol to induce tag switch. Saturated cultures were re-inoculated into fresh medium to release cells into the cell cycle in the presence of β-estradiol. Cells were arrested in G1 using α-factor. Cells before addition of β-estradiol (pre) and β-estradiol-treated G1 cells (post) were collected for whole-cell lysate preparation and detection of old (V5) and new (Flag) histone H3. The levels of H3K4me1, H3K4me2, H3K4me3 and total histone H3 in the indicated strains are shown. 2Flag , two copies of Flag tag; asterisk, truncated H3. See Supplementary Fig. 22 for full- length image of blots. ( d ) The average mean profile for histone H3 turnover across 800 bp region upstream (−) or downstream (+) of the TSSs (0) at all yeast genes. ( e , f ) Mean profiles for histone H3 turnover across regions (as described for d ) at genes co-repressed ( e ) or co-activated ( f ) by Set1 and Jhd2 in WT, set1Δ and jhd2Δ strains.

Techniques Used: Western Blot, Expressing, FLAG-tag

Set1 and Jhd2 co-regulate chromatin structure and nucleosomal turnover genome-wide during transcriptional regulation in yeast. ( a ) Nucleosome occupancy ratios (Δnucleosome occupancy) between jhd2Δ and control WT across all yeast genes are shown as a heat map, where gain or loss in nucleosome occupancy are in red and blue, respectively. Genes in rows are organized into six k-means clusters. Columns represent 50 bp windows over ±800 bp regions relative to the TSS. Relative positions for −1, +1 and other coding regions nucleosome are indicated at the bottom. Windows overlapping neighbouring genes were excluded. ( b ) Heat map for Δnucleosome occupancy between set1Δ and WT across all yeast genes. Heat map details are as described for a . ( c ) Native nucleosome occupancy for genes in the six k -means clusters generated from reads obtained in WT are shown. Nucleosome occupancy above global mean is in brown. ( d , e ) Heat maps for Set1 and Jhd2 occupancy within the six clusters are shown. ( f ) Heat map for Rpb3 occupancy within the six k -means clusters, using published data 51 , is shown. Pol2 subunit occupancy above global mean is in green ( Supplementary Fig. 14b ). ( g ) The average mean profile for histone H3 turnover across 800 bp region upstream (−) or downstream (+) of the TSSs (0) at all yeast genes in WT , set1Δ and jhd2Δ are shown.
Figure Legend Snippet: Set1 and Jhd2 co-regulate chromatin structure and nucleosomal turnover genome-wide during transcriptional regulation in yeast. ( a ) Nucleosome occupancy ratios (Δnucleosome occupancy) between jhd2Δ and control WT across all yeast genes are shown as a heat map, where gain or loss in nucleosome occupancy are in red and blue, respectively. Genes in rows are organized into six k-means clusters. Columns represent 50 bp windows over ±800 bp regions relative to the TSS. Relative positions for −1, +1 and other coding regions nucleosome are indicated at the bottom. Windows overlapping neighbouring genes were excluded. ( b ) Heat map for Δnucleosome occupancy between set1Δ and WT across all yeast genes. Heat map details are as described for a . ( c ) Native nucleosome occupancy for genes in the six k -means clusters generated from reads obtained in WT are shown. Nucleosome occupancy above global mean is in brown. ( d , e ) Heat maps for Set1 and Jhd2 occupancy within the six clusters are shown. ( f ) Heat map for Rpb3 occupancy within the six k -means clusters, using published data 51 , is shown. Pol2 subunit occupancy above global mean is in green ( Supplementary Fig. 14b ). ( g ) The average mean profile for histone H3 turnover across 800 bp region upstream (−) or downstream (+) of the TSSs (0) at all yeast genes in WT , set1Δ and jhd2Δ are shown.

Techniques Used: Genome Wide, Generated

Model for Set1 and Jhd2-mediated co-regulation of chromatin structure and RNA Pol2 functions. ( a ) Target genes co-repressed by Set1 and Jhd2 are packaged into a well-defined chromatin organization. At these genes, Set1 and Jhd2 activities via H3K4 methylation–demethylation impact histone turnover at promoters leading to nucleosome retention, which restricts activator and/or RNA Pol2 binding. Thus, Set1 and Jhd2 are proposed to primarily co-regulate transcription initiation at these target genes. ( b ) Target genes co-activated by Set1 and Jhd2 are present in a poorly organized chromatin configuration, contain high levels of H3K4me2 and undergo high nucleosomal turnover over coding region. Set1 and Jhd2 activities via modulating H3K4 methylation levels impact nucleosomal turnover over coding regions, and proposed to remove +1 nucleosomes in front of RNA Pol2 and stabilize or retain nucleosomes behind the elongating RNA Pol2. Thus, Set1 and Jhd2 are proposed to co-regulate transcription elongation at these target genes. P , phosphorylated form of RNA Pol2 engaged in elongation.
Figure Legend Snippet: Model for Set1 and Jhd2-mediated co-regulation of chromatin structure and RNA Pol2 functions. ( a ) Target genes co-repressed by Set1 and Jhd2 are packaged into a well-defined chromatin organization. At these genes, Set1 and Jhd2 activities via H3K4 methylation–demethylation impact histone turnover at promoters leading to nucleosome retention, which restricts activator and/or RNA Pol2 binding. Thus, Set1 and Jhd2 are proposed to primarily co-regulate transcription initiation at these target genes. ( b ) Target genes co-activated by Set1 and Jhd2 are present in a poorly organized chromatin configuration, contain high levels of H3K4me2 and undergo high nucleosomal turnover over coding region. Set1 and Jhd2 activities via modulating H3K4 methylation levels impact nucleosomal turnover over coding regions, and proposed to remove +1 nucleosomes in front of RNA Pol2 and stabilize or retain nucleosomes behind the elongating RNA Pol2. Thus, Set1 and Jhd2 are proposed to co-regulate transcription elongation at these target genes. P , phosphorylated form of RNA Pol2 engaged in elongation.

Techniques Used: Methylation, Binding Assay

15) Product Images from "The COMPASS Family of H3K4 Methylases in Drosophila ▿"

Article Title: The COMPASS Family of H3K4 Methylases in Drosophila ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.06092-11

Subunit composition for the histone H3K4 methyltransferase COMPASS and COMPASS-like complexes in yeast and Drosophila . Yeast Set1/COMPASS, the founding member of the H3K4 methylases, is represented as a direct descendant, reflecting the similarity of
Figure Legend Snippet: Subunit composition for the histone H3K4 methyltransferase COMPASS and COMPASS-like complexes in yeast and Drosophila . Yeast Set1/COMPASS, the founding member of the H3K4 methylases, is represented as a direct descendant, reflecting the similarity of

Techniques Used:

16) Product Images from "NEC is likely a NETs dependent process and markers of NETosis are predictive of NEC in mice and humans"

Article Title: NEC is likely a NETs dependent process and markers of NETosis are predictive of NEC in mice and humans

Journal: Scientific Reports

doi: 10.1038/s41598-018-31087-0

NEC induces neutrophil activation and possibly NET formation. ( A,E ) Quantification of extracellular DNA, including neutrophil extracellular traps (NETs), was visualized by SYTOX Orange staining. NEC animals demonstrated a relevant increase in NETs, which was significantly reduced in the absence of PAD (blue). Human subjects with NEC had similar NET scores. ( B,C,F ) Neutrophil elastase (NE) and myeloperoxidase (MPO), both markers of neutrophil activation, increased with time in NEC animals in comparison to controls, and were reduced in animals with PAD inhibition (blue). ( D,G ) Quantification of the NET marker citrullinated histone H3 (H3cit) showed an elevation in animals, as well as humans with NEC. The surrogate marker was significantly reduced in the PAD inhibition group (blue), with H3cit levels in animals with PAD inhibition being close to controls (p = 0.075). Total number of subjects: controls n = 10, 1d NEC n = 6, 2d NEC n = 8, 4d NEC n = 10, 6d NEC n = 7, PAD inhibition n = 10, humans n = 9. Statistics: Mann-Whitney test and ANOVA.
Figure Legend Snippet: NEC induces neutrophil activation and possibly NET formation. ( A,E ) Quantification of extracellular DNA, including neutrophil extracellular traps (NETs), was visualized by SYTOX Orange staining. NEC animals demonstrated a relevant increase in NETs, which was significantly reduced in the absence of PAD (blue). Human subjects with NEC had similar NET scores. ( B,C,F ) Neutrophil elastase (NE) and myeloperoxidase (MPO), both markers of neutrophil activation, increased with time in NEC animals in comparison to controls, and were reduced in animals with PAD inhibition (blue). ( D,G ) Quantification of the NET marker citrullinated histone H3 (H3cit) showed an elevation in animals, as well as humans with NEC. The surrogate marker was significantly reduced in the PAD inhibition group (blue), with H3cit levels in animals with PAD inhibition being close to controls (p = 0.075). Total number of subjects: controls n = 10, 1d NEC n = 6, 2d NEC n = 8, 4d NEC n = 10, 6d NEC n = 7, PAD inhibition n = 10, humans n = 9. Statistics: Mann-Whitney test and ANOVA.

Techniques Used: Activation Assay, Staining, Inhibition, Marker, MANN-WHITNEY

17) Product Images from "Deacetylase inhibitors repress STAT5-mediated transcription by interfering with bromodomain and extra-terminal (BET) protein function"

Article Title: Deacetylase inhibitors repress STAT5-mediated transcription by interfering with bromodomain and extra-terminal (BET) protein function

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv188

Histone acetylation is rapidly and specifically increased upon treatment with deacetylase inhibitors that inhibit STAT5 activity. Ba/F3 cells were treated for 0–60 min with 200 nM TSA, 3 mM VPA, 500 nM apicidin, 1 μM MGCD0103 or 5 μM MS-275. Freeze-thaw protein lysates were analysed by western blot using antibodies directed against acetylated histone H3 (Ac-H3), acetylated histone H4 (Ac-H4) and total histone H3 (H3) as loading control.
Figure Legend Snippet: Histone acetylation is rapidly and specifically increased upon treatment with deacetylase inhibitors that inhibit STAT5 activity. Ba/F3 cells were treated for 0–60 min with 200 nM TSA, 3 mM VPA, 500 nM apicidin, 1 μM MGCD0103 or 5 μM MS-275. Freeze-thaw protein lysates were analysed by western blot using antibodies directed against acetylated histone H3 (Ac-H3), acetylated histone H4 (Ac-H4) and total histone H3 (H3) as loading control.

Techniques Used: Histone Deacetylase Assay, Activity Assay, Mass Spectrometry, Western Blot

Histone occupancy is reduced and histone acetylation is altered all along the Cis gene upon TSA treatment. Rested Ba/F3 cells were pre-treated 30 min with 200 nM TSA or 0.02% DMSO (vehicle) and further stimulated 30 min with IL-3 before being processed for ChIP using antibodies directed against total histone H3, acetylated histone H3 (Ac-H3) and acetylated histone H4 (Ac-H4). Co-precipitated genomic DNA was analysed by quantitative PCR using primers specific for the -800 to +4000 Cis gene locus (Supplementary Table S1). Positions of amplicons investigated and of the four STAT5 binding sites arranged in two clusters are indicated along the x axis. Non-normalized Ac-H3 and Ac-H4 ChIP data are shown in Supplementary Figure S6.
Figure Legend Snippet: Histone occupancy is reduced and histone acetylation is altered all along the Cis gene upon TSA treatment. Rested Ba/F3 cells were pre-treated 30 min with 200 nM TSA or 0.02% DMSO (vehicle) and further stimulated 30 min with IL-3 before being processed for ChIP using antibodies directed against total histone H3, acetylated histone H3 (Ac-H3) and acetylated histone H4 (Ac-H4). Co-precipitated genomic DNA was analysed by quantitative PCR using primers specific for the -800 to +4000 Cis gene locus (Supplementary Table S1). Positions of amplicons investigated and of the four STAT5 binding sites arranged in two clusters are indicated along the x axis. Non-normalized Ac-H3 and Ac-H4 ChIP data are shown in Supplementary Figure S6.

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

Alterations in histone acetylation by TSA at different gene loci differentially affect gene activation. Rested Ba/F3 cells were pre-treated 30 min with 200 nM TSA or 0.02% DMSO (vehicle) and further stimulated 30 min with IL-3 before being processed for ChIP using antibodies directed against RNA polymerase II (RNA Pol II), histone H3, acetylated histone H3 (Ac-H3) and acetylated histone H4 (Ac-H4), as before. Co-precipitated genomic DNA was analysed by quantitative PCR at STAT5 target ( Cis, Osm ) and control ( c-Fos, p21 ) genes, using the same primers as in Figure 6 . Non-normalized Ac-H3 and Ac-H4 ChIP data are shown in Supplementary Figure S7.
Figure Legend Snippet: Alterations in histone acetylation by TSA at different gene loci differentially affect gene activation. Rested Ba/F3 cells were pre-treated 30 min with 200 nM TSA or 0.02% DMSO (vehicle) and further stimulated 30 min with IL-3 before being processed for ChIP using antibodies directed against RNA polymerase II (RNA Pol II), histone H3, acetylated histone H3 (Ac-H3) and acetylated histone H4 (Ac-H4), as before. Co-precipitated genomic DNA was analysed by quantitative PCR at STAT5 target ( Cis, Osm ) and control ( c-Fos, p21 ) genes, using the same primers as in Figure 6 . Non-normalized Ac-H3 and Ac-H4 ChIP data are shown in Supplementary Figure S7.

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

Histone occupancy is reduced at promoters of both STAT5-dependent and -independent genes upon TSA treatment. Ba/F3 cells were withdrawn from IL-3 for 10 h and treated with 200 nM TSA or 0.02% DMSO (vehicle) for 5, 15, 30 and 60 min. As a control for IL-3 stimulation, Ba/F3 cells were stimulated with IL-3 for 30 min following a 30 min TSA pre-treatment (hence treated 60 min with TSA). Cells were harvested and processed for ChIP, as described in Materials and Methods section. ChIP was performed using antibodies directed against total histone H3 or RNA polymerase II (RNA Pol II). Co-precipitated genomic DNA was analysed by quantitative PCR using primers specific for proximal promoter regions (histone H3 ChIP) or transcription start sites (RNA Pol II ChIP) of STAT5 target ( Cis, Osm ) and control ( c-Fos, p21 ) genes. Cis and Osm promoter amplicons overlap their respective STAT5-responsive elements (Supplementary Table S1).
Figure Legend Snippet: Histone occupancy is reduced at promoters of both STAT5-dependent and -independent genes upon TSA treatment. Ba/F3 cells were withdrawn from IL-3 for 10 h and treated with 200 nM TSA or 0.02% DMSO (vehicle) for 5, 15, 30 and 60 min. As a control for IL-3 stimulation, Ba/F3 cells were stimulated with IL-3 for 30 min following a 30 min TSA pre-treatment (hence treated 60 min with TSA). Cells were harvested and processed for ChIP, as described in Materials and Methods section. ChIP was performed using antibodies directed against total histone H3 or RNA polymerase II (RNA Pol II). Co-precipitated genomic DNA was analysed by quantitative PCR using primers specific for proximal promoter regions (histone H3 ChIP) or transcription start sites (RNA Pol II ChIP) of STAT5 target ( Cis, Osm ) and control ( c-Fos, p21 ) genes. Cis and Osm promoter amplicons overlap their respective STAT5-responsive elements (Supplementary Table S1).

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

The nuclear BET protein Brd2 is rapidly relocalized to the chromatin fraction together with acetylated histones upon treatment with deacetylase inhibitors that inhibit STAT5 activity. ( A ) Ba/F3 cells were treated with 200 nM TSA, 10 μM MG132, both (T+MG) or vehicle (0.12% DMSO final in all conditions). Cells were treated for a total duration of ∼2 h, with a 45 min MG132 pre-treatment followed by a 90 min TSA treatment. Soluble and insoluble nuclear fractions were analysed by western blot using antibodies against Brd2 and the nuclear marker HDAC1 as loading control. Similar results were obtained upon 3 h pre-treatment with 10 μM MG132 followed by 90 min incubation with 200 nM TSA (4.5 h treatment in total; not shown). ( B ) Ba/F3 cells were rested for 6 h and stimulated with IL-3 for 60 min. Prior to IL-3 stimulation, cells were pre-treated 3.5 h with 10 μM MG132, 30 min with 200 nM TSA, both (3h MG132 followed by 30 min TSA) or vehicle (0.12% DMSO final in all conditions). Hence cells were incubated 4.5 h in total with 10 μM MG132 and 90 min with 200 nM TSA. Expression of the STAT5 target gene Cis , of the MG132-regulated gene hsp70 and of Brd2 and 36b4 genes as controls was monitored by quantitative RT-PCR. ( C ) Ba/F3 cells were treated for the indicated times with 200 nM TSA, 3 mM valproic acid (VPA), 500 nM apicidin (Api.), 1 μM MGCD0103 (MGC), 5 μM MS-275 (MS), or vehicle (Veh.; 0.02% DMSO final in all conditions). Cytosolic as well as soluble and insoluble nuclear fractions were analysed by western blot using the indicated antibodies. As before, α-tubulin and HDAC1 served as cytosolic and nuclear markers, respectively, to control cell fractionation.
Figure Legend Snippet: The nuclear BET protein Brd2 is rapidly relocalized to the chromatin fraction together with acetylated histones upon treatment with deacetylase inhibitors that inhibit STAT5 activity. ( A ) Ba/F3 cells were treated with 200 nM TSA, 10 μM MG132, both (T+MG) or vehicle (0.12% DMSO final in all conditions). Cells were treated for a total duration of ∼2 h, with a 45 min MG132 pre-treatment followed by a 90 min TSA treatment. Soluble and insoluble nuclear fractions were analysed by western blot using antibodies against Brd2 and the nuclear marker HDAC1 as loading control. Similar results were obtained upon 3 h pre-treatment with 10 μM MG132 followed by 90 min incubation with 200 nM TSA (4.5 h treatment in total; not shown). ( B ) Ba/F3 cells were rested for 6 h and stimulated with IL-3 for 60 min. Prior to IL-3 stimulation, cells were pre-treated 3.5 h with 10 μM MG132, 30 min with 200 nM TSA, both (3h MG132 followed by 30 min TSA) or vehicle (0.12% DMSO final in all conditions). Hence cells were incubated 4.5 h in total with 10 μM MG132 and 90 min with 200 nM TSA. Expression of the STAT5 target gene Cis , of the MG132-regulated gene hsp70 and of Brd2 and 36b4 genes as controls was monitored by quantitative RT-PCR. ( C ) Ba/F3 cells were treated for the indicated times with 200 nM TSA, 3 mM valproic acid (VPA), 500 nM apicidin (Api.), 1 μM MGCD0103 (MGC), 5 μM MS-275 (MS), or vehicle (Veh.; 0.02% DMSO final in all conditions). Cytosolic as well as soluble and insoluble nuclear fractions were analysed by western blot using the indicated antibodies. As before, α-tubulin and HDAC1 served as cytosolic and nuclear markers, respectively, to control cell fractionation.

Techniques Used: Histone Deacetylase Assay, Activity Assay, Western Blot, Marker, Incubation, Expressing, Quantitative RT-PCR, Mass Spectrometry, Cell Fractionation

18) Product Images from "Deacetylase inhibitors repress STAT5-mediated transcription by interfering with bromodomain and extra-terminal (BET) protein function"

Article Title: Deacetylase inhibitors repress STAT5-mediated transcription by interfering with bromodomain and extra-terminal (BET) protein function

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv188

Histone acetylation is rapidly and specifically increased upon treatment with deacetylase inhibitors that inhibit STAT5 activity. Ba/F3 cells were treated for 0–60 min with 200 nM TSA, 3 mM VPA, 500 nM apicidin, 1 μM MGCD0103 or 5 μM MS-275. Freeze-thaw protein lysates were analysed by western blot using antibodies directed against acetylated histone H3 (Ac-H3), acetylated histone H4 (Ac-H4) and total histone H3 (H3) as loading control.
Figure Legend Snippet: Histone acetylation is rapidly and specifically increased upon treatment with deacetylase inhibitors that inhibit STAT5 activity. Ba/F3 cells were treated for 0–60 min with 200 nM TSA, 3 mM VPA, 500 nM apicidin, 1 μM MGCD0103 or 5 μM MS-275. Freeze-thaw protein lysates were analysed by western blot using antibodies directed against acetylated histone H3 (Ac-H3), acetylated histone H4 (Ac-H4) and total histone H3 (H3) as loading control.

Techniques Used: Histone Deacetylase Assay, Activity Assay, Mass Spectrometry, Western Blot

Histone occupancy is reduced and histone acetylation is altered all along the Cis gene upon TSA treatment. Rested Ba/F3 cells were pre-treated 30 min with 200 nM TSA or 0.02% DMSO (vehicle) and further stimulated 30 min with IL-3 before being processed for ChIP using antibodies directed against total histone H3, acetylated histone H3 (Ac-H3) and acetylated histone H4 (Ac-H4). Co-precipitated genomic DNA was analysed by quantitative PCR using primers specific for the -800 to +4000 Cis gene locus (Supplementary Table S1). Positions of amplicons investigated and of the four STAT5 binding sites arranged in two clusters are indicated along the x axis. Non-normalized Ac-H3 and Ac-H4 ChIP data are shown in Supplementary Figure S6.
Figure Legend Snippet: Histone occupancy is reduced and histone acetylation is altered all along the Cis gene upon TSA treatment. Rested Ba/F3 cells were pre-treated 30 min with 200 nM TSA or 0.02% DMSO (vehicle) and further stimulated 30 min with IL-3 before being processed for ChIP using antibodies directed against total histone H3, acetylated histone H3 (Ac-H3) and acetylated histone H4 (Ac-H4). Co-precipitated genomic DNA was analysed by quantitative PCR using primers specific for the -800 to +4000 Cis gene locus (Supplementary Table S1). Positions of amplicons investigated and of the four STAT5 binding sites arranged in two clusters are indicated along the x axis. Non-normalized Ac-H3 and Ac-H4 ChIP data are shown in Supplementary Figure S6.

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

Alterations in histone acetylation by TSA at different gene loci differentially affect gene activation. Rested Ba/F3 cells were pre-treated 30 min with 200 nM TSA or 0.02% DMSO (vehicle) and further stimulated 30 min with IL-3 before being processed for ChIP using antibodies directed against RNA polymerase II (RNA Pol II), histone H3, acetylated histone H3 (Ac-H3) and acetylated histone H4 (Ac-H4), as before. Co-precipitated genomic DNA was analysed by quantitative PCR at STAT5 target ( Cis, Osm ) and control ( c-Fos, p21 ) genes, using the same primers as in Figure 6 . Non-normalized Ac-H3 and Ac-H4 ChIP data are shown in Supplementary Figure S7.
Figure Legend Snippet: Alterations in histone acetylation by TSA at different gene loci differentially affect gene activation. Rested Ba/F3 cells were pre-treated 30 min with 200 nM TSA or 0.02% DMSO (vehicle) and further stimulated 30 min with IL-3 before being processed for ChIP using antibodies directed against RNA polymerase II (RNA Pol II), histone H3, acetylated histone H3 (Ac-H3) and acetylated histone H4 (Ac-H4), as before. Co-precipitated genomic DNA was analysed by quantitative PCR at STAT5 target ( Cis, Osm ) and control ( c-Fos, p21 ) genes, using the same primers as in Figure 6 . Non-normalized Ac-H3 and Ac-H4 ChIP data are shown in Supplementary Figure S7.

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

Histone occupancy is reduced at promoters of both STAT5-dependent and -independent genes upon TSA treatment. Ba/F3 cells were withdrawn from IL-3 for 10 h and treated with 200 nM TSA or 0.02% DMSO (vehicle) for 5, 15, 30 and 60 min. As a control for IL-3 stimulation, Ba/F3 cells were stimulated with IL-3 for 30 min following a 30 min TSA pre-treatment (hence treated 60 min with TSA). Cells were harvested and processed for ChIP, as described in Materials and Methods section. ChIP was performed using antibodies directed against total histone H3 or RNA polymerase II (RNA Pol II). Co-precipitated genomic DNA was analysed by quantitative PCR using primers specific for proximal promoter regions (histone H3 ChIP) or transcription start sites (RNA Pol II ChIP) of STAT5 target ( Cis, Osm ) and control ( c-Fos, p21 ) genes. Cis and Osm promoter amplicons overlap their respective STAT5-responsive elements (Supplementary Table S1).
Figure Legend Snippet: Histone occupancy is reduced at promoters of both STAT5-dependent and -independent genes upon TSA treatment. Ba/F3 cells were withdrawn from IL-3 for 10 h and treated with 200 nM TSA or 0.02% DMSO (vehicle) for 5, 15, 30 and 60 min. As a control for IL-3 stimulation, Ba/F3 cells were stimulated with IL-3 for 30 min following a 30 min TSA pre-treatment (hence treated 60 min with TSA). Cells were harvested and processed for ChIP, as described in Materials and Methods section. ChIP was performed using antibodies directed against total histone H3 or RNA polymerase II (RNA Pol II). Co-precipitated genomic DNA was analysed by quantitative PCR using primers specific for proximal promoter regions (histone H3 ChIP) or transcription start sites (RNA Pol II ChIP) of STAT5 target ( Cis, Osm ) and control ( c-Fos, p21 ) genes. Cis and Osm promoter amplicons overlap their respective STAT5-responsive elements (Supplementary Table S1).

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

The nuclear BET protein Brd2 is rapidly relocalized to the chromatin fraction together with acetylated histones upon treatment with deacetylase inhibitors that inhibit STAT5 activity. ( A ) Ba/F3 cells were treated with 200 nM TSA, 10 μM MG132, both (T+MG) or vehicle (0.12% DMSO final in all conditions). Cells were treated for a total duration of ∼2 h, with a 45 min MG132 pre-treatment followed by a 90 min TSA treatment. Soluble and insoluble nuclear fractions were analysed by western blot using antibodies against Brd2 and the nuclear marker HDAC1 as loading control. Similar results were obtained upon 3 h pre-treatment with 10 μM MG132 followed by 90 min incubation with 200 nM TSA (4.5 h treatment in total; not shown). ( B ) Ba/F3 cells were rested for 6 h and stimulated with IL-3 for 60 min. Prior to IL-3 stimulation, cells were pre-treated 3.5 h with 10 μM MG132, 30 min with 200 nM TSA, both (3h MG132 followed by 30 min TSA) or vehicle (0.12% DMSO final in all conditions). Hence cells were incubated 4.5 h in total with 10 μM MG132 and 90 min with 200 nM TSA. Expression of the STAT5 target gene Cis , of the MG132-regulated gene hsp70 and of Brd2 and 36b4 genes as controls was monitored by quantitative RT-PCR. ( C ) Ba/F3 cells were treated for the indicated times with 200 nM TSA, 3 mM valproic acid (VPA), 500 nM apicidin (Api.), 1 μM MGCD0103 (MGC), 5 μM MS-275 (MS), or vehicle (Veh.; 0.02% DMSO final in all conditions). Cytosolic as well as soluble and insoluble nuclear fractions were analysed by western blot using the indicated antibodies. As before, α-tubulin and HDAC1 served as cytosolic and nuclear markers, respectively, to control cell fractionation.
Figure Legend Snippet: The nuclear BET protein Brd2 is rapidly relocalized to the chromatin fraction together with acetylated histones upon treatment with deacetylase inhibitors that inhibit STAT5 activity. ( A ) Ba/F3 cells were treated with 200 nM TSA, 10 μM MG132, both (T+MG) or vehicle (0.12% DMSO final in all conditions). Cells were treated for a total duration of ∼2 h, with a 45 min MG132 pre-treatment followed by a 90 min TSA treatment. Soluble and insoluble nuclear fractions were analysed by western blot using antibodies against Brd2 and the nuclear marker HDAC1 as loading control. Similar results were obtained upon 3 h pre-treatment with 10 μM MG132 followed by 90 min incubation with 200 nM TSA (4.5 h treatment in total; not shown). ( B ) Ba/F3 cells were rested for 6 h and stimulated with IL-3 for 60 min. Prior to IL-3 stimulation, cells were pre-treated 3.5 h with 10 μM MG132, 30 min with 200 nM TSA, both (3h MG132 followed by 30 min TSA) or vehicle (0.12% DMSO final in all conditions). Hence cells were incubated 4.5 h in total with 10 μM MG132 and 90 min with 200 nM TSA. Expression of the STAT5 target gene Cis , of the MG132-regulated gene hsp70 and of Brd2 and 36b4 genes as controls was monitored by quantitative RT-PCR. ( C ) Ba/F3 cells were treated for the indicated times with 200 nM TSA, 3 mM valproic acid (VPA), 500 nM apicidin (Api.), 1 μM MGCD0103 (MGC), 5 μM MS-275 (MS), or vehicle (Veh.; 0.02% DMSO final in all conditions). Cytosolic as well as soluble and insoluble nuclear fractions were analysed by western blot using the indicated antibodies. As before, α-tubulin and HDAC1 served as cytosolic and nuclear markers, respectively, to control cell fractionation.

Techniques Used: Histone Deacetylase Assay, Activity Assay, Western Blot, Marker, Incubation, Expressing, Quantitative RT-PCR, Mass Spectrometry, Cell Fractionation

19) Product Images from "Identification of Histone Mutants That Are Defective for Transcription-Coupled Nucleosome Occupancy ▿"

Article Title: Identification of Histone Mutants That Are Defective for Transcription-Coupled Nucleosome Occupancy ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.05195-11

Correlation between MNase protection of SRG1 and SER3 expression. The extent of MNase protection across the SRG1 -transcribed unit for wild-type and histone mutant strains () was plotted against the relative level of SER3 expression in these strains
Figure Legend Snippet: Correlation between MNase protection of SRG1 and SER3 expression. The extent of MNase protection across the SRG1 -transcribed unit for wild-type and histone mutant strains () was plotted against the relative level of SER3 expression in these strains

Techniques Used: Expressing, Mutagenesis

Effect of histone mutants on nucleosome positions at SER3 . (A) Diagram of the SER3 locus. The gray ovals mark the position of nucleosomes when wild-type cells are grown in SER3 -repressing conditions (YPD). The block arrow indicates SRG1 transcription.
Figure Legend Snippet: Effect of histone mutants on nucleosome positions at SER3 . (A) Diagram of the SER3 locus. The gray ovals mark the position of nucleosomes when wild-type cells are grown in SER3 -repressing conditions (YPD). The block arrow indicates SRG1 transcription.

Techniques Used: Blocking Assay

Single-amino-acid substitutions in histones H3 and H4 strongly derepress SER3 . (A) Northern blot analysis examining the effect of histone mutants on SER3 , SRG1 , and SCR1 (loading control). Total RNA was isolated from a wild-type strain (FY4) and derivatives
Figure Legend Snippet: Single-amino-acid substitutions in histones H3 and H4 strongly derepress SER3 . (A) Northern blot analysis examining the effect of histone mutants on SER3 , SRG1 , and SCR1 (loading control). Total RNA was isolated from a wild-type strain (FY4) and derivatives

Techniques Used: Northern Blot, Isolation

Effect of histone mutants on cryptic intragenic transcription and posttranslational histone modifications. (A) Northern analysis of FLO8 , STE11 , and SYF1 for cryptic intragenic transcription. Total RNA was isolated from ( hht1-hhf1 ) Δ strains that
Figure Legend Snippet: Effect of histone mutants on cryptic intragenic transcription and posttranslational histone modifications. (A) Northern analysis of FLO8 , STE11 , and SYF1 for cryptic intragenic transcription. Total RNA was isolated from ( hht1-hhf1 ) Δ strains that

Techniques Used: Northern Blot, Isolation

Effect of histone mutants on histone H3 occupancy over the coding regions of a subset of yeast genes. (A) Histone H3 ChIP analysis was performed on chromatin prepared from ( hht1-hhf1 ) Δ strains expressing HHTS-HHFS alleles (JDY86, YJ927, and YJ928)
Figure Legend Snippet: Effect of histone mutants on histone H3 occupancy over the coding regions of a subset of yeast genes. (A) Histone H3 ChIP analysis was performed on chromatin prepared from ( hht1-hhf1 ) Δ strains expressing HHTS-HHFS alleles (JDY86, YJ927, and YJ928)

Techniques Used: Chromatin Immunoprecipitation, Expressing

Identification of histone mutations that derepress SER3 .
Figure Legend Snippet: Identification of histone mutations that derepress SER3 .

Techniques Used:

Mapping of the eight H3/H4 histone residues that strongly derepress SER3 onto the yeast nucleosome crystal structure. (A) A surface representation of the yeast nucleosome core particle viewed down the DNA superhelical axis. Histone proteins are color
Figure Legend Snippet: Mapping of the eight H3/H4 histone residues that strongly derepress SER3 onto the yeast nucleosome crystal structure. (A) A surface representation of the yeast nucleosome core particle viewed down the DNA superhelical axis. Histone proteins are color

Techniques Used:

Analysis of histone mutants for sin phenotype. Wild-type (FY4), snf2 Δ (YJ112), HHTS-HHFS snf2 Δ (YJ1049), hhts-T1181 snf2 Δ (YJ1081), hhts-K122A snf2 Δ (YJ1051), hhts-K122R snf2 Δ (YJ1054), hhts-K122Q snf2 Δ
Figure Legend Snippet: Analysis of histone mutants for sin phenotype. Wild-type (FY4), snf2 Δ (YJ112), HHTS-HHFS snf2 Δ (YJ1049), hhts-T1181 snf2 Δ (YJ1081), hhts-K122A snf2 Δ (YJ1051), hhts-K122R snf2 Δ (YJ1054), hhts-K122Q snf2 Δ

Techniques Used:

Relative occupancy of histone H3 in histone mutants over SER3 . Histone H3 ChIP was performed on chromatin isolated from ( hht1-hhf1 )Δ strains expressing HHTS-HHFS alleles (JDY86, YJ927, and YJ928) or the indicated histone mutant alleles (JDY86
Figure Legend Snippet: Relative occupancy of histone H3 in histone mutants over SER3 . Histone H3 ChIP was performed on chromatin isolated from ( hht1-hhf1 )Δ strains expressing HHTS-HHFS alleles (JDY86, YJ927, and YJ928) or the indicated histone mutant alleles (JDY86

Techniques Used: Chromatin Immunoprecipitation, Isolation, Expressing, Mutagenesis

20) Product Images from "Circulating Histones Are Mediators of Trauma-associated Lung Injury"

Article Title: Circulating Histones Are Mediators of Trauma-associated Lung Injury

Journal: American Journal of Respiratory and Critical Care Medicine

doi: 10.1164/rccm.201206-1037OC

Histone-induced toxicity in mice. ( A ) Hematoxylin and eosin (H E)-stained sections: ( a ) lung from an untreated mouse and ( b ) lung, ( c ) liver, and ( d ) kidney from a mouse 4 hours after infusion with histones (60 mg/kg). Obvious pathological changes were found in the lungs, such as edema ( black arrows ), microvascular congestion ( red arrow ), and hemorrhage ( blue arrow ) ( b ). Less obvious changes were found in ( c ) the liver and ( d ) kidneys. ( B ) Survival curves of mice injected with calf thymus (Cth) histones (75 mg/kg; a lethal dose) preincubated without (none of seven survived) or with anti-histone scFv (ahscFv, 10 mg/kg) (seven of seven survived) and control scFv (cscFv, 10 mg/ml) (none of seven survived); log-rank test, P
Figure Legend Snippet: Histone-induced toxicity in mice. ( A ) Hematoxylin and eosin (H E)-stained sections: ( a ) lung from an untreated mouse and ( b ) lung, ( c ) liver, and ( d ) kidney from a mouse 4 hours after infusion with histones (60 mg/kg). Obvious pathological changes were found in the lungs, such as edema ( black arrows ), microvascular congestion ( red arrow ), and hemorrhage ( blue arrow ) ( b ). Less obvious changes were found in ( c ) the liver and ( d ) kidneys. ( B ) Survival curves of mice injected with calf thymus (Cth) histones (75 mg/kg; a lethal dose) preincubated without (none of seven survived) or with anti-histone scFv (ahscFv, 10 mg/kg) (seven of seven survived) and control scFv (cscFv, 10 mg/ml) (none of seven survived); log-rank test, P

Techniques Used: Mouse Assay, Staining, Injection

Membrane binding and calcium influx determine histone toxicity in endothelial cells. ( A ) Confocal images of EA.hy926 cells 10 minutes after incubation with fluorescein isothiocyanate (FITC)–labeled histones (10 μg/ml) alone ( left ) or FITC-labeled histones preincubated with anti-histone scFv (ahscFv; 100 μg/ml) ( right ). Arrows indicate the membrane associated with FITC-labeled histones. Scale bar: 20 μm. ( B ) Immunohistochemical staining of histone H3 in tissues from a mouse 4 hours after infusion with histones at 60 mg/kg ( left ) and an untreated mouse ( right ). Red arrows indicate endothelial nuclei and blue arrows point to a continuous line between endothelial nuclei stained with anti-histones to indicate association of histones with the plasma membranes of endothelial cells. Scale bar: 20 μm. ( C ) Representative whole-cell currents recorded from EA.hy926 cells when histones (20 μg/ml) were applied to the extracellular bathing solution. Currents generated by application of histones were reversible by washing after a short exposure (30–60 s). ( D ) Example of elevation of intracellular Ca 2+ recorded with a Hitachi F-7000 fluorescence spectrometer when EA.hy926 cells were exposed to various histone concentrations. ( E ) Example of intracellular Ca 2+ elevation triggered by histones (20 μg/ml) that was nearly abolished by removal of Ca 2+ from extracellular medium. ( F ) Survival rates (mean ± SD) of cells incubated for 1 hour with medium containing 0–3 mM Ca 2+ in the presence or absence of histones (20 μg/ml), from three independent experiments. *Significant reduction in cell survival rate compared with that without Ca 2+ (analysis of variance test, P
Figure Legend Snippet: Membrane binding and calcium influx determine histone toxicity in endothelial cells. ( A ) Confocal images of EA.hy926 cells 10 minutes after incubation with fluorescein isothiocyanate (FITC)–labeled histones (10 μg/ml) alone ( left ) or FITC-labeled histones preincubated with anti-histone scFv (ahscFv; 100 μg/ml) ( right ). Arrows indicate the membrane associated with FITC-labeled histones. Scale bar: 20 μm. ( B ) Immunohistochemical staining of histone H3 in tissues from a mouse 4 hours after infusion with histones at 60 mg/kg ( left ) and an untreated mouse ( right ). Red arrows indicate endothelial nuclei and blue arrows point to a continuous line between endothelial nuclei stained with anti-histones to indicate association of histones with the plasma membranes of endothelial cells. Scale bar: 20 μm. ( C ) Representative whole-cell currents recorded from EA.hy926 cells when histones (20 μg/ml) were applied to the extracellular bathing solution. Currents generated by application of histones were reversible by washing after a short exposure (30–60 s). ( D ) Example of elevation of intracellular Ca 2+ recorded with a Hitachi F-7000 fluorescence spectrometer when EA.hy926 cells were exposed to various histone concentrations. ( E ) Example of intracellular Ca 2+ elevation triggered by histones (20 μg/ml) that was nearly abolished by removal of Ca 2+ from extracellular medium. ( F ) Survival rates (mean ± SD) of cells incubated for 1 hour with medium containing 0–3 mM Ca 2+ in the presence or absence of histones (20 μg/ml), from three independent experiments. *Significant reduction in cell survival rate compared with that without Ca 2+ (analysis of variance test, P

Techniques Used: Binding Assay, Incubation, Labeling, Immunohistochemistry, Staining, Generated, Fluorescence

Histone-induced coagulation activation in vivo . ( A ) Box plot shows the medians of thrombin–anti-thrombin (TAT) levels in 20 healthy donors (Normal) and 52 patients with severe nonthoracic trauma. *Median test, P = 0.001. ( B ) Mean ± SD of TAT in mice infused with saline (Control), calf thymus histones (Histones, 50 mg/ml), or histones plus anti-histone scFv (ahscFv, 10 mg/kg) (Hist+ahscFv) (10 mice per group). *Analysis of variance (ANOVA) test, P
Figure Legend Snippet: Histone-induced coagulation activation in vivo . ( A ) Box plot shows the medians of thrombin–anti-thrombin (TAT) levels in 20 healthy donors (Normal) and 52 patients with severe nonthoracic trauma. *Median test, P = 0.001. ( B ) Mean ± SD of TAT in mice infused with saline (Control), calf thymus histones (Histones, 50 mg/ml), or histones plus anti-histone scFv (ahscFv, 10 mg/kg) (Hist+ahscFv) (10 mice per group). *Analysis of variance (ANOVA) test, P

Techniques Used: Coagulation, Activation Assay, In Vivo, Mouse Assay

Histone-triggered cytokine release. ( A ) Circulating IL-6 levels in both the mouse trauma model (Trauma) and the histone infusion model with or without the coinfusion of anti-histone scFv (ahscFv, 10 mg/kg). With 10 mice per group, the means ± SD of IL-6 are shown. *Analysis of variance (ANOVA) test shows a significant increase compared with that before treatment (Before), P
Figure Legend Snippet: Histone-triggered cytokine release. ( A ) Circulating IL-6 levels in both the mouse trauma model (Trauma) and the histone infusion model with or without the coinfusion of anti-histone scFv (ahscFv, 10 mg/kg). With 10 mice per group, the means ± SD of IL-6 are shown. *Analysis of variance (ANOVA) test shows a significant increase compared with that before treatment (Before), P

Techniques Used: Mouse Assay

21) Product Images from "Sustained activation of STAT5 is essential for chromatin remodeling and maintenance of mammary-specific function"

Article Title: Sustained activation of STAT5 is essential for chromatin remodeling and maintenance of mammary-specific function

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200807021

Constitutively active STAT5 induces chromatin remodeling and β-casein expression in the absence of laminin-111 signals. (A–C) Introducing constitutively activated STAT5A/B (STAT5A/B 1*6) is sufficient to induce chromatin remodeling and β-casein transcription in the absence of lrECM on polyHEMA. EpH4 cells infected with retrovirus (vector control) or expressing STAT5A/B 1*6 were treated with Prl and 2% lrECM on polyHEMA for 24 h. (A) Phosphorylation of STAT5 was detected by Western blot assays. (B) ChIP analysis of levels of acetylated histone H4 at β-casein promoter. (C) The mRNA levels of β-casein were measured by real-time RT-PCR.
Figure Legend Snippet: Constitutively active STAT5 induces chromatin remodeling and β-casein expression in the absence of laminin-111 signals. (A–C) Introducing constitutively activated STAT5A/B (STAT5A/B 1*6) is sufficient to induce chromatin remodeling and β-casein transcription in the absence of lrECM on polyHEMA. EpH4 cells infected with retrovirus (vector control) or expressing STAT5A/B 1*6 were treated with Prl and 2% lrECM on polyHEMA for 24 h. (A) Phosphorylation of STAT5 was detected by Western blot assays. (B) ChIP analysis of levels of acetylated histone H4 at β-casein promoter. (C) The mRNA levels of β-casein were measured by real-time RT-PCR.

Techniques Used: Expressing, Infection, Plasmid Preparation, Western Blot, Chromatin Immunoprecipitation, Quantitative RT-PCR

Sustained activation of STAT5 is required for histone acetylation in the β-casein promoter. (A and B) ChIP assays measuring the acetylated histone levels in the β-casein promoter. (A) EpH4 cells on polyHEMA were treated with Prl or Prl plus 2% lrECM for different times before the analysis. (B) EpH4 cells were treated with AG490 to block the sustained STAT5 activation as shown in Fig. 4 A before the ChIP analysis. The PCR results were quantified by AlphaEaseFC software, and the values of ChIP DNA were normalized to input DNA. Fold enrichments were determined by dividing the normalized values from treated cells by that of untreated cells.
Figure Legend Snippet: Sustained activation of STAT5 is required for histone acetylation in the β-casein promoter. (A and B) ChIP assays measuring the acetylated histone levels in the β-casein promoter. (A) EpH4 cells on polyHEMA were treated with Prl or Prl plus 2% lrECM for different times before the analysis. (B) EpH4 cells were treated with AG490 to block the sustained STAT5 activation as shown in Fig. 4 A before the ChIP analysis. The PCR results were quantified by AlphaEaseFC software, and the values of ChIP DNA were normalized to input DNA. Fold enrichments were determined by dividing the normalized values from treated cells by that of untreated cells.

Techniques Used: Activation Assay, Chromatin Immunoprecipitation, Blocking Assay, Polymerase Chain Reaction, Software

22) Product Images from "TIEG1 Inhibits Breast Cancer Invasion and Metastasis by Inhibition of Epidermal Growth Factor Receptor (EGFR) Transcription and the EGFR Signaling Pathway"

Article Title: TIEG1 Inhibits Breast Cancer Invasion and Metastasis by Inhibition of Epidermal Growth Factor Receptor (EGFR) Transcription and the EGFR Signaling Pathway

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.06152-11

Overexpression of TIEG1 attenuates EGFR expression and histone acetylation of the EGFR promoter in MDA-MB-231HM cells. (A and B) TIEG1 expression vector or control vector was transfected into MDA-MB-231HM cells and generated stable transfectants, MDA-MB-231HM/TIEG1
Figure Legend Snippet: Overexpression of TIEG1 attenuates EGFR expression and histone acetylation of the EGFR promoter in MDA-MB-231HM cells. (A and B) TIEG1 expression vector or control vector was transfected into MDA-MB-231HM cells and generated stable transfectants, MDA-MB-231HM/TIEG1

Techniques Used: Over Expression, Expressing, Multiple Displacement Amplification, Plasmid Preparation, Transfection, Generated

Binding status of TIEG1 complex and histone acetylation on the EGFR promoter in MDA-MB-231 and MDA-MB-231HM cells. (A and B) Relative TIEG1 and EGFR mRNA levels were detected in MDA-MB-231 and MDA-MB-231HM cells. Total RNA was extracted from the cells
Figure Legend Snippet: Binding status of TIEG1 complex and histone acetylation on the EGFR promoter in MDA-MB-231 and MDA-MB-231HM cells. (A and B) Relative TIEG1 and EGFR mRNA levels were detected in MDA-MB-231 and MDA-MB-231HM cells. Total RNA was extracted from the cells

Techniques Used: Binding Assay, Multiple Displacement Amplification

23) Product Images from "Subcellular localisation modulates ubiquitylation and degradation of Ascl1"

Article Title: Subcellular localisation modulates ubiquitylation and degradation of Ascl1

Journal: Scientific Reports

doi: 10.1038/s41598-018-23056-4

Ascl1 is ubiquitylated by Huwe1 in the cytoplasm. ( A ) Schematic representation of experimental design. Adenovirally-mediated Cre expression was used to delete Huwe1 from NSCs harbouring the Flox-Huwe1 allele and cells were harvested after 1 day in differentiation media. ( B ) Flox-Huwe1 NSCs at day 5 were incubated +/− MG132 for 2 hours, and cytoplasmic and chromatin fractions were processed for western blot analysis to detect Huwe1 as well as α-Tubulin (loading control for cytoplasmic fraction) and histone H3 (loading control for chromatin fraction). ( C ) Cytoplasmic fractions with and without cre-mediated knockdown of Huwe1 (see panel B), were incubated with and without MG132 for 2 hours and western blotted to detect free and ubiquitin-conjugated (blue bracket) forms of Ascl1. Top panel is long exposure to reveal high molecular weight ubiquitylated forms of Ascl1 (blue bracket), bottom Ascl1 panel is lower exposure (cropped from the same blot) to show stabilisation of Ascl1 after Huwe1 knockdown, α-Tubulin (loading control for cytoplasmic fraction, cropped to show specific band of correct molecular weight), n = 3.
Figure Legend Snippet: Ascl1 is ubiquitylated by Huwe1 in the cytoplasm. ( A ) Schematic representation of experimental design. Adenovirally-mediated Cre expression was used to delete Huwe1 from NSCs harbouring the Flox-Huwe1 allele and cells were harvested after 1 day in differentiation media. ( B ) Flox-Huwe1 NSCs at day 5 were incubated +/− MG132 for 2 hours, and cytoplasmic and chromatin fractions were processed for western blot analysis to detect Huwe1 as well as α-Tubulin (loading control for cytoplasmic fraction) and histone H3 (loading control for chromatin fraction). ( C ) Cytoplasmic fractions with and without cre-mediated knockdown of Huwe1 (see panel B), were incubated with and without MG132 for 2 hours and western blotted to detect free and ubiquitin-conjugated (blue bracket) forms of Ascl1. Top panel is long exposure to reveal high molecular weight ubiquitylated forms of Ascl1 (blue bracket), bottom Ascl1 panel is lower exposure (cropped from the same blot) to show stabilisation of Ascl1 after Huwe1 knockdown, α-Tubulin (loading control for cytoplasmic fraction, cropped to show specific band of correct molecular weight), n = 3.

Techniques Used: Expressing, Incubation, Western Blot, Molecular Weight

Chromatin-bound Ascl1 is more stable than cytoplasmic Ascl1. ( A . Error bars = SEM; n = 3 independent experiments. ( B ) P19 cells transfected with (human) Ascl1 and transferred the day after to differentiation media for 24 hours. Cellular fractionation was performed after 2 hours treatment with or without MG132 and ubiquitin-bound proteins were isolated using the TUBEs method. Western blotting for Ascl1 compared input, resin-unbound and resin-bound fractions. Blue bracket, Ascl1 with long ubiquitin chains; black bracket, Ascl1 with short ubiquitin chains. Also shown are α-Tubulin (control for cytoplasmic fractionation) and histone H3 (control for chromatin fractionation). ( C . Error bars = SEM; n = 3 independent experiments. ( D ) As ( C .
Figure Legend Snippet: Chromatin-bound Ascl1 is more stable than cytoplasmic Ascl1. ( A . Error bars = SEM; n = 3 independent experiments. ( B ) P19 cells transfected with (human) Ascl1 and transferred the day after to differentiation media for 24 hours. Cellular fractionation was performed after 2 hours treatment with or without MG132 and ubiquitin-bound proteins were isolated using the TUBEs method. Western blotting for Ascl1 compared input, resin-unbound and resin-bound fractions. Blue bracket, Ascl1 with long ubiquitin chains; black bracket, Ascl1 with short ubiquitin chains. Also shown are α-Tubulin (control for cytoplasmic fractionation) and histone H3 (control for chromatin fractionation). ( C . Error bars = SEM; n = 3 independent experiments. ( D ) As ( C .

Techniques Used: Transfection, Cell Fractionation, Isolation, Western Blot, Fractionation

Sites available for ubiquitylation differ in cytoplasmic and chromatin bound Ascl1. ( A ) Schematic representation of human Ascl1 indicating mutation of lysines into arginines at the N-terminus and within the bHLH domain. ( B ) P19 cells transfected with wild-type Ascl1, N-term K > R_Ascl1, bHLH K > R_Ascl1 or Full K > R_Ascl1 were transferred the day after to differentiation media for 24 hours. Cellular fractionation was performed, and ubiquitylated proteins were isolated using the TUBEs method. Western blotting for Ascl1 compared input, resin-unbound and resin-bound fractions from cytoplasmic and chromatin compartments as labelled. Black bracket, Ascl1 with short ubiquitin chains in the chromatin fraction; blue bracket, Ascl1 with long ubiquitin chains in the cytoplasmic fraction. Also shown are α-Tubulin (control for cytoplasmic fractionation) and histone H3 (control for chromatin fractionation). ( C ) As ( B ) above, except cells were incubated with or without MG132 before harvesting to inhibit ubiquitin-mediated proteolysis. Western blots for loading control were cropped to show specific band of correct molecular weight.
Figure Legend Snippet: Sites available for ubiquitylation differ in cytoplasmic and chromatin bound Ascl1. ( A ) Schematic representation of human Ascl1 indicating mutation of lysines into arginines at the N-terminus and within the bHLH domain. ( B ) P19 cells transfected with wild-type Ascl1, N-term K > R_Ascl1, bHLH K > R_Ascl1 or Full K > R_Ascl1 were transferred the day after to differentiation media for 24 hours. Cellular fractionation was performed, and ubiquitylated proteins were isolated using the TUBEs method. Western blotting for Ascl1 compared input, resin-unbound and resin-bound fractions from cytoplasmic and chromatin compartments as labelled. Black bracket, Ascl1 with short ubiquitin chains in the chromatin fraction; blue bracket, Ascl1 with long ubiquitin chains in the cytoplasmic fraction. Also shown are α-Tubulin (control for cytoplasmic fractionation) and histone H3 (control for chromatin fractionation). ( C ) As ( B ) above, except cells were incubated with or without MG132 before harvesting to inhibit ubiquitin-mediated proteolysis. Western blots for loading control were cropped to show specific band of correct molecular weight.

Techniques Used: Mutagenesis, Transfection, Cell Fractionation, Isolation, Western Blot, Fractionation, Incubation, Molecular Weight

Preventing ubiquitylation of Ascl1 enhances its transcriptional activation. ( A ) P19 cells transfected with wild-type Ascl1 and were transferred the day after to differentiation media for 24 hours, then treated with 20 μg/ml α-Amanitin for 4 hours. Chromatin-bound proteins were isolated and western blotted for Ascl1, top Ascl1 panel long exposure, bottom Ascl1 panel, cropped lower exposure of the same blot to show decrease in Ascl1 protein due to block of ongoing transcription and histone H3 (chromatin marker, cropped to show specific band of correct molecular weight). ( B ) Gene reporter assay in P19 cells transfected with wild-type Ascl1 and mutants thereof as labelled to assay Ascl1 transcriptional activity. Fold activity relative to vector-only controls. Error bars = SEM; n = 3 independent experiments; two-tailed unpaired t-test (*p
Figure Legend Snippet: Preventing ubiquitylation of Ascl1 enhances its transcriptional activation. ( A ) P19 cells transfected with wild-type Ascl1 and were transferred the day after to differentiation media for 24 hours, then treated with 20 μg/ml α-Amanitin for 4 hours. Chromatin-bound proteins were isolated and western blotted for Ascl1, top Ascl1 panel long exposure, bottom Ascl1 panel, cropped lower exposure of the same blot to show decrease in Ascl1 protein due to block of ongoing transcription and histone H3 (chromatin marker, cropped to show specific band of correct molecular weight). ( B ) Gene reporter assay in P19 cells transfected with wild-type Ascl1 and mutants thereof as labelled to assay Ascl1 transcriptional activity. Fold activity relative to vector-only controls. Error bars = SEM; n = 3 independent experiments; two-tailed unpaired t-test (*p

Techniques Used: Activation Assay, Transfection, Isolation, Western Blot, Blocking Assay, Marker, Molecular Weight, Reporter Assay, Activity Assay, Plasmid Preparation, Two Tailed Test

24) Product Images from "Dynamic reprogramming of DNA methylation in SETD2-deregulated renal cell carcinoma"

Article Title: Dynamic reprogramming of DNA methylation in SETD2-deregulated renal cell carcinoma

Journal: Oncotarget

doi:

Reduction and redistribution of H3K36me3 upon inactivation of SETD2 in 786-O ccRCC cells A. Western blot of H3K36 methylation in parental 786-O cells and two independent SETD2 KO clones. Histone H3 is a loading control. B. Distribution of H3K36me3 ChIP-seq peaks genome-wide in 786-O ccRCC cells. C. Box plots for H3K36me3 peak length across genomic features. Mean line is represented in red. D. Number of genes with ≥ 2-fold-change in H3K36me3 level in SETD2 KO clone 2. E. Representative browser shots demonstrating loss and gain of H3K36me3 in SETD2 KO2 clone. Top panel represents intergenic regions that gain H3K36me3. Bent arrows = TSS, Green bars = CpG islands. See also Figure S1, S2 .
Figure Legend Snippet: Reduction and redistribution of H3K36me3 upon inactivation of SETD2 in 786-O ccRCC cells A. Western blot of H3K36 methylation in parental 786-O cells and two independent SETD2 KO clones. Histone H3 is a loading control. B. Distribution of H3K36me3 ChIP-seq peaks genome-wide in 786-O ccRCC cells. C. Box plots for H3K36me3 peak length across genomic features. Mean line is represented in red. D. Number of genes with ≥ 2-fold-change in H3K36me3 level in SETD2 KO clone 2. E. Representative browser shots demonstrating loss and gain of H3K36me3 in SETD2 KO2 clone. Top panel represents intergenic regions that gain H3K36me3. Bent arrows = TSS, Green bars = CpG islands. See also Figure S1, S2 .

Techniques Used: Western Blot, Methylation, Clone Assay, Chromatin Immunoprecipitation, Genome Wide

25) Product Images from "Invadolysin acts genetically via the SAGA complex to modulate chromosome structure"

Article Title: Invadolysin acts genetically via the SAGA complex to modulate chromosome structure

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv211

nonstop interacts genetically with invadolysin and exhibits similar phenotypes to invadolysin mutants. ( A ) Drosophila Nonstop is a highly conserved protein belonging to the C19 class of DUB cysteine proteases, with a peptidase domain at the C-terminus, and a zinc finger motif in the central region. Drosophila nonstop exhibits homology to UBP8 from Saccharomyces cerevisiae and human USP22. The lengths of the proteins and locations of the relevant domains are shown. ( B ) invadolysin and nonstop mutants exhibit similar mitotic chromosome phenotypes after staining with DAPI. Both invadolysin and nonstop mitotic chromosomes are hypercondensed in length, yet fuzzy in appearance (scale bar = 5 μm). ( C ) The mitotic index was decreased in both invadolysin and nonstop mutant brains. Cells were counted from 10 fields each from three brains for all the genotypes shown. The mitotic index (following immunostaining for phospho-histone H3S10) of mutants ( invadolysin and nonstop ) was significantly lower than in wild-type neuroblasts. The p-values are: IX-14 4y7 P = 0.002, not 1 P = 0.005 and not 2 P = 0.01.
Figure Legend Snippet: nonstop interacts genetically with invadolysin and exhibits similar phenotypes to invadolysin mutants. ( A ) Drosophila Nonstop is a highly conserved protein belonging to the C19 class of DUB cysteine proteases, with a peptidase domain at the C-terminus, and a zinc finger motif in the central region. Drosophila nonstop exhibits homology to UBP8 from Saccharomyces cerevisiae and human USP22. The lengths of the proteins and locations of the relevant domains are shown. ( B ) invadolysin and nonstop mutants exhibit similar mitotic chromosome phenotypes after staining with DAPI. Both invadolysin and nonstop mitotic chromosomes are hypercondensed in length, yet fuzzy in appearance (scale bar = 5 μm). ( C ) The mitotic index was decreased in both invadolysin and nonstop mutant brains. Cells were counted from 10 fields each from three brains for all the genotypes shown. The mitotic index (following immunostaining for phospho-histone H3S10) of mutants ( invadolysin and nonstop ) was significantly lower than in wild-type neuroblasts. The p-values are: IX-14 4y7 P = 0.002, not 1 P = 0.005 and not 2 P = 0.01.

Techniques Used: Staining, Mutagenesis, Immunostaining

invadolysin and nonstop homozygous, and IX-14 1 /not 1 transheterozygous third instar larvae accumulate similar histone modifications. Over-expression of the Bre1 ubiquitin ligase phenocopies this effect. ( A ) Immunoblotting of invadolysin, nonstop, sgf11, ada2b and gcn5 homozygous third instar larval extracts using various antibodies revealed changes to histone modification. Mono-ubiquitinated histone H2B, di- and tri-methylated histone H3 at lysine 4, and acetylated H3K9/14 were increased in invadolysin and nonstop larval protein extracts. ( B ) Immunoblotting of IX-14 1 /not 1 transheterozygous third instar larval extracts using the same antibodies as in (A) revealed a similar accumulation of mono-ubiquitinated histone H2B, di- and tri-methylated histone H3 at lysine 4, and acetylated H3K9/14 in transheterozygous larval protein extracts as well. The levels of non-SAGA dependent modified histones in (A) and (B) remain unchanged (*H3K4me1 and *H3K18ac). ( C ) Immunoblotting of dBre1 overexpressing (using an actin-Gal4 driver) third instar larval extracts revealed the accumulation of ubH2B and H3K4me3. Loading controls for panels (A)–(C) are represented by histone H2B, histone H3 and α-tubulin.
Figure Legend Snippet: invadolysin and nonstop homozygous, and IX-14 1 /not 1 transheterozygous third instar larvae accumulate similar histone modifications. Over-expression of the Bre1 ubiquitin ligase phenocopies this effect. ( A ) Immunoblotting of invadolysin, nonstop, sgf11, ada2b and gcn5 homozygous third instar larval extracts using various antibodies revealed changes to histone modification. Mono-ubiquitinated histone H2B, di- and tri-methylated histone H3 at lysine 4, and acetylated H3K9/14 were increased in invadolysin and nonstop larval protein extracts. ( B ) Immunoblotting of IX-14 1 /not 1 transheterozygous third instar larval extracts using the same antibodies as in (A) revealed a similar accumulation of mono-ubiquitinated histone H2B, di- and tri-methylated histone H3 at lysine 4, and acetylated H3K9/14 in transheterozygous larval protein extracts as well. The levels of non-SAGA dependent modified histones in (A) and (B) remain unchanged (*H3K4me1 and *H3K18ac). ( C ) Immunoblotting of dBre1 overexpressing (using an actin-Gal4 driver) third instar larval extracts revealed the accumulation of ubH2B and H3K4me3. Loading controls for panels (A)–(C) are represented by histone H2B, histone H3 and α-tubulin.

Techniques Used: Over Expression, Modification, Methylation

Model for the interaction between invadolysin, nonstop and bre1 . We hypothesize that invadolysin affects higher order chromosome architecture through an effect on the balance of histone modification. Ubiquitination of histone H2B is accomplished through the concerted action of the bre1 ubiquitin ligase and the nonstop ubiquitinating protease. In the absence of invadolysin, we speculate there is decreased activity of nonstop, which would result in the accumulation of ubiquitinated H2B. In nonstop and invadolysin mutants, and in animals where Bre1 is overexpressed, ubH2B, H3K4me3, H3K4me2 and H3K9/14ac accumulate and abnormally-structured chromosomes are observed.
Figure Legend Snippet: Model for the interaction between invadolysin, nonstop and bre1 . We hypothesize that invadolysin affects higher order chromosome architecture through an effect on the balance of histone modification. Ubiquitination of histone H2B is accomplished through the concerted action of the bre1 ubiquitin ligase and the nonstop ubiquitinating protease. In the absence of invadolysin, we speculate there is decreased activity of nonstop, which would result in the accumulation of ubiquitinated H2B. In nonstop and invadolysin mutants, and in animals where Bre1 is overexpressed, ubH2B, H3K4me3, H3K4me2 and H3K9/14ac accumulate and abnormally-structured chromosomes are observed.

Techniques Used: Modification, Activity Assay

26) Product Images from "Peptidylarginine Deiminase Inhibition Reduces Vascular Damage and Modulates Innate Immune Responses in Murine Models of Atherosclerosis"

Article Title: Peptidylarginine Deiminase Inhibition Reduces Vascular Damage and Modulates Innate Immune Responses in Murine Models of Atherosclerosis

Journal: Circulation research

doi: 10.1161/CIRCRESAHA.114.303312

Apoe −/− mice demonstrate enhanced neutrophil extracellular trap (NET) formation and develop autoantibodies to NETs A and B, Representative immunofluorescence staining of nonpermeabilized Apoe −/− neutrophils for citrullinated histone H3 (H3-Cit; A) and MPO (B). DNA is stained blue, and the indicated protein green. Scale bars=10 μm. C, Bone marrow neutrophils were isolated from 8-week-old Apoe −/− mice. Neutrophils were incubated in the presence of 10% serum from the indicated mice for 4 hours (n=5 mice per group). *** P
Figure Legend Snippet: Apoe −/− mice demonstrate enhanced neutrophil extracellular trap (NET) formation and develop autoantibodies to NETs A and B, Representative immunofluorescence staining of nonpermeabilized Apoe −/− neutrophils for citrullinated histone H3 (H3-Cit; A) and MPO (B). DNA is stained blue, and the indicated protein green. Scale bars=10 μm. C, Bone marrow neutrophils were isolated from 8-week-old Apoe −/− mice. Neutrophils were incubated in the presence of 10% serum from the indicated mice for 4 hours (n=5 mice per group). *** P

Techniques Used: Mouse Assay, Immunofluorescence, Staining, Isolation, Incubation

Interferon expression and histone citrullination are upregulated in atherosclerotic lesions A, Apoe −/− mice were placed on high-fat chow beginning at 8 weeks of age. RNA was isolated from aortic arches of 8- and 18-week-old Apoe −/− mice. Fold change in gene expression was calculated for 18-week-old mice, relative to 8-week-old mice (n=5). Mean and SEM are plotted. * P
Figure Legend Snippet: Interferon expression and histone citrullination are upregulated in atherosclerotic lesions A, Apoe −/− mice were placed on high-fat chow beginning at 8 weeks of age. RNA was isolated from aortic arches of 8- and 18-week-old Apoe −/− mice. Fold change in gene expression was calculated for 18-week-old mice, relative to 8-week-old mice (n=5). Mean and SEM are plotted. * P

Techniques Used: Expressing, Mouse Assay, Isolation

27) Product Images from "ARABIDOPSIS TRITHORAX-RELATED7 Is Required for Methylation of Lysine 4 of Histone H3 and for Transcriptional Activation of FLOWERING LOCUS C [C] [C] [W]"

Article Title: ARABIDOPSIS TRITHORAX-RELATED7 Is Required for Methylation of Lysine 4 of Histone H3 and for Transcriptional Activation of FLOWERING LOCUS C [C] [C] [W]

Journal: The Plant Cell

doi: 10.1105/tpc.109.070060

Schematic Model of Transcriptional Activation through Histone Modifications. Summary of the relationship among the factors involved in the transcriptional activation of the yeast GAL1 gene ( [A] ) and the FLC gene (B) . Circles, histone octamers; K4, Lys 4 of histone H3 (H3K4); K36, Lys 36 of histone H3 (H3K36); me, methyl groups on Lys residues. (A) In yeast, the Paf1 complex is required for the recruitment of both Set1 and Set2, which are H3K4 and H3K36 methylases, respectively. Both trimethylation of H3K4 (H3K4me3) and dimethylation of H3K36 (H3K36me2) are involved in the transcriptional activation of certain genes including GAL1. (B) In Arabidopsis , H3K4me3 and H3K36me2 are also essential for the transcriptional activation of FLC . Both Set1-class (ATXR7) and Trx-class (ATX1/2) H3K4 methylases are required for full H3K4 methylation and, thus, FLC activation. The Paf1 complex (including ELF7 and VIP4) is required for the function of ATXR7 and possibly ATX1/2, but not EFS (a Set2 ortholog). [See online article for color version of this figure.]
Figure Legend Snippet: Schematic Model of Transcriptional Activation through Histone Modifications. Summary of the relationship among the factors involved in the transcriptional activation of the yeast GAL1 gene ( [A] ) and the FLC gene (B) . Circles, histone octamers; K4, Lys 4 of histone H3 (H3K4); K36, Lys 36 of histone H3 (H3K36); me, methyl groups on Lys residues. (A) In yeast, the Paf1 complex is required for the recruitment of both Set1 and Set2, which are H3K4 and H3K36 methylases, respectively. Both trimethylation of H3K4 (H3K4me3) and dimethylation of H3K36 (H3K36me2) are involved in the transcriptional activation of certain genes including GAL1. (B) In Arabidopsis , H3K4me3 and H3K36me2 are also essential for the transcriptional activation of FLC . Both Set1-class (ATXR7) and Trx-class (ATX1/2) H3K4 methylases are required for full H3K4 methylation and, thus, FLC activation. The Paf1 complex (including ELF7 and VIP4) is required for the function of ATXR7 and possibly ATX1/2, but not EFS (a Set2 ortholog). [See online article for color version of this figure.]

Techniques Used: Activation Assay, Methylation

28) Product Images from "TET2- and TDG-mediated changes are required for the acquisition of distinct histone modifications in divergent terminal differentiation of myeloid cells"

Article Title: TET2- and TDG-mediated changes are required for the acquisition of distinct histone modifications in divergent terminal differentiation of myeloid cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx666

TET2 and TDG regulate active DNA demethylation and gene expression in MAC and OC differentiation. ( A ) A schematic representation depicting the participation of enzymes including DNA methyltransferases (DNMT1, DNMT3A and DNMT3B), methylcytosine dioxygenases (TET2), thymine DNA glycosylases (TDG) and the base excision repair machinery (BER pathway) in the oxidation and demethylation of cytosines. ( B ) Gene expression levels of several enzymes (or related family members) associated with active DNA demethylation are evaluated in MOs and differentiated MACs and OCs at 5 and 20 days of differentiation. These enzymes are grouped as 5mC hydroxylases, cytidine deaminases, or glycosylases based on their described activity during the demethylation process. ( C ) mRNA levels of AICDA , and mRNA and protein levels of TET2 and TDG are evaluated in MOs and differentiated MACs and OCs during 20 days. Quantitative RT-PCR data is normalized against RPL38 . Histone H3 and actin levels are used as a loading control in the western blot. ( D ) The effects of siRNA-mediated downregulation of TET2, TDG and AID are evaluated in OCs and MACs. TRAP staining and dentin resorption assays (left panels) are performed in OCs after 20 days of differentiation. Phagocytosis assays and FACS analyses of surface markers, including CD163, CD206, MSR1, CD11b and CD33 (right panels) are performed in MACs after 5 days of differentiation. ( E ) Effects of TET2, TDG and AID downregulation on 5mC and 5hmC in OC differentiated during 20 days. Percentage of 5mC and 5hmC are evaluated in ANXA2, TM4SF19 and ADAM12 following siRNA downregulation of TET2 (upper rows), TDG (third row) and AID (lower row). ( F ) Analyses of gene expression of ANXA2, TM4SF9 and ADAM12 by RT-qPCR following siRNA downregulation of TET2 (upper row), TDG (middle row) and AID (lower row). Relative mRNA is normalized against RPL38 .
Figure Legend Snippet: TET2 and TDG regulate active DNA demethylation and gene expression in MAC and OC differentiation. ( A ) A schematic representation depicting the participation of enzymes including DNA methyltransferases (DNMT1, DNMT3A and DNMT3B), methylcytosine dioxygenases (TET2), thymine DNA glycosylases (TDG) and the base excision repair machinery (BER pathway) in the oxidation and demethylation of cytosines. ( B ) Gene expression levels of several enzymes (or related family members) associated with active DNA demethylation are evaluated in MOs and differentiated MACs and OCs at 5 and 20 days of differentiation. These enzymes are grouped as 5mC hydroxylases, cytidine deaminases, or glycosylases based on their described activity during the demethylation process. ( C ) mRNA levels of AICDA , and mRNA and protein levels of TET2 and TDG are evaluated in MOs and differentiated MACs and OCs during 20 days. Quantitative RT-PCR data is normalized against RPL38 . Histone H3 and actin levels are used as a loading control in the western blot. ( D ) The effects of siRNA-mediated downregulation of TET2, TDG and AID are evaluated in OCs and MACs. TRAP staining and dentin resorption assays (left panels) are performed in OCs after 20 days of differentiation. Phagocytosis assays and FACS analyses of surface markers, including CD163, CD206, MSR1, CD11b and CD33 (right panels) are performed in MACs after 5 days of differentiation. ( E ) Effects of TET2, TDG and AID downregulation on 5mC and 5hmC in OC differentiated during 20 days. Percentage of 5mC and 5hmC are evaluated in ANXA2, TM4SF19 and ADAM12 following siRNA downregulation of TET2 (upper rows), TDG (third row) and AID (lower row). ( F ) Analyses of gene expression of ANXA2, TM4SF9 and ADAM12 by RT-qPCR following siRNA downregulation of TET2 (upper row), TDG (middle row) and AID (lower row). Relative mRNA is normalized against RPL38 .

Techniques Used: Expressing, Magnetic Cell Separation, Activity Assay, Quantitative RT-PCR, Western Blot, Staining, FACS

29) Product Images from "The LRS and SIN Domains: Two Structurally Equivalent but Functionally Distinct Nucleosomal Surfaces Required for Transcriptional Silencing ▿"

Article Title: The LRS and SIN Domains: Two Structurally Equivalent but Functionally Distinct Nucleosomal Surfaces Required for Transcriptional Silencing ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00248-06

Sin − and Lrs − alterations do not generally disrupt H3-K79 methylation. (A) JPY12 cells expressing either wild-type, Lrs − , or Sin − histones as the sole source of histone H3 or H4 were grown to mid-log phase in YEPD medium. Cells were lysed in Laemmli buffer and lysates were analyzed by SDS-PAGE, followed by Western blot analysis with antibodies raised against histone H3 dimethyl K79 and TBP (loading control; upper panel) or histone H3 trimethyl K79 and total histone H3 (loading control; lower panel). (B) Soluble chromatin was isolated from wild-type (JPY12), dot1Δ (EMHY234), and a series of dot1Δ Lrs − strains and incubated with recombinant Dot1p and [ 3 H] S -adenosyl-methionine. Reaction products were separated by SDS-PAGE, and methylated histone H3 was detected by fluorography (upper panel). Histone H3 was detected by immunoblot analysis to normalize input levels of histone proteins in each reaction (lower panel).
Figure Legend Snippet: Sin − and Lrs − alterations do not generally disrupt H3-K79 methylation. (A) JPY12 cells expressing either wild-type, Lrs − , or Sin − histones as the sole source of histone H3 or H4 were grown to mid-log phase in YEPD medium. Cells were lysed in Laemmli buffer and lysates were analyzed by SDS-PAGE, followed by Western blot analysis with antibodies raised against histone H3 dimethyl K79 and TBP (loading control; upper panel) or histone H3 trimethyl K79 and total histone H3 (loading control; lower panel). (B) Soluble chromatin was isolated from wild-type (JPY12), dot1Δ (EMHY234), and a series of dot1Δ Lrs − strains and incubated with recombinant Dot1p and [ 3 H] S -adenosyl-methionine. Reaction products were separated by SDS-PAGE, and methylated histone H3 was detected by fluorography (upper panel). Histone H3 was detected by immunoblot analysis to normalize input levels of histone proteins in each reaction (lower panel).

Techniques Used: Methylation, Expressing, SDS Page, Western Blot, Isolation, Incubation, Recombinant

Both Lrs − and Sin − alterations disrupt telomeric silencing. Silencing was measured in strain ANY34 by assaying for expression of two reporter genes: (A) the ADE2 reporter gene integrated at the V-R telomere and (B) the URA3 reporter gene integrated at the VII-L telomere. Mutant with wild type designates the ANY34 strain containing a wild-type HHT1-HHF1 pJP11 plasmid and the indicated wild-type (WT) or mutant histone HHT2-HHF2 pDM18 plasmid (Lys + Trp + cells). Mutant alone designates the ANY34 strain containing only the mutant histone pDM18 plasmid (Lys − Trp + cells). Control strains include the wild type (WT) containing one or two wild-type histone plasmids and the histone H3 Lrs − mutant A75V, which displays loss of telomeric silencing.
Figure Legend Snippet: Both Lrs − and Sin − alterations disrupt telomeric silencing. Silencing was measured in strain ANY34 by assaying for expression of two reporter genes: (A) the ADE2 reporter gene integrated at the V-R telomere and (B) the URA3 reporter gene integrated at the VII-L telomere. Mutant with wild type designates the ANY34 strain containing a wild-type HHT1-HHF1 pJP11 plasmid and the indicated wild-type (WT) or mutant histone HHT2-HHF2 pDM18 plasmid (Lys + Trp + cells). Mutant alone designates the ANY34 strain containing only the mutant histone pDM18 plasmid (Lys − Trp + cells). Control strains include the wild type (WT) containing one or two wild-type histone plasmids and the histone H3 Lrs − mutant A75V, which displays loss of telomeric silencing.

Techniques Used: Expressing, Mutagenesis, Plasmid Preparation

Lrs − alterations do not show a Sin − phenotype in vivo. (A) Lrs − alterations do not suppress swi/snf defects in HO-lacZ transcription. Strains CY232 ( SWI ) and CY240 ( swi1 ), both containing an HO-lacZ reporter gene, were transformed with plasmids expressing either wild-type or Lrs − histone H3 or the Sin − histone H4-R45C. Four clones of each were grown on SC-Trp plates, and HO-lacZ reporter gene expression was analyzed using a β-galactosidase filter assay. (B) Relative levels of HO-lacZ and ACT1 gene expression in panel A were determined by RT-PCR for an swi1Δ strain (CY240) transformed with plasmids expressing either wild-type histone H4, histone H3-R83A, or histone H4-R45C. Similar results were observed for strains expressing other Lrs − histones. (C) Lrs − alterations do not lead to derepression of PHO5 gene expression in high-phosphate media. JPY12 cells expressing either wild-type, Lrs − , or Sin − histones as the sole source of histone H3 or H4 were grown to mid-log phase in YEPD medium and harvested for RNA. The level of PHO5 and ACT1 gene expression was analyzed by RT-PCR. PHO5 expression was normalized to ACT1 expression in each strain, with expression levels in the wild-type strain set to 1.0. The bottom panels show raw data from a representative experiment. Quantified data are graphed above and reflect the average of four independent experiments with standard deviations. WT, wild type.
Figure Legend Snippet: Lrs − alterations do not show a Sin − phenotype in vivo. (A) Lrs − alterations do not suppress swi/snf defects in HO-lacZ transcription. Strains CY232 ( SWI ) and CY240 ( swi1 ), both containing an HO-lacZ reporter gene, were transformed with plasmids expressing either wild-type or Lrs − histone H3 or the Sin − histone H4-R45C. Four clones of each were grown on SC-Trp plates, and HO-lacZ reporter gene expression was analyzed using a β-galactosidase filter assay. (B) Relative levels of HO-lacZ and ACT1 gene expression in panel A were determined by RT-PCR for an swi1Δ strain (CY240) transformed with plasmids expressing either wild-type histone H4, histone H3-R83A, or histone H4-R45C. Similar results were observed for strains expressing other Lrs − histones. (C) Lrs − alterations do not lead to derepression of PHO5 gene expression in high-phosphate media. JPY12 cells expressing either wild-type, Lrs − , or Sin − histones as the sole source of histone H3 or H4 were grown to mid-log phase in YEPD medium and harvested for RNA. The level of PHO5 and ACT1 gene expression was analyzed by RT-PCR. PHO5 expression was normalized to ACT1 expression in each strain, with expression levels in the wild-type strain set to 1.0. The bottom panels show raw data from a representative experiment. Quantified data are graphed above and reflect the average of four independent experiments with standard deviations. WT, wild type.

Techniques Used: In Vivo, Transformation Assay, Expressing, Clone Assay, Reverse Transcription Polymerase Chain Reaction

Sir2 overexpression does not rescue the ribosomal DNA-silencing phenotype of lrs or sin mutant alleles. JPY12 strains harboring both sin and lrs mutant alleles were transformed with a 2-μm vector containing SIR2 or an empty vector, and silencing of the mURA3 reporter gene inserted in the 5′ region of the 35S rRNA gene was assayed by measuring growth on SC-Ura and SC-His plus 0.1% 5-FOA. Mutant with wild type designates the ANY34 strain containing a wild-type HHT1-HHF1 pJP11 plasmid and the indicated wild-type (WT) or mutant histone HHT2-HHF2 pDM18 plasmid (Lys + Trp + cells). Mutant alone designates the ANY34 strain containing only the mutant histone pDM18 plasmid (Lys − Trp + cells).
Figure Legend Snippet: Sir2 overexpression does not rescue the ribosomal DNA-silencing phenotype of lrs or sin mutant alleles. JPY12 strains harboring both sin and lrs mutant alleles were transformed with a 2-μm vector containing SIR2 or an empty vector, and silencing of the mURA3 reporter gene inserted in the 5′ region of the 35S rRNA gene was assayed by measuring growth on SC-Ura and SC-His plus 0.1% 5-FOA. Mutant with wild type designates the ANY34 strain containing a wild-type HHT1-HHF1 pJP11 plasmid and the indicated wild-type (WT) or mutant histone HHT2-HHF2 pDM18 plasmid (Lys + Trp + cells). Mutant alone designates the ANY34 strain containing only the mutant histone pDM18 plasmid (Lys − Trp + cells).

Techniques Used: Over Expression, Mutagenesis, Transformation Assay, Plasmid Preparation

Lrs − alterations do not disrupt nucleosomal array folding in vitro. Histone octamers reconstituted from recombinant histones H2A, H2B, H4, and either wild-type H3 or H3-R83A were deposited onto 208-11 DNA templates by salt dialysis. (A) R83A Lrs − nucleosomes are indistinguishable by native PAGE. Arrays harboring either the wild type or the Lrs − R83A version of histone H3 were cleaved with EcoRI, electrophoretically separated on a native 4% PAGE, and stained with ethidium bromide. The mononucleosome (Nuc) and naked DNA (Naked) bands are indicated. (B) R83A Lrs − nucleosomal arrays show normal intramolecular, salt-dependent folding as shown by sedimentation velocity analysis of 208-11 arrays in the presence or absence of Mg 2+ . The G(s) distributions are depicted for the indicated arrays sedimented in either TE (10 mM Tris [pH 8.0], 0.25 mM EDTA) or TE with 1.75 mM MgCl 2 . S 20,w is the sedimentation coefficient corrected to water at 20°C. (C) Intermolecular oligomerization is not altered by the histone H3-R83A mutation. Nucleosomal arrays were incubated in TE with varying concentrations of MgCl 2 at room temperature for 15 min, followed by centrifugation in a microcentrifuge at 14,000 × g for 10 min. The percentage of array remaining in the supernatant is plotted as a function of MgCl 2 concentration. (D) rDNA silencing of the lrs mutant allele H3-R83A was determined by assaying for growth on SC-Ura to measure expression of the mURA/His reporter and plating on Pb 2+ -containing media to assay expression of MET15 reporter, both integrated into the rDNA locus. WT, wild type. R, ratio.
Figure Legend Snippet: Lrs − alterations do not disrupt nucleosomal array folding in vitro. Histone octamers reconstituted from recombinant histones H2A, H2B, H4, and either wild-type H3 or H3-R83A were deposited onto 208-11 DNA templates by salt dialysis. (A) R83A Lrs − nucleosomes are indistinguishable by native PAGE. Arrays harboring either the wild type or the Lrs − R83A version of histone H3 were cleaved with EcoRI, electrophoretically separated on a native 4% PAGE, and stained with ethidium bromide. The mononucleosome (Nuc) and naked DNA (Naked) bands are indicated. (B) R83A Lrs − nucleosomal arrays show normal intramolecular, salt-dependent folding as shown by sedimentation velocity analysis of 208-11 arrays in the presence or absence of Mg 2+ . The G(s) distributions are depicted for the indicated arrays sedimented in either TE (10 mM Tris [pH 8.0], 0.25 mM EDTA) or TE with 1.75 mM MgCl 2 . S 20,w is the sedimentation coefficient corrected to water at 20°C. (C) Intermolecular oligomerization is not altered by the histone H3-R83A mutation. Nucleosomal arrays were incubated in TE with varying concentrations of MgCl 2 at room temperature for 15 min, followed by centrifugation in a microcentrifuge at 14,000 × g for 10 min. The percentage of array remaining in the supernatant is plotted as a function of MgCl 2 concentration. (D) rDNA silencing of the lrs mutant allele H3-R83A was determined by assaying for growth on SC-Ura to measure expression of the mURA/His reporter and plating on Pb 2+ -containing media to assay expression of MET15 reporter, both integrated into the rDNA locus. WT, wild type. R, ratio.

Techniques Used: In Vitro, Recombinant, Clear Native PAGE, Polyacrylamide Gel Electrophoresis, Staining, Sedimentation, Mutagenesis, Incubation, Centrifugation, Concentration Assay, Expressing

Sin − alterations do not disrupt ribosomal DNA silencing. Silencing was measured by assaying for expression of two RNA polymerase II-transcribed reporter genes: (A) the MET15 reporter inserted in the NTS2 region and (B) the mURA3 reporter in the 5′ region of the 35S rRNA gene. Mutant with wild-type designates the JPY12 strain containing a wild-type HHT1-HHF1 pJP11 plasmid and the indicated wild-type (WT) or mutant histone HHT2-HHF2 pDM18 plasmid (Lys + Trp + cells). Mutant alone designates the JPY12 strain containing only the mutant histone pDM18 plasmid (Lys − Trp + cells). Control strains include the wild type (WT) containing one or two wild-type histone plasmids and the histone H3 Lrs − mutant A75V.
Figure Legend Snippet: Sin − alterations do not disrupt ribosomal DNA silencing. Silencing was measured by assaying for expression of two RNA polymerase II-transcribed reporter genes: (A) the MET15 reporter inserted in the NTS2 region and (B) the mURA3 reporter in the 5′ region of the 35S rRNA gene. Mutant with wild-type designates the JPY12 strain containing a wild-type HHT1-HHF1 pJP11 plasmid and the indicated wild-type (WT) or mutant histone HHT2-HHF2 pDM18 plasmid (Lys + Trp + cells). Mutant alone designates the JPY12 strain containing only the mutant histone pDM18 plasmid (Lys − Trp + cells). Control strains include the wild type (WT) containing one or two wild-type histone plasmids and the histone H3 Lrs − mutant A75V.

Techniques Used: Expressing, Mutagenesis, Plasmid Preparation

Sin − and Lrs − histones disrupt Sir2p and Sir4p binding to telomeric chromatin. JPY12 cells expressing either wild-type, Lrs − , or Sin − histones as the sole source of histone H3 or H4 were grown to mid-log phase in YEPD medium and then processed for Western immunoblot analysis (A) or chromatin immunoprecipitation (B to E). (A) Western blot analysis shows that Lrs − and Sin − histone alterations do not affect Sir2p protein levels. (B to D) ChIP analysis of Sir2p or Sir4p recruitment to the telomere, 500 bp (B) and 70 bp (C) from the end of the right arm of chromosome VI and to the nonspecific PHO5 promoter (D). An additional JPY12 strain containing an SIR4 deletion ( Sir4Δ ), was used to determine background signals in this assay. Quantification shown below the panels indicates the percent immunoprecipitated telomeric DNA (IP/Input) normalized to the percent IP from the nonspecific PHO5 locus (D) to normalize for IP efficiency. The normalized value for the wild-type strain was set at 1.0. Quantification below panel D indicates the percent immunoprecipitated PHO5 DNA (IP/Input), with the value for wild-type cells set at 1.0. (E) Rap1p binding 70 bp from the end of the right arm of chromosome VI was analyzed using antibodies to Rap1p. Similar results were observed in an independent experiment. Chr., chromosome.
Figure Legend Snippet: Sin − and Lrs − histones disrupt Sir2p and Sir4p binding to telomeric chromatin. JPY12 cells expressing either wild-type, Lrs − , or Sin − histones as the sole source of histone H3 or H4 were grown to mid-log phase in YEPD medium and then processed for Western immunoblot analysis (A) or chromatin immunoprecipitation (B to E). (A) Western blot analysis shows that Lrs − and Sin − histone alterations do not affect Sir2p protein levels. (B to D) ChIP analysis of Sir2p or Sir4p recruitment to the telomere, 500 bp (B) and 70 bp (C) from the end of the right arm of chromosome VI and to the nonspecific PHO5 promoter (D). An additional JPY12 strain containing an SIR4 deletion ( Sir4Δ ), was used to determine background signals in this assay. Quantification shown below the panels indicates the percent immunoprecipitated telomeric DNA (IP/Input) normalized to the percent IP from the nonspecific PHO5 locus (D) to normalize for IP efficiency. The normalized value for the wild-type strain was set at 1.0. Quantification below panel D indicates the percent immunoprecipitated PHO5 DNA (IP/Input), with the value for wild-type cells set at 1.0. (E) Rap1p binding 70 bp from the end of the right arm of chromosome VI was analyzed using antibodies to Rap1p. Similar results were observed in an independent experiment. Chr., chromosome.

Techniques Used: Binding Assay, Expressing, Western Blot, Chromatin Immunoprecipitation, Immunoprecipitation

30) Product Images from "Structural basis for the interaction of Asf1 with histone H3 and its functional implications"

Article Title: Structural basis for the interaction of Asf1 with histone H3 and its functional implications

Journal:

doi: 10.1073/pnas.0500149102

Interaction of human Asf1a (1-156) with histone H3 (122-135). ( a ) Superposition of the HSQC spectrum of human Asf1a (1-156) (80 μM) with (red) and without (black) an excess of histone H3 (122-135) (160 μM) at 298 K. ( b ) Mean-square chemical
Figure Legend Snippet: Interaction of human Asf1a (1-156) with histone H3 (122-135). ( a ) Superposition of the HSQC spectrum of human Asf1a (1-156) (80 μM) with (red) and without (black) an excess of histone H3 (122-135) (160 μM) at 298 K. ( b ) Mean-square chemical

Techniques Used:

In vitro  and  in vivo  analysis of Asf1 mutants. ( a ) V94R, D54R, and R108E are involved in the binding of Asf1 to the histone H3/H4 complex. Equal amounts of purified recombinant (His) 6 -GST, (His) 6 -GST-Asf1a (1-156), and the indicated mutants were bound
Figure Legend Snippet: In vitro and in vivo analysis of Asf1 mutants. ( a ) V94R, D54R, and R108E are involved in the binding of Asf1 to the histone H3/H4 complex. Equal amounts of purified recombinant (His) 6 -GST, (His) 6 -GST-Asf1a (1-156), and the indicated mutants were bound

Techniques Used: In Vitro, In Vivo, Binding Assay, Purification, Recombinant

31) Product Images from "Association of ATRX with pericentric heterochromatin and the Y chromosome of neonatal mouse spermatogonia"

Article Title: Association of ATRX with pericentric heterochromatin and the Y chromosome of neonatal mouse spermatogonia

Journal: BMC Molecular Biology

doi: 10.1186/1471-2199-9-29

Model for the establishment of a transcriptionally repressive chromatin environment at centromeric heterochromatin and the Y chromosome in neonatal spermatogonia . The levels of CpG methylation previously observed at major and minor satellite sequences following genome reprogramming in fetal germ cells [2–4] are maintained within a centromeric heterochromatin environment lacking global methylation as determined by 5-mC staining [10]. Establishment of repressive histone and chromatin modifications such as H3K9 me3 and ATRX are essential for the maintenance of a repressive chromatin configuration and might contribute to the mechanisms responsible for maintaining the transcriptional quiescence of potentially deleterious repetitive elements at tandem repeats. Histone methylation (H3K9 me3 ) and association with chromatin remodeling proteins such as ATRX may precede the establishment of chromosomal 5-mC patterns as a mechanism to maintain a repressive chromatin environment at constitutive heterochromatin domains in neonatal spermatogonia. Although pericentric heterochromatin and the Y chromosome share similar chromatin marks, differences might exist on the mechanisms imposing these chromatin modifications as the Y chromosome lacks chromosomal 5-mC in both germ cells and somatic cells.
Figure Legend Snippet: Model for the establishment of a transcriptionally repressive chromatin environment at centromeric heterochromatin and the Y chromosome in neonatal spermatogonia . The levels of CpG methylation previously observed at major and minor satellite sequences following genome reprogramming in fetal germ cells [2–4] are maintained within a centromeric heterochromatin environment lacking global methylation as determined by 5-mC staining [10]. Establishment of repressive histone and chromatin modifications such as H3K9 me3 and ATRX are essential for the maintenance of a repressive chromatin configuration and might contribute to the mechanisms responsible for maintaining the transcriptional quiescence of potentially deleterious repetitive elements at tandem repeats. Histone methylation (H3K9 me3 ) and association with chromatin remodeling proteins such as ATRX may precede the establishment of chromosomal 5-mC patterns as a mechanism to maintain a repressive chromatin environment at constitutive heterochromatin domains in neonatal spermatogonia. Although pericentric heterochromatin and the Y chromosome share similar chromatin marks, differences might exist on the mechanisms imposing these chromatin modifications as the Y chromosome lacks chromosomal 5-mC in both germ cells and somatic cells.

Techniques Used: CpG Methylation Assay, Methylation, Staining

Chromosomal localization of ATRX and histone H3 tri-methylation on lysine 9 (H3K9 me3 ) in neonatal spermatogonia . A) Analysis of histone methylation patterns revealed that in spite of the absence of global DNA methylation (red) at pericentric heterochromatin (thin arrow), H3K9 me3 (green) remained associated with centromeric domains in the chromosomes of neonatal spermatogonia. Importantly, H3K9 me3 is also preferentially associated with one of the unmethylated chromosomes on each spread analyzed (bold arrow). B) In contrast, H3K4 me2 (green) remains associated with entire chromatids in the majority of chromosomes (thin arrow). However, one of the unmethylated chromosomes was consistently found to be completely devoid of H3K4 me2 (bold arrow). C) Notably, the chromatin remodeling protein ATRX (green) remains associated with pericentric heterochromatin and consistently marks a single unmethylated chromosome on each metaphase spread analyzed. Scale bar = 10 μm.
Figure Legend Snippet: Chromosomal localization of ATRX and histone H3 tri-methylation on lysine 9 (H3K9 me3 ) in neonatal spermatogonia . A) Analysis of histone methylation patterns revealed that in spite of the absence of global DNA methylation (red) at pericentric heterochromatin (thin arrow), H3K9 me3 (green) remained associated with centromeric domains in the chromosomes of neonatal spermatogonia. Importantly, H3K9 me3 is also preferentially associated with one of the unmethylated chromosomes on each spread analyzed (bold arrow). B) In contrast, H3K4 me2 (green) remains associated with entire chromatids in the majority of chromosomes (thin arrow). However, one of the unmethylated chromosomes was consistently found to be completely devoid of H3K4 me2 (bold arrow). C) Notably, the chromatin remodeling protein ATRX (green) remains associated with pericentric heterochromatin and consistently marks a single unmethylated chromosome on each metaphase spread analyzed. Scale bar = 10 μm.

Techniques Used: Methylation, DNA Methylation Assay

Repressive histone and chromatin modifications in the absence of global DNA methylation in the Y chromosome of neonatal spermatogonia . A-C) Immuno-FISH analysis of neonatal spermatogonia revealed that the ATRX protein (green) as well as H3K9 me3 (green; D-F ) consistently associate with pericentric heterochromatin domains in autosomes (thin arrows). In addition to their centromeric localization, ATRX as well as H3K9 me3 preferentially associate with both arms of the Y chromosome (red; bold arrows). G-I) In contrast, a transcriptionally permissive histone modification such as H3K4 me2 (green) exhibited an inverted chromosomal distribution whereby constitutive heterochromatin (i.e. pericentric heterochromatin and the Y chromosome; red) are completely devoid of H3K4 me2 . The Y chromosome in neonatal spermatogonia occupies a transcriptionally quiescent nuclear domain during interphase. Transcription run-on assays after incorporation of Br-UTP (green) in permeabilized interphase nuclei of somatic (J-L) and neonatal spermatogonia (M-O) revealed that global transcriptional activity is undetectable in the nuclear domain occupied by the Y chromosome (red; see inset) in both somatic cell and germ cell nuclei. Neonatal spermatogonial cell nuclei were identified by their unique global DNA methylation patterns (5-mC; green). Scale bar = 10 μm.
Figure Legend Snippet: Repressive histone and chromatin modifications in the absence of global DNA methylation in the Y chromosome of neonatal spermatogonia . A-C) Immuno-FISH analysis of neonatal spermatogonia revealed that the ATRX protein (green) as well as H3K9 me3 (green; D-F ) consistently associate with pericentric heterochromatin domains in autosomes (thin arrows). In addition to their centromeric localization, ATRX as well as H3K9 me3 preferentially associate with both arms of the Y chromosome (red; bold arrows). G-I) In contrast, a transcriptionally permissive histone modification such as H3K4 me2 (green) exhibited an inverted chromosomal distribution whereby constitutive heterochromatin (i.e. pericentric heterochromatin and the Y chromosome; red) are completely devoid of H3K4 me2 . The Y chromosome in neonatal spermatogonia occupies a transcriptionally quiescent nuclear domain during interphase. Transcription run-on assays after incorporation of Br-UTP (green) in permeabilized interphase nuclei of somatic (J-L) and neonatal spermatogonia (M-O) revealed that global transcriptional activity is undetectable in the nuclear domain occupied by the Y chromosome (red; see inset) in both somatic cell and germ cell nuclei. Neonatal spermatogonial cell nuclei were identified by their unique global DNA methylation patterns (5-mC; green). Scale bar = 10 μm.

Techniques Used: DNA Methylation Assay, Fluorescence In Situ Hybridization, Modification, Activity Assay

32) Product Images from "An evolutionarily 'young' lysine residue in histone H3 attenuates transcriptional output in Saccharomyces cerevisiae"

Article Title: An evolutionarily 'young' lysine residue in histone H3 attenuates transcriptional output in Saccharomyces cerevisiae

Journal: Genes & Development

doi: 10.1101/gad.2050311

Histone H3-K42A is a pleiotropic mutation, which disrupts a structurally important nucleosome surface. ( A ) The crystal structure of the S. cerevisiae nucleosome highlighting histone H3 Lys 42 (orange) positioned at the DNA entry and exit points. The figure
Figure Legend Snippet: Histone H3-K42A is a pleiotropic mutation, which disrupts a structurally important nucleosome surface. ( A ) The crystal structure of the S. cerevisiae nucleosome highlighting histone H3 Lys 42 (orange) positioned at the DNA entry and exit points. The figure

Techniques Used: Mutagenesis

Histone H3-K42me2 may play a role in transcription. ( A ) Strains expressing the indicated histone H3 allele were assayed for their sensitivity to 6AU to detect transcription elongation defects. ppr1Δ acts as a positive control for comparison. (
Figure Legend Snippet: Histone H3-K42me2 may play a role in transcription. ( A ) Strains expressing the indicated histone H3 allele were assayed for their sensitivity to 6AU to detect transcription elongation defects. ppr1Δ acts as a positive control for comparison. (

Techniques Used: Expressing, Positive Control

Histone H3-K42 is dimethylated in  S. cerevisiae . ( A ) Electrospray tandem mass spectrum (MS/MS) of the doubly charged dimethylated peptide: YK(dimet.)PGTVALR,  m/z  516.869, recorded using a quadrupole time-of-flight mass spectrometer in an LC-MS/MS experiment
Figure Legend Snippet: Histone H3-K42 is dimethylated in S. cerevisiae . ( A ) Electrospray tandem mass spectrum (MS/MS) of the doubly charged dimethylated peptide: YK(dimet.)PGTVALR, m/z 516.869, recorded using a quadrupole time-of-flight mass spectrometer in an LC-MS/MS experiment

Techniques Used: Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Histone H3-K42 is an important residue during transcription elongation. ( A ) Strains with the indicated genotypes were plated as described in the Materials and Methods to detect transcriptional elongation defects. ( B ) Quantitative RT–PCR analysis
Figure Legend Snippet: Histone H3-K42 is an important residue during transcription elongation. ( A ) Strains with the indicated genotypes were plated as described in the Materials and Methods to detect transcriptional elongation defects. ( B ) Quantitative RT–PCR analysis

Techniques Used: Quantitative RT-PCR

Histone H3-K42me2 may regulate transcription
Figure Legend Snippet: Histone H3-K42me2 may regulate transcription

Techniques Used:

Histone H3-K42A alters the  S. cerevisiae  transcriptome. ( A ) Kinetics of appearance of  MET16  mRNA in cells expressing wild-type (WT) or K42A histone H3, as described in the Materials and Methods. Error bars represent the standard deviation between two
Figure Legend Snippet: Histone H3-K42A alters the S. cerevisiae transcriptome. ( A ) Kinetics of appearance of MET16 mRNA in cells expressing wild-type (WT) or K42A histone H3, as described in the Materials and Methods. Error bars represent the standard deviation between two

Techniques Used: Expressing, Standard Deviation

33) Product Images from "Deimination of linker histones links neutrophil extracellular trap release with autoantibodies in systemic autoimmunity"

Article Title: Deimination of linker histones links neutrophil extracellular trap release with autoantibodies in systemic autoimmunity

Journal: The FASEB Journal

doi: 10.1096/fj.13-247254

NETosis and histone deimination. A–D ) NETosis was induced by treating purified neutrophils with calcium ionophore and chelerythrine for 1 h. Cells were fixed and bound by antibodies to deiminated histone H3 (dH3; A ), to deiminated histone H4 (dH4;
Figure Legend Snippet: NETosis and histone deimination. A–D ) NETosis was induced by treating purified neutrophils with calcium ionophore and chelerythrine for 1 h. Cells were fixed and bound by antibodies to deiminated histone H3 (dH3; A ), to deiminated histone H4 (dH4;

Techniques Used: Purification

MS/MS analysis of H1 peptides from activated neutrophils A–F ) MS/MS spectra of peptides yielding doubly charged precursor ions of H1 histone from Ca 2+ ionophore-treated neutrophils. Fragmented ions (b series and y series) are marked on the peptide
Figure Legend Snippet: MS/MS analysis of H1 peptides from activated neutrophils A–F ) MS/MS spectra of peptides yielding doubly charged precursor ions of H1 histone from Ca 2+ ionophore-treated neutrophils. Fragmented ions (b series and y series) are marked on the peptide

Techniques Used: Mass Spectrometry

MS/MS analysis of in vitro deiminated recombinant H1.2. MS/MS spectra of doubly charged precursor ions of peptides obtained by LysC protease digestion of deiminated histone H1.2. Fragmented ions (b series and y series) are marked on respective peptide
Figure Legend Snippet: MS/MS analysis of in vitro deiminated recombinant H1.2. MS/MS spectra of doubly charged precursor ions of peptides obtained by LysC protease digestion of deiminated histone H1.2. Fragmented ions (b series and y series) are marked on respective peptide

Techniques Used: Mass Spectrometry, In Vitro, Recombinant

Analysis of linker histone transcripts from purified neutrophils. Relative mRNA levels of different H1 subtypes were determined from total mRNA extracted from neutrophils. cDNA was prepared by reverse transcription, and expression levels were measured
Figure Legend Snippet: Analysis of linker histone transcripts from purified neutrophils. Relative mRNA levels of different H1 subtypes were determined from total mRNA extracted from neutrophils. cDNA was prepared by reverse transcription, and expression levels were measured

Techniques Used: Purification, Expressing

MS/MS analysis of H1 histones from activated neutrophils
Figure Legend Snippet: MS/MS analysis of H1 histones from activated neutrophils

Techniques Used: Mass Spectrometry

34) Product Images from "Deimination of linker histones links neutrophil extracellular trap release with autoantibodies in systemic autoimmunity"

Article Title: Deimination of linker histones links neutrophil extracellular trap release with autoantibodies in systemic autoimmunity

Journal: The FASEB Journal

doi: 10.1096/fj.13-247254

NETosis and histone deimination. A–D ) NETosis was induced by treating purified neutrophils with calcium ionophore and chelerythrine for 1 h. Cells were fixed and bound by antibodies to deiminated histone H3 (dH3; A ), to deiminated histone H4 (dH4;
Figure Legend Snippet: NETosis and histone deimination. A–D ) NETosis was induced by treating purified neutrophils with calcium ionophore and chelerythrine for 1 h. Cells were fixed and bound by antibodies to deiminated histone H3 (dH3; A ), to deiminated histone H4 (dH4;

Techniques Used: Purification

MS/MS analysis of H1 peptides from activated neutrophils A–F ) MS/MS spectra of peptides yielding doubly charged precursor ions of H1 histone from Ca 2+ ionophore-treated neutrophils. Fragmented ions (b series and y series) are marked on the peptide
Figure Legend Snippet: MS/MS analysis of H1 peptides from activated neutrophils A–F ) MS/MS spectra of peptides yielding doubly charged precursor ions of H1 histone from Ca 2+ ionophore-treated neutrophils. Fragmented ions (b series and y series) are marked on the peptide

Techniques Used: Mass Spectrometry

MS/MS analysis of in vitro deiminated recombinant H1.2. MS/MS spectra of doubly charged precursor ions of peptides obtained by LysC protease digestion of deiminated histone H1.2. Fragmented ions (b series and y series) are marked on respective peptide
Figure Legend Snippet: MS/MS analysis of in vitro deiminated recombinant H1.2. MS/MS spectra of doubly charged precursor ions of peptides obtained by LysC protease digestion of deiminated histone H1.2. Fragmented ions (b series and y series) are marked on respective peptide

Techniques Used: Mass Spectrometry, In Vitro, Recombinant

Analysis of linker histone transcripts from purified neutrophils. Relative mRNA levels of different H1 subtypes were determined from total mRNA extracted from neutrophils. cDNA was prepared by reverse transcription, and expression levels were measured
Figure Legend Snippet: Analysis of linker histone transcripts from purified neutrophils. Relative mRNA levels of different H1 subtypes were determined from total mRNA extracted from neutrophils. cDNA was prepared by reverse transcription, and expression levels were measured

Techniques Used: Purification, Expressing

MS/MS analysis of H1 histones from activated neutrophils
Figure Legend Snippet: MS/MS analysis of H1 histones from activated neutrophils

Techniques Used: Mass Spectrometry

35) Product Images from "Withaferin A Associated Differential Regulation of Inflammatory Cytokines"

Article Title: Withaferin A Associated Differential Regulation of Inflammatory Cytokines

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00195

Inhibition of nuclear factor-kappa B (NF-κB) activation by withaferin A (WFA). Cell lysates from the cytoplasmic and nuclear fractions of lipopolysaccharide (LPS)-stimulated THP-1 cells following WFA (5, 10, 20 µM) treatment were subjected to protein expression analysis with antibodies for NF-κB, β-actin, and Histone H3 (a nuclear marker). NF-κB signaling molecules at the expected molecular weights of 50 and 65 kDa were observed both in both the cytosolic and nuclear fractions. The estimated size of 18 kDa for Histone H3 protein in nuclear lysate serves as internal control, whereas a non-specific band at ~15 kDa in the cytoplasmic fraction was also observed. Beta-actin (43 kDa) serves as an internal control for the cytoplasmic lysates. The experiment was performed twice with similar observations from two independent cell preparations.
Figure Legend Snippet: Inhibition of nuclear factor-kappa B (NF-κB) activation by withaferin A (WFA). Cell lysates from the cytoplasmic and nuclear fractions of lipopolysaccharide (LPS)-stimulated THP-1 cells following WFA (5, 10, 20 µM) treatment were subjected to protein expression analysis with antibodies for NF-κB, β-actin, and Histone H3 (a nuclear marker). NF-κB signaling molecules at the expected molecular weights of 50 and 65 kDa were observed both in both the cytosolic and nuclear fractions. The estimated size of 18 kDa for Histone H3 protein in nuclear lysate serves as internal control, whereas a non-specific band at ~15 kDa in the cytoplasmic fraction was also observed. Beta-actin (43 kDa) serves as an internal control for the cytoplasmic lysates. The experiment was performed twice with similar observations from two independent cell preparations.

Techniques Used: Inhibition, Activation Assay, Expressing, Marker

36) Product Images from "Superoxide Induces Neutrophil Extracellular Trap Formation in a TLR-4 and NOX-Dependent Mechanism"

Article Title: Superoxide Induces Neutrophil Extracellular Trap Formation in a TLR-4 and NOX-Dependent Mechanism

Journal: Molecular Medicine

doi: 10.2119/molmed.2016.00054

TLR-4 mediates NET induction by extracellular superoxide. (A) Flow cytometry analysis of citrullinated Histone H3 in WT neutrophils after treatment with media alone (control), eritoran TLR-4 antagonist (8 ng/mL), xanthine oxidase (10 mU/mL) and
Figure Legend Snippet: TLR-4 mediates NET induction by extracellular superoxide. (A) Flow cytometry analysis of citrullinated Histone H3 in WT neutrophils after treatment with media alone (control), eritoran TLR-4 antagonist (8 ng/mL), xanthine oxidase (10 mU/mL) and

Techniques Used: Flow Cytometry, Cytometry

37) Product Images from "Neonatal NET-inhibitory factor and related peptides inhibit neutrophil extracellular trap formation"

Article Title: Neonatal NET-inhibitory factor and related peptides inhibit neutrophil extracellular trap formation

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI83873

NRPs inhibit nuclear decondensation and histone citrullination in activated neutrophils.
Figure Legend Snippet: NRPs inhibit nuclear decondensation and histone citrullination in activated neutrophils.

Techniques Used:

38) Product Images from "A phase I study of danusertib (PHA-739358) in adult patients with accelerated or blastic phase chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia resistant or intolerant to imatinib and/or other second generation c-ABL therapy"

Article Title: A phase I study of danusertib (PHA-739358) in adult patients with accelerated or blastic phase chronic myeloid leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia resistant or intolerant to imatinib and/or other second generation c-ABL therapy

Journal: Haematologica

doi: 10.3324/haematol.2014.115279

Histone H3 (upper panel) and Crkl phosphorylation (lower panel) in peripheral blood mononucleated cells (schedule A).
Figure Legend Snippet: Histone H3 (upper panel) and Crkl phosphorylation (lower panel) in peripheral blood mononucleated cells (schedule A).

Techniques Used:

39) Product Images from "Ginsenoside compound K protects human umbilical vein endothelial cells against oxidized low-density lipoprotein-induced injury via inhibition of nuclear factor-κB, p38, and JNK MAPK pathways"

Article Title: Ginsenoside compound K protects human umbilical vein endothelial cells against oxidized low-density lipoprotein-induced injury via inhibition of nuclear factor-κB, p38, and JNK MAPK pathways

Journal: Journal of Ginseng Research

doi: 10.1016/j.jgr.2017.09.004

CK reduces ox-LDL-induced HUVECs inflammation through inhibiting the NF-κB pathway. (A) HUVECs were pretreated with CK (2.5μM) for 12 h, followed by treatment with ox-LDL (80 μg/mL) for another 24 h. NF-κB p65 immunoreactivity was observed by immunofluorescence assay. (B) HUVECs were treated as described in (A). LOX-1, p-IKKβ, p-IκBα, IκBα, NF-κB p65 (nuclear), NF-κB p65 (cytoplasm), NF-κB p65, Histone H3, and β-actin were evaluated by Western blot analysis. (C) Densitometric analysis was used to quantify the levels of LOX-1, p-IKKα/β, p-IκB and IκB. (D) Densitometric analysis was used to quantify the levels of NF-κB p65. Values are expressed as the mean ± SD, n = 3. # p
Figure Legend Snippet: CK reduces ox-LDL-induced HUVECs inflammation through inhibiting the NF-κB pathway. (A) HUVECs were pretreated with CK (2.5μM) for 12 h, followed by treatment with ox-LDL (80 μg/mL) for another 24 h. NF-κB p65 immunoreactivity was observed by immunofluorescence assay. (B) HUVECs were treated as described in (A). LOX-1, p-IKKβ, p-IκBα, IκBα, NF-κB p65 (nuclear), NF-κB p65 (cytoplasm), NF-κB p65, Histone H3, and β-actin were evaluated by Western blot analysis. (C) Densitometric analysis was used to quantify the levels of LOX-1, p-IKKα/β, p-IκB and IκB. (D) Densitometric analysis was used to quantify the levels of NF-κB p65. Values are expressed as the mean ± SD, n = 3. # p

Techniques Used: Immunofluorescence, Western Blot

40) Product Images from "Localized Histone Acetylation and Deacetylation Triggered by the Homologous Recombination Pathway of Double-Strand DNA Repair"

Article Title: Localized Histone Acetylation and Deacetylation Triggered by the Homologous Recombination Pathway of Double-Strand DNA Repair

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.25.12.4903-4913.2005

Levels of histone acetylation before, during, and after repair of a double-strand break by homologous recombination. A. Schematic of the mating-type loci in yeast. The locations of PCR primers used to quantify HO cleavage and repair are shown, where the reverse primer is specific to the MAT locus and the forward primers recognize the X sequence. If the mating type is a , then the PCR product is 1.0 kb, and if it is α, then the product is 1.1 kb. Also shown are the positions of primer pairs used to assay the amount of acetylation 0.6 kb and 2 kb away from the HO endonuclease site. Chrom.III, chromosome III. B. Cutting and repair of the HO lesion in wild-type yeast. HO endonuclease was induced at time zero in strain BAT009 (wild type) containing the plasmid pGAL-HO-URA3 by the addition of galactose and was repressed at 2 h by the addition of glucose. Samples were analyzed throughout the time course with the primers shown in panel A and control primers. The MAT a and MAT α products were quantified from the gel and were normalized to the control product. The amount of MAT product at time zero was normalized to 1. C. ChIP analysis of acetylated lysines on histones H4 0.6 kb and 2.0 kb away from the HO site. HO endonuclease was induced at time zero in strain BAT009 containing the plasmid pGAL-HO-URA3 by the addition of galactose and was repressed at 2 h by the addition of glucose as described above for panel B. Relative percent immunoprecipitation (IP) was quantitated by taking the ratio of the MAT product to the SMC2 control product. To obtain the IP/input values ( y axis), the amount of immunoprecipitated DNA was divided by the amount of the input DNA. Values were normalized so that the IP/input ratio was equal to 1 at time zero. The averages and standard errors of the means (error bars) are plotted for at least six independent quantitative real-time PCR experiments from two independent ChIPs. The asterisks represent a significant P value where one asterisk represents a P value of 0.05 to 0.01, two asterisks 0.009 to 0.001, and three asterisks ≤0.0009. Black asterisks indicate a significant change from the previous time point, and small grey circles indicate a significant change from the value before HO induction at time zero. H4AcK5, H4 with lysine 5 acetylated; H3 C-term, H3 C terminus. D. ChIP analysis of histone H3 levels and acetylated lysines on histone H3, as described for panel C.
Figure Legend Snippet: Levels of histone acetylation before, during, and after repair of a double-strand break by homologous recombination. A. Schematic of the mating-type loci in yeast. The locations of PCR primers used to quantify HO cleavage and repair are shown, where the reverse primer is specific to the MAT locus and the forward primers recognize the X sequence. If the mating type is a , then the PCR product is 1.0 kb, and if it is α, then the product is 1.1 kb. Also shown are the positions of primer pairs used to assay the amount of acetylation 0.6 kb and 2 kb away from the HO endonuclease site. Chrom.III, chromosome III. B. Cutting and repair of the HO lesion in wild-type yeast. HO endonuclease was induced at time zero in strain BAT009 (wild type) containing the plasmid pGAL-HO-URA3 by the addition of galactose and was repressed at 2 h by the addition of glucose. Samples were analyzed throughout the time course with the primers shown in panel A and control primers. The MAT a and MAT α products were quantified from the gel and were normalized to the control product. The amount of MAT product at time zero was normalized to 1. C. ChIP analysis of acetylated lysines on histones H4 0.6 kb and 2.0 kb away from the HO site. HO endonuclease was induced at time zero in strain BAT009 containing the plasmid pGAL-HO-URA3 by the addition of galactose and was repressed at 2 h by the addition of glucose as described above for panel B. Relative percent immunoprecipitation (IP) was quantitated by taking the ratio of the MAT product to the SMC2 control product. To obtain the IP/input values ( y axis), the amount of immunoprecipitated DNA was divided by the amount of the input DNA. Values were normalized so that the IP/input ratio was equal to 1 at time zero. The averages and standard errors of the means (error bars) are plotted for at least six independent quantitative real-time PCR experiments from two independent ChIPs. The asterisks represent a significant P value where one asterisk represents a P value of 0.05 to 0.01, two asterisks 0.009 to 0.001, and three asterisks ≤0.0009. Black asterisks indicate a significant change from the previous time point, and small grey circles indicate a significant change from the value before HO induction at time zero. H4AcK5, H4 with lysine 5 acetylated; H3 C-term, H3 C terminus. D. ChIP analysis of histone H3 levels and acetylated lysines on histone H3, as described for panel C.

Techniques Used: Homologous Recombination, Polymerase Chain Reaction, Sequencing, Plasmid Preparation, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction

The N-terminal tails of histones H3 and H4 and the acetylatable lysines of histone H4 confer resistance to the HO endonuclease. A. Sensitivity to prolonged exposure to HO endonuclease. S. cerevisiae strains RMY200 (wild type [WT]), RMY430 ( H3 tail Δ), BAT039 ( H4 tail Δ), FLY722 (H4K5,8,12,16R), FLY821 (H4K5,8,12R), and BAT038 ( rad52 Δ) containing the pGAL-HO-ADE2 plasmid were serially diluted onto plates containing increasing amounts of galactose (GAL) in order to induce increasing amounts of the HO endonuclease. B. Sensitivity to limited exposure to HO endonuclease. The viability of yeast strains used in panel A was determined after induction of the HO endonuclease for 0, 1, 2, or 3 h. The average viability ± standard deviation (error bar) of three independent ChIP experiments is shown. Viability at 0 h is normalized to 100 percent.
Figure Legend Snippet: The N-terminal tails of histones H3 and H4 and the acetylatable lysines of histone H4 confer resistance to the HO endonuclease. A. Sensitivity to prolonged exposure to HO endonuclease. S. cerevisiae strains RMY200 (wild type [WT]), RMY430 ( H3 tail Δ), BAT039 ( H4 tail Δ), FLY722 (H4K5,8,12,16R), FLY821 (H4K5,8,12R), and BAT038 ( rad52 Δ) containing the pGAL-HO-ADE2 plasmid were serially diluted onto plates containing increasing amounts of galactose (GAL) in order to induce increasing amounts of the HO endonuclease. B. Sensitivity to limited exposure to HO endonuclease. The viability of yeast strains used in panel A was determined after induction of the HO endonuclease for 0, 1, 2, or 3 h. The average viability ± standard deviation (error bar) of three independent ChIP experiments is shown. Viability at 0 h is normalized to 100 percent.

Techniques Used: Plasmid Preparation, Standard Deviation, Chromatin Immunoprecipitation

Related Articles

Proximity Ligation Assay:

Article Title: MDC1 methylation mediated by lysine methyltransferases EHMT1 and EHMT2 regulates active ATM accumulation flanking DNA damage sites
Article Snippet: .. 53BP1 (Santa Cruz, sc22760; IF, 1:1,000); Conjugated ubiquitin (FK2; Enzo Life Sciences, BML-PW8800; IF, 1:10,000); RAP80 (Novus Biologicals, NBP1-87156; IF, 1:300); γ-H2AX (Millipore, 05–636, IF, 1:1000; WB, 1:500); Flag (WAKO, 018–22381; WB, 1:5000, Medical & Biological Laboratories Co., Ltd. (MBL), PM020; WB, 1:2000, IP, 1:200, M185-3L (FLA-1); IF and PLA, 1:5000); MDC1 (Abcam, ab11171; IF and PLA, 1:2,000, WB, 1:5000, IP, 1:300,); EHMT1 (MBL, D220-3; WB, 1:1000); EHMT2 (Cell signaling, 3306; WB, 1:500); histone H3 (Abcam, ab1791; WB: 1:5,000); H3K9me2 (Abcam, ab1220; WB: 1:1,000); HA (MBL, 561; WB, 1:2000, IP, 1:500); ATM (Santa Cruz, sc23921; WB, 1:200); MRE11 (Abcam, ab214; WB 1:500); NBS1 (GeneTex, GTX70224; WB 1:2000); H2A (Millipore, 07–146; WB, 1:1000); phospholyrated ATM (Ser1981) (Abcam, ab81292; WB, 1:5000, Cell signaling, 4526; IF and PLA, 1:500); TRF1 (GeneTex, GTX10579; PLA, 1:200). .. To generate a methyl-specific antibody against dimethyl-K45 of MDC1, dimethyl-K45 peptide NH2-C + AHGPE(Lys[Me]2)DFPLH-COOH was conjugated with keyhole-limpet hemocyanin and used to immunize rabbits.

Immunostaining:

Article Title: Activation of the oncogenic transcription factor B-Myb via multisite phosphorylation and prolyl cis/trans isomerization
Article Snippet: .. Primary antibodies for immunostaining were as follows: mouse anti-B-Myb (Lx015.1, 1:10) , mouse anti-Myb-DBD (5E11, 1:10) , rabbit anti-phospho-B-Myb(T487) (Abcam, ab76009, 1:3 000–10 000), mouse anti β-actin (Sigma-Aldrich, AC-15, 1:10 000), mouse anti-GFP (Sigma-Aldrich, 7.1 + 13.1, 1:5000), mouse anti-Plk1 (Santa Cruz Biotechnology, F-8, 1:1000–5000), mouse anti-Pin1 (Santa Cruz Biotechnology, G-8, 1:500–5000), rabbit anti-cyclin A (Santa Cruz Biotechnology, H-432, 1:500), mouse anti-Cdk2 (Santa Cruz Biotechnology, D-12, 1:1000), mouse anti-Myc-tag (9E10, 1:500–5000), mouse anti-HA.11-tag (6B12, 1:5000), rabbit anti-phospho-PLK binding motif (SpTP) (Cell Signaling Technology, D73F6, 1:3000), anti-histone H3 (Abcam, ab1791, 1:1000), rabbit anti-GST (i11) ( ). .. In vitro protein kinase assays were performed as described ( ).

Incubation:

Article Title: Comprehensive Analysis of the Palindromic Motif TCTCGCGAGA: A Regulatory Element of the HNRNPK Promoter
Article Snippet: .. The wells were cleared, then incubated with 0.5 µg antibody [anti-RNAPII-CTD (Santa Cruz Biotechnology, Santa Cruz, CA, USA), sc-47701; anti-Histone H3, Abcam, ab1791; anti-Histone H3K4me3, Abcam, ab8580; anti-Histone H3K27me3 (Millipore, Billerica, MA, USA), 07-449; anti-Histone H3Ac(Lys9/18), Millipore, 07-593; anti-PARP, Abcam, ab6079] diluted in 100 µl blocking buffer/well for 60 min at room temperature. .. Chromatin samples (4 µl chromatin/100 µl blocking buffer) were added to the wells (100 µl/well), and the plates were floated in a 4°C ultrasonic water bath for 1 h in order to accelerate protein-antibody binding.

Blocking Assay:

Article Title: Comprehensive Analysis of the Palindromic Motif TCTCGCGAGA: A Regulatory Element of the HNRNPK Promoter
Article Snippet: .. The wells were cleared, then incubated with 0.5 µg antibody [anti-RNAPII-CTD (Santa Cruz Biotechnology, Santa Cruz, CA, USA), sc-47701; anti-Histone H3, Abcam, ab1791; anti-Histone H3K4me3, Abcam, ab8580; anti-Histone H3K27me3 (Millipore, Billerica, MA, USA), 07-449; anti-Histone H3Ac(Lys9/18), Millipore, 07-593; anti-PARP, Abcam, ab6079] diluted in 100 µl blocking buffer/well for 60 min at room temperature. .. Chromatin samples (4 µl chromatin/100 µl blocking buffer) were added to the wells (100 µl/well), and the plates were floated in a 4°C ultrasonic water bath for 1 h in order to accelerate protein-antibody binding.

Western Blot:

Article Title: MDC1 methylation mediated by lysine methyltransferases EHMT1 and EHMT2 regulates active ATM accumulation flanking DNA damage sites
Article Snippet: .. 53BP1 (Santa Cruz, sc22760; IF, 1:1,000); Conjugated ubiquitin (FK2; Enzo Life Sciences, BML-PW8800; IF, 1:10,000); RAP80 (Novus Biologicals, NBP1-87156; IF, 1:300); γ-H2AX (Millipore, 05–636, IF, 1:1000; WB, 1:500); Flag (WAKO, 018–22381; WB, 1:5000, Medical & Biological Laboratories Co., Ltd. (MBL), PM020; WB, 1:2000, IP, 1:200, M185-3L (FLA-1); IF and PLA, 1:5000); MDC1 (Abcam, ab11171; IF and PLA, 1:2,000, WB, 1:5000, IP, 1:300,); EHMT1 (MBL, D220-3; WB, 1:1000); EHMT2 (Cell signaling, 3306; WB, 1:500); histone H3 (Abcam, ab1791; WB: 1:5,000); H3K9me2 (Abcam, ab1220; WB: 1:1,000); HA (MBL, 561; WB, 1:2000, IP, 1:500); ATM (Santa Cruz, sc23921; WB, 1:200); MRE11 (Abcam, ab214; WB 1:500); NBS1 (GeneTex, GTX70224; WB 1:2000); H2A (Millipore, 07–146; WB, 1:1000); phospholyrated ATM (Ser1981) (Abcam, ab81292; WB, 1:5000, Cell signaling, 4526; IF and PLA, 1:500); TRF1 (GeneTex, GTX10579; PLA, 1:200). .. To generate a methyl-specific antibody against dimethyl-K45 of MDC1, dimethyl-K45 peptide NH2-C + AHGPE(Lys[Me]2)DFPLH-COOH was conjugated with keyhole-limpet hemocyanin and used to immunize rabbits.

Binding Assay:

Article Title: Activation of the oncogenic transcription factor B-Myb via multisite phosphorylation and prolyl cis/trans isomerization
Article Snippet: .. Primary antibodies for immunostaining were as follows: mouse anti-B-Myb (Lx015.1, 1:10) , mouse anti-Myb-DBD (5E11, 1:10) , rabbit anti-phospho-B-Myb(T487) (Abcam, ab76009, 1:3 000–10 000), mouse anti β-actin (Sigma-Aldrich, AC-15, 1:10 000), mouse anti-GFP (Sigma-Aldrich, 7.1 + 13.1, 1:5000), mouse anti-Plk1 (Santa Cruz Biotechnology, F-8, 1:1000–5000), mouse anti-Pin1 (Santa Cruz Biotechnology, G-8, 1:500–5000), rabbit anti-cyclin A (Santa Cruz Biotechnology, H-432, 1:500), mouse anti-Cdk2 (Santa Cruz Biotechnology, D-12, 1:1000), mouse anti-Myc-tag (9E10, 1:500–5000), mouse anti-HA.11-tag (6B12, 1:5000), rabbit anti-phospho-PLK binding motif (SpTP) (Cell Signaling Technology, D73F6, 1:3000), anti-histone H3 (Abcam, ab1791, 1:1000), rabbit anti-GST (i11) ( ). .. In vitro protein kinase assays were performed as described ( ).

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  • h2a z  (Abcam)
    93
    Abcam h2a z
    Acetylation of <t>H2A.Z</t> is required for upregulation of Hes1 Notch target gene. ( A ) Schematic representation of the Flag-RBP-J/Tip60 fusion proteins used in Figure 4B and C and in Supplementary Figures S9 and S10 . Amino acid numbering is accordingly to accession NP_033061.3 for RBP-J and NP_874368.1 for Tip60. RBP-J domain: LAG1-DNAbind, LAG1 DNA binding (CDD:255260); Tip60 domain: MOZ/SAS, MOZ/SAS family (CDD:250916). ( B ) RBP-J/Tip60 wildtype (wt) but not its catalytic dead (cd) mutant upregulates Hes1 expression in MT cells. MT cells were infected with retroviral particles delivering plasmids encoding Flag-tagged RBP-J/Tip60-wt, cd mutant or empty vector (Control). Total RNA was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp or Hes1 . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD ([**] P
    H2a Z, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 56 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/h2a z/product/Abcam
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    85
    Abcam eef2 fraction
    Co-immune precipitations reveal <t>eEF2</t> interactions with Dph6 and Dph5. (A) eEF2 interacts with Dph6 in a fashion that is independent of Dph7. (B) eEF2 interaction with Dph5 is dramatically enhanced by elimination of Dph7 or Dph1. Yeast strains co-expressing (His) 6 -tagged eEF2 with Dph6-HA (A) or Dph5-HA (B) in the background of wild-type (A: DPH7 and B: wt) and dph mutant strains (A: dph7 ; B: dph1 , dph6 and dph7 ) were subjected to immune precipitations (IP) using the anti-HA antibody. Strains expressing (His) 6 -tagged eEF2 on their own served as IP controls (A and B: no HA-tag). Subsequently, the precipitates were probed with anti-HA (A: top left panel; B: first panel) and anti-(His) 6 antibodies (A: bottom left panel) to check for the content of Dph6-HA (A) and Dph5-HA (B), respectively (all indicated by arrows). The content of HA-tagged Dph6 (A) and Dph5 (B) as well as (His) 6 -marked eEF2 (A and B) in the protein extracts prior to IP (pre-IP) was examined on individual Western blots using anti-HA (A: top right panel; B: fourth panel) and anti-(His) 6 antibodies (A: bottom right panel; B: third panel), respectively. While absence of Dph7 hardly affected the Dph6•eEF2 interaction (A), Dph5•eEF2 interaction was strongly enhanced by inactivating DPH7 or DPH1 (B).
    Eef2 Fraction, supplied by Abcam, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Acetylation of H2A.Z is required for upregulation of Hes1 Notch target gene. ( A ) Schematic representation of the Flag-RBP-J/Tip60 fusion proteins used in Figure 4B and C and in Supplementary Figures S9 and S10 . Amino acid numbering is accordingly to accession NP_033061.3 for RBP-J and NP_874368.1 for Tip60. RBP-J domain: LAG1-DNAbind, LAG1 DNA binding (CDD:255260); Tip60 domain: MOZ/SAS, MOZ/SAS family (CDD:250916). ( B ) RBP-J/Tip60 wildtype (wt) but not its catalytic dead (cd) mutant upregulates Hes1 expression in MT cells. MT cells were infected with retroviral particles delivering plasmids encoding Flag-tagged RBP-J/Tip60-wt, cd mutant or empty vector (Control). Total RNA was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp or Hes1 . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD ([**] P

    Journal: Nucleic Acids Research

    Article Title: Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response

    doi: 10.1093/nar/gky551

    Figure Lengend Snippet: Acetylation of H2A.Z is required for upregulation of Hes1 Notch target gene. ( A ) Schematic representation of the Flag-RBP-J/Tip60 fusion proteins used in Figure 4B and C and in Supplementary Figures S9 and S10 . Amino acid numbering is accordingly to accession NP_033061.3 for RBP-J and NP_874368.1 for Tip60. RBP-J domain: LAG1-DNAbind, LAG1 DNA binding (CDD:255260); Tip60 domain: MOZ/SAS, MOZ/SAS family (CDD:250916). ( B ) RBP-J/Tip60 wildtype (wt) but not its catalytic dead (cd) mutant upregulates Hes1 expression in MT cells. MT cells were infected with retroviral particles delivering plasmids encoding Flag-tagged RBP-J/Tip60-wt, cd mutant or empty vector (Control). Total RNA was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp or Hes1 . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD ([**] P

    Article Snippet: In order to evaluate whether Tip60 is responsible for the acetylation of H2A.Z in higher eukaryotes and to evaluate the transcriptional consequences of the Tip60-mediated acetylation of H2A.Z in the context of Notch signaling, we directly fused the transcription factor RBP-J to Tip60 wildtype (wt) or its catalytic-dead mutant [(cd, ( ) and Figure A].

    Techniques: Binding Assay, Mutagenesis, Expressing, Infection, Plasmid Preparation, Real-time Polymerase Chain Reaction

    Depletion of Drosophila His2Av/H2A.Z, dTip60 and domino/p400 leads to an asymmetric response to Delta-dependent Notch signaling and reveals a strong requirement for these factors in Notch responsive cells. ( A ) Drosophila homologs of RBP-J and p400 proteins, Su(H) and Domino respectively, physically interact with each other. GST pull-down experiments were performed using bacterially purified GST-Su(H) and in vitro transcribed/translated Domino aa 1557–2352 corresponding to the human RBP-J interacting p400–2 fragment (Figure 3D ). ( B ) Scheme of a Drosophila wing disc with the dorsal wing pouch highlighted in green. Notch activation by its ligands Delta/DLL and Serrate/Jagged at the dorsal (d)/ventral (v) boundary results in activation of target genes, such as wingless ( wg , in red; a: anterior, p: posterior, N act: activated Notch). Graph shows that the relative area of dorsal wing pouch is reduced in wing discs bearing clones of Delta -expressing cells that are depleted of His2Av/H2A.Z, dTip60 or domino/p400 . Data are mean ± SD ([**] P

    Journal: Nucleic Acids Research

    Article Title: Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response

    doi: 10.1093/nar/gky551

    Figure Lengend Snippet: Depletion of Drosophila His2Av/H2A.Z, dTip60 and domino/p400 leads to an asymmetric response to Delta-dependent Notch signaling and reveals a strong requirement for these factors in Notch responsive cells. ( A ) Drosophila homologs of RBP-J and p400 proteins, Su(H) and Domino respectively, physically interact with each other. GST pull-down experiments were performed using bacterially purified GST-Su(H) and in vitro transcribed/translated Domino aa 1557–2352 corresponding to the human RBP-J interacting p400–2 fragment (Figure 3D ). ( B ) Scheme of a Drosophila wing disc with the dorsal wing pouch highlighted in green. Notch activation by its ligands Delta/DLL and Serrate/Jagged at the dorsal (d)/ventral (v) boundary results in activation of target genes, such as wingless ( wg , in red; a: anterior, p: posterior, N act: activated Notch). Graph shows that the relative area of dorsal wing pouch is reduced in wing discs bearing clones of Delta -expressing cells that are depleted of His2Av/H2A.Z, dTip60 or domino/p400 . Data are mean ± SD ([**] P

    Article Snippet: In order to evaluate whether Tip60 is responsible for the acetylation of H2A.Z in higher eukaryotes and to evaluate the transcriptional consequences of the Tip60-mediated acetylation of H2A.Z in the context of Notch signaling, we directly fused the transcription factor RBP-J to Tip60 wildtype (wt) or its catalytic-dead mutant [(cd, ( ) and Figure A].

    Techniques: Purification, In Vitro, Activation Assay, Activated Clotting Time Assay, Clone Assay, Expressing

    H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. ( A ) Schematic representation of the NICD-inducible system established in MT cells. The NICD was fused to the estrogen receptor binding domain (NICD-ER) and retrovirally introduced into MT cells. The NICD-ER fusion protein is retained into the cytoplasm unless cells are treated with ( Z )-4-hydroxytamoxifen (4-OHT) that induces its nuclear translocation and activation of Notch target genes. ( B ) Hes1 and Il2ra Notch target genes are induced upon 4-OHT treatment of MT NICD-ER cells. Total RNA from MT NICD-ER cells, treated for 24 h with 4-OHT or EtOH as control, was reverse transcribed into cDNA and analyzed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of three independent experiments. ( C ) H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. MT NICD-ER cells were treated for 24 h with 4-OHT or EtOH as control and subjected to ChIP analysis using antibodies against H2A.Z, H2A.Zac, H3 or IgG as control. The qPCR analysis was focused at the Notch-dependent enhancers (red squares) represented on the left ( Hes1 +0.6 kb and Il2ra -26 kb ). Chrom X was used as negative control ( Control ). Data were normalized to the positive control ( GAPDH 0 kb ) and, in the case of H2A.Zac/H2A.Z, the H2A.Zac signals were further normalized to H2A.Z. Shown is the mean ± SD of two independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response

    doi: 10.1093/nar/gky551

    Figure Lengend Snippet: H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. ( A ) Schematic representation of the NICD-inducible system established in MT cells. The NICD was fused to the estrogen receptor binding domain (NICD-ER) and retrovirally introduced into MT cells. The NICD-ER fusion protein is retained into the cytoplasm unless cells are treated with ( Z )-4-hydroxytamoxifen (4-OHT) that induces its nuclear translocation and activation of Notch target genes. ( B ) Hes1 and Il2ra Notch target genes are induced upon 4-OHT treatment of MT NICD-ER cells. Total RNA from MT NICD-ER cells, treated for 24 h with 4-OHT or EtOH as control, was reverse transcribed into cDNA and analyzed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of three independent experiments. ( C ) H2A.Z acetylation (H2A.Zac) but not H2A.Z occupancy positively correlates with activation of Notch target genes. MT NICD-ER cells were treated for 24 h with 4-OHT or EtOH as control and subjected to ChIP analysis using antibodies against H2A.Z, H2A.Zac, H3 or IgG as control. The qPCR analysis was focused at the Notch-dependent enhancers (red squares) represented on the left ( Hes1 +0.6 kb and Il2ra -26 kb ). Chrom X was used as negative control ( Control ). Data were normalized to the positive control ( GAPDH 0 kb ) and, in the case of H2A.Zac/H2A.Z, the H2A.Zac signals were further normalized to H2A.Z. Shown is the mean ± SD of two independent experiments.

    Article Snippet: In order to evaluate whether Tip60 is responsible for the acetylation of H2A.Z in higher eukaryotes and to evaluate the transcriptional consequences of the Tip60-mediated acetylation of H2A.Z in the context of Notch signaling, we directly fused the transcription factor RBP-J to Tip60 wildtype (wt) or its catalytic-dead mutant [(cd, ( ) and Figure A].

    Techniques: Activation Assay, Binding Assay, Translocation Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control, Positive Control

    Histone variant H2A.Z has a negative impact on the expression of Notch target genes. ( A ) Histone Variant H2A.Z is efficiently depleted by CRISPR/Cas9 in MT cells. Whole Cell Extract (WCE) was prepared from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells and analysed by Western blotting. GAPDH was used as loading control. ( B ) Hes1 and Il2ra Notch target genes are upregulated upon depletion of H2A.Z. Total RNA from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of five independent experiments ([*] P

    Journal: Nucleic Acids Research

    Article Title: Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response

    doi: 10.1093/nar/gky551

    Figure Lengend Snippet: Histone variant H2A.Z has a negative impact on the expression of Notch target genes. ( A ) Histone Variant H2A.Z is efficiently depleted by CRISPR/Cas9 in MT cells. Whole Cell Extract (WCE) was prepared from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells and analysed by Western blotting. GAPDH was used as loading control. ( B ) Hes1 and Il2ra Notch target genes are upregulated upon depletion of H2A.Z. Total RNA from wildtype ( Control ) or H2A.Z depleted (clones sgH2afv/H2afz #12 and sgH2afv/H2afz #20 ) MT cells was reverse transcribed into cDNA and analysed by qPCR using primers specific for Tbp, Hes1 or Il2ra . Data were normalized to the housekeeping gene GusB ( glucuronidase β ). Shown is the mean ± SD of five independent experiments ([*] P

    Article Snippet: In order to evaluate whether Tip60 is responsible for the acetylation of H2A.Z in higher eukaryotes and to evaluate the transcriptional consequences of the Tip60-mediated acetylation of H2A.Z in the context of Notch signaling, we directly fused the transcription factor RBP-J to Tip60 wildtype (wt) or its catalytic-dead mutant [(cd, ( ) and Figure A].

    Techniques: Variant Assay, Expressing, CRISPR, Western Blot, Real-time Polymerase Chain Reaction

    Schematic summary representing the link between H2A.Z and Notch signaling. In absence of Notch signaling (OFF, left side), H2A.Z occupancy increases while its acetylation (H2A.Zac) is low. Upon Notch activation (ON, right side), H2A.Z occupancy is reduced while its acetylation increases.

    Journal: Nucleic Acids Research

    Article Title: Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response

    doi: 10.1093/nar/gky551

    Figure Lengend Snippet: Schematic summary representing the link between H2A.Z and Notch signaling. In absence of Notch signaling (OFF, left side), H2A.Z occupancy increases while its acetylation (H2A.Zac) is low. Upon Notch activation (ON, right side), H2A.Z occupancy is reduced while its acetylation increases.

    Article Snippet: In order to evaluate whether Tip60 is responsible for the acetylation of H2A.Z in higher eukaryotes and to evaluate the transcriptional consequences of the Tip60-mediated acetylation of H2A.Z in the context of Notch signaling, we directly fused the transcription factor RBP-J to Tip60 wildtype (wt) or its catalytic-dead mutant [(cd, ( ) and Figure A].

    Techniques: Activation Assay

    Extrahepatic transmission of epigenetic modifications and evidence for modifications in DNA methylation at fibrogenic regulator gene associated with liver disease progression in humans a–b) ChIP analysis of trimethylated H3 lysine 27 (H3K27me3) and histone variant H2A.Z enrichment at the rat PPAR-γ gene promoter in mature sperm collected from male adult rats that were given chronic CCl 4 or olive oil (control) for 4 weeks, then recovered for 2 weeks (n=5). All ChIP results in a) to e) are expressed as fold control isotype matched antibody. b) ChIP analysis as in a) was carried out on mature sperm isolated from male adult rats that underwent bile duct ligation (BDL) or sham operation (control) 15 days previously. c) ChIP analysis as in a) was carried out on mature sperm isolated from rats that received twice weekly intravenous serum transfers (for four weeks total) from control or rats that were given CCl 4 for 4 weeks and serum collected 48hrs following last injection (n=6) d) ChIP analysis as in a) was carried out on primary rat mesenchymal stem cells which were treated with control or 48hrs conditioned activated HSC media for 72hrs (n=3). e) ChIP analysis as in a) was carried out on human PPAR-γ gene promoter in primary human mesenchymal stem cells which were treated with quiescent (day 1) or activated HSC (day 15) conditioned media for 72hrs (n=3). f) DNA methylation at particular CG dinucleotides within human PPAR-γ promoter in NAFLD patients liver biopsy tissues was determined by pyrosequencing. Position of the differentially methylated CGs is shown in the schematic drawing above the graphs. Differences are expressed as percentage DNA methylation Statistical analysis; Mann Whitney test, where p=0.0013 for CpG1 and p=0.0047 for CpG2.

    Journal: Nature medicine

    Article Title: Multigenerational Epigenetic Adaptation of the Hepatic Wound-Healing Response

    doi: 10.1038/nm.2893

    Figure Lengend Snippet: Extrahepatic transmission of epigenetic modifications and evidence for modifications in DNA methylation at fibrogenic regulator gene associated with liver disease progression in humans a–b) ChIP analysis of trimethylated H3 lysine 27 (H3K27me3) and histone variant H2A.Z enrichment at the rat PPAR-γ gene promoter in mature sperm collected from male adult rats that were given chronic CCl 4 or olive oil (control) for 4 weeks, then recovered for 2 weeks (n=5). All ChIP results in a) to e) are expressed as fold control isotype matched antibody. b) ChIP analysis as in a) was carried out on mature sperm isolated from male adult rats that underwent bile duct ligation (BDL) or sham operation (control) 15 days previously. c) ChIP analysis as in a) was carried out on mature sperm isolated from rats that received twice weekly intravenous serum transfers (for four weeks total) from control or rats that were given CCl 4 for 4 weeks and serum collected 48hrs following last injection (n=6) d) ChIP analysis as in a) was carried out on primary rat mesenchymal stem cells which were treated with control or 48hrs conditioned activated HSC media for 72hrs (n=3). e) ChIP analysis as in a) was carried out on human PPAR-γ gene promoter in primary human mesenchymal stem cells which were treated with quiescent (day 1) or activated HSC (day 15) conditioned media for 72hrs (n=3). f) DNA methylation at particular CG dinucleotides within human PPAR-γ promoter in NAFLD patients liver biopsy tissues was determined by pyrosequencing. Position of the differentially methylated CGs is shown in the schematic drawing above the graphs. Differences are expressed as percentage DNA methylation Statistical analysis; Mann Whitney test, where p=0.0013 for CpG1 and p=0.0047 for CpG2.

    Article Snippet: Precleared chromatin was incubated with anti H2A.Z, anti H3K27me3 or isotype matched control antibody overnight at 4 degrees C. We added one hundred microliters of blocked Staph A membranes to ChIP reactions for two hours at 4 degrees C, then samples washed, eluted and genomic DNA purified as previously outlined .

    Techniques: Transmission Assay, DNA Methylation Assay, Chromatin Immunoprecipitation, Variant Assay, Isolation, Ligation, Injection, Methylation, MANN-WHITNEY

    Co-immune precipitations reveal eEF2 interactions with Dph6 and Dph5. (A) eEF2 interacts with Dph6 in a fashion that is independent of Dph7. (B) eEF2 interaction with Dph5 is dramatically enhanced by elimination of Dph7 or Dph1. Yeast strains co-expressing (His) 6 -tagged eEF2 with Dph6-HA (A) or Dph5-HA (B) in the background of wild-type (A: DPH7 and B: wt) and dph mutant strains (A: dph7 ; B: dph1 , dph6 and dph7 ) were subjected to immune precipitations (IP) using the anti-HA antibody. Strains expressing (His) 6 -tagged eEF2 on their own served as IP controls (A and B: no HA-tag). Subsequently, the precipitates were probed with anti-HA (A: top left panel; B: first panel) and anti-(His) 6 antibodies (A: bottom left panel) to check for the content of Dph6-HA (A) and Dph5-HA (B), respectively (all indicated by arrows). The content of HA-tagged Dph6 (A) and Dph5 (B) as well as (His) 6 -marked eEF2 (A and B) in the protein extracts prior to IP (pre-IP) was examined on individual Western blots using anti-HA (A: top right panel; B: fourth panel) and anti-(His) 6 antibodies (A: bottom right panel; B: third panel), respectively. While absence of Dph7 hardly affected the Dph6•eEF2 interaction (A), Dph5•eEF2 interaction was strongly enhanced by inactivating DPH7 or DPH1 (B).

    Journal: PLoS Genetics

    Article Title: The Amidation Step of Diphthamide Biosynthesis in Yeast Requires DPH6, a Gene Identified through Mining the DPH1-DPH5 Interaction Network

    doi: 10.1371/journal.pgen.1003334

    Figure Lengend Snippet: Co-immune precipitations reveal eEF2 interactions with Dph6 and Dph5. (A) eEF2 interacts with Dph6 in a fashion that is independent of Dph7. (B) eEF2 interaction with Dph5 is dramatically enhanced by elimination of Dph7 or Dph1. Yeast strains co-expressing (His) 6 -tagged eEF2 with Dph6-HA (A) or Dph5-HA (B) in the background of wild-type (A: DPH7 and B: wt) and dph mutant strains (A: dph7 ; B: dph1 , dph6 and dph7 ) were subjected to immune precipitations (IP) using the anti-HA antibody. Strains expressing (His) 6 -tagged eEF2 on their own served as IP controls (A and B: no HA-tag). Subsequently, the precipitates were probed with anti-HA (A: top left panel; B: first panel) and anti-(His) 6 antibodies (A: bottom left panel) to check for the content of Dph6-HA (A) and Dph5-HA (B), respectively (all indicated by arrows). The content of HA-tagged Dph6 (A) and Dph5 (B) as well as (His) 6 -marked eEF2 (A and B) in the protein extracts prior to IP (pre-IP) was examined on individual Western blots using anti-HA (A: top right panel; B: fourth panel) and anti-(His) 6 antibodies (A: bottom right panel; B: third panel), respectively. While absence of Dph7 hardly affected the Dph6•eEF2 interaction (A), Dph5•eEF2 interaction was strongly enhanced by inactivating DPH7 or DPH1 (B).

    Article Snippet: The identity of purified eEF2 fraction was confirmed by SDS-PAGE and Western blot analysis using an anti-(His)6 antibody (Abcam, #ab18184).

    Techniques: Expressing, Mutagenesis, Western Blot

    The biosynthetic pathway for modification of eEF2 by diphthamide. For roles played by the bona fide diphthamide genes DPH1–DPH5 in steps 1 and 2 of the pathway, see main text. The ill-defined step 3, conversion of diphthine to diphthamide by amidation, is highlighted (red label). It likely involves ATP and ammonium cofactors in a reaction catalyzed by unidentified DPH gene product(s). Step 4 indicates diphthamide can be hijacked for eEF2 inactivation and cell death induction by antifungals, i.e. sordarin and bacterial ADP ribosylase toxins (ADPRtox); alternatively, it has been reported to undergo cell growth related physiological ADP ribosylation (ADPRphys?) by elusive cellular modifier(s). ACP, 2-[3-amino-carboxyl-propyl]-histidine; SAM: S-adenosylmethionine.

    Journal: PLoS Genetics

    Article Title: The Amidation Step of Diphthamide Biosynthesis in Yeast Requires DPH6, a Gene Identified through Mining the DPH1-DPH5 Interaction Network

    doi: 10.1371/journal.pgen.1003334

    Figure Lengend Snippet: The biosynthetic pathway for modification of eEF2 by diphthamide. For roles played by the bona fide diphthamide genes DPH1–DPH5 in steps 1 and 2 of the pathway, see main text. The ill-defined step 3, conversion of diphthine to diphthamide by amidation, is highlighted (red label). It likely involves ATP and ammonium cofactors in a reaction catalyzed by unidentified DPH gene product(s). Step 4 indicates diphthamide can be hijacked for eEF2 inactivation and cell death induction by antifungals, i.e. sordarin and bacterial ADP ribosylase toxins (ADPRtox); alternatively, it has been reported to undergo cell growth related physiological ADP ribosylation (ADPRphys?) by elusive cellular modifier(s). ACP, 2-[3-amino-carboxyl-propyl]-histidine; SAM: S-adenosylmethionine.

    Article Snippet: The identity of purified eEF2 fraction was confirmed by SDS-PAGE and Western blot analysis using an anti-(His)6 antibody (Abcam, #ab18184).

    Techniques: Modification

    DPH6 and DPH7 deletion strains copy traits typically related to the bona fide diphthamide mutants dph1-dph5 . (A) Sordarin resistance. Ten-fold serial cell dilutions of the indicated yeast strains, BY4741 wild-type (wt) background and its dph1-dph7 gene deletion derivatives (upper panels) as well an MKK-derived eft1 eft2 double deletion background maintaining plasmid p EFT2 wild-type or H 699 substitution (H 699 N and H 699 I) alleles of EFT2 (lower panels), were grown on YPD plates in the absence (control) or presence (+sor) of 10 µg ml −1 sordarin. Growth was assayed for 3 d at 30°C. Sordarin resistant (R) and sensitive (S) responses are indicated. (B) Lack of in vitro ADP ribose acceptor activity of eEF2. Cell extracts obtained from dph1 , dph5 , dph6 and dph7 mutant and wild-type (wt) strains were incubated with (+DT) or without (−DT) 20 nM diphtheria toxin in the presence of biotin-NAD (10 µM) at 37°C for 1 hour. The transfer of biotin-ADP-ribose to eEF2 was detected by Western blotting using a streptavidin-conjugate. Two unknown non-specific bands (indicated by *) served as internal controls for even sample loading. (C) DT phenotype. As indicated, yeast dph mutants and wild-type control (wt) were tested for sensitivity to intracellular expression of DTA, the cytotoxic ADP ribosylase fragment of DT. This in vivo assay involved galactose-inducible expression from vector pSU8 (see Materials and Methods ). Serial cell dilutions were replica spotted onto raffinose (2% raf) and galactose-inducing conditions using concentrations (2, 0.2 and 0.1% gal) suited to achieve gradual down-regulation of DTA toxicity. Growth was for 3 days at 30°C. DTA sensitive (S) resistant (R), partially resistant (PR) and reduced sensitive (RS) phenotypes are indicated.

    Journal: PLoS Genetics

    Article Title: The Amidation Step of Diphthamide Biosynthesis in Yeast Requires DPH6, a Gene Identified through Mining the DPH1-DPH5 Interaction Network

    doi: 10.1371/journal.pgen.1003334

    Figure Lengend Snippet: DPH6 and DPH7 deletion strains copy traits typically related to the bona fide diphthamide mutants dph1-dph5 . (A) Sordarin resistance. Ten-fold serial cell dilutions of the indicated yeast strains, BY4741 wild-type (wt) background and its dph1-dph7 gene deletion derivatives (upper panels) as well an MKK-derived eft1 eft2 double deletion background maintaining plasmid p EFT2 wild-type or H 699 substitution (H 699 N and H 699 I) alleles of EFT2 (lower panels), were grown on YPD plates in the absence (control) or presence (+sor) of 10 µg ml −1 sordarin. Growth was assayed for 3 d at 30°C. Sordarin resistant (R) and sensitive (S) responses are indicated. (B) Lack of in vitro ADP ribose acceptor activity of eEF2. Cell extracts obtained from dph1 , dph5 , dph6 and dph7 mutant and wild-type (wt) strains were incubated with (+DT) or without (−DT) 20 nM diphtheria toxin in the presence of biotin-NAD (10 µM) at 37°C for 1 hour. The transfer of biotin-ADP-ribose to eEF2 was detected by Western blotting using a streptavidin-conjugate. Two unknown non-specific bands (indicated by *) served as internal controls for even sample loading. (C) DT phenotype. As indicated, yeast dph mutants and wild-type control (wt) were tested for sensitivity to intracellular expression of DTA, the cytotoxic ADP ribosylase fragment of DT. This in vivo assay involved galactose-inducible expression from vector pSU8 (see Materials and Methods ). Serial cell dilutions were replica spotted onto raffinose (2% raf) and galactose-inducing conditions using concentrations (2, 0.2 and 0.1% gal) suited to achieve gradual down-regulation of DTA toxicity. Growth was for 3 days at 30°C. DTA sensitive (S) resistant (R), partially resistant (PR) and reduced sensitive (RS) phenotypes are indicated.

    Article Snippet: The identity of purified eEF2 fraction was confirmed by SDS-PAGE and Western blot analysis using an anti-(His)6 antibody (Abcam, #ab18184).

    Techniques: Derivative Assay, Plasmid Preparation, In Vitro, Activity Assay, Mutagenesis, Incubation, TNKS1 Histone Ribosylation Assay, Western Blot, Expressing, In Vivo

    Model for the diphthamide pathway incorporating the proposed novel roles of Dph5, Dph6, and Dph7. (A) Diphthamide pathway showing interaction of Dph5 with unmodified eEF2 and the proposed role of Dph7 in displacement of Dph5 prior to diphthine amidation. (B) Elimination of the trimethylamino group in the absence of the proposed amidase Dph6 suggesting lability of diphthine in its absence.

    Journal: PLoS Genetics

    Article Title: The Amidation Step of Diphthamide Biosynthesis in Yeast Requires DPH6, a Gene Identified through Mining the DPH1-DPH5 Interaction Network

    doi: 10.1371/journal.pgen.1003334

    Figure Lengend Snippet: Model for the diphthamide pathway incorporating the proposed novel roles of Dph5, Dph6, and Dph7. (A) Diphthamide pathway showing interaction of Dph5 with unmodified eEF2 and the proposed role of Dph7 in displacement of Dph5 prior to diphthine amidation. (B) Elimination of the trimethylamino group in the absence of the proposed amidase Dph6 suggesting lability of diphthine in its absence.

    Article Snippet: The identity of purified eEF2 fraction was confirmed by SDS-PAGE and Western blot analysis using an anti-(His)6 antibody (Abcam, #ab18184).

    Techniques: