mnase digestion buffer  (New England Biolabs)


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    New England Biolabs mnase digestion buffer
    Nucleosome positioning is altered at the TSSs of genes in Δ dim-1 strain. (A) Southern blots of <t>DNA</t> from 20-min time course micrococcal nuclease <t>(MNase)</t> digest with WT and Δ dim-1 nuclei, probed for the indicated regions; the “NCU04771up start” probe covers the intergenic promoter region of gene NCU04771, including the nucleosome-free region. Arrowheads indicate the time points (8 and 10 min) from which mono- and di-nucleosomes and intervening DNA was purified for paired-end high-throughput sequencing (below). Cartoons at right show interpretation of nucleosome patterns leading to smallest fragments. (B) Average nucleosome enrichment profiles of MNase-sequencing data from WT and Δ dim-1 strains normalized to the average signal across the 5′ end of all Neurospora genes, spanning 400 bp upstream to 600 bp downstream of the TSS; numbers below indicate the peak apices, in base pairs from the transcriptional start site (TSS) in WT and Δ dim-1 strains, and the difference in the apex position, for the +1, +2, and +3 nucleosomes. (C) Representative examples of nucleosome positions in individual genes with changed (NCU08052), or unchanged (NCU04402; dim-5 ) expression in a Δ dim-1 background. Red arrows highlight nucleosome disorder in a Δ dim-1 strain of two nucleosomes that are well-positioned in a WT strain.
    Mnase Digestion Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 153 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Nucleosome Positioning by an Evolutionarily Conserved Chromatin Remodeler Prevents Aberrant DNA Methylation in Neurospora"

    Article Title: Nucleosome Positioning by an Evolutionarily Conserved Chromatin Remodeler Prevents Aberrant DNA Methylation in Neurospora

    Journal: Genetics

    doi: 10.1534/genetics.118.301711

    Nucleosome positioning is altered at the TSSs of genes in Δ dim-1 strain. (A) Southern blots of DNA from 20-min time course micrococcal nuclease (MNase) digest with WT and Δ dim-1 nuclei, probed for the indicated regions; the “NCU04771up start” probe covers the intergenic promoter region of gene NCU04771, including the nucleosome-free region. Arrowheads indicate the time points (8 and 10 min) from which mono- and di-nucleosomes and intervening DNA was purified for paired-end high-throughput sequencing (below). Cartoons at right show interpretation of nucleosome patterns leading to smallest fragments. (B) Average nucleosome enrichment profiles of MNase-sequencing data from WT and Δ dim-1 strains normalized to the average signal across the 5′ end of all Neurospora genes, spanning 400 bp upstream to 600 bp downstream of the TSS; numbers below indicate the peak apices, in base pairs from the transcriptional start site (TSS) in WT and Δ dim-1 strains, and the difference in the apex position, for the +1, +2, and +3 nucleosomes. (C) Representative examples of nucleosome positions in individual genes with changed (NCU08052), or unchanged (NCU04402; dim-5 ) expression in a Δ dim-1 background. Red arrows highlight nucleosome disorder in a Δ dim-1 strain of two nucleosomes that are well-positioned in a WT strain.
    Figure Legend Snippet: Nucleosome positioning is altered at the TSSs of genes in Δ dim-1 strain. (A) Southern blots of DNA from 20-min time course micrococcal nuclease (MNase) digest with WT and Δ dim-1 nuclei, probed for the indicated regions; the “NCU04771up start” probe covers the intergenic promoter region of gene NCU04771, including the nucleosome-free region. Arrowheads indicate the time points (8 and 10 min) from which mono- and di-nucleosomes and intervening DNA was purified for paired-end high-throughput sequencing (below). Cartoons at right show interpretation of nucleosome patterns leading to smallest fragments. (B) Average nucleosome enrichment profiles of MNase-sequencing data from WT and Δ dim-1 strains normalized to the average signal across the 5′ end of all Neurospora genes, spanning 400 bp upstream to 600 bp downstream of the TSS; numbers below indicate the peak apices, in base pairs from the transcriptional start site (TSS) in WT and Δ dim-1 strains, and the difference in the apex position, for the +1, +2, and +3 nucleosomes. (C) Representative examples of nucleosome positions in individual genes with changed (NCU08052), or unchanged (NCU04402; dim-5 ) expression in a Δ dim-1 background. Red arrows highlight nucleosome disorder in a Δ dim-1 strain of two nucleosomes that are well-positioned in a WT strain.

    Techniques Used: Purification, Next-Generation Sequencing, Sequencing, Expressing

    2) Product Images from "An Improved Strategy to Recover Large Fragments of Functional Human Neutrophil Extracellular Traps"

    Article Title: An Improved Strategy to Recover Large Fragments of Functional Human Neutrophil Extracellular Traps

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2013.00166

    DNA nucleases induce NET digestion . (A) Migration profile of pure λDNA after digestion with 4 U/mL DNase, MNase, or Alu-I. (B) Alu-I, DNase, and MNase dose-effects on NET dsDNA obtained after A23187 stimulation of PMN. Incubation with the restriction enzymes lasted 20 min at 37°C. DNA migration took place in 0.8% agarose gel containing ethidium bromide.
    Figure Legend Snippet: DNA nucleases induce NET digestion . (A) Migration profile of pure λDNA after digestion with 4 U/mL DNase, MNase, or Alu-I. (B) Alu-I, DNase, and MNase dose-effects on NET dsDNA obtained after A23187 stimulation of PMN. Incubation with the restriction enzymes lasted 20 min at 37°C. DNA migration took place in 0.8% agarose gel containing ethidium bromide.

    Techniques Used: Migration, Incubation, Agarose Gel Electrophoresis

    3) Product Images from "RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF"

    Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky562

    Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or DNA sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for MNAse digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.
    Figure Legend Snippet: Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or DNA sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for MNAse digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.

    Techniques Used: Binding Assay, Genome Wide, Sequencing, Immunoprecipitation

    4) Product Images from "Regulatory architecture of the RCA gene cluster captures an intragenic TAD boundary and enhancer elements in B cells"

    Article Title: Regulatory architecture of the RCA gene cluster captures an intragenic TAD boundary and enhancer elements in B cells

    Journal: bioRxiv

    doi: 10.1101/2020.02.16.941070

    BEN-2 shows B cell-specific nucleosome occupancy, chromatin accessibility and enrichment for the H3K27ac active enhancer histone mark across a panel of B cell lines. A. Nucleosome occupancy at BEN-2 as measured by ChART-PCR with MNase digestion. Data was normalised to the inaccessible SFTPA2 gene promoter such that a value of 1.0 represents fully compacted nucleosomes, and lower values indicate less compacted nucleosomes. B. Chromatin accessibility at BEN-2 as measured by ChART-PCR with DNase I digestion. Data have been normalised to the inaccessible SFTPA2 gene promoter. C. H3K27ac enrichment at BEN-2 as determined by ChIP-qPCR using the percent input method. Grey bars indicate H3K27ac enrichment at the target locus, and black bars show enrichment using a non-specific IgG control antibody. All data are presented as mean ± SEM from at least 3 biological replicates.
    Figure Legend Snippet: BEN-2 shows B cell-specific nucleosome occupancy, chromatin accessibility and enrichment for the H3K27ac active enhancer histone mark across a panel of B cell lines. A. Nucleosome occupancy at BEN-2 as measured by ChART-PCR with MNase digestion. Data was normalised to the inaccessible SFTPA2 gene promoter such that a value of 1.0 represents fully compacted nucleosomes, and lower values indicate less compacted nucleosomes. B. Chromatin accessibility at BEN-2 as measured by ChART-PCR with DNase I digestion. Data have been normalised to the inaccessible SFTPA2 gene promoter. C. H3K27ac enrichment at BEN-2 as determined by ChIP-qPCR using the percent input method. Grey bars indicate H3K27ac enrichment at the target locus, and black bars show enrichment using a non-specific IgG control antibody. All data are presented as mean ± SEM from at least 3 biological replicates.

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

    5) Product Images from "Identification of a DNA N6-adenine methyltransferase complex and its impact on chromatin organization"

    Article Title: Identification of a DNA N6-adenine methyltransferase complex and its impact on chromatin organization

    Journal: Cell

    doi: 10.1016/j.cell.2019.04.028

    Quantitative modulation of nucleosome occupancy by 6mA (A) Experimental workflow. Chromatin is assembled using either salt dialysis or the NAP1 histone chaperone. Italicized blue steps are selectively included. (B) Tiling qPCR analysis of synthetic chromosome with cognate 6mA sites. Horizontal grey box represents annotated gene, and vertical black lines depict native 6mA positions. Horizontal blue bars span ~100bp regions amplified by qPCR. Red horizontal lines represent the region containing 6mA. ‘Hemi methyl’ chromosomes contain 6mA on the antisense and sense strands, respectively, while the ‘Full methyl’ chromosome has 6mA on both strands. Black arrowheads: decrease in nucleosome occupancy specifically at the 6mA cluster. (C) Tiling qPCR analysis of ectopically methylated synthetic chromosome. Vertical black lines illustrate possible 6mA sites installed enzymatically. Red arrowheads: decrease in nucleosome occupancy in the ectopically methylated region. Black arrowheads: position of cognate 6mA sites (not in this construct). (D) Tiling qPCR analysis of chromatin from panel B that is subsequently incubated with ACF and/or ATP. ACF equalizes nucleosome occupancy between the 6mA cluster and flanking regions in the presence of ATP (black line). Nucleosome occupancy at the methylated region is not restored to the same level as the unmethylated control (black arrowheads). (E) MNase-seq analysis of chromatin is assembled on native gDNA (“+” 6mA) and mini-genome DNA (“−“ 6mA) using NAP1 +/− ACF and ATP. P -values were calculated using a two-sample unequal variance t-test.
    Figure Legend Snippet: Quantitative modulation of nucleosome occupancy by 6mA (A) Experimental workflow. Chromatin is assembled using either salt dialysis or the NAP1 histone chaperone. Italicized blue steps are selectively included. (B) Tiling qPCR analysis of synthetic chromosome with cognate 6mA sites. Horizontal grey box represents annotated gene, and vertical black lines depict native 6mA positions. Horizontal blue bars span ~100bp regions amplified by qPCR. Red horizontal lines represent the region containing 6mA. ‘Hemi methyl’ chromosomes contain 6mA on the antisense and sense strands, respectively, while the ‘Full methyl’ chromosome has 6mA on both strands. Black arrowheads: decrease in nucleosome occupancy specifically at the 6mA cluster. (C) Tiling qPCR analysis of ectopically methylated synthetic chromosome. Vertical black lines illustrate possible 6mA sites installed enzymatically. Red arrowheads: decrease in nucleosome occupancy in the ectopically methylated region. Black arrowheads: position of cognate 6mA sites (not in this construct). (D) Tiling qPCR analysis of chromatin from panel B that is subsequently incubated with ACF and/or ATP. ACF equalizes nucleosome occupancy between the 6mA cluster and flanking regions in the presence of ATP (black line). Nucleosome occupancy at the methylated region is not restored to the same level as the unmethylated control (black arrowheads). (E) MNase-seq analysis of chromatin is assembled on native gDNA (“+” 6mA) and mini-genome DNA (“−“ 6mA) using NAP1 +/− ACF and ATP. P -values were calculated using a two-sample unequal variance t-test.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Methylation, Construct, Incubation

    6) Product Images from "Identification of a DNA N6-adenine methyltransferase complex and its impact on chromatin organization"

    Article Title: Identification of a DNA N6-adenine methyltransferase complex and its impact on chromatin organization

    Journal: Cell

    doi: 10.1016/j.cell.2019.04.028

    Quantitative modulation of nucleosome occupancy by 6mA (A) Experimental workflow. Chromatin is assembled using either salt dialysis or the NAP1 histone chaperone. Italicized blue steps are selectively included. (B) Tiling qPCR analysis of synthetic chromosome with cognate 6mA sites. Horizontal grey box represents annotated gene, and vertical black lines depict native 6mA positions. Horizontal blue bars span ~100bp regions amplified by qPCR. Red horizontal lines represent the region containing 6mA. ‘Hemi methyl’ chromosomes contain 6mA on the antisense and sense strands, respectively, while the ‘Full methyl’ chromosome has 6mA on both strands. Black arrowheads: decrease in nucleosome occupancy specifically at the 6mA cluster. (C) Tiling qPCR analysis of ectopically methylated synthetic chromosome. Vertical black lines illustrate possible 6mA sites installed enzymatically. Red arrowheads: decrease in nucleosome occupancy in the ectopically methylated region. Black arrowheads: position of cognate 6mA sites (not in this construct). (D) Tiling qPCR analysis of chromatin from panel B that is subsequently incubated with ACF and/or ATP. ACF equalizes nucleosome occupancy between the 6mA cluster and flanking regions in the presence of ATP (black line). Nucleosome occupancy at the methylated region is not restored to the same level as the unmethylated control (black arrowheads). (E) MNase-seq analysis of chromatin is assembled on native gDNA (“+” 6mA) and mini-genome DNA (“−“ 6mA) using NAP1 +/− ACF and ATP. P -values were calculated using a two-sample unequal variance t-test.
    Figure Legend Snippet: Quantitative modulation of nucleosome occupancy by 6mA (A) Experimental workflow. Chromatin is assembled using either salt dialysis or the NAP1 histone chaperone. Italicized blue steps are selectively included. (B) Tiling qPCR analysis of synthetic chromosome with cognate 6mA sites. Horizontal grey box represents annotated gene, and vertical black lines depict native 6mA positions. Horizontal blue bars span ~100bp regions amplified by qPCR. Red horizontal lines represent the region containing 6mA. ‘Hemi methyl’ chromosomes contain 6mA on the antisense and sense strands, respectively, while the ‘Full methyl’ chromosome has 6mA on both strands. Black arrowheads: decrease in nucleosome occupancy specifically at the 6mA cluster. (C) Tiling qPCR analysis of ectopically methylated synthetic chromosome. Vertical black lines illustrate possible 6mA sites installed enzymatically. Red arrowheads: decrease in nucleosome occupancy in the ectopically methylated region. Black arrowheads: position of cognate 6mA sites (not in this construct). (D) Tiling qPCR analysis of chromatin from panel B that is subsequently incubated with ACF and/or ATP. ACF equalizes nucleosome occupancy between the 6mA cluster and flanking regions in the presence of ATP (black line). Nucleosome occupancy at the methylated region is not restored to the same level as the unmethylated control (black arrowheads). (E) MNase-seq analysis of chromatin is assembled on native gDNA (“+” 6mA) and mini-genome DNA (“−“ 6mA) using NAP1 +/− ACF and ATP. P -values were calculated using a two-sample unequal variance t-test.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Methylation, Construct, Incubation

    7) Product Images from "Identification and characterization of DNA sequences that prevent glucocorticoid receptor binding to nearby response elements"

    Article Title: Identification and characterization of DNA sequences that prevent glucocorticoid receptor binding to nearby response elements

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw203

    Characterization of the NRS sequences. ( A ) EMSA comparing binding of the DNA binding domain of GR to a GBS sequence flanked by either the control sequence (left) or by NRS2 (right). ( B ) DNase I accessibility assay was performed with populations of transgenic cells with stably integrated reporters as indicated. Regions of interest were analyzed by qPCR ( FKBP5 : control accessible region; IGFBP1 : control inaccessible region; integr. GBS: integrated reporter region). Results are shown as % of input remaining after DNAse I digestion ±SEM (n = 3). ( C ) Nucleosome occupancy was analyzed using micrococcal nuclease (MNase) assays for populations of transgenic cells with stably integrated reporters as indicated. Regions of interest were analyzed by qPCR. Integr. GBS: integrated reporter region. Results are shown as % of input remaining after MNase digestion ±SEM (n = 3).
    Figure Legend Snippet: Characterization of the NRS sequences. ( A ) EMSA comparing binding of the DNA binding domain of GR to a GBS sequence flanked by either the control sequence (left) or by NRS2 (right). ( B ) DNase I accessibility assay was performed with populations of transgenic cells with stably integrated reporters as indicated. Regions of interest were analyzed by qPCR ( FKBP5 : control accessible region; IGFBP1 : control inaccessible region; integr. GBS: integrated reporter region). Results are shown as % of input remaining after DNAse I digestion ±SEM (n = 3). ( C ) Nucleosome occupancy was analyzed using micrococcal nuclease (MNase) assays for populations of transgenic cells with stably integrated reporters as indicated. Regions of interest were analyzed by qPCR. Integr. GBS: integrated reporter region. Results are shown as % of input remaining after MNase digestion ±SEM (n = 3).

    Techniques Used: Binding Assay, Sequencing, Transgenic Assay, Stable Transfection, Real-time Polymerase Chain Reaction

    8) Product Images from "Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum"

    Article Title: Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum

    Journal: EMBO Reports

    doi: 10.15252/embr.201846331

    Ectopically expressed histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p‐HA in ring (R), trophozoite (T), and schizont (S) stages of Plasmodium falciparum asexual growth. PfH3p‐HA was detected using anti‐HA antibodies (green) and endogenous histone H3 with anti‐histone H3 N‐terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. Nuclei isolated from wild‐type (WT) or PfH3p‐HA‐expressing (WT + PfH3p‐HA) schizont‐stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 min of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant Blue (C.B.) or visualized by immunoblotting with anti‐HA (α‐HA) or anti‐C‐terminal histone H3 (α‐H3c) antibodies. Co‐immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild‐type (WT) or transfected (WT + PfH3p‐HA) schizont‐stage parasites were performed with either anti‐HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti‐HA or anti‐histone H4 antibodies. Source data are available online for this figure.
    Figure Legend Snippet: Ectopically expressed histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p‐HA in ring (R), trophozoite (T), and schizont (S) stages of Plasmodium falciparum asexual growth. PfH3p‐HA was detected using anti‐HA antibodies (green) and endogenous histone H3 with anti‐histone H3 N‐terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. Nuclei isolated from wild‐type (WT) or PfH3p‐HA‐expressing (WT + PfH3p‐HA) schizont‐stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 min of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant Blue (C.B.) or visualized by immunoblotting with anti‐HA (α‐HA) or anti‐C‐terminal histone H3 (α‐H3c) antibodies. Co‐immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild‐type (WT) or transfected (WT + PfH3p‐HA) schizont‐stage parasites were performed with either anti‐HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti‐HA or anti‐histone H4 antibodies. Source data are available online for this figure.

    Techniques Used: Immunofluorescence, Staining, Isolation, Expressing, Purification, Agarose Gel Electrophoresis, Polyacrylamide Gel Electrophoresis, Immunoprecipitation, Transfection

    9) Product Images from "Identification of a DNA N6-adenine methyltransferase complex and its impact on chromatin organization"

    Article Title: Identification of a DNA N6-adenine methyltransferase complex and its impact on chromatin organization

    Journal: Cell

    doi: 10.1016/j.cell.2019.04.028

    Quantitative modulation of nucleosome occupancy by 6mA (A) Experimental workflow. Chromatin is assembled using either salt dialysis or the NAP1 histone chaperone. Italicized blue steps are selectively included. (B) Tiling qPCR analysis of synthetic chromosome with cognate 6mA sites. Horizontal grey box represents annotated gene, and vertical black lines depict native 6mA positions. Horizontal blue bars span ~100bp regions amplified by qPCR. Red horizontal lines represent the region containing 6mA. ‘Hemi methyl’ chromosomes contain 6mA on the antisense and sense strands, respectively, while the ‘Full methyl’ chromosome has 6mA on both strands. Black arrowheads: decrease in nucleosome occupancy specifically at the 6mA cluster. (C) Tiling qPCR analysis of ectopically methylated synthetic chromosome. Vertical black lines illustrate possible 6mA sites installed enzymatically. Red arrowheads: decrease in nucleosome occupancy in the ectopically methylated region. Black arrowheads: position of cognate 6mA sites (not in this construct). (D) Tiling qPCR analysis of chromatin from panel B that is subsequently incubated with ACF and/or ATP. ACF equalizes nucleosome occupancy between the 6mA cluster and flanking regions in the presence of ATP (black line). Nucleosome occupancy at the methylated region is not restored to the same level as the unmethylated control (black arrowheads). (E) MNase-seq analysis of chromatin is assembled on native gDNA (“+” 6mA) and mini-genome DNA (“−“ 6mA) using NAP1 +/− ACF and ATP. P -values were calculated using a two-sample unequal variance t-test.
    Figure Legend Snippet: Quantitative modulation of nucleosome occupancy by 6mA (A) Experimental workflow. Chromatin is assembled using either salt dialysis or the NAP1 histone chaperone. Italicized blue steps are selectively included. (B) Tiling qPCR analysis of synthetic chromosome with cognate 6mA sites. Horizontal grey box represents annotated gene, and vertical black lines depict native 6mA positions. Horizontal blue bars span ~100bp regions amplified by qPCR. Red horizontal lines represent the region containing 6mA. ‘Hemi methyl’ chromosomes contain 6mA on the antisense and sense strands, respectively, while the ‘Full methyl’ chromosome has 6mA on both strands. Black arrowheads: decrease in nucleosome occupancy specifically at the 6mA cluster. (C) Tiling qPCR analysis of ectopically methylated synthetic chromosome. Vertical black lines illustrate possible 6mA sites installed enzymatically. Red arrowheads: decrease in nucleosome occupancy in the ectopically methylated region. Black arrowheads: position of cognate 6mA sites (not in this construct). (D) Tiling qPCR analysis of chromatin from panel B that is subsequently incubated with ACF and/or ATP. ACF equalizes nucleosome occupancy between the 6mA cluster and flanking regions in the presence of ATP (black line). Nucleosome occupancy at the methylated region is not restored to the same level as the unmethylated control (black arrowheads). (E) MNase-seq analysis of chromatin is assembled on native gDNA (“+” 6mA) and mini-genome DNA (“−“ 6mA) using NAP1 +/− ACF and ATP. P -values were calculated using a two-sample unequal variance t-test.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Methylation, Construct, Incubation

    10) Product Images from "A high-resolution map of transcriptional repression"

    Article Title: A high-resolution map of transcriptional repression

    Journal: eLife

    doi: 10.7554/eLife.22767

    Histone deacetylation contributes to stable gene silencing. ( A ) RT-PCR showed that 1 ng/ml TSA for 24 hr significantly relieved Ikaros-induced reduced repression of Igll and Myc primary transcripts. Mean ± SE, 3 independent biological replicates. ( B ) MNase PCR showed that 1 ng/ml TSA for 24 hr did not significantly affect protection of 80–120 bp amplicons (short, left) but significantly reduced protection of 130–140 bp amplicons (long, right) at the Igll1 promoter. Mean ± SE, 3 independent biological replicates. ( C ) ChIP-PCR to assess Ikaros-induced recruitment of histone H2B to the Igll1 promoter between control cells and cells treated with 1 ng/ml TSA for 24 hr. Enrichment was normalised to total H3. Mean ± SE, 3 independent biological replicates. TSA significantly blunted the Ikaros-induced increase the H2B/H3 ratio at the Igll1 promoter and TSS. ( D ) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, cells were treated over night with TSA (1 ng/ml) and/or 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of TSA was statistically significant across replicates (p=9.54 × 10-18 GLM binomial logit). DOI: http://dx.doi.org/10.7554/eLife.22767.021 10.7554/eLife.22767.022 Numerical data used to generate Figure 6A,B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.022
    Figure Legend Snippet: Histone deacetylation contributes to stable gene silencing. ( A ) RT-PCR showed that 1 ng/ml TSA for 24 hr significantly relieved Ikaros-induced reduced repression of Igll and Myc primary transcripts. Mean ± SE, 3 independent biological replicates. ( B ) MNase PCR showed that 1 ng/ml TSA for 24 hr did not significantly affect protection of 80–120 bp amplicons (short, left) but significantly reduced protection of 130–140 bp amplicons (long, right) at the Igll1 promoter. Mean ± SE, 3 independent biological replicates. ( C ) ChIP-PCR to assess Ikaros-induced recruitment of histone H2B to the Igll1 promoter between control cells and cells treated with 1 ng/ml TSA for 24 hr. Enrichment was normalised to total H3. Mean ± SE, 3 independent biological replicates. TSA significantly blunted the Ikaros-induced increase the H2B/H3 ratio at the Igll1 promoter and TSS. ( D ) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, cells were treated over night with TSA (1 ng/ml) and/or 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of TSA was statistically significant across replicates (p=9.54 × 10-18 GLM binomial logit). DOI: http://dx.doi.org/10.7554/eLife.22767.021 10.7554/eLife.22767.022 Numerical data used to generate Figure 6A,B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.022

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Fluorescence In Situ Hybridization

    Interdependence of silencing mechanisms leveraged by Ikaros. ( A ) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, control or sh Chd4 cells were treated over night with 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of Chd4 knockdown was statistically significant across replicates (p=5.54×10-38 GLM binomial logit). ( B ) ChIP kinetics of Ikaros and EBF binding to the Igll1 promoter in control (left) and sh Chd4 cells (right). Increased binding of Ikaros to the Igll1 promoter was significant for both control and sh Chd4 cells, decreased binding of EBF1 was significant in control, but not in sh Chd4 cells. Mean ± SE, 3 independent biological replicates. Ikaros and EBF1 binding at 15, 30 and 120 min were significantly higher in sh Chd4 than control cells. ( C ) MNase-seq data from 3 independent biological replicates were integrated with Ikaros ChIP-seq data to show nucleosome occupancy at Ikaros binding peaks before and 6 hr after nuclear translocation of Ikaros. ( D ) Dynamics of Ikaros binding, RNAP2 eviction, loss of primary transcripts, nucleosome invasion, and histone deacetylation. DOI: http://dx.doi.org/10.7554/eLife.22767.023 10.7554/eLife.22767.024 Numerical data used to generate Figure 7A and B . DOI: http://dx.doi.org/10.7554/eLife.22767.024
    Figure Legend Snippet: Interdependence of silencing mechanisms leveraged by Ikaros. ( A ) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, control or sh Chd4 cells were treated over night with 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of Chd4 knockdown was statistically significant across replicates (p=5.54×10-38 GLM binomial logit). ( B ) ChIP kinetics of Ikaros and EBF binding to the Igll1 promoter in control (left) and sh Chd4 cells (right). Increased binding of Ikaros to the Igll1 promoter was significant for both control and sh Chd4 cells, decreased binding of EBF1 was significant in control, but not in sh Chd4 cells. Mean ± SE, 3 independent biological replicates. Ikaros and EBF1 binding at 15, 30 and 120 min were significantly higher in sh Chd4 than control cells. ( C ) MNase-seq data from 3 independent biological replicates were integrated with Ikaros ChIP-seq data to show nucleosome occupancy at Ikaros binding peaks before and 6 hr after nuclear translocation of Ikaros. ( D ) Dynamics of Ikaros binding, RNAP2 eviction, loss of primary transcripts, nucleosome invasion, and histone deacetylation. DOI: http://dx.doi.org/10.7554/eLife.22767.023 10.7554/eLife.22767.024 Numerical data used to generate Figure 7A and B . DOI: http://dx.doi.org/10.7554/eLife.22767.024

    Techniques Used: Fluorescence In Situ Hybridization, Chromatin Immunoprecipitation, Binding Assay, Translocation Assay

    11) Product Images from "A high-resolution map of transcriptional repression"

    Article Title: A high-resolution map of transcriptional repression

    Journal: eLife

    doi: 10.7554/eLife.22767

    Histone deacetylation contributes to stable gene silencing. ( A ) RT-PCR showed that 1 ng/ml TSA for 24 hr significantly relieved Ikaros-induced reduced repression of Igll and Myc primary transcripts. Mean ± SE, 3 independent biological replicates. ( B ) MNase PCR showed that 1 ng/ml TSA for 24 hr did not significantly affect protection of 80–120 bp amplicons (short, left) but significantly reduced protection of 130–140 bp amplicons (long, right) at the Igll1 promoter. Mean ± SE, 3 independent biological replicates. ( C ) ChIP-PCR to assess Ikaros-induced recruitment of histone H2B to the Igll1 promoter between control cells and cells treated with 1 ng/ml TSA for 24 hr. Enrichment was normalised to total H3. Mean ± SE, 3 independent biological replicates. TSA significantly blunted the Ikaros-induced increase the H2B/H3 ratio at the Igll1 promoter and TSS. ( D ) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, cells were treated over night with TSA (1 ng/ml) and/or 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of TSA was statistically significant across replicates (p=9.54 × 10-18 GLM binomial logit). DOI: http://dx.doi.org/10.7554/eLife.22767.021 10.7554/eLife.22767.022 Numerical data used to generate Figure 6A,B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.022
    Figure Legend Snippet: Histone deacetylation contributes to stable gene silencing. ( A ) RT-PCR showed that 1 ng/ml TSA for 24 hr significantly relieved Ikaros-induced reduced repression of Igll and Myc primary transcripts. Mean ± SE, 3 independent biological replicates. ( B ) MNase PCR showed that 1 ng/ml TSA for 24 hr did not significantly affect protection of 80–120 bp amplicons (short, left) but significantly reduced protection of 130–140 bp amplicons (long, right) at the Igll1 promoter. Mean ± SE, 3 independent biological replicates. ( C ) ChIP-PCR to assess Ikaros-induced recruitment of histone H2B to the Igll1 promoter between control cells and cells treated with 1 ng/ml TSA for 24 hr. Enrichment was normalised to total H3. Mean ± SE, 3 independent biological replicates. TSA significantly blunted the Ikaros-induced increase the H2B/H3 ratio at the Igll1 promoter and TSS. ( D ) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, cells were treated over night with TSA (1 ng/ml) and/or 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of TSA was statistically significant across replicates (p=9.54 × 10-18 GLM binomial logit). DOI: http://dx.doi.org/10.7554/eLife.22767.021 10.7554/eLife.22767.022 Numerical data used to generate Figure 6A,B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.022

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Fluorescence In Situ Hybridization

    Interdependence of silencing mechanisms leveraged by Ikaros. ( A ) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, control or sh Chd4 cells were treated over night with 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of Chd4 knockdown was statistically significant across replicates (p=5.54×10-38 GLM binomial logit). ( B ) ChIP kinetics of Ikaros and EBF binding to the Igll1 promoter in control (left) and sh Chd4 cells (right). Increased binding of Ikaros to the Igll1 promoter was significant for both control and sh Chd4 cells, decreased binding of EBF1 was significant in control, but not in sh Chd4 cells. Mean ± SE, 3 independent biological replicates. Ikaros and EBF1 binding at 15, 30 and 120 min were significantly higher in sh Chd4 than control cells. ( C ) MNase-seq data from 3 independent biological replicates were integrated with Ikaros ChIP-seq data to show nucleosome occupancy at Ikaros binding peaks before and 6 hr after nuclear translocation of Ikaros. ( D ) Dynamics of Ikaros binding, RNAP2 eviction, loss of primary transcripts, nucleosome invasion, and histone deacetylation. DOI: http://dx.doi.org/10.7554/eLife.22767.023 10.7554/eLife.22767.024 Numerical data used to generate Figure 7A and B . DOI: http://dx.doi.org/10.7554/eLife.22767.024
    Figure Legend Snippet: Interdependence of silencing mechanisms leveraged by Ikaros. ( A ) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, control or sh Chd4 cells were treated over night with 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of Chd4 knockdown was statistically significant across replicates (p=5.54×10-38 GLM binomial logit). ( B ) ChIP kinetics of Ikaros and EBF binding to the Igll1 promoter in control (left) and sh Chd4 cells (right). Increased binding of Ikaros to the Igll1 promoter was significant for both control and sh Chd4 cells, decreased binding of EBF1 was significant in control, but not in sh Chd4 cells. Mean ± SE, 3 independent biological replicates. Ikaros and EBF1 binding at 15, 30 and 120 min were significantly higher in sh Chd4 than control cells. ( C ) MNase-seq data from 3 independent biological replicates were integrated with Ikaros ChIP-seq data to show nucleosome occupancy at Ikaros binding peaks before and 6 hr after nuclear translocation of Ikaros. ( D ) Dynamics of Ikaros binding, RNAP2 eviction, loss of primary transcripts, nucleosome invasion, and histone deacetylation. DOI: http://dx.doi.org/10.7554/eLife.22767.023 10.7554/eLife.22767.024 Numerical data used to generate Figure 7A and B . DOI: http://dx.doi.org/10.7554/eLife.22767.024

    Techniques Used: Fluorescence In Situ Hybridization, Chromatin Immunoprecipitation, Binding Assay, Translocation Assay

    12) Product Images from "Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics"

    Article Title: Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky526

    Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.
    Figure Legend Snippet: Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.

    Techniques Used: Clear Native PAGE, Electrophoresis, Polyacrylamide Gel Electrophoresis, Staining

    13) Product Images from "HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism"

    Article Title: HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004652

    Isolation of cross-linked ORF57-RNA complexes for HITS-CLIP analysis. Top Nitrocellulose membrane from an ORF57 immunoprecipitation performed under HITS-CLIP conditions. Cross-linked RNA fragments were end-labeled with 32 P to be visualized by Phosphoimager. Cells were induced to undergo lytic reactivation, exposed to UV and/or treated with high or low concentrations of MNase as indicated. The samples lacking an ORF57 antibody (lane 1) were precipitated with pre-bleed antibodies from the same rabbit. Lanes 6 and 7 are a dark exposure of lanes 4 and 5. The dashed white box indicates the position of the ORF57 complex cut from the membrane for library preparation. Bottom Western blot of the same HITS-CLIP samples shown in the top panel. Affinity purified rabbit anti-ORF57 was used to detect ORF57. Positions of molecular weight markers are shown on the left. On the right, a single asterisk marks the position of a contaminating ~37 kDa protein; double and triple asterisks mark positions of putative ORF57 homodimers and homotrimers bound to the same RNA. The doublet is likely due to an ORF57 cleavage product [ 104 ].
    Figure Legend Snippet: Isolation of cross-linked ORF57-RNA complexes for HITS-CLIP analysis. Top Nitrocellulose membrane from an ORF57 immunoprecipitation performed under HITS-CLIP conditions. Cross-linked RNA fragments were end-labeled with 32 P to be visualized by Phosphoimager. Cells were induced to undergo lytic reactivation, exposed to UV and/or treated with high or low concentrations of MNase as indicated. The samples lacking an ORF57 antibody (lane 1) were precipitated with pre-bleed antibodies from the same rabbit. Lanes 6 and 7 are a dark exposure of lanes 4 and 5. The dashed white box indicates the position of the ORF57 complex cut from the membrane for library preparation. Bottom Western blot of the same HITS-CLIP samples shown in the top panel. Affinity purified rabbit anti-ORF57 was used to detect ORF57. Positions of molecular weight markers are shown on the left. On the right, a single asterisk marks the position of a contaminating ~37 kDa protein; double and triple asterisks mark positions of putative ORF57 homodimers and homotrimers bound to the same RNA. The doublet is likely due to an ORF57 cleavage product [ 104 ].

    Techniques Used: Isolation, Cross-linking Immunoprecipitation, Immunoprecipitation, Labeling, Western Blot, Affinity Purification, Molecular Weight

    14) Product Images from "Characterizing the nuclease accessibility of DNA in human cells to map higher order structures of chromatin"

    Article Title: Characterizing the nuclease accessibility of DNA in human cells to map higher order structures of chromatin

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1203

    MNase sequence preferences dominate differential chromatin fragmentation. ( A ) Genome browser plot showing the nucleosome occupancy distribution along chromosome 6. Differences of high- and low-MNase digestions were calculated as log 2 fold changes (log FC) of the average profile intensity in 1 Mb non-overlapping windows. GC content variation is displayed as the deviation from the genome wide average (40.5%). R is the Pearson correlation coefficient determined for the log FC (high/low) and the GC content deviation. The mappability track at the bottom illustrates how uniquely 100mer sequences align to a region of the genome ( 41 ). ( B ) Average GC content of isolated mono- and di-nucleosomal fragments. The genome wide GC average is indicated by the dashed line. (C–E) Genome wide correlation of GC content und nucleosome occupancy profiles. Dots represent 1 Mb non-overlapping windows; the coefficient of determination (R 2 ) refers to the result of a simple linear regression between GC content and the average signal of ( C ) log FC of high-MNase versus low-MNase, ( D ) high-MNase, ( E ) low-MNase.
    Figure Legend Snippet: MNase sequence preferences dominate differential chromatin fragmentation. ( A ) Genome browser plot showing the nucleosome occupancy distribution along chromosome 6. Differences of high- and low-MNase digestions were calculated as log 2 fold changes (log FC) of the average profile intensity in 1 Mb non-overlapping windows. GC content variation is displayed as the deviation from the genome wide average (40.5%). R is the Pearson correlation coefficient determined for the log FC (high/low) and the GC content deviation. The mappability track at the bottom illustrates how uniquely 100mer sequences align to a region of the genome ( 41 ). ( B ) Average GC content of isolated mono- and di-nucleosomal fragments. The genome wide GC average is indicated by the dashed line. (C–E) Genome wide correlation of GC content und nucleosome occupancy profiles. Dots represent 1 Mb non-overlapping windows; the coefficient of determination (R 2 ) refers to the result of a simple linear regression between GC content and the average signal of ( C ) log FC of high-MNase versus low-MNase, ( D ) high-MNase, ( E ) low-MNase.

    Techniques Used: Sequencing, Genome Wide, Isolation

    Differences of low- and high-MNase analysis at local sites. ( A ) Genome browser snapshot around an accessible enhancer marked by DNase I HS peak (ENCODE, ENCFF567PRQ). (B–E) Averaged MNase-seq profiles around: ( B ) DNase I HS sites (ENCODE, ENCFF692NCU); average G/C—di-nucleotide content is indicated by the grey line. ( C ) TSS of highly expressed genes (left panel) and not expressed genes (right panel). FPKM values determined by RNA-Seq analysis (ENCODE, ENCFF000DNW) were used to estimate the level of gene expression. Genes not detected in RNA-Seq analysis (FPKM = 0) were considered as not expressed and those genes showing the 25% highest FPKM values were considered as highly expressed. ( D ) Active enhancer sites in HeLa cells as defined in ( 49 ). ( E ) CTCF sites (ENCODE, ENCFF002DCW). ( F ) Hea tmaps of high-MNase (left panel) and low-MNase (right panel) are centred on CTCF binding sites and sorted in descending order of ChIP-Seq density at detected CTCF sites. ( G ) Simulation of low- and high-MNase digestions of a nucleosome array exhibiting nucleosomes with variable MNase sensitivity and hyper-accessible DNA sites. A higher intra-nucleosome cleavage probability p cut ( nuc, AT+ ) was assigned to a domain, simulating an AT-rich region in the middle of the chromatin strand. In the centre of the nucleosomal array an hyper-accessible site was introduced, representing a fragile nucleosome with markedly increased intra-nucleosome cleavage probability p cut ( nuc, fragile ), and the two adjacent DNA linker sites were assigned with enhanced inter-nucleosomal cleavage probability p cut ( linker, hyperaccessible ). Chosen cleavage probabilities: p cut ( nuc, AT+ ) = 2· p cut ( nuc ), p cut ( nuc, fragile ) = 4· p cut ( nuc ), p cut (linker) = 10· p cut ( nuc ), p cut (linker, hyper-accessible) = 12· p cut ( nuc ). ( H ) Schematic model summarising the results of this study.
    Figure Legend Snippet: Differences of low- and high-MNase analysis at local sites. ( A ) Genome browser snapshot around an accessible enhancer marked by DNase I HS peak (ENCODE, ENCFF567PRQ). (B–E) Averaged MNase-seq profiles around: ( B ) DNase I HS sites (ENCODE, ENCFF692NCU); average G/C—di-nucleotide content is indicated by the grey line. ( C ) TSS of highly expressed genes (left panel) and not expressed genes (right panel). FPKM values determined by RNA-Seq analysis (ENCODE, ENCFF000DNW) were used to estimate the level of gene expression. Genes not detected in RNA-Seq analysis (FPKM = 0) were considered as not expressed and those genes showing the 25% highest FPKM values were considered as highly expressed. ( D ) Active enhancer sites in HeLa cells as defined in ( 49 ). ( E ) CTCF sites (ENCODE, ENCFF002DCW). ( F ) Hea tmaps of high-MNase (left panel) and low-MNase (right panel) are centred on CTCF binding sites and sorted in descending order of ChIP-Seq density at detected CTCF sites. ( G ) Simulation of low- and high-MNase digestions of a nucleosome array exhibiting nucleosomes with variable MNase sensitivity and hyper-accessible DNA sites. A higher intra-nucleosome cleavage probability p cut ( nuc, AT+ ) was assigned to a domain, simulating an AT-rich region in the middle of the chromatin strand. In the centre of the nucleosomal array an hyper-accessible site was introduced, representing a fragile nucleosome with markedly increased intra-nucleosome cleavage probability p cut ( nuc, fragile ), and the two adjacent DNA linker sites were assigned with enhanced inter-nucleosomal cleavage probability p cut ( linker, hyperaccessible ). Chosen cleavage probabilities: p cut ( nuc, AT+ ) = 2· p cut ( nuc ), p cut ( nuc, fragile ) = 4· p cut ( nuc ), p cut (linker) = 10· p cut ( nuc ), p cut (linker, hyper-accessible) = 12· p cut ( nuc ). ( H ) Schematic model summarising the results of this study.

    Techniques Used: RNA Sequencing Assay, Expressing, Binding Assay, Chromatin Immunoprecipitation

    Isolation of nucleosomal DNA after differential MNase hydrolysis. ( A ) Nucleosomal DNA ladder of HeLa cells incubated with increasing concentrations of MNase. The agarose gel is showing the analysis of the purified DNA after MNase hydrolysis. The molecular weight marker is shown in lane 9 and the positions of subnucleosomal, mono-, di- and tri-nucleosomal DNA are indicated. The scheme below illustrates the hypothetical results of a partial and full hydrolysis of differentially accessible chromatin domains. ( B ) Simulation of low- and high-MNase digestions (see Materials and Methods for details) of an equally accessible linker DNA with p cut ( linker ) = constant (left panel) and of a nucleosomal array containing a central compacted chromatin domain with reduced linker accessibility p cut ( linker, compact ) = 0.8· p cut ( linker ), (right panel). The relative sensitivity of intra-nucleosomal (intra) and inter-nucleosomal (inter) cleavage is indicated. ( C ) Large scale low- and high-MNase hydrolysis of chromatin (lanes 2 and 3) and subsequent isolation of nucleosomal DNA corresponding to the mono-, di-, and tri-nucleosomes (lanes 5 to 10). (D, E) 2D and 3D FISH experiments with isolated tri-nucleosomal DNA. Metaphase FISH ( D ) and 3D FISH ( E ) were performed with Cy3/FITC labelled tri-nucleosomal DNA derived from high-/low-MNase digestions, as indicated. ( F ) Low- ad high-digested mono- and di-nucleosomal DNA were subjected to library preparation and high throughput sequencing. The fragment size distributions of the mapped paired-end reads for mono- (left panel) and di-nucleosomal DNA (right panel) are shown. The shaded boxes highlight the fraction of isolated mono-nucleosomal (140–200 bp) and di-nucleosomal (250–500 bp) fragments.
    Figure Legend Snippet: Isolation of nucleosomal DNA after differential MNase hydrolysis. ( A ) Nucleosomal DNA ladder of HeLa cells incubated with increasing concentrations of MNase. The agarose gel is showing the analysis of the purified DNA after MNase hydrolysis. The molecular weight marker is shown in lane 9 and the positions of subnucleosomal, mono-, di- and tri-nucleosomal DNA are indicated. The scheme below illustrates the hypothetical results of a partial and full hydrolysis of differentially accessible chromatin domains. ( B ) Simulation of low- and high-MNase digestions (see Materials and Methods for details) of an equally accessible linker DNA with p cut ( linker ) = constant (left panel) and of a nucleosomal array containing a central compacted chromatin domain with reduced linker accessibility p cut ( linker, compact ) = 0.8· p cut ( linker ), (right panel). The relative sensitivity of intra-nucleosomal (intra) and inter-nucleosomal (inter) cleavage is indicated. ( C ) Large scale low- and high-MNase hydrolysis of chromatin (lanes 2 and 3) and subsequent isolation of nucleosomal DNA corresponding to the mono-, di-, and tri-nucleosomes (lanes 5 to 10). (D, E) 2D and 3D FISH experiments with isolated tri-nucleosomal DNA. Metaphase FISH ( D ) and 3D FISH ( E ) were performed with Cy3/FITC labelled tri-nucleosomal DNA derived from high-/low-MNase digestions, as indicated. ( F ) Low- ad high-digested mono- and di-nucleosomal DNA were subjected to library preparation and high throughput sequencing. The fragment size distributions of the mapped paired-end reads for mono- (left panel) and di-nucleosomal DNA (right panel) are shown. The shaded boxes highlight the fraction of isolated mono-nucleosomal (140–200 bp) and di-nucleosomal (250–500 bp) fragments.

    Techniques Used: Isolation, Incubation, Agarose Gel Electrophoresis, Purification, Molecular Weight, Marker, Fluorescence In Situ Hybridization, Derivative Assay, Next-Generation Sequencing

    Nucleosomes in AT-rich regions are partially depleted in high-, but not in low-MNase conditions. ( A ) Average G/C-di-nucleotide frequency, deduced from GG, GC, CG, CC occurrences, centred on mapped mono-nucleosomal fragment midpoints. A random distribution (grey) was simulated on 1 million randomly generated fragments of 147 bp lengths. Vertical lines indicate nucleosome boundaries (±73 bp). ( B ) Genome browser snapshot showing the GC bias of nucleosome annotation after high MNase digestions. Nucleosome positions, which have been called with the DANPOS2 toolkit ( 80 ), are represented by blue boxes. Arrows indicate over-digested nucleosomes in high MNase condition. GC content was calculated in 50 bp windows with 10 bp sliding steps. The genome wide GC average is given by the dashed line. ( C ) Genome wide correlation of GC content and quantity of nucleosome prediction. Dots represent 1 Mb non-overlapping windows; ( D ) Kernel density plot of the nucleosome count in 1 Mb sized windows. ( E ) Simulation of low- and high-MNase digestions of a nucleosome strand exhibiting nucleosomes with variable MNase sensitivity. A higher intra-nucleosome cleavage probability p cut ( nuc, AT+ ) was assigned to a stretch simulating an AT-rich region in the middle of the chromatin strand. Used cleavage probabilities: p cut ( nuc, AT+ ) = 2· p cut ( nuc ) and p cut (linker) = 10· p cut ( nuc ).
    Figure Legend Snippet: Nucleosomes in AT-rich regions are partially depleted in high-, but not in low-MNase conditions. ( A ) Average G/C-di-nucleotide frequency, deduced from GG, GC, CG, CC occurrences, centred on mapped mono-nucleosomal fragment midpoints. A random distribution (grey) was simulated on 1 million randomly generated fragments of 147 bp lengths. Vertical lines indicate nucleosome boundaries (±73 bp). ( B ) Genome browser snapshot showing the GC bias of nucleosome annotation after high MNase digestions. Nucleosome positions, which have been called with the DANPOS2 toolkit ( 80 ), are represented by blue boxes. Arrows indicate over-digested nucleosomes in high MNase condition. GC content was calculated in 50 bp windows with 10 bp sliding steps. The genome wide GC average is given by the dashed line. ( C ) Genome wide correlation of GC content and quantity of nucleosome prediction. Dots represent 1 Mb non-overlapping windows; ( D ) Kernel density plot of the nucleosome count in 1 Mb sized windows. ( E ) Simulation of low- and high-MNase digestions of a nucleosome strand exhibiting nucleosomes with variable MNase sensitivity. A higher intra-nucleosome cleavage probability p cut ( nuc, AT+ ) was assigned to a stretch simulating an AT-rich region in the middle of the chromatin strand. Used cleavage probabilities: p cut ( nuc, AT+ ) = 2· p cut ( nuc ) and p cut (linker) = 10· p cut ( nuc ).

    Techniques Used: Generated, Genome Wide

    15) Product Images from "Mechanistic insights on the mode of action of an antiproliferative thiosemicarbazone-nickel complex revealed by an integrated chemogenomic profiling study"

    Article Title: Mechanistic insights on the mode of action of an antiproliferative thiosemicarbazone-nickel complex revealed by an integrated chemogenomic profiling study

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-67439-y

    Ni(S-tcitr) 2 affects chromatin remodeling and microtubule cytoskeleton functionality. ( a ) Effect of Ni(S-tcitr) 2 treatment on in vivo chromatin remodeling. Equal amounts of spheroplasts from yeast cells treated with Ni(S-tcitr) 2 or DMSO (vehicle) were subjected to chromatin digestion with micrococcal nuclease (MNase); purified DNA was then fractionated by agarose gel electrophoresis and visualized by ethidium bromide staining (see ‘ Methods ’ for details). A representative gel picture shows different patterns of MNase digestion products. ( b,c ) Relative quantification of nucleosome-size fragments obtained from digestion of Ni(S-tcitr) 2 -treated and control (DMSO) chromatin samples with high (10 U; b ) or low (2.5 U; c ) amounts of MNase. ( d ) Synergistic toxicity of Ni(S-tcitr) 2 and the antimicrotubule drug benomyl. Ten-fold serial dilutions of wild-type (WT) cells and yeast mutant strains deleted in genes coding for chromatin remodeling ( arp6Δ and arp8Δ ), microtubule ( alf1Δ ) or cell cycle checkpoint components ( bub1Δ ) were plated onto YPD agar plates containing sublethal concentrations of benomyl (17 µM) and /or Ni(S-tcitr) 2 (10 µM) as indicated, and incubated for 2 days at 28 °C.
    Figure Legend Snippet: Ni(S-tcitr) 2 affects chromatin remodeling and microtubule cytoskeleton functionality. ( a ) Effect of Ni(S-tcitr) 2 treatment on in vivo chromatin remodeling. Equal amounts of spheroplasts from yeast cells treated with Ni(S-tcitr) 2 or DMSO (vehicle) were subjected to chromatin digestion with micrococcal nuclease (MNase); purified DNA was then fractionated by agarose gel electrophoresis and visualized by ethidium bromide staining (see ‘ Methods ’ for details). A representative gel picture shows different patterns of MNase digestion products. ( b,c ) Relative quantification of nucleosome-size fragments obtained from digestion of Ni(S-tcitr) 2 -treated and control (DMSO) chromatin samples with high (10 U; b ) or low (2.5 U; c ) amounts of MNase. ( d ) Synergistic toxicity of Ni(S-tcitr) 2 and the antimicrotubule drug benomyl. Ten-fold serial dilutions of wild-type (WT) cells and yeast mutant strains deleted in genes coding for chromatin remodeling ( arp6Δ and arp8Δ ), microtubule ( alf1Δ ) or cell cycle checkpoint components ( bub1Δ ) were plated onto YPD agar plates containing sublethal concentrations of benomyl (17 µM) and /or Ni(S-tcitr) 2 (10 µM) as indicated, and incubated for 2 days at 28 °C.

    Techniques Used: In Vivo, Purification, Agarose Gel Electrophoresis, Staining, Mutagenesis, Incubation

    16) Product Images from "Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans"

    Article Title: Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-465

    DAM accessibility correlates with nucleosome positions . This figure shows the superimposition of average DAM accessibility versus average MNase accessibility around the transcription start site (TSS) of 3,904 strongly expressed C. elegans genes. The dotted red curve represents a moving average of the muscle profile with a sliding window of 400 nucleotides. Positioned H3K4me2/3 nucleosomes are represented by ovals above the picture of the generic gene. Numbers on the nucleosomes indicate their positions relative to the TSS. [NDR = nucleosome depleted region]
    Figure Legend Snippet: DAM accessibility correlates with nucleosome positions . This figure shows the superimposition of average DAM accessibility versus average MNase accessibility around the transcription start site (TSS) of 3,904 strongly expressed C. elegans genes. The dotted red curve represents a moving average of the muscle profile with a sliding window of 400 nucleotides. Positioned H3K4me2/3 nucleosomes are represented by ovals above the picture of the generic gene. Numbers on the nucleosomes indicate their positions relative to the TSS. [NDR = nucleosome depleted region]

    Techniques Used:

    17) Product Images from "No Overt Nucleosome Eviction at Deprotected Telomeres ▿No Overt Nucleosome Eviction at Deprotected Telomeres ▿ †"

    Article Title: No Overt Nucleosome Eviction at Deprotected Telomeres ▿No Overt Nucleosome Eviction at Deprotected Telomeres ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01764-07

    No disruption of telomeric chromatin after POT1 depletion. (A) Immunoblot confirming deletion of POT1a and POT1b after retroviral infection of POT1a STOP/FLOX POT1b STOP/FLOX cells with pWzl-Cre followed by 5 days of selection with hygromycin. γ-Tubulin is shown as a loading control. (B) Bulk nucleosomes in cells with and without POT1a and -b detected by ethidium bromide staining of DNA from nuclei digested with MNase and fractionated on 1% agarose gels. (C) Telomeric nucleosomes detected by Southern blot hybridization with a 32 P-(CCCTAA) 4 probe. Roman numerals represent oligonucleosomes formed by partial digestion. MNase concentrations in U/ml are shown. S/F, STOP/FLOX; Vec, vector; +, present; −, absent.
    Figure Legend Snippet: No disruption of telomeric chromatin after POT1 depletion. (A) Immunoblot confirming deletion of POT1a and POT1b after retroviral infection of POT1a STOP/FLOX POT1b STOP/FLOX cells with pWzl-Cre followed by 5 days of selection with hygromycin. γ-Tubulin is shown as a loading control. (B) Bulk nucleosomes in cells with and without POT1a and -b detected by ethidium bromide staining of DNA from nuclei digested with MNase and fractionated on 1% agarose gels. (C) Telomeric nucleosomes detected by Southern blot hybridization with a 32 P-(CCCTAA) 4 probe. Roman numerals represent oligonucleosomes formed by partial digestion. MNase concentrations in U/ml are shown. S/F, STOP/FLOX; Vec, vector; +, present; −, absent.

    Techniques Used: Infection, Selection, Staining, Southern Blot, Hybridization, Plasmid Preparation

    No disruption of telomeric chromatin after TRF2 depletion. (A) Immunoblot confirming loss of TRF2 and Rap1 and phosphorylation of Chk2 at 90 h after retrovirus-mediated Hit Run-Cre expression in TRF2 FLOX/− p53 −/− Lig4 −/− MEFs. The nonspecific band on the TRF2 blot served as a loading control. (B) Bulk nucleosomes in cells with and without TRF2 detected by ethidium bromide staining of DNA from nuclei digested with MNase and fractionated on a 1% agarose gel. (C) Telomeric nucleosomes detected by Southern blot hybridization with a 32 P-(CCCTAA) 4 probe. Roman numerals represent oligonucleosomes formed by partial digestion. MNase concentrations in U/ml are shown. F, FLOX.
    Figure Legend Snippet: No disruption of telomeric chromatin after TRF2 depletion. (A) Immunoblot confirming loss of TRF2 and Rap1 and phosphorylation of Chk2 at 90 h after retrovirus-mediated Hit Run-Cre expression in TRF2 FLOX/− p53 −/− Lig4 −/− MEFs. The nonspecific band on the TRF2 blot served as a loading control. (B) Bulk nucleosomes in cells with and without TRF2 detected by ethidium bromide staining of DNA from nuclei digested with MNase and fractionated on a 1% agarose gel. (C) Telomeric nucleosomes detected by Southern blot hybridization with a 32 P-(CCCTAA) 4 probe. Roman numerals represent oligonucleosomes formed by partial digestion. MNase concentrations in U/ml are shown. F, FLOX.

    Techniques Used: Expressing, Staining, Agarose Gel Electrophoresis, Southern Blot, Hybridization

    A novel assay for the nucleosomal organization near the telomere terminus. (A) Scheme of the last nucleosome assay. Nuclei are treated with MNase, and the isolated DNA fragments are incubated with a biotinylated oligonucleotide representing the C-rich telomeric DNA strand, biotin-(CCCTAA) 6 . Magnetic streptavidin beads are used to pull down DNA fragments containing a telomeric (TTAGGG) n 3′ overhang. The supernatant contains fragments of bulk nucleosomes, while the pulldown contains nucleosomal fragments ending at the telomere terminus. Fragments are fractionated on a 1% agarose gel and subjected to Southern blot hybridization with a 32 P-(CCCTAA) 4 probe. (B) Scheme for the construction of a model telomeric fragment to test the last nucleosome assay. A BglII/KpnI fragment excised from pSXneo.25(T 2 AG 3 ) (dsTel) was annealed and ligated to a single-stranded telomeric oligonucleotide (ssTel) containing the complementary BglII recognition sequence at its 5′ end. CIP, calf intestinal phosphatase. (C) Southern blot detection of the telomeric signal ligation products of dsTel and ssTel (lane 1), dsTel only (lane 2), or ssTel only (lane 3). (D) Southern blot detection of the telomeric signal after annealing the DNA products from lanes 1 and 2 shown in panel C with biotin-(CCCTAA) 6 and separating the supernatant (sup) and pulldown (ppt) following incubation with streptavidin beads. Two percent of the supernatant was loaded next to 50% of the total pulldown. Sizes in base pairs are marked to the left of panels C and D. (E) Detection of the telomeric signal associated with the last nucleosomes in wild-type MEFs. The last nucleosome assay was performed as described above, and the supernatant (2% of total fraction) containing the bulk nucleosomes was loaded in the first eight lanes, while nucleosomal fragments containing the telomeric overhang (50% of total pulldown) were loaded in the last eight lanes. Roman numerals represent oligonucleosomes formed by partial digestion. MNase concentrations in U/ml are shown. (F) Detection of the telomeric signal pulled down by the last nucleosome assay following ExoI treatment of DNA from MNase-digested nuclei. DNA fragments isolated after MNase digestion were mixed and treated with ExoI (300 U in 300 μl), removing the 3′ overhang. ExoI-treated and untreated samples were then subjected to the last nucleosome assay. Two percent of the supernatant (bulk nucleosomes) was loaded next to 50% of the total pulldown (last nucleosomes). Percentage of total TTAGGG signal pulled down was calculated with the formula (2 × pulldown signal)/[(2 × pulldown signal) + (50 × supernatant signal)]. +, present; −, absent.
    Figure Legend Snippet: A novel assay for the nucleosomal organization near the telomere terminus. (A) Scheme of the last nucleosome assay. Nuclei are treated with MNase, and the isolated DNA fragments are incubated with a biotinylated oligonucleotide representing the C-rich telomeric DNA strand, biotin-(CCCTAA) 6 . Magnetic streptavidin beads are used to pull down DNA fragments containing a telomeric (TTAGGG) n 3′ overhang. The supernatant contains fragments of bulk nucleosomes, while the pulldown contains nucleosomal fragments ending at the telomere terminus. Fragments are fractionated on a 1% agarose gel and subjected to Southern blot hybridization with a 32 P-(CCCTAA) 4 probe. (B) Scheme for the construction of a model telomeric fragment to test the last nucleosome assay. A BglII/KpnI fragment excised from pSXneo.25(T 2 AG 3 ) (dsTel) was annealed and ligated to a single-stranded telomeric oligonucleotide (ssTel) containing the complementary BglII recognition sequence at its 5′ end. CIP, calf intestinal phosphatase. (C) Southern blot detection of the telomeric signal ligation products of dsTel and ssTel (lane 1), dsTel only (lane 2), or ssTel only (lane 3). (D) Southern blot detection of the telomeric signal after annealing the DNA products from lanes 1 and 2 shown in panel C with biotin-(CCCTAA) 6 and separating the supernatant (sup) and pulldown (ppt) following incubation with streptavidin beads. Two percent of the supernatant was loaded next to 50% of the total pulldown. Sizes in base pairs are marked to the left of panels C and D. (E) Detection of the telomeric signal associated with the last nucleosomes in wild-type MEFs. The last nucleosome assay was performed as described above, and the supernatant (2% of total fraction) containing the bulk nucleosomes was loaded in the first eight lanes, while nucleosomal fragments containing the telomeric overhang (50% of total pulldown) were loaded in the last eight lanes. Roman numerals represent oligonucleosomes formed by partial digestion. MNase concentrations in U/ml are shown. (F) Detection of the telomeric signal pulled down by the last nucleosome assay following ExoI treatment of DNA from MNase-digested nuclei. DNA fragments isolated after MNase digestion were mixed and treated with ExoI (300 U in 300 μl), removing the 3′ overhang. ExoI-treated and untreated samples were then subjected to the last nucleosome assay. Two percent of the supernatant (bulk nucleosomes) was loaded next to 50% of the total pulldown (last nucleosomes). Percentage of total TTAGGG signal pulled down was calculated with the formula (2 × pulldown signal)/[(2 × pulldown signal) + (50 × supernatant signal)]. +, present; −, absent.

    Techniques Used: Isolation, Incubation, Agarose Gel Electrophoresis, Southern Blot, Hybridization, Sequencing, Ligation

    18) Product Images from "CAL1 is the Drosophila CENP-A assembly factor"

    Article Title: CAL1 is the Drosophila CENP-A assembly factor

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201305036

    CAL1 is a CENP-A–specific nucleosome assembly factor. (A) CENP-A 101–225 –H4 was assembled on circular relaxed plasmid (pCR2.1-4 [CEN3 + CEN6]) using increasing amounts of CAL1 1–96 , CAL1 1–132 , or CAL1 1–160 in the presence of topoisomerase I. The extracted DNA samples were analyzed on agarose gel and stained with SYBR gold. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lane 3: CENP-A 101–225 –H4, H2A–H2B but no CAL1; lanes 4–7: relaxed plasmid and CENP-A 101–225 –H4, H2A–H2B with CAL1 1–96 ; lanes 8–11: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–132 ; lanes 12–15: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–160 . (B) CENP-A–containing nucleosomes and histone H3 nucleosomes were assembled using histone proteins, CAL1 1–160 , or yeast Nap1 and pGEM3Z-601 plasmid. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lanes 3, 6, and 9: mock reaction without CAL1 1–160 or Nap1; lanes 4 and 5: H3–H4, H2A–H2B with CAL1 1–160 ; lanes 7 and 8: CENP-A 101–225 –H4, H2A-H2B with CAL1 1–160 ; lanes 10 and 11: H3–H4, H2A–H2B with Nap1; lanes 12 and 13: CENP-A 101–225 –H4, H2A–H2B with Nap1. (C) CAL1-assembled CENP-A nucleosomes are negatively supercoiled. CENP-A nucleosomes (lane CENP-A) were assembled by CAL1 1–160 in the presence of topoisomerase I and H2A–H2B dimers, whereas histone H3 nucleosomes (lane H3) were assembled by Nap1. Controls are: supercoiled plasmid (superc.), relaxed plasmid (relaxed), and relaxed plasmid without histones or Nap1, but treated and processed as in the H3 and CENP-A nucleosome assembly reactions (mock). The samples were analyzed on agarose gels with or without 1 µg/ml chloroquine. Boxes highlight the decrease in migration that occurs in the presence of chloroquine in both H3 and CENP-A nucleosomes. (A–C) S, supercoiled; R, relaxed plasmid. (D) Diagram depicting the expected migration patterns for negatively and positively supercoiled DNA separated by 2D gel electrophoresis ( Tachiwana et al., 2011 ). Left gel: H3 nucleosomes assembled with Nap1; right gel: CENP-A nucleosomes assembled with CAL1 1–160 . (E) Mononucleosomes were assembled on 147-bp Widom DNA with Nap1 or CAL1 1–160 and digested with MNase for 30 or 120 s. Native PAGE of samples shown. Lanes 1–3: DNA only; lanes 4 and 5: H3 nucleosomes assembled by Nap1; lanes 6 and 7: CENP-A nucleosomes assembled by Nap1; lanes 8 and 9: CENP-A nucleosomes assembled by CAL1 1–160 .
    Figure Legend Snippet: CAL1 is a CENP-A–specific nucleosome assembly factor. (A) CENP-A 101–225 –H4 was assembled on circular relaxed plasmid (pCR2.1-4 [CEN3 + CEN6]) using increasing amounts of CAL1 1–96 , CAL1 1–132 , or CAL1 1–160 in the presence of topoisomerase I. The extracted DNA samples were analyzed on agarose gel and stained with SYBR gold. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lane 3: CENP-A 101–225 –H4, H2A–H2B but no CAL1; lanes 4–7: relaxed plasmid and CENP-A 101–225 –H4, H2A–H2B with CAL1 1–96 ; lanes 8–11: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–132 ; lanes 12–15: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–160 . (B) CENP-A–containing nucleosomes and histone H3 nucleosomes were assembled using histone proteins, CAL1 1–160 , or yeast Nap1 and pGEM3Z-601 plasmid. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lanes 3, 6, and 9: mock reaction without CAL1 1–160 or Nap1; lanes 4 and 5: H3–H4, H2A–H2B with CAL1 1–160 ; lanes 7 and 8: CENP-A 101–225 –H4, H2A-H2B with CAL1 1–160 ; lanes 10 and 11: H3–H4, H2A–H2B with Nap1; lanes 12 and 13: CENP-A 101–225 –H4, H2A–H2B with Nap1. (C) CAL1-assembled CENP-A nucleosomes are negatively supercoiled. CENP-A nucleosomes (lane CENP-A) were assembled by CAL1 1–160 in the presence of topoisomerase I and H2A–H2B dimers, whereas histone H3 nucleosomes (lane H3) were assembled by Nap1. Controls are: supercoiled plasmid (superc.), relaxed plasmid (relaxed), and relaxed plasmid without histones or Nap1, but treated and processed as in the H3 and CENP-A nucleosome assembly reactions (mock). The samples were analyzed on agarose gels with or without 1 µg/ml chloroquine. Boxes highlight the decrease in migration that occurs in the presence of chloroquine in both H3 and CENP-A nucleosomes. (A–C) S, supercoiled; R, relaxed plasmid. (D) Diagram depicting the expected migration patterns for negatively and positively supercoiled DNA separated by 2D gel electrophoresis ( Tachiwana et al., 2011 ). Left gel: H3 nucleosomes assembled with Nap1; right gel: CENP-A nucleosomes assembled with CAL1 1–160 . (E) Mononucleosomes were assembled on 147-bp Widom DNA with Nap1 or CAL1 1–160 and digested with MNase for 30 or 120 s. Native PAGE of samples shown. Lanes 1–3: DNA only; lanes 4 and 5: H3 nucleosomes assembled by Nap1; lanes 6 and 7: CENP-A nucleosomes assembled by Nap1; lanes 8 and 9: CENP-A nucleosomes assembled by CAL1 1–160 .

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Staining, Migration, Two-Dimensional Gel Electrophoresis, Electrophoresis, Clear Native PAGE

    19) Product Images from "The RSC complex remodels nucleosomes in transcribed coding sequences and promotes transcription in Saccharomyces cerevisiae"

    Article Title: The RSC complex remodels nucleosomes in transcribed coding sequences and promotes transcription in Saccharomyces cerevisiae

    Journal: bioRxiv

    doi: 10.1101/2020.03.11.987974

    Nucleosomes in transcribed coding Sequences are more susceptible to MNase digestion. A) Fragment length distribution of MNase-digested DNA sequences from two biological replicates. B) Heatmaps depicting average nucleosome occupancies of the two replicates +/- 1000 bp relative to the transcription start sites (TSS). The genes were sorted on the basis of average Pol II (Rpb3) ChIP-seq occupancies in their CDSs. C) Heatmap depicting Pol II occupancies, which were determined by ChIP-seq for Rpb3 using sonicated chromatin (n=5746). D) Schematics showing potential generation of MNase protected DNA fragments (MPDFs) from nucleosomes. MNase digestion of a wrapped nucleosome would likely generate MPDFs ∼130-160 bp, whereas a remodeled nucleosomes might be more accessible, leading to shorter MPDFs (
    Figure Legend Snippet: Nucleosomes in transcribed coding Sequences are more susceptible to MNase digestion. A) Fragment length distribution of MNase-digested DNA sequences from two biological replicates. B) Heatmaps depicting average nucleosome occupancies of the two replicates +/- 1000 bp relative to the transcription start sites (TSS). The genes were sorted on the basis of average Pol II (Rpb3) ChIP-seq occupancies in their CDSs. C) Heatmap depicting Pol II occupancies, which were determined by ChIP-seq for Rpb3 using sonicated chromatin (n=5746). D) Schematics showing potential generation of MNase protected DNA fragments (MPDFs) from nucleosomes. MNase digestion of a wrapped nucleosome would likely generate MPDFs ∼130-160 bp, whereas a remodeled nucleosomes might be more accessible, leading to shorter MPDFs (

    Techniques Used: Chromatin Immunoprecipitation, Sonication

    20) Product Images from "The regulatory landscape of early maize inflorescence development"

    Article Title: The regulatory landscape of early maize inflorescence development

    Journal: bioRxiv

    doi: 10.1101/870378

    MNase HS as a proxy for TF-DNA binding. (a) Overlap of high confidence peaks from FEA4 and KN1 ChIP-seq experiments with MNase HS regions based on LCS data. Less than two percent of co-bound sites and six percent of all sites do not overlap MNase HS. (b) Read density (reads per million) of LCS are plotted around a consensus TSS of genes bound by KN1 and/or FEA4. Genes cobound by both TFs in the proximal promoter show a wider range of accessibility. Data are shown for tassel; similar profiles are shown for ear presented in Figure S9. (c) Examples of intergenic MNase HS sites in tassel, ear, and/or shoot that are co-bound by KN 1 and FEA4: a known enhancer element 65 kb upstream of tb1, and a site 6 kb upstream of the gnarley 1 (gn1) locus. (d) Fragment densities from the light MNase digest plotted around high-confidence FEA4 ChIP-seq peaks show strong enrichment at the consensus FEA4 binding motif, (e) Density of ABI3-like motifs were plotted in relation to experimentally validated FEA4 binding sites. The ABI3-like motif was discovered by mining HS regions called by iSeg that overlapped consensus FEA4 binding sites. ABI3-like motifs are most densely positioned within 50 bp of FEA4 motifs. (f) GO terms overrepresented among genes within 1 kb of FEA4-ABI3-like motif modules (ABI3-like motifs within 50 bp of FEA binding sites) and compared with genes within 1 kb of random FEA4 motifs of equal number.
    Figure Legend Snippet: MNase HS as a proxy for TF-DNA binding. (a) Overlap of high confidence peaks from FEA4 and KN1 ChIP-seq experiments with MNase HS regions based on LCS data. Less than two percent of co-bound sites and six percent of all sites do not overlap MNase HS. (b) Read density (reads per million) of LCS are plotted around a consensus TSS of genes bound by KN1 and/or FEA4. Genes cobound by both TFs in the proximal promoter show a wider range of accessibility. Data are shown for tassel; similar profiles are shown for ear presented in Figure S9. (c) Examples of intergenic MNase HS sites in tassel, ear, and/or shoot that are co-bound by KN 1 and FEA4: a known enhancer element 65 kb upstream of tb1, and a site 6 kb upstream of the gnarley 1 (gn1) locus. (d) Fragment densities from the light MNase digest plotted around high-confidence FEA4 ChIP-seq peaks show strong enrichment at the consensus FEA4 binding motif, (e) Density of ABI3-like motifs were plotted in relation to experimentally validated FEA4 binding sites. The ABI3-like motif was discovered by mining HS regions called by iSeg that overlapped consensus FEA4 binding sites. ABI3-like motifs are most densely positioned within 50 bp of FEA4 motifs. (f) GO terms overrepresented among genes within 1 kb of FEA4-ABI3-like motif modules (ABI3-like motifs within 50 bp of FEA binding sites) and compared with genes within 1 kb of random FEA4 motifs of equal number.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation

    21) Product Images from "Organization of DNA in a bacterial nucleoid"

    Article Title: Organization of DNA in a bacterial nucleoid

    Journal: BMC Microbiology

    doi: 10.1186/s12866-016-0637-3

    A possible model of low-level nucleoid organization. a A fragment of EM image of E. coli nucleoid (adapted from [ 21 ]). b Genomic DNA (gray) forms loops with A-tract clusters (cyan) located at apexes and MNase resistant fragments (black) occupying loop ‘stems’. c A blow-up of a DNA loop, containing several A-tracts (cyan) and two MNase resistant fragments (black)
    Figure Legend Snippet: A possible model of low-level nucleoid organization. a A fragment of EM image of E. coli nucleoid (adapted from [ 21 ]). b Genomic DNA (gray) forms loops with A-tract clusters (cyan) located at apexes and MNase resistant fragments (black) occupying loop ‘stems’. c A blow-up of a DNA loop, containing several A-tracts (cyan) and two MNase resistant fragments (black)

    Techniques Used:

    Analysis of the DNA fragments from in vivo MNase digestion of nucleoid in wild type E. coli ( a ) and in vitro MNase digestion of purified genomic DNA ( b ) in agarose gel. a Wild type cells with the empty vector (lanes 2, 3) and MNase-expressing vector (lanes 4-8) were supplemented with arabinose (lane 6), CaCl 2 (lane 5) or both (lanes 3, 7, 8). Digestion reactions were stopped 1 minute (lane 7) or 5 minutes (lanes 3, 5, 8) after CaCl 2 was added. Lanes 1 and 9 show DNA molecular weight marker. b Lane 1, purified wild type genomic DNA; lanes 2-6, a time course of in vitro MNase digestion of the wild type genomic DNA; lane 7, DNA molecular marker
    Figure Legend Snippet: Analysis of the DNA fragments from in vivo MNase digestion of nucleoid in wild type E. coli ( a ) and in vitro MNase digestion of purified genomic DNA ( b ) in agarose gel. a Wild type cells with the empty vector (lanes 2, 3) and MNase-expressing vector (lanes 4-8) were supplemented with arabinose (lane 6), CaCl 2 (lane 5) or both (lanes 3, 7, 8). Digestion reactions were stopped 1 minute (lane 7) or 5 minutes (lanes 3, 5, 8) after CaCl 2 was added. Lanes 1 and 9 show DNA molecular weight marker. b Lane 1, purified wild type genomic DNA; lanes 2-6, a time course of in vitro MNase digestion of the wild type genomic DNA; lane 7, DNA molecular marker

    Techniques Used: In Vivo, In Vitro, Purification, Agarose Gel Electrophoresis, Plasmid Preparation, Expressing, Molecular Weight, Marker

    Genome-wide distribution of sequenced tags. a Tag frequencies for the entire E. coli genome. Frequencies of tags mapped on the positive and negative strands are shown with red and blue bars respectively. a , c Schematic illustrations of tag cross-correlation ( b ) and auto-correlation analyses ( c ). MNase resistant fragments are shown with grey rectangles. Vertical red and blue arrows represent 5’-ends of the digestion fragments mapping to the DNA positive and negative strands respectively
    Figure Legend Snippet: Genome-wide distribution of sequenced tags. a Tag frequencies for the entire E. coli genome. Frequencies of tags mapped on the positive and negative strands are shown with red and blue bars respectively. a , c Schematic illustrations of tag cross-correlation ( b ) and auto-correlation analyses ( c ). MNase resistant fragments are shown with grey rectangles. Vertical red and blue arrows represent 5’-ends of the digestion fragments mapping to the DNA positive and negative strands respectively

    Techniques Used: Genome Wide

    22) Product Images from "The RSC complex remodels nucleosomes in transcribed coding sequences and promotes transcription in Saccharomyces cerevisiae"

    Article Title: The RSC complex remodels nucleosomes in transcribed coding sequences and promotes transcription in Saccharomyces cerevisiae

    Journal: bioRxiv

    doi: 10.1101/2020.03.11.987974

    Nucleosomes in transcribed coding Sequences are more susceptible to MNase digestion. A) Fragment length distribution of MNase-digested DNA sequences from two biological replicates. B) Heatmaps depicting average nucleosome occupancies of the two replicates +/- 1000 bp relative to the transcription start sites (TSS). The genes were sorted on the basis of average Pol II (Rpb3) ChIP-seq occupancies in their CDSs. C) Heatmap depicting Pol II occupancies, which were determined by ChIP-seq for Rpb3 using sonicated chromatin (n=5746). D) Schematics showing potential generation of MNase protected DNA fragments (MPDFs) from nucleosomes. MNase digestion of a wrapped nucleosome would likely generate MPDFs ∼130-160 bp, whereas a remodeled nucleosomes might be more accessible, leading to shorter MPDFs (
    Figure Legend Snippet: Nucleosomes in transcribed coding Sequences are more susceptible to MNase digestion. A) Fragment length distribution of MNase-digested DNA sequences from two biological replicates. B) Heatmaps depicting average nucleosome occupancies of the two replicates +/- 1000 bp relative to the transcription start sites (TSS). The genes were sorted on the basis of average Pol II (Rpb3) ChIP-seq occupancies in their CDSs. C) Heatmap depicting Pol II occupancies, which were determined by ChIP-seq for Rpb3 using sonicated chromatin (n=5746). D) Schematics showing potential generation of MNase protected DNA fragments (MPDFs) from nucleosomes. MNase digestion of a wrapped nucleosome would likely generate MPDFs ∼130-160 bp, whereas a remodeled nucleosomes might be more accessible, leading to shorter MPDFs (

    Techniques Used: Chromatin Immunoprecipitation, Sonication

    23) Product Images from "Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication"

    Article Title: Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication

    Journal: bioRxiv

    doi: 10.1101/789578

    Fluorescent nucleosomes on λ DNA are discretely distributed in a ‘beads-on-a-string’ manner. (A and B) Native EMSA (top) and MNase assay (bottom) for nucleosomes labelled at H2A-K119C with Cy5 (A) and H4-E63C with AlexaFluor647 (B) reconstituted on λ DNA at increasing octamer:DNA ratios. Left panels show SYBR Gold staining of the DNA (magenta), central panels show Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panels are the composites of both detection modes. Deposition of increasing amounts of histone octamer on λ DNA leads to gradual increase in the template size and slower migration through 0.5 % agarose in EMSA. The larger the template size the slower the migration, as manifested by the more prominent shift of the DNA band. The observed template size increase results from higher density of correctly folded nucleosomes as indicated by the presence of mono-, di- and tri-nucleosomes in the corresponding native MNase protection assays. The apparent loss of H4-E63C A647 signal in EMSA is most likely due to self-quenching of histone fluorescence, caused by structural arrangement of high-density nucleosomes. (C and D) Single-molecule imaging of nucleosomes labelled at H2A-K119C with Cy5 (C) and H4-E63C with AlexaFluor647 (D) reconstituted on λ DNA at increasing nucleosome density. Left panels show SYTOX Orange staining of the DNA (magenta), central panels show Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panels are the composites of both detection modes. For details of experimental set up see panel E. Fluorescent nucleosomes reconstituted on λ DNA by salt dialysis show the characteristic ‘bead-on-a-string’ appearance. Nucleosome formation on λ DNA leads to apparent shortening of the DNA template, consistent with its wrapping around the octameric histone core. (E) Schematic of the DNA immobilized in the microfluidic device for single-molecule imaging. Fluorescent nucleosomes are pre-assembled on λ DNA by salt dialysis. The nucleosomal DNA template is stretched under flow and doubly tethered to the PEGylated glass surface of the microfluidic device via biotin-streptavidin interactions. The imaging is carried out in TIRF mode using 561- and 640-nm lasers to visualize SYTOX Orange-stained DNA (magenta) and Cy5/AlexaFluor647-labelled histones (yellow), respectively. (F and G) Single-molecule quantification of the DNA contour length for nucleosomes labelled at H2A-K119C with Cy5 (F) and H4-E63C with AlexaFluor647 (G) reconstituted on λ DNA at increasing octamer:DNA ratios. The four species presented on each graph correspond to the four samples shown in panels A and B. The DNA length of individual molecules was measured based on SYTOX Orange staining of the DNA (approximately 400 molecules at each histone octamer concentration). As illustrated in panels C and D, deposition of nucleosomes on λ DNA results in apparent shortening of the DNA template. The higher the octamer content in the reconstitution reaction, the shorter the mean DNA contour lengths and the broader the DNA length distributions were observed.
    Figure Legend Snippet: Fluorescent nucleosomes on λ DNA are discretely distributed in a ‘beads-on-a-string’ manner. (A and B) Native EMSA (top) and MNase assay (bottom) for nucleosomes labelled at H2A-K119C with Cy5 (A) and H4-E63C with AlexaFluor647 (B) reconstituted on λ DNA at increasing octamer:DNA ratios. Left panels show SYBR Gold staining of the DNA (magenta), central panels show Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panels are the composites of both detection modes. Deposition of increasing amounts of histone octamer on λ DNA leads to gradual increase in the template size and slower migration through 0.5 % agarose in EMSA. The larger the template size the slower the migration, as manifested by the more prominent shift of the DNA band. The observed template size increase results from higher density of correctly folded nucleosomes as indicated by the presence of mono-, di- and tri-nucleosomes in the corresponding native MNase protection assays. The apparent loss of H4-E63C A647 signal in EMSA is most likely due to self-quenching of histone fluorescence, caused by structural arrangement of high-density nucleosomes. (C and D) Single-molecule imaging of nucleosomes labelled at H2A-K119C with Cy5 (C) and H4-E63C with AlexaFluor647 (D) reconstituted on λ DNA at increasing nucleosome density. Left panels show SYTOX Orange staining of the DNA (magenta), central panels show Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panels are the composites of both detection modes. For details of experimental set up see panel E. Fluorescent nucleosomes reconstituted on λ DNA by salt dialysis show the characteristic ‘bead-on-a-string’ appearance. Nucleosome formation on λ DNA leads to apparent shortening of the DNA template, consistent with its wrapping around the octameric histone core. (E) Schematic of the DNA immobilized in the microfluidic device for single-molecule imaging. Fluorescent nucleosomes are pre-assembled on λ DNA by salt dialysis. The nucleosomal DNA template is stretched under flow and doubly tethered to the PEGylated glass surface of the microfluidic device via biotin-streptavidin interactions. The imaging is carried out in TIRF mode using 561- and 640-nm lasers to visualize SYTOX Orange-stained DNA (magenta) and Cy5/AlexaFluor647-labelled histones (yellow), respectively. (F and G) Single-molecule quantification of the DNA contour length for nucleosomes labelled at H2A-K119C with Cy5 (F) and H4-E63C with AlexaFluor647 (G) reconstituted on λ DNA at increasing octamer:DNA ratios. The four species presented on each graph correspond to the four samples shown in panels A and B. The DNA length of individual molecules was measured based on SYTOX Orange staining of the DNA (approximately 400 molecules at each histone octamer concentration). As illustrated in panels C and D, deposition of nucleosomes on λ DNA results in apparent shortening of the DNA template. The higher the octamer content in the reconstitution reaction, the shorter the mean DNA contour lengths and the broader the DNA length distributions were observed.

    Techniques Used: Staining, Fluorescence, Migration, Imaging, Concentration Assay

    Assembly of fluorescent nucleosomes on λ DNA. (A) Crystal structure of the Xenopus nucleosome (PDB 1AOI) illustrating the location and type of fluorescent dye (Cy5 or AlexaFluor647 – abbreviated as A647) used to label histones. Histones are color-coded (H2A – green, H2B – grey, H3 – blue and H4 – magenta) and the two chains of the same histone type can be distinguished by different color shading. For clarity, only one of the two histones of the same type is marked and labelled. (B) SDS-PAGE analysis of wild-type (WT) and fluorescently-labelled histones and histone octamers. Top panel shows Coomassie Brilliant Blue (CBB) staining whereas bottom panel illustrates fluorescence signal of histones labelled with Cy5 or AlexaFluor647. (C) Electrophoretic mobility shift assay (EMSA) for WT and fluorescently-labelled nucleosomes reconstituted on λ DNA. Left panel shows SYBR Gold staining of the DNA (magenta), central panel shows Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panel is the composite of both detection modes. Naked λ DNA (∼48.5 kbp, first lane) migrates through 0.5 % agarose faster than nucleosomal λ templates, containing either WT or fluorescently-labelled histones. (D) Native micrococcal nuclease (MNase) protection assay for WT and fluorescently-labelled nucleosomes reconstituted on λ DNA. MNase preferentially digests unprotected DNA in linker regions between nucleosomes (see also panel F). Products of MNase digest were resolved in 1.5 % agarose under native conditions revealing intact mono- and di-nucleosomes for nucleosomal templates and complete digest of naked λ DNA (first lane). Signal detection as in panel C. (E) Denaturing micrococcal nuclease (MNase) protection assay for WT and fluorescently-labelled nucleosomes reconstituted on λ DNA. Here, products of MNase digest were first deproteinated with proteinase K (see also panel F) in the presence of SDS and then resolved in 1.5 % agarose, yielding DNA fragments protected by mono-(∼150 bp band) and di-nucleosomes (∼300 bp band) for nucleosomal templates, and short (
    Figure Legend Snippet: Assembly of fluorescent nucleosomes on λ DNA. (A) Crystal structure of the Xenopus nucleosome (PDB 1AOI) illustrating the location and type of fluorescent dye (Cy5 or AlexaFluor647 – abbreviated as A647) used to label histones. Histones are color-coded (H2A – green, H2B – grey, H3 – blue and H4 – magenta) and the two chains of the same histone type can be distinguished by different color shading. For clarity, only one of the two histones of the same type is marked and labelled. (B) SDS-PAGE analysis of wild-type (WT) and fluorescently-labelled histones and histone octamers. Top panel shows Coomassie Brilliant Blue (CBB) staining whereas bottom panel illustrates fluorescence signal of histones labelled with Cy5 or AlexaFluor647. (C) Electrophoretic mobility shift assay (EMSA) for WT and fluorescently-labelled nucleosomes reconstituted on λ DNA. Left panel shows SYBR Gold staining of the DNA (magenta), central panel shows Cy5 and AlexaFluor647 fluorescence signal (yellow) of labelled histones and right panel is the composite of both detection modes. Naked λ DNA (∼48.5 kbp, first lane) migrates through 0.5 % agarose faster than nucleosomal λ templates, containing either WT or fluorescently-labelled histones. (D) Native micrococcal nuclease (MNase) protection assay for WT and fluorescently-labelled nucleosomes reconstituted on λ DNA. MNase preferentially digests unprotected DNA in linker regions between nucleosomes (see also panel F). Products of MNase digest were resolved in 1.5 % agarose under native conditions revealing intact mono- and di-nucleosomes for nucleosomal templates and complete digest of naked λ DNA (first lane). Signal detection as in panel C. (E) Denaturing micrococcal nuclease (MNase) protection assay for WT and fluorescently-labelled nucleosomes reconstituted on λ DNA. Here, products of MNase digest were first deproteinated with proteinase K (see also panel F) in the presence of SDS and then resolved in 1.5 % agarose, yielding DNA fragments protected by mono-(∼150 bp band) and di-nucleosomes (∼300 bp band) for nucleosomal templates, and short (

    Techniques Used: SDS Page, Staining, Fluorescence, Electrophoretic Mobility Shift Assay

    24) Product Images from "Micromanipulation of prophase I chromosomes from mouse spermatocytes reveals high stiffness and gel-like chromatin organization"

    Article Title: Micromanipulation of prophase I chromosomes from mouse spermatocytes reveals high stiffness and gel-like chromatin organization

    Journal: bioRxiv

    doi: 10.1101/2020.08.31.276402

    Mitotic and meiotic chromosomes have a contiguous DNA connection, which is dissolved by 4 bp restriction enzymes, but only weakened by 6 bp restriction enzymes. Image pairs show pipette positions untreated (native isolated) chromosomes when relaxed and stretched (a) , and chromosomes following enzyme treatments (b-d) . Vertical blue lines mark positions of force pipettes. Force pipette deflection by pulling (horizontal blue lines) indicates mechanical connection; no movement (no horizontal blue line) indicates no mechanical connection. Red notches mark positions of stiff pipettes. Bars are 5 μm. ( b ) Both mitotic and meiotic chromosomes were weakened, but not fully digested after treatment with PvuII (cut sequence CAG ˅ CTG). ( c ) Both mitotic and meiotic chromosomes lost connectivity after treatment with AluI (cut sequence AG ˅ CT; for 1 of 4 trials meiotic chromosomes were not fully digested by AluI). ( d ) Both mitotic and meiotic chromosomes lost connectivity when treated with MNase (cleaves all DNA sequences). ( e ) Quantification of chromosome stretching elasticity after no treatment or after being treated with PvuII, AluI, and MNase. No treatment caused a 13 ± 4% weakening of mitotic chromosomes (N=10) and a 1 ± 4% weakening of meiotic chromosomes (N=10). PvuII treatment caused a 70 ± 8% reduction in stiffness for MEF chromosomes (N=4) and 70 ± 9% reduction in stiffness for meiotic chromosomes (N=4). One of four AluI treatments of meiotic chromosomes caused a 90% reduction in stiffness (rather than fully digesting), while AluI treatment digested 4 of 4 mitotic chromosomes. All MNase treatments caused full digestion of mitotic and meiotic chromosomes (N=4 in both cases). All averages are reported as mean value ± SEM. Bars are 5 μm.
    Figure Legend Snippet: Mitotic and meiotic chromosomes have a contiguous DNA connection, which is dissolved by 4 bp restriction enzymes, but only weakened by 6 bp restriction enzymes. Image pairs show pipette positions untreated (native isolated) chromosomes when relaxed and stretched (a) , and chromosomes following enzyme treatments (b-d) . Vertical blue lines mark positions of force pipettes. Force pipette deflection by pulling (horizontal blue lines) indicates mechanical connection; no movement (no horizontal blue line) indicates no mechanical connection. Red notches mark positions of stiff pipettes. Bars are 5 μm. ( b ) Both mitotic and meiotic chromosomes were weakened, but not fully digested after treatment with PvuII (cut sequence CAG ˅ CTG). ( c ) Both mitotic and meiotic chromosomes lost connectivity after treatment with AluI (cut sequence AG ˅ CT; for 1 of 4 trials meiotic chromosomes were not fully digested by AluI). ( d ) Both mitotic and meiotic chromosomes lost connectivity when treated with MNase (cleaves all DNA sequences). ( e ) Quantification of chromosome stretching elasticity after no treatment or after being treated with PvuII, AluI, and MNase. No treatment caused a 13 ± 4% weakening of mitotic chromosomes (N=10) and a 1 ± 4% weakening of meiotic chromosomes (N=10). PvuII treatment caused a 70 ± 8% reduction in stiffness for MEF chromosomes (N=4) and 70 ± 9% reduction in stiffness for meiotic chromosomes (N=4). One of four AluI treatments of meiotic chromosomes caused a 90% reduction in stiffness (rather than fully digesting), while AluI treatment digested 4 of 4 mitotic chromosomes. All MNase treatments caused full digestion of mitotic and meiotic chromosomes (N=4 in both cases). All averages are reported as mean value ± SEM. Bars are 5 μm.

    Techniques Used: Transferring, Isolation, Sequencing

    25) Product Images from "CAL1 is the Drosophila CENP-A assembly factor"

    Article Title: CAL1 is the Drosophila CENP-A assembly factor

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201305036

    CAL1 is a CENP-A–specific nucleosome assembly factor. (A) CENP-A 101–225 –H4 was assembled on circular relaxed plasmid (pCR2.1-4 [CEN3 + CEN6]) using increasing amounts of CAL1 1–96 , CAL1 1–132 , or CAL1 1–160 in the presence of topoisomerase I. The extracted DNA samples were analyzed on agarose gel and stained with SYBR gold. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lane 3: CENP-A 101–225 –H4, H2A–H2B but no CAL1; lanes 4–7: relaxed plasmid and CENP-A 101–225 –H4, H2A–H2B with CAL1 1–96 ; lanes 8–11: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–132 ; lanes 12–15: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–160 . (B) CENP-A–containing nucleosomes and histone H3 nucleosomes were assembled using histone proteins, CAL1 1–160 , or yeast Nap1 and pGEM3Z-601 plasmid. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lanes 3, 6, and 9: mock reaction without CAL1 1–160 or Nap1; lanes 4 and 5: H3–H4, H2A–H2B with CAL1 1–160 ; lanes 7 and 8: CENP-A 101–225 –H4, H2A-H2B with CAL1 1–160 ; lanes 10 and 11: H3–H4, H2A–H2B with Nap1; lanes 12 and 13: CENP-A 101–225 –H4, H2A–H2B with Nap1. (C) CAL1-assembled CENP-A nucleosomes are negatively supercoiled. CENP-A nucleosomes (lane CENP-A) were assembled by CAL1 1–160 in the presence of topoisomerase I and H2A–H2B dimers, whereas histone H3 nucleosomes (lane H3) were assembled by Nap1. Controls are: supercoiled plasmid (superc.), relaxed plasmid (relaxed), and relaxed plasmid without histones or Nap1, but treated and processed as in the H3 and CENP-A nucleosome assembly reactions (mock). The samples were analyzed on agarose gels with or without 1 µg/ml chloroquine. Boxes highlight the decrease in migration that occurs in the presence of chloroquine in both H3 and CENP-A nucleosomes. (A–C) S, supercoiled; R, relaxed plasmid. (D) Diagram depicting the expected migration patterns for negatively and positively supercoiled DNA separated by 2D gel electrophoresis ( Tachiwana et al., 2011 ). Left gel: H3 nucleosomes assembled with Nap1; right gel: CENP-A nucleosomes assembled with CAL1 1–160 . (E) Mononucleosomes were assembled on 147-bp Widom DNA with Nap1 or CAL1 1–160 and digested with MNase for 30 or 120 s. Native PAGE of samples shown. Lanes 1–3: DNA only; lanes 4 and 5: H3 nucleosomes assembled by Nap1; lanes 6 and 7: CENP-A nucleosomes assembled by Nap1; lanes 8 and 9: CENP-A nucleosomes assembled by CAL1 1–160 .
    Figure Legend Snippet: CAL1 is a CENP-A–specific nucleosome assembly factor. (A) CENP-A 101–225 –H4 was assembled on circular relaxed plasmid (pCR2.1-4 [CEN3 + CEN6]) using increasing amounts of CAL1 1–96 , CAL1 1–132 , or CAL1 1–160 in the presence of topoisomerase I. The extracted DNA samples were analyzed on agarose gel and stained with SYBR gold. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lane 3: CENP-A 101–225 –H4, H2A–H2B but no CAL1; lanes 4–7: relaxed plasmid and CENP-A 101–225 –H4, H2A–H2B with CAL1 1–96 ; lanes 8–11: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–132 ; lanes 12–15: CENP-A 101–225 –H4, H2A–H2B with CAL1 1–160 . (B) CENP-A–containing nucleosomes and histone H3 nucleosomes were assembled using histone proteins, CAL1 1–160 , or yeast Nap1 and pGEM3Z-601 plasmid. Lane 1: supercoiled plasmid; lane 2: relaxed plasmid; lanes 3, 6, and 9: mock reaction without CAL1 1–160 or Nap1; lanes 4 and 5: H3–H4, H2A–H2B with CAL1 1–160 ; lanes 7 and 8: CENP-A 101–225 –H4, H2A-H2B with CAL1 1–160 ; lanes 10 and 11: H3–H4, H2A–H2B with Nap1; lanes 12 and 13: CENP-A 101–225 –H4, H2A–H2B with Nap1. (C) CAL1-assembled CENP-A nucleosomes are negatively supercoiled. CENP-A nucleosomes (lane CENP-A) were assembled by CAL1 1–160 in the presence of topoisomerase I and H2A–H2B dimers, whereas histone H3 nucleosomes (lane H3) were assembled by Nap1. Controls are: supercoiled plasmid (superc.), relaxed plasmid (relaxed), and relaxed plasmid without histones or Nap1, but treated and processed as in the H3 and CENP-A nucleosome assembly reactions (mock). The samples were analyzed on agarose gels with or without 1 µg/ml chloroquine. Boxes highlight the decrease in migration that occurs in the presence of chloroquine in both H3 and CENP-A nucleosomes. (A–C) S, supercoiled; R, relaxed plasmid. (D) Diagram depicting the expected migration patterns for negatively and positively supercoiled DNA separated by 2D gel electrophoresis ( Tachiwana et al., 2011 ). Left gel: H3 nucleosomes assembled with Nap1; right gel: CENP-A nucleosomes assembled with CAL1 1–160 . (E) Mononucleosomes were assembled on 147-bp Widom DNA with Nap1 or CAL1 1–160 and digested with MNase for 30 or 120 s. Native PAGE of samples shown. Lanes 1–3: DNA only; lanes 4 and 5: H3 nucleosomes assembled by Nap1; lanes 6 and 7: CENP-A nucleosomes assembled by Nap1; lanes 8 and 9: CENP-A nucleosomes assembled by CAL1 1–160 .

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Staining, Migration, Two-Dimensional Gel Electrophoresis, Electrophoresis, Clear Native PAGE

    26) Product Images from "Histone H2A variants confer specific properties to nucleosomes and impact on chromatin accessibility"

    Article Title: Histone H2A variants confer specific properties to nucleosomes and impact on chromatin accessibility

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky540

    Arabidopsis H2A nucleosome are homotypic. ( A ) DNA isolated from MNase digested nuclei demonstrating almost complete digestion of chromatin into mononucleosomes (*). Small proportion of chromatin was digested into dinucleosomes (**). ( B ) Extracts from MNase digested nuclei were immunoprecipitated with antibodies against H2A.W.6, H2A.W.7, H2A.1/13, H2A.X and H2A.Z.9 histone H2A variants and analyzed by western blotting with indicated antibodies. Detection of H3 is used as a control for nucleosome integrity. In this particular experiment we observed that H2A.W.6 nucleosomes contained 19% of heterotypic nucleosomes with H2A.W.7 and H2A.W.7 nucleosomes contained 15% of heterotypic nucleosomes with H2A.W.6. (Note that antibody against the canonical H2A recognizes two variants H2A.1 and H2A.13 ( 22 ).)
    Figure Legend Snippet: Arabidopsis H2A nucleosome are homotypic. ( A ) DNA isolated from MNase digested nuclei demonstrating almost complete digestion of chromatin into mononucleosomes (*). Small proportion of chromatin was digested into dinucleosomes (**). ( B ) Extracts from MNase digested nuclei were immunoprecipitated with antibodies against H2A.W.6, H2A.W.7, H2A.1/13, H2A.X and H2A.Z.9 histone H2A variants and analyzed by western blotting with indicated antibodies. Detection of H3 is used as a control for nucleosome integrity. In this particular experiment we observed that H2A.W.6 nucleosomes contained 19% of heterotypic nucleosomes with H2A.W.7 and H2A.W.7 nucleosomes contained 15% of heterotypic nucleosomes with H2A.W.6. (Note that antibody against the canonical H2A recognizes two variants H2A.1 and H2A.13 ( 22 ).)

    Techniques Used: Isolation, Immunoprecipitation, Western Blot

    Analysis of H2A.W.6 nucleosomes from transgenic plants expressing H2A.W.6 mutants. ( A ) Schematic presentation of H2A.W.6 mutants in L1 loop and the docking domain. The L1 and docking domain sequences of H2A.W.6 and H2A.Z are also indicated. ( B ) Immunoprecipitation of H2A.W.6 after digestion of nuclei with MNase. Western blotting was performed with antibodies against H2A.W.6, H2A.Z.9, H2A.1/13, H3 and H2B.4/9/11. The numbers below H3 and H2B blots represent their enrichment levels relative to H2A.W.6. The ratios for wild type were set to one and others are expressed relative to wild type. ( C ) Immunoprecipitation of H2A.Z.9 nucleosomes from transgenic plants expressing indicated H2A.W.6 mutants. The numbers below H3 and H2B blots indicate their levels normalized to the enrichment of H2A.Z.9 in each IP, demonstrating that instability of H2A.W.6 mutant nucleosomes is not due to the general effect of the experimental conditions.
    Figure Legend Snippet: Analysis of H2A.W.6 nucleosomes from transgenic plants expressing H2A.W.6 mutants. ( A ) Schematic presentation of H2A.W.6 mutants in L1 loop and the docking domain. The L1 and docking domain sequences of H2A.W.6 and H2A.Z are also indicated. ( B ) Immunoprecipitation of H2A.W.6 after digestion of nuclei with MNase. Western blotting was performed with antibodies against H2A.W.6, H2A.Z.9, H2A.1/13, H3 and H2B.4/9/11. The numbers below H3 and H2B blots represent their enrichment levels relative to H2A.W.6. The ratios for wild type were set to one and others are expressed relative to wild type. ( C ) Immunoprecipitation of H2A.Z.9 nucleosomes from transgenic plants expressing indicated H2A.W.6 mutants. The numbers below H3 and H2B blots indicate their levels normalized to the enrichment of H2A.Z.9 in each IP, demonstrating that instability of H2A.W.6 mutant nucleosomes is not due to the general effect of the experimental conditions.

    Techniques Used: Transgenic Assay, Expressing, Immunoprecipitation, Western Blot, Mutagenesis

    MNase treatment assay. ( A ) In vitro assembled nucleosomes containing indicated H2A variants and H2A.W.6 mutants were treated with MNase for indicated times at 25°C and digested DNA was analyzed by native PAGE. ( B ) Native-PAGE analyses of DNA fragments before (left panel) or after 15 min MNase digestion (right panel) of nucleosomes containing indicated H2A variants or H2A.W.6 mutants. The red and blue arrow indicate the top and bottom bands, respectively used for the quantification shown in panel C. ( C ) Graphical presentation of DNA fragments distribution after 15 min digestion with MNase shown in panel B. ( D ) Schematic representation of the 165, 155 or 145 bp fragment obtained by MNase treatment. ( E and F ) Graphical representation of the band intensity of ∼165 bp (panel E) or ∼155 bp DNA (panel F) fragments obtained by addition of MNase at indicated times. The intensity of each band was normalized to 193 base-pair DNA fragment corresponding to time point 0 in panel A.
    Figure Legend Snippet: MNase treatment assay. ( A ) In vitro assembled nucleosomes containing indicated H2A variants and H2A.W.6 mutants were treated with MNase for indicated times at 25°C and digested DNA was analyzed by native PAGE. ( B ) Native-PAGE analyses of DNA fragments before (left panel) or after 15 min MNase digestion (right panel) of nucleosomes containing indicated H2A variants or H2A.W.6 mutants. The red and blue arrow indicate the top and bottom bands, respectively used for the quantification shown in panel C. ( C ) Graphical presentation of DNA fragments distribution after 15 min digestion with MNase shown in panel B. ( D ) Schematic representation of the 165, 155 or 145 bp fragment obtained by MNase treatment. ( E and F ) Graphical representation of the band intensity of ∼165 bp (panel E) or ∼155 bp DNA (panel F) fragments obtained by addition of MNase at indicated times. The intensity of each band was normalized to 193 base-pair DNA fragment corresponding to time point 0 in panel A.

    Techniques Used: In Vitro, Clear Native PAGE

    27) Product Images from "Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure"

    Article Title: Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku150

    Nucleosome duplexes are decoupled into mononucleosomes. ( A ) EMSA of purified nucleosome duplexes containing H3(T118ph) HO and mp2-187 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S4 ). ( B ) Quantification of fraction of nucleosome duplexes (squares), nucleosome (circles) and free DNA (diamond) species for the gel in (A) versus time. Error bars are the standard deviation of three independent experiments. ( C ) EMSA of purified nucleosome duplexes and altosomes containing H3(T118ph) HO and mp2-247 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert in part to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S5 ). ( D ) Quantification of the fraction of nucleosome duplexes (squares), altosomes (triangles), nucleosome (circles) and free DNA (diamond) species for the gel in (C) versus time. Error bars are the standard deviation of three independent experiments.
    Figure Legend Snippet: Nucleosome duplexes are decoupled into mononucleosomes. ( A ) EMSA of purified nucleosome duplexes containing H3(T118ph) HO and mp2-187 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S4 ). ( B ) Quantification of fraction of nucleosome duplexes (squares), nucleosome (circles) and free DNA (diamond) species for the gel in (A) versus time. Error bars are the standard deviation of three independent experiments. ( C ) EMSA of purified nucleosome duplexes and altosomes containing H3(T118ph) HO and mp2-247 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert in part to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S5 ). ( D ) Quantification of the fraction of nucleosome duplexes (squares), altosomes (triangles), nucleosome (circles) and free DNA (diamond) species for the gel in (C) versus time. Error bars are the standard deviation of three independent experiments.

    Techniques Used: Purification, Standard Deviation

    28) Product Images from "Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages"

    Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093971

    PolII and TBP binding in the fraction of IL12B and IL1A promoters in a population of induced BMDMs that is nucleosome-free. (A and B), ChIP experiments were performed as described in the legend of Figure 2F with antibodies that detect (A) PolII or (B) TBP in BMDMs before (dark blue bars), and upon 1.5 h (yellow) or 3 h (red) LPS induction. Cross-linked chromatin was either untreated (solid bars), or lightly digested with MNase (hatched bars) as described in the Materials and Methods . The data was normalized to a region in the KIT promoter and genomic locations are indicated. The experiment was performed twice and error bars indicating the SEM are shown.
    Figure Legend Snippet: PolII and TBP binding in the fraction of IL12B and IL1A promoters in a population of induced BMDMs that is nucleosome-free. (A and B), ChIP experiments were performed as described in the legend of Figure 2F with antibodies that detect (A) PolII or (B) TBP in BMDMs before (dark blue bars), and upon 1.5 h (yellow) or 3 h (red) LPS induction. Cross-linked chromatin was either untreated (solid bars), or lightly digested with MNase (hatched bars) as described in the Materials and Methods . The data was normalized to a region in the KIT promoter and genomic locations are indicated. The experiment was performed twice and error bars indicating the SEM are shown.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation

    29) Product Images from "A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions"

    Article Title: A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp037

    Overview of the experimental steps required to create and analyse a chromatin accessibility library. ( A ) Step 1: fungal mycelia pre-grown under specific conditions or isolated DNA ( in vitro controls) are processed as described in Materials and methods section and digested with MNase or restriction enzymes of choice. Step 2: digested DNA is blunt-ended and phosphorylated by subsequent treatment of the chromatin with Klenow fragment polymerase, T4 polynucleotide kinase. This step produces blunt-ended DNA fragments for ligation with adaptors. Step 3: DNA fragments are ligated with double-stranded adaptors A and B, originating from oligonucleotides Adaptor-A short and Adaptor-A long or Adaptor-B short and Adaptor-B long , where adaptor oligonucleotide B long is biotinylated for later retention on the streptavidin beads. In this step, fragments containing all adaptor combinations (A-A, A-B and B-B) are generated. Step 4: the ligation step leaves nicks at the 3′-terminus that are repaired by Bst polymerase treatment. Step 5: all fragments containing biotinylated adaptor B are captured on streptavidin-coated magnetic beads. At this step, adaptor A-A fragments are lost. Step 6: after a washing step, the retained fragments (adaptors A-B and B-B fragments) are denatured at 95°C. The denaturation step results in the release of single strands which exclusively carry A-B adaptor fragments. Step 7: the single-stranded A-B adaptor fragment library is amplified by a nested PCR approach to give the final A-B fragment library. The input and output fragment libraries are quality controlled by amplification with single A and B, as well as mixed A-B primers. Only the A-B primer mix should result in the amplification of fragments in the range of 200–1000 bp (see Panel B). Step 8: the resulting A-B adaptor fragment library is diluted and aliquots are used for analytical PCR amplifications for fragment size analysis of specific loci of interest. In the final analytical PCR step, either gene-specific or adaptor-specific primers can be labelled for subsequent capillary sequencer analysis. The chromatograms are finally analysed by image analysis software. ( B ) Example of quality control of A-B adaptor fragment libraries. Two input chromatin fragment libraries without adaptor ligation (lanes 1 and 2) are compared to two output libraries with adaptor ligation as described in Materials and methods section (lanes 3 and 4). Libraries originating from nitrate-grown cells (lanes 1 and 3) as well as from ammonium-grown cells (lanes 2 and 4) are shown as an example. M, DNA size marker.
    Figure Legend Snippet: Overview of the experimental steps required to create and analyse a chromatin accessibility library. ( A ) Step 1: fungal mycelia pre-grown under specific conditions or isolated DNA ( in vitro controls) are processed as described in Materials and methods section and digested with MNase or restriction enzymes of choice. Step 2: digested DNA is blunt-ended and phosphorylated by subsequent treatment of the chromatin with Klenow fragment polymerase, T4 polynucleotide kinase. This step produces blunt-ended DNA fragments for ligation with adaptors. Step 3: DNA fragments are ligated with double-stranded adaptors A and B, originating from oligonucleotides Adaptor-A short and Adaptor-A long or Adaptor-B short and Adaptor-B long , where adaptor oligonucleotide B long is biotinylated for later retention on the streptavidin beads. In this step, fragments containing all adaptor combinations (A-A, A-B and B-B) are generated. Step 4: the ligation step leaves nicks at the 3′-terminus that are repaired by Bst polymerase treatment. Step 5: all fragments containing biotinylated adaptor B are captured on streptavidin-coated magnetic beads. At this step, adaptor A-A fragments are lost. Step 6: after a washing step, the retained fragments (adaptors A-B and B-B fragments) are denatured at 95°C. The denaturation step results in the release of single strands which exclusively carry A-B adaptor fragments. Step 7: the single-stranded A-B adaptor fragment library is amplified by a nested PCR approach to give the final A-B fragment library. The input and output fragment libraries are quality controlled by amplification with single A and B, as well as mixed A-B primers. Only the A-B primer mix should result in the amplification of fragments in the range of 200–1000 bp (see Panel B). Step 8: the resulting A-B adaptor fragment library is diluted and aliquots are used for analytical PCR amplifications for fragment size analysis of specific loci of interest. In the final analytical PCR step, either gene-specific or adaptor-specific primers can be labelled for subsequent capillary sequencer analysis. The chromatograms are finally analysed by image analysis software. ( B ) Example of quality control of A-B adaptor fragment libraries. Two input chromatin fragment libraries without adaptor ligation (lanes 1 and 2) are compared to two output libraries with adaptor ligation as described in Materials and methods section (lanes 3 and 4). Libraries originating from nitrate-grown cells (lanes 1 and 3) as well as from ammonium-grown cells (lanes 2 and 4) are shown as an example. M, DNA size marker.

    Techniques Used: Isolation, In Vitro, Ligation, Generated, Magnetic Beads, Amplification, Nested PCR, Polymerase Chain Reaction, Software, Marker

    Nucleosome positioning analysis at the areA gene promoter. ( A ) Overview of the 700 bp promoter region of areA . The FAM-labelled gene-specific primers used for analytical PCR and subsequent fragment analysis are shown as horizontal arrows and named according to their relative position within the promoter sequence. The colours of the primers represent the colour code of the fragment size profiles. Blue arrowheads indicate the position of predicted AreA binding GATA sites in the promoter of this positively autoregulated gene. The start of the areA coding region (ORF) is indicated by the bent arrow. A summary of MNase hypersensitive sites obtained from fragment size analysis of the in vitro control DNA is presented (indicated by vertical blue arrows). Numbers below the blue arrows indicate the exact nucleotide position of the MNase cut in the areA promoter region. Overlapping fragment size profiles in the areA promoter obtained by fragment size analysis of the in vitro MNase digest library using the primers indicted above are shown below the locus overview. As in the other figures, ‘P’ indicates signals originating from non-incorporated labelled primers. ( B ) Overlapping fragment size profiles in the areA promoter obtained by fragment size analysis of the same MNase digest libraries as used for the analysis of the niiA-niaD region ( Figure 2 ). Analytical PCRs were carried out using labelled primers indicated in panel A. The areA promoter profiles of the two libraries (induced, repressed) are shown here. The nucleosome positioned within the reading frame of areA is depicted as orf 1 and nucleosomes in the promoter region are designated −1 and −2. Under conditions in which AreA is active on its own promoter (induced) additional MNase cutting sites are revealed in nucleosome −2 at position ∼650 bp, at the end of nucleosome −1 at position ∼930 bp as well as in the orf1 nucleosome. ‘P’ indicates non-incorporated primer signals.
    Figure Legend Snippet: Nucleosome positioning analysis at the areA gene promoter. ( A ) Overview of the 700 bp promoter region of areA . The FAM-labelled gene-specific primers used for analytical PCR and subsequent fragment analysis are shown as horizontal arrows and named according to their relative position within the promoter sequence. The colours of the primers represent the colour code of the fragment size profiles. Blue arrowheads indicate the position of predicted AreA binding GATA sites in the promoter of this positively autoregulated gene. The start of the areA coding region (ORF) is indicated by the bent arrow. A summary of MNase hypersensitive sites obtained from fragment size analysis of the in vitro control DNA is presented (indicated by vertical blue arrows). Numbers below the blue arrows indicate the exact nucleotide position of the MNase cut in the areA promoter region. Overlapping fragment size profiles in the areA promoter obtained by fragment size analysis of the in vitro MNase digest library using the primers indicted above are shown below the locus overview. As in the other figures, ‘P’ indicates signals originating from non-incorporated labelled primers. ( B ) Overlapping fragment size profiles in the areA promoter obtained by fragment size analysis of the same MNase digest libraries as used for the analysis of the niiA-niaD region ( Figure 2 ). Analytical PCRs were carried out using labelled primers indicated in panel A. The areA promoter profiles of the two libraries (induced, repressed) are shown here. The nucleosome positioned within the reading frame of areA is depicted as orf 1 and nucleosomes in the promoter region are designated −1 and −2. Under conditions in which AreA is active on its own promoter (induced) additional MNase cutting sites are revealed in nucleosome −2 at position ∼650 bp, at the end of nucleosome −1 at position ∼930 bp as well as in the orf1 nucleosome. ‘P’ indicates non-incorporated primer signals.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Binding Assay, In Vitro

    MNase accessibility assay of the A. nidulans niiA-niaD locus employing the library-based chromatin analysis method. ( A ) Overview of the bidirectional promoter driving gene expression of niiA and niaD (the transcriptional start points of the genes are indicated by bent arrows). A summary of MNase hypersensitive sites obtained from in vitro digested control DNA of the region is presented (indicated by vertical arrows). The MNase sites determined by indirect end-labelling and hybridization ( 29 ) are shown in the upper part of this summary by blue vertical arrows and sites determined with the library approach in this work are shown in the lower part by green arrows. Numbers below the green arrows indicate the exact nucleotide position of the MNase cut in the niiA-niaD region. The fluorescently labelled gene-specific primers used for analytical PCR and subsequent fragment analysis are shown as horizontal arrows. The colour code of the primers represents the colour code of the fragment size profiles. Below the locus overview, we show the overlapping fragment size profiles of this region composed by processing the original sequencer chromatograms as described in Materials and methods section. Signals originating from the labelled primers still present in the fragment analysis reaction mixture after the analytical PCR reaction (not incorporated primers), are indicated by a ‘P’ directly above the corresponding peak. ( B ) Overlapping fragment size profiles in the niiA-niaD region obtained by PCR amplification of MNase digest libraries with labelled locus-specific primers. Nucleosomes are numbered consecutively from the central nfr between nucleosome −1 and +1 [according to Muro-Pastor et al. ( 29 )]. Nucleosomes positioned within the reading frame of niiA are depicted as orf 1 and orf 2. Two libraries are compared: (i) ‘induced’ indicates the profiles obtained from the library constructed from chromatin digestion of cells treated with nitrate as inducing agent; and (ii) ‘repressed’ indicates the profiles obtained from the library constructed from chromatin digestion of cells treated with ammonium as repressing agent. ‘P’ indicates signals originating from the labelled primers. Vertical dotted lines are drawn to highlight the highly accessible regions at the borders of positioned nucleosomes.
    Figure Legend Snippet: MNase accessibility assay of the A. nidulans niiA-niaD locus employing the library-based chromatin analysis method. ( A ) Overview of the bidirectional promoter driving gene expression of niiA and niaD (the transcriptional start points of the genes are indicated by bent arrows). A summary of MNase hypersensitive sites obtained from in vitro digested control DNA of the region is presented (indicated by vertical arrows). The MNase sites determined by indirect end-labelling and hybridization ( 29 ) are shown in the upper part of this summary by blue vertical arrows and sites determined with the library approach in this work are shown in the lower part by green arrows. Numbers below the green arrows indicate the exact nucleotide position of the MNase cut in the niiA-niaD region. The fluorescently labelled gene-specific primers used for analytical PCR and subsequent fragment analysis are shown as horizontal arrows. The colour code of the primers represents the colour code of the fragment size profiles. Below the locus overview, we show the overlapping fragment size profiles of this region composed by processing the original sequencer chromatograms as described in Materials and methods section. Signals originating from the labelled primers still present in the fragment analysis reaction mixture after the analytical PCR reaction (not incorporated primers), are indicated by a ‘P’ directly above the corresponding peak. ( B ) Overlapping fragment size profiles in the niiA-niaD region obtained by PCR amplification of MNase digest libraries with labelled locus-specific primers. Nucleosomes are numbered consecutively from the central nfr between nucleosome −1 and +1 [according to Muro-Pastor et al. ( 29 )]. Nucleosomes positioned within the reading frame of niiA are depicted as orf 1 and orf 2. Two libraries are compared: (i) ‘induced’ indicates the profiles obtained from the library constructed from chromatin digestion of cells treated with nitrate as inducing agent; and (ii) ‘repressed’ indicates the profiles obtained from the library constructed from chromatin digestion of cells treated with ammonium as repressing agent. ‘P’ indicates signals originating from the labelled primers. Vertical dotted lines are drawn to highlight the highly accessible regions at the borders of positioned nucleosomes.

    Techniques Used: Expressing, In Vitro, Hybridization, Polymerase Chain Reaction, Amplification, Construct

    30) Product Images from "ARGONAUTE2 cooperates with SWI/SNF complex to determine nucleosome occupancy at human Transcription Start Sites"

    Article Title: ARGONAUTE2 cooperates with SWI/SNF complex to determine nucleosome occupancy at human Transcription Start Sites

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1387

    AGO2 knock-down affects nucleosome occupancy at TSSs bound by SWI/SNF. ( a ) HeLaS3 cells were transfected with a control siRNA (siCTRL) or a pool of AGO2 siRNA (siAGO2). Down-regulation of AGO2 protein was verified by western blot. GAPDH was used as loading control. ( b ) Chromatin from siCTRL- or siAGO2-treated HeLaS3 cells was digested by MNase and recovered DNA fragments were sequenced. Nucleosome occupancy profile for siCTRL and siAGO2 cells was plotted for TSSs with at least 30 swiRNAs (siCTRL, black line; siAGO2, green line). The occupancy at the nucleosome +1 (arrow) is reduced in AGO2 knock-down cells. ( c ) Bars height represents percent reduction of nucleosome occupancy (siAGO2 versus siCTRL) at TSS ±150 nt overlapped by at least the indicated number of swiRNAs (green), IgG-IP ‘other sRNAs’ (black) and AGO1-associated ‘other sRNAs’ (purple). ** P value
    Figure Legend Snippet: AGO2 knock-down affects nucleosome occupancy at TSSs bound by SWI/SNF. ( a ) HeLaS3 cells were transfected with a control siRNA (siCTRL) or a pool of AGO2 siRNA (siAGO2). Down-regulation of AGO2 protein was verified by western blot. GAPDH was used as loading control. ( b ) Chromatin from siCTRL- or siAGO2-treated HeLaS3 cells was digested by MNase and recovered DNA fragments were sequenced. Nucleosome occupancy profile for siCTRL and siAGO2 cells was plotted for TSSs with at least 30 swiRNAs (siCTRL, black line; siAGO2, green line). The occupancy at the nucleosome +1 (arrow) is reduced in AGO2 knock-down cells. ( c ) Bars height represents percent reduction of nucleosome occupancy (siAGO2 versus siCTRL) at TSS ±150 nt overlapped by at least the indicated number of swiRNAs (green), IgG-IP ‘other sRNAs’ (black) and AGO1-associated ‘other sRNAs’ (purple). ** P value

    Techniques Used: Transfection, Western Blot

    31) Product Images from "A high-resolution map of transcriptional repression"

    Article Title: A high-resolution map of transcriptional repression

    Journal: eLife

    doi: 10.7554/eLife.22767

    Histone deacetylation contributes to stable gene silencing. ( A ) RT-PCR showed that 1 ng/ml TSA for 24 hr significantly relieved Ikaros-induced reduced repression of Igll and Myc primary transcripts. Mean ± SE, 3 independent biological replicates. ( B ) MNase PCR showed that 1 ng/ml TSA for 24 hr did not significantly affect protection of 80–120 bp amplicons (short, left) but significantly reduced protection of 130–140 bp amplicons (long, right) at the Igll1 promoter. Mean ± SE, 3 independent biological replicates. ( C ) ChIP-PCR to assess Ikaros-induced recruitment of histone H2B to the Igll1 promoter between control cells and cells treated with 1 ng/ml TSA for 24 hr. Enrichment was normalised to total H3. Mean ± SE, 3 independent biological replicates. TSA significantly blunted the Ikaros-induced increase the H2B/H3 ratio at the Igll1 promoter and TSS. ( D ) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, cells were treated over night with TSA (1 ng/ml) and/or 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of TSA was statistically significant across replicates (p=9.54 × 10-18 GLM binomial logit). DOI: http://dx.doi.org/10.7554/eLife.22767.021 10.7554/eLife.22767.022 Numerical data used to generate Figure 6A,B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.022
    Figure Legend Snippet: Histone deacetylation contributes to stable gene silencing. ( A ) RT-PCR showed that 1 ng/ml TSA for 24 hr significantly relieved Ikaros-induced reduced repression of Igll and Myc primary transcripts. Mean ± SE, 3 independent biological replicates. ( B ) MNase PCR showed that 1 ng/ml TSA for 24 hr did not significantly affect protection of 80–120 bp amplicons (short, left) but significantly reduced protection of 130–140 bp amplicons (long, right) at the Igll1 promoter. Mean ± SE, 3 independent biological replicates. ( C ) ChIP-PCR to assess Ikaros-induced recruitment of histone H2B to the Igll1 promoter between control cells and cells treated with 1 ng/ml TSA for 24 hr. Enrichment was normalised to total H3. Mean ± SE, 3 independent biological replicates. TSA significantly blunted the Ikaros-induced increase the H2B/H3 ratio at the Igll1 promoter and TSS. ( D ) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, cells were treated over night with TSA (1 ng/ml) and/or 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of TSA was statistically significant across replicates (p=9.54 × 10-18 GLM binomial logit). DOI: http://dx.doi.org/10.7554/eLife.22767.021 10.7554/eLife.22767.022 Numerical data used to generate Figure 6A,B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.022

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Fluorescence In Situ Hybridization

    Ikaros controls promoter accessibility through NuRD-associated chromatin remodeling. ( A ) Left: CHD4 expression in control and Chd4 shRNA cells by western blotting. Tubulin is a loading control. One of 5 independent biological replicates. Right: Experimental outline. ( B ) MNase-PCR at the Igll1 and Myc promoters in control (black) or Chd4 shRNA cells (red) at the indicated times after 4-OHT. Mean ± SE, 3 independent biological replicates. Chd4 shRNA significantly reduced the Ikaros-induced increase in nucleosome occupancy at 15, 30 and 120 min at the Igll1 promoter and at 30 and 120 min at the Myc promoter. ( C ) RNAP2 ChIP-PCR (top) and MNase-PCR (bottom) at the Igll1 and Myc promoters after 4-OHT in control (black) or Chd4 shRNA cells (red). Mean ± SE, 3 independent biological replicates. RNAP2 binding was significantly reduced in control cells but not in Chd4 shRNA-treated cells from 5 to 120 min after 4-OHT at the Igll1 and the Myc promoter. Primary transcripts were significantly reduced in control but not in Chd4 shRNA-treated cells at 15 and 30 min for Igll1 and at 30 and 120 min for Myc . ( D ) ChIP-PCR for CHD4 (black), MBD3 (grey) and BRG1 (orange) at the Igll1 promoters at the indicated times after 4-OHT. Mean ± SE, 5 independent biological replicates for CHD4 and BRG1, 3 independent biological replicates for MBD3. CHD4 and MBD3 binding at the Igll1 promoter were significantly increased from 5 to 60 min. BRG1 binding was significantly decreased from 30 to 120 min. DOI: http://dx.doi.org/10.7554/eLife.22767.015 10.7554/eLife.22767.016 Numerical data used to generate Figure 4B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.016
    Figure Legend Snippet: Ikaros controls promoter accessibility through NuRD-associated chromatin remodeling. ( A ) Left: CHD4 expression in control and Chd4 shRNA cells by western blotting. Tubulin is a loading control. One of 5 independent biological replicates. Right: Experimental outline. ( B ) MNase-PCR at the Igll1 and Myc promoters in control (black) or Chd4 shRNA cells (red) at the indicated times after 4-OHT. Mean ± SE, 3 independent biological replicates. Chd4 shRNA significantly reduced the Ikaros-induced increase in nucleosome occupancy at 15, 30 and 120 min at the Igll1 promoter and at 30 and 120 min at the Myc promoter. ( C ) RNAP2 ChIP-PCR (top) and MNase-PCR (bottom) at the Igll1 and Myc promoters after 4-OHT in control (black) or Chd4 shRNA cells (red). Mean ± SE, 3 independent biological replicates. RNAP2 binding was significantly reduced in control cells but not in Chd4 shRNA-treated cells from 5 to 120 min after 4-OHT at the Igll1 and the Myc promoter. Primary transcripts were significantly reduced in control but not in Chd4 shRNA-treated cells at 15 and 30 min for Igll1 and at 30 and 120 min for Myc . ( D ) ChIP-PCR for CHD4 (black), MBD3 (grey) and BRG1 (orange) at the Igll1 promoters at the indicated times after 4-OHT. Mean ± SE, 5 independent biological replicates for CHD4 and BRG1, 3 independent biological replicates for MBD3. CHD4 and MBD3 binding at the Igll1 promoter were significantly increased from 5 to 60 min. BRG1 binding was significantly decreased from 30 to 120 min. DOI: http://dx.doi.org/10.7554/eLife.22767.015 10.7554/eLife.22767.016 Numerical data used to generate Figure 4B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.016

    Techniques Used: Expressing, shRNA, Western Blot, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Binding Assay

    Nuclear translocation of Ikaros by proteolytic cleavage of Ikaros fusion proteins. ( A ) Schematic representation of Ikaros translocation induced by the proteolytic cleavage of Ikaros-TEV-ERt2 by Tobacco Etch Virus (TEV) protease. Ikaros-TEV-ERt2 fusion proteins are retained in the cytompasm until cleavage. The separate N- and C-termini of TEV protease are fused to FKBP and FRB, respectively. TEV activity is restored by the addition of rapamycin (R) ( Wehr et al., 2006 ). Cleavage separates Ikaros from ERt2. The efficiency of inducible cleavage after rapamycin (2 hr, 25 nM) is monitored by western blotting using Ikaros antibodies (right). Actin was used as loading control. Representative of 3 independent biological replicates. ( B ) MNase digestion followed by PCR was used to determine changes in the accessibility of the Igll1 and Myc promoters after triptolide-induced removal of RNAP2. The experiment complements the data shown in Figure 3B and C , but Split-TEV was used instead of 4-OHT-induced nuclear translocation of Ikaros. Split-TEV was activated by the addition of rapamycin at 25 nM for 2 hr. Mean ± SE, 3 independent biological replicates. Ikaros induction and tripolide-mediated RNAP2 depletion may synergise in increasing the nucleosome occupancy of the Igll1 promoter. DOI: http://dx.doi.org/10.7554/eLife.22767.013 10.7554/eLife.22767.014 Numerical data used to generate Figure 3—figure supplement 1A,B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.014
    Figure Legend Snippet: Nuclear translocation of Ikaros by proteolytic cleavage of Ikaros fusion proteins. ( A ) Schematic representation of Ikaros translocation induced by the proteolytic cleavage of Ikaros-TEV-ERt2 by Tobacco Etch Virus (TEV) protease. Ikaros-TEV-ERt2 fusion proteins are retained in the cytompasm until cleavage. The separate N- and C-termini of TEV protease are fused to FKBP and FRB, respectively. TEV activity is restored by the addition of rapamycin (R) ( Wehr et al., 2006 ). Cleavage separates Ikaros from ERt2. The efficiency of inducible cleavage after rapamycin (2 hr, 25 nM) is monitored by western blotting using Ikaros antibodies (right). Actin was used as loading control. Representative of 3 independent biological replicates. ( B ) MNase digestion followed by PCR was used to determine changes in the accessibility of the Igll1 and Myc promoters after triptolide-induced removal of RNAP2. The experiment complements the data shown in Figure 3B and C , but Split-TEV was used instead of 4-OHT-induced nuclear translocation of Ikaros. Split-TEV was activated by the addition of rapamycin at 25 nM for 2 hr. Mean ± SE, 3 independent biological replicates. Ikaros induction and tripolide-mediated RNAP2 depletion may synergise in increasing the nucleosome occupancy of the Igll1 promoter. DOI: http://dx.doi.org/10.7554/eLife.22767.013 10.7554/eLife.22767.014 Numerical data used to generate Figure 3—figure supplement 1A,B,C and D . DOI: http://dx.doi.org/10.7554/eLife.22767.014

    Techniques Used: Translocation Assay, Activity Assay, Western Blot, Polymerase Chain Reaction

    Interdependence of silencing mechanisms leveraged by Ikaros. ( A ) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, control or sh Chd4 cells were treated over night with 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of Chd4 knockdown was statistically significant across replicates (p=5.54×10-38 GLM binomial logit). ( B ) ChIP kinetics of Ikaros and EBF binding to the Igll1 promoter in control (left) and sh Chd4 cells (right). Increased binding of Ikaros to the Igll1 promoter was significant for both control and sh Chd4 cells, decreased binding of EBF1 was significant in control, but not in sh Chd4 cells. Mean ± SE, 3 independent biological replicates. Ikaros and EBF1 binding at 15, 30 and 120 min were significantly higher in sh Chd4 than control cells. ( C ) MNase-seq data from 3 independent biological replicates were integrated with Ikaros ChIP-seq data to show nucleosome occupancy at Ikaros binding peaks before and 6 hr after nuclear translocation of Ikaros. ( D ) Dynamics of Ikaros binding, RNAP2 eviction, loss of primary transcripts, nucleosome invasion, and histone deacetylation. DOI: http://dx.doi.org/10.7554/eLife.22767.023 10.7554/eLife.22767.024 Numerical data used to generate Figure 7A and B . DOI: http://dx.doi.org/10.7554/eLife.22767.024
    Figure Legend Snippet: Interdependence of silencing mechanisms leveraged by Ikaros. ( A ) 3D DNA-FISH to monitor the position of Igll1 alleles (green) relative to γ-satellite DNA (red, blue is DAPI). The percentage of Igll1 alleles associated with γ-satellite DNA is shown as mean ± SE. Where indicated, control or sh Chd4 cells were treated over night with 4-OHT. At least 300 Igll1 alleles were scored for each experimental condition across 3 independent biological replicates. The impact of Chd4 knockdown was statistically significant across replicates (p=5.54×10-38 GLM binomial logit). ( B ) ChIP kinetics of Ikaros and EBF binding to the Igll1 promoter in control (left) and sh Chd4 cells (right). Increased binding of Ikaros to the Igll1 promoter was significant for both control and sh Chd4 cells, decreased binding of EBF1 was significant in control, but not in sh Chd4 cells. Mean ± SE, 3 independent biological replicates. Ikaros and EBF1 binding at 15, 30 and 120 min were significantly higher in sh Chd4 than control cells. ( C ) MNase-seq data from 3 independent biological replicates were integrated with Ikaros ChIP-seq data to show nucleosome occupancy at Ikaros binding peaks before and 6 hr after nuclear translocation of Ikaros. ( D ) Dynamics of Ikaros binding, RNAP2 eviction, loss of primary transcripts, nucleosome invasion, and histone deacetylation. DOI: http://dx.doi.org/10.7554/eLife.22767.023 10.7554/eLife.22767.024 Numerical data used to generate Figure 7A and B . DOI: http://dx.doi.org/10.7554/eLife.22767.024

    Techniques Used: Fluorescence In Situ Hybridization, Chromatin Immunoprecipitation, Binding Assay, Translocation Assay

    32) Product Images from "Atomic-resolution mapping of transcription factor-DNA interactions by femtosecond laser crosslinking and mass spectrometry"

    Article Title: Atomic-resolution mapping of transcription factor-DNA interactions by femtosecond laser crosslinking and mass spectrometry

    Journal: Nature Communications

    doi: 10.1038/s41467-020-16837-x

    Schematic workflow of the fliX-MS pipeline. a A pulsed laser beam was generated using a femtosecond fiber laser with 515 nm wavelength, repetition rate of 0.5 MHz, and pulse duration of 500 fs. The wavelength was doubled to 258 nm by second harmonic generation (SHG) over a beta barium borate (BBO) crystal and the laser beam adjusted to fit the inner diameter of a regular 1.5 ml Eppendorf tube. b Protein–DNA complexes were irradiated or left untreated as control. Samples were denatured, DNA digested to mono/short oligonucleotides by a mix of Mnase, DNase I, and Benzonase, and proteins digested to peptides by trypsin and Lys-C. Peptides and peptide–nucleotide cross-links were separated from free DNA on C18 StageTips 25 , and cross-links subsequently enriched with TiO 2 beads. c Peptides were measured by LC–MS/MS and data analyzed with the RNP(xl) software package implemented in the proteome discoverer software 50 followed by manual annotation of candidate spectra.
    Figure Legend Snippet: Schematic workflow of the fliX-MS pipeline. a A pulsed laser beam was generated using a femtosecond fiber laser with 515 nm wavelength, repetition rate of 0.5 MHz, and pulse duration of 500 fs. The wavelength was doubled to 258 nm by second harmonic generation (SHG) over a beta barium borate (BBO) crystal and the laser beam adjusted to fit the inner diameter of a regular 1.5 ml Eppendorf tube. b Protein–DNA complexes were irradiated or left untreated as control. Samples were denatured, DNA digested to mono/short oligonucleotides by a mix of Mnase, DNase I, and Benzonase, and proteins digested to peptides by trypsin and Lys-C. Peptides and peptide–nucleotide cross-links were separated from free DNA on C18 StageTips 25 , and cross-links subsequently enriched with TiO 2 beads. c Peptides were measured by LC–MS/MS and data analyzed with the RNP(xl) software package implemented in the proteome discoverer software 50 followed by manual annotation of candidate spectra.

    Techniques Used: Generated, Irradiation, Liquid Chromatography with Mass Spectroscopy, Software

    33) Product Images from "Formation of Mobile Chromatin-Associated Nuclear Foci Containing HIV-1 Vpr and VPRBP Is Critical for the Induction of G2 Cell Cycle Arrest"

    Article Title: Formation of Mobile Chromatin-Associated Nuclear Foci Containing HIV-1 Vpr and VPRBP Is Critical for the Induction of G2 Cell Cycle Arrest

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1001080

    Association of Vpr with chromatin correlates with the formation of nuclear foci. A ) HeLa cells were transfected with plasmids expressing HA-tagged Vpr (WT) or an empty plasmid used as negative control. Forty-eight hours after transfection, cells were harvested and lysed with 0.5% Triton X-100. The soluble fraction was used as input control (Soluble). Insoluble debris containing chromatin was treated with microccocal nuclease (+MNase) or with buffer alone (−MNase). The resulting solubilized fractions and input controls were resolved by SDS-PAGE and analyzed by western blot. Specific monoclonal antibodies were used to detect GAPDH (cytoplasmic marker) and HA-Vpr. Histone 3 (chromatin marker) and VPRBP were detected using rabbit polyclonal antibodies. * Denotes a non-specific band detected with the anti-HA antibody. B ) HeLa cells were transfected with plasmids expressing HA-tagged Vpr (WT), Vpr (Q65R), Vpr (R80A), and Vpr (1–78). Cell extracts were processed and analysed as in A). C ) HeLa cells were first transfected with scrambled siRNA or siRNA targeting VPRBP. Twenty-four hours after transfection, cells were transfected with a plasmid expressing HA-Vpr (WT) or an empty plasmid as negative control. Cell extracts were processed and analyzed as in A).
    Figure Legend Snippet: Association of Vpr with chromatin correlates with the formation of nuclear foci. A ) HeLa cells were transfected with plasmids expressing HA-tagged Vpr (WT) or an empty plasmid used as negative control. Forty-eight hours after transfection, cells were harvested and lysed with 0.5% Triton X-100. The soluble fraction was used as input control (Soluble). Insoluble debris containing chromatin was treated with microccocal nuclease (+MNase) or with buffer alone (−MNase). The resulting solubilized fractions and input controls were resolved by SDS-PAGE and analyzed by western blot. Specific monoclonal antibodies were used to detect GAPDH (cytoplasmic marker) and HA-Vpr. Histone 3 (chromatin marker) and VPRBP were detected using rabbit polyclonal antibodies. * Denotes a non-specific band detected with the anti-HA antibody. B ) HeLa cells were transfected with plasmids expressing HA-tagged Vpr (WT), Vpr (Q65R), Vpr (R80A), and Vpr (1–78). Cell extracts were processed and analysed as in A). C ) HeLa cells were first transfected with scrambled siRNA or siRNA targeting VPRBP. Twenty-four hours after transfection, cells were transfected with a plasmid expressing HA-Vpr (WT) or an empty plasmid as negative control. Cell extracts were processed and analyzed as in A).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Negative Control, SDS Page, Western Blot, Marker

    34) Product Images from "The regulatory landscape of early maize inflorescence development"

    Article Title: The regulatory landscape of early maize inflorescence development

    Journal: Genome Biology

    doi: 10.1186/s13059-020-02070-8

    MNase HS as a proxy for TF-DNA binding. (a) Overlap of high confidence peaks from FEA4 and KN1 ChIP-seq experiments with MNase HS regions based on LCS data. Less than 2 % of co-bound sites and 6 % of all sites do not overlap MNase HS. (b) Read density (reads per million) of LCS are plotted around a consensus TSS of genes bound by KN1 and/or FEA4. Genes co-bound by both TFs in the proximal promoter show a wider range of accessibility. Data are shown for tassel; similar profiles are shown for ear presented in Fig. S9. (c) Examples of intergenic MNase HS sites in tassel, ear, and/or shoot that are co-bound by KN1 and FEA4: a known enhancer element 65 kb upstream of tb1, and a site 6 kb upstream of the gnarley 1 ( gn1 ) locus. (d) Fragment densities from the light MNase digest plotted around high-confidence FEA4 ChIP-seq peaks show strong enrichment at the consensus FEA4 binding motif. (e) Density of ABI3-like motifs were plotted in relation to experimentally validated FEA4 binding sites. The ABI3-like motif was discovered by mining HS regions called by iSeg that overlapped consensus FEA4 binding sites. ABI3-like motifs are most densely positioned within 50 bp of FEA4 motifs. (f) GO terms overrepresented among genes within 1 kb of FEA4-ABI3-like motif modules (ABI3-like motifs within 50 bp of FEA binding sites) and compared with genes within 1 kb of random FEA4 motifs of equal number
    Figure Legend Snippet: MNase HS as a proxy for TF-DNA binding. (a) Overlap of high confidence peaks from FEA4 and KN1 ChIP-seq experiments with MNase HS regions based on LCS data. Less than 2 % of co-bound sites and 6 % of all sites do not overlap MNase HS. (b) Read density (reads per million) of LCS are plotted around a consensus TSS of genes bound by KN1 and/or FEA4. Genes co-bound by both TFs in the proximal promoter show a wider range of accessibility. Data are shown for tassel; similar profiles are shown for ear presented in Fig. S9. (c) Examples of intergenic MNase HS sites in tassel, ear, and/or shoot that are co-bound by KN1 and FEA4: a known enhancer element 65 kb upstream of tb1, and a site 6 kb upstream of the gnarley 1 ( gn1 ) locus. (d) Fragment densities from the light MNase digest plotted around high-confidence FEA4 ChIP-seq peaks show strong enrichment at the consensus FEA4 binding motif. (e) Density of ABI3-like motifs were plotted in relation to experimentally validated FEA4 binding sites. The ABI3-like motif was discovered by mining HS regions called by iSeg that overlapped consensus FEA4 binding sites. ABI3-like motifs are most densely positioned within 50 bp of FEA4 motifs. (f) GO terms overrepresented among genes within 1 kb of FEA4-ABI3-like motif modules (ABI3-like motifs within 50 bp of FEA binding sites) and compared with genes within 1 kb of random FEA4 motifs of equal number

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation

    35) Product Images from "Micromanipulation of prophase I chromosomes from mouse spermatocytes reveals high stiffness and gel-like chromatin organization"

    Article Title: Micromanipulation of prophase I chromosomes from mouse spermatocytes reveals high stiffness and gel-like chromatin organization

    Journal: bioRxiv

    doi: 10.1101/2020.08.31.276402

    Mitotic and meiotic chromosomes have a contiguous DNA connection, which is dissolved by 4 bp restriction enzymes, but only weakened by 6 bp restriction enzymes. Image pairs show pipette positions untreated (native isolated) chromosomes when relaxed and stretched (a) , and chromosomes following enzyme treatments (b-d) . Vertical blue lines mark positions of force pipettes. Force pipette deflection by pulling (horizontal blue lines) indicates mechanical connection; no movement (no horizontal blue line) indicates no mechanical connection. Red notches mark positions of stiff pipettes. Bars are 5 μm. ( b ) Both mitotic and meiotic chromosomes were weakened, but not fully digested after treatment with PvuII (cut sequence CAG ˅ CTG). ( c ) Both mitotic and meiotic chromosomes lost connectivity after treatment with AluI (cut sequence AG ˅ CT; for 1 of 4 trials meiotic chromosomes were not fully digested by AluI). ( d ) Both mitotic and meiotic chromosomes lost connectivity when treated with MNase (cleaves all DNA sequences). ( e ) Quantification of chromosome stretching elasticity after no treatment or after being treated with PvuII, AluI, and MNase. No treatment caused a 13 ± 4% weakening of mitotic chromosomes (N=10) and a 1 ± 4% weakening of meiotic chromosomes (N=10). PvuII treatment caused a 70 ± 8% reduction in stiffness for MEF chromosomes (N=4) and 70 ± 9% reduction in stiffness for meiotic chromosomes (N=4). One of four AluI treatments of meiotic chromosomes caused a 90% reduction in stiffness (rather than fully digesting), while AluI treatment digested 4 of 4 mitotic chromosomes. All MNase treatments caused full digestion of mitotic and meiotic chromosomes (N=4 in both cases). All averages are reported as mean value ± SEM. Bars are 5 μm.
    Figure Legend Snippet: Mitotic and meiotic chromosomes have a contiguous DNA connection, which is dissolved by 4 bp restriction enzymes, but only weakened by 6 bp restriction enzymes. Image pairs show pipette positions untreated (native isolated) chromosomes when relaxed and stretched (a) , and chromosomes following enzyme treatments (b-d) . Vertical blue lines mark positions of force pipettes. Force pipette deflection by pulling (horizontal blue lines) indicates mechanical connection; no movement (no horizontal blue line) indicates no mechanical connection. Red notches mark positions of stiff pipettes. Bars are 5 μm. ( b ) Both mitotic and meiotic chromosomes were weakened, but not fully digested after treatment with PvuII (cut sequence CAG ˅ CTG). ( c ) Both mitotic and meiotic chromosomes lost connectivity after treatment with AluI (cut sequence AG ˅ CT; for 1 of 4 trials meiotic chromosomes were not fully digested by AluI). ( d ) Both mitotic and meiotic chromosomes lost connectivity when treated with MNase (cleaves all DNA sequences). ( e ) Quantification of chromosome stretching elasticity after no treatment or after being treated with PvuII, AluI, and MNase. No treatment caused a 13 ± 4% weakening of mitotic chromosomes (N=10) and a 1 ± 4% weakening of meiotic chromosomes (N=10). PvuII treatment caused a 70 ± 8% reduction in stiffness for MEF chromosomes (N=4) and 70 ± 9% reduction in stiffness for meiotic chromosomes (N=4). One of four AluI treatments of meiotic chromosomes caused a 90% reduction in stiffness (rather than fully digesting), while AluI treatment digested 4 of 4 mitotic chromosomes. All MNase treatments caused full digestion of mitotic and meiotic chromosomes (N=4 in both cases). All averages are reported as mean value ± SEM. Bars are 5 μm.

    Techniques Used: Transferring, Isolation, Sequencing

    36) Product Images from "Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics"

    Article Title: Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky526

    Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.
    Figure Legend Snippet: Dynamics of H2B K34ub nucleosomes. ( A ) Nucleosomes assembled on 147 bp 601 DNA were resolved in native PAGE under ionic and temperature conditions indicated on top. For the middle panel, although electrophoresis was performed at ∼26°C, the temperature at the glass surface was ∼35–37°C due to higher conductivity of the buffer. All gels were pre-electrophoresed in the relevant buffer. ( B ) Unmodified and H2B K34ub nucleosomes /hexasomes assembled on 177 bp 601 were digested with MNase at 26°C or 37°C and DNA was resolved in 6.5% PAGE and stained with SYBR Gold.

    Techniques Used: Clear Native PAGE, Electrophoresis, Polyacrylamide Gel Electrophoresis, Staining

    37) Product Images from "RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF"

    Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky562

    Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or DNA sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for MNAse digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.
    Figure Legend Snippet: Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or DNA sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for MNAse digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.

    Techniques Used: Binding Assay, Genome Wide, Sequencing, Immunoprecipitation

    38) Product Images from "Comprehensive nucleosome mapping of the human genome in cancer progression"

    Article Title: Comprehensive nucleosome mapping of the human genome in cancer progression

    Journal: Oncotarget

    doi: 10.18632/oncotarget.6811

    The newly developed mTSS-Capture method combined with paired-end sequencing maps genome-wide nucleosome distribution in primary patient samples and identifies bona fide nucleosome characteristics, concordant with other human nucleosome mapping studies A . Work-flow of the mTSS-seq method. Following MNase digestion using a titration of MNase, populations of mononucleosomally protected DNA and subnucleosomal fragments are isolated, and prepared as libraries for Illumina sequencing. Solution-based sequence capture is performed using biotinylated oligos, enabling the enrichment of fragments within 2kb of each transcription start site in the human genome. Paired-end 50bp sequencing was then performed on each index. B . Alignment of the mTSS-seq midpoints to the human genome using the UCSC genome browser for LAC patient #4137 Normal tissue is shown for chr11, hg19 ( http://genome.ucsc.edu ). Zooming in twice at 100X allows for further visualization of the sequence capture oligos surrounding the TSS in a 500kb and a 5kb region showing the ATM locus. C . Averaged, normalized reads per million (y-axis) from mTSS-seq plotted as fragments (gray) and midpoints (black), centered on and surrounding 2kb of the TSS for ~22,000 open reading frames in hg19 (x-axis). DNase I-hypersensitivity (GSM736580; green) and RNA polymerase II from ChIP-seq (GSM935299; blue) data from A549 cells are shown. (D) LAC patient 4137 Normal nucleosomal midpoints (blue track) were plotted in the UCSC genome browser against the published human lymphocyte nucleosome distribution maps by Gaffney et. al. (green track) for the ZNF451 and CCDC97 loci. Sequence capture oligos and corresponding RefSeq gene models are shown for each locus. Correlations are shown for ZNF451 and CCDC87, respectively.
    Figure Legend Snippet: The newly developed mTSS-Capture method combined with paired-end sequencing maps genome-wide nucleosome distribution in primary patient samples and identifies bona fide nucleosome characteristics, concordant with other human nucleosome mapping studies A . Work-flow of the mTSS-seq method. Following MNase digestion using a titration of MNase, populations of mononucleosomally protected DNA and subnucleosomal fragments are isolated, and prepared as libraries for Illumina sequencing. Solution-based sequence capture is performed using biotinylated oligos, enabling the enrichment of fragments within 2kb of each transcription start site in the human genome. Paired-end 50bp sequencing was then performed on each index. B . Alignment of the mTSS-seq midpoints to the human genome using the UCSC genome browser for LAC patient #4137 Normal tissue is shown for chr11, hg19 ( http://genome.ucsc.edu ). Zooming in twice at 100X allows for further visualization of the sequence capture oligos surrounding the TSS in a 500kb and a 5kb region showing the ATM locus. C . Averaged, normalized reads per million (y-axis) from mTSS-seq plotted as fragments (gray) and midpoints (black), centered on and surrounding 2kb of the TSS for ~22,000 open reading frames in hg19 (x-axis). DNase I-hypersensitivity (GSM736580; green) and RNA polymerase II from ChIP-seq (GSM935299; blue) data from A549 cells are shown. (D) LAC patient 4137 Normal nucleosomal midpoints (blue track) were plotted in the UCSC genome browser against the published human lymphocyte nucleosome distribution maps by Gaffney et. al. (green track) for the ZNF451 and CCDC97 loci. Sequence capture oligos and corresponding RefSeq gene models are shown for each locus. Correlations are shown for ZNF451 and CCDC87, respectively.

    Techniques Used: Sequencing, Genome Wide, Titration, Isolation, Chromatin Immunoprecipitation

    Related Articles

    Isolation:

    Article Title: ARGONAUTE2 cooperates with SWI/SNF complex to determine nucleosome occupancy at human Transcription Start Sites
    Article Snippet: .. Isolation of nucleosomal DNA by micrococcal nuclease (MNase) digestion Digestion of chromatin from untreated, siCTRL- or siAGO2-treated HeLa S3 cells (2 × 106 ) was performed with 50 U of MNase (New England Biolabs) in 300 μl of permeabilization buffer (15 mM Tris–HCl pH 7.4, 300 mM sucrose, 60 mM KCl, 15 mM NaCl, 4 mM CaCl2 , 0.5 mM EGTA, 0.2% NP-40, 0.5 mM β-mercaptoethanol) for 20 min at 37°C. ..

    Protease Inhibitor:

    Article Title: Epigenomic profiling discovers trans-lineage SOX2 partnerships driving tumor heterogeneity in lung squamous cell carcinoma
    Article Snippet: .. For Brn2 ChIP, cells were crosslinked with 2 mM disuccinimidyl glutarate in PBS with 1 mM MgCl2 for 45 min at room temperature and 1% formaldehyde in PBS for 11 min at room temperature, washed in 5 mg/ml BSA in PBS and then in just cold PBS, re-suspended in nuclear extraction buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3mM MgCl2 , 0.1 % IGEPAL CA-630, 1× protease inhibitor cocktail) and incubated on ice for 10 min. Extracted nuclei were then washed and resuspended in micrococcal nuclease digestion buffer (0.3 M sucrose, 20 mM Tris-HCl pH 7.5, 3 mM CaCl2, 1× protease inhibitor cocktail) and incubated with 12,000 gel units/ml micrococcal nuclease (NEB, M0247S) at 37 °C with frequently mixing to digest chromatin to lengths of approximately 100-to-1,000 bp. .. Digestion was stopped with 25 mM EDTA pH 8.0 and nuclei was re-suspended in lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, 1× protease inhibitor cocktail) and sonicated with the Diagenode Bioruptor sonicator (low intensity, 30 sec on and 30 sec off, 2 cycles).

    Incubation:

    Article Title: Epigenomic profiling discovers trans-lineage SOX2 partnerships driving tumor heterogeneity in lung squamous cell carcinoma
    Article Snippet: .. For Brn2 ChIP, cells were crosslinked with 2 mM disuccinimidyl glutarate in PBS with 1 mM MgCl2 for 45 min at room temperature and 1% formaldehyde in PBS for 11 min at room temperature, washed in 5 mg/ml BSA in PBS and then in just cold PBS, re-suspended in nuclear extraction buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3mM MgCl2 , 0.1 % IGEPAL CA-630, 1× protease inhibitor cocktail) and incubated on ice for 10 min. Extracted nuclei were then washed and resuspended in micrococcal nuclease digestion buffer (0.3 M sucrose, 20 mM Tris-HCl pH 7.5, 3 mM CaCl2, 1× protease inhibitor cocktail) and incubated with 12,000 gel units/ml micrococcal nuclease (NEB, M0247S) at 37 °C with frequently mixing to digest chromatin to lengths of approximately 100-to-1,000 bp. .. Digestion was stopped with 25 mM EDTA pH 8.0 and nuclei was re-suspended in lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, 1× protease inhibitor cocktail) and sonicated with the Diagenode Bioruptor sonicator (low intensity, 30 sec on and 30 sec off, 2 cycles).

    Sonication:

    Article Title: The orphan nuclear receptor NR4A2 is part of a p53–microRNA-34 network
    Article Snippet: .. Cell extracts were digested for 10 min with 50 units of micrococcal nuclease (New England Biolabs, Ipswich, MA) at 37 °C and further sonicated to yield sheared DNA fragments with an average length of 200 to 1000 base pairs. .. The sonicated samples were centrifuged to pellet the cell debris, and the supernatant was diluted 7-fold with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, and protease inhibitor cocktail).

    Sequencing:

    Article Title: EPIGENETIC ANALYSIS OF SV40 MINICHROMOSOMES
    Article Snippet: .. We presently prepare our libraries for sequencing from micrococcal nuclease digested SV40 chromatin and ChIP analyses using an NEBNEXT kit designed to work with the Illumina sequencing system. ..

    Lysis:

    Article Title: Analysis of neonatal brain lacking ATRX or MeCP2 reveals changes in nucleosome density, CTCF binding and chromatin looping
    Article Snippet: .. Cells were resuspended in lysis buffer [0.34 M sucrose, 60 mM KCl, 15 mM Tris–HCl, 15 mM NaCl, 0.5% NP-40 and 1× protease inhibitors (Sigma-Aldrich)] and flash-frozen and thawed three times, nuclei were centrifuged and resuspended in micrococcal nuclease digestion buffer (NEB). .. Micrococcal nuclease (2 U; NEB) was added and incubated at 37°C for 5 min, then quenched with EDTA.

    Chromatin Immunoprecipitation:

    Article Title: EPIGENETIC ANALYSIS OF SV40 MINICHROMOSOMES
    Article Snippet: .. We presently prepare our libraries for sequencing from micrococcal nuclease digested SV40 chromatin and ChIP analyses using an NEBNEXT kit designed to work with the Illumina sequencing system. ..

    Article Title: Epigenomic profiling discovers trans-lineage SOX2 partnerships driving tumor heterogeneity in lung squamous cell carcinoma
    Article Snippet: .. For Brn2 ChIP, cells were crosslinked with 2 mM disuccinimidyl glutarate in PBS with 1 mM MgCl2 for 45 min at room temperature and 1% formaldehyde in PBS for 11 min at room temperature, washed in 5 mg/ml BSA in PBS and then in just cold PBS, re-suspended in nuclear extraction buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3mM MgCl2 , 0.1 % IGEPAL CA-630, 1× protease inhibitor cocktail) and incubated on ice for 10 min. Extracted nuclei were then washed and resuspended in micrococcal nuclease digestion buffer (0.3 M sucrose, 20 mM Tris-HCl pH 7.5, 3 mM CaCl2, 1× protease inhibitor cocktail) and incubated with 12,000 gel units/ml micrococcal nuclease (NEB, M0247S) at 37 °C with frequently mixing to digest chromatin to lengths of approximately 100-to-1,000 bp. .. Digestion was stopped with 25 mM EDTA pH 8.0 and nuclei was re-suspended in lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS, 1× protease inhibitor cocktail) and sonicated with the Diagenode Bioruptor sonicator (low intensity, 30 sec on and 30 sec off, 2 cycles).

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    5 hydroxythymidine DNA Kinase 1 000 units
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    New England Biolabs mnase digestion buffer
    Nucleosome positioning is altered at the TSSs of genes in Δ dim-1 strain. (A) Southern blots of <t>DNA</t> from 20-min time course micrococcal nuclease <t>(MNase)</t> digest with WT and Δ dim-1 nuclei, probed for the indicated regions; the “NCU04771up start” probe covers the intergenic promoter region of gene NCU04771, including the nucleosome-free region. Arrowheads indicate the time points (8 and 10 min) from which mono- and di-nucleosomes and intervening DNA was purified for paired-end high-throughput sequencing (below). Cartoons at right show interpretation of nucleosome patterns leading to smallest fragments. (B) Average nucleosome enrichment profiles of MNase-sequencing data from WT and Δ dim-1 strains normalized to the average signal across the 5′ end of all Neurospora genes, spanning 400 bp upstream to 600 bp downstream of the TSS; numbers below indicate the peak apices, in base pairs from the transcriptional start site (TSS) in WT and Δ dim-1 strains, and the difference in the apex position, for the +1, +2, and +3 nucleosomes. (C) Representative examples of nucleosome positions in individual genes with changed (NCU08052), or unchanged (NCU04402; dim-5 ) expression in a Δ dim-1 background. Red arrows highlight nucleosome disorder in a Δ dim-1 strain of two nucleosomes that are well-positioned in a WT strain.
    Mnase Digestion Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs mnase
    DNA nucleases induce NET digestion . (A) Migration profile of pure λDNA after digestion with 4 U/mL <t>DNase,</t> <t>MNase,</t> or Alu-I. (B) Alu-I, DNase, and MNase dose-effects on NET dsDNA obtained after A23187 stimulation of PMN. Incubation with the restriction enzymes lasted 20 min at 37°C. DNA migration took place in 0.8% agarose gel containing ethidium bromide.
    Mnase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Nucleosome positioning is altered at the TSSs of genes in Δ dim-1 strain. (A) Southern blots of DNA from 20-min time course micrococcal nuclease (MNase) digest with WT and Δ dim-1 nuclei, probed for the indicated regions; the “NCU04771up start” probe covers the intergenic promoter region of gene NCU04771, including the nucleosome-free region. Arrowheads indicate the time points (8 and 10 min) from which mono- and di-nucleosomes and intervening DNA was purified for paired-end high-throughput sequencing (below). Cartoons at right show interpretation of nucleosome patterns leading to smallest fragments. (B) Average nucleosome enrichment profiles of MNase-sequencing data from WT and Δ dim-1 strains normalized to the average signal across the 5′ end of all Neurospora genes, spanning 400 bp upstream to 600 bp downstream of the TSS; numbers below indicate the peak apices, in base pairs from the transcriptional start site (TSS) in WT and Δ dim-1 strains, and the difference in the apex position, for the +1, +2, and +3 nucleosomes. (C) Representative examples of nucleosome positions in individual genes with changed (NCU08052), or unchanged (NCU04402; dim-5 ) expression in a Δ dim-1 background. Red arrows highlight nucleosome disorder in a Δ dim-1 strain of two nucleosomes that are well-positioned in a WT strain.

    Journal: Genetics

    Article Title: Nucleosome Positioning by an Evolutionarily Conserved Chromatin Remodeler Prevents Aberrant DNA Methylation in Neurospora

    doi: 10.1534/genetics.118.301711

    Figure Lengend Snippet: Nucleosome positioning is altered at the TSSs of genes in Δ dim-1 strain. (A) Southern blots of DNA from 20-min time course micrococcal nuclease (MNase) digest with WT and Δ dim-1 nuclei, probed for the indicated regions; the “NCU04771up start” probe covers the intergenic promoter region of gene NCU04771, including the nucleosome-free region. Arrowheads indicate the time points (8 and 10 min) from which mono- and di-nucleosomes and intervening DNA was purified for paired-end high-throughput sequencing (below). Cartoons at right show interpretation of nucleosome patterns leading to smallest fragments. (B) Average nucleosome enrichment profiles of MNase-sequencing data from WT and Δ dim-1 strains normalized to the average signal across the 5′ end of all Neurospora genes, spanning 400 bp upstream to 600 bp downstream of the TSS; numbers below indicate the peak apices, in base pairs from the transcriptional start site (TSS) in WT and Δ dim-1 strains, and the difference in the apex position, for the +1, +2, and +3 nucleosomes. (C) Representative examples of nucleosome positions in individual genes with changed (NCU08052), or unchanged (NCU04402; dim-5 ) expression in a Δ dim-1 background. Red arrows highlight nucleosome disorder in a Δ dim-1 strain of two nucleosomes that are well-positioned in a WT strain.

    Article Snippet: Nuclei containing 16 µg of DNA were adjusted to 400 µl with MNase digestion buffer, CaCl2 was added to a final concentration of 2 mM, MNase (New England Biolabs, Beverly, MA) was added to a final concentration of 0.5 unit/µl, and the reaction was carried out at 37° for 20 min. At 2-min intervals, 30 µl aliquots (corresponding to 1.2 µg of DNA) were removed and mixed with 8 µl of 100 mM EGTA to stop the reaction and stored on ice.

    Techniques: Purification, Next-Generation Sequencing, Sequencing, Expressing

    DNA nucleases induce NET digestion . (A) Migration profile of pure λDNA after digestion with 4 U/mL DNase, MNase, or Alu-I. (B) Alu-I, DNase, and MNase dose-effects on NET dsDNA obtained after A23187 stimulation of PMN. Incubation with the restriction enzymes lasted 20 min at 37°C. DNA migration took place in 0.8% agarose gel containing ethidium bromide.

    Journal: Frontiers in Immunology

    Article Title: An Improved Strategy to Recover Large Fragments of Functional Human Neutrophil Extracellular Traps

    doi: 10.3389/fimmu.2013.00166

    Figure Lengend Snippet: DNA nucleases induce NET digestion . (A) Migration profile of pure λDNA after digestion with 4 U/mL DNase, MNase, or Alu-I. (B) Alu-I, DNase, and MNase dose-effects on NET dsDNA obtained after A23187 stimulation of PMN. Incubation with the restriction enzymes lasted 20 min at 37°C. DNA migration took place in 0.8% agarose gel containing ethidium bromide.

    Article Snippet: NET digestion by nucleases In a first set of experiments, 4 μg of purified λDNA (Invitrogen) was treated with 4 U/mL DNase I (Sigma Aldrich), 4 U/mL MNase (New England BioLabs, France), or 4 U/mL Alu I (New England BioLabs) for 20 min at 37°C.

    Techniques: Migration, Incubation, Agarose Gel Electrophoresis

    Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or DNA sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for MNAse digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.

    Journal: Nucleic Acids Research

    Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF

    doi: 10.1093/nar/gky562

    Figure Lengend Snippet: Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or DNA sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for MNAse digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.

    Article Snippet: Cells were fixed with 0.7% formaldehyde and DNA was digested by 10 ul MNAse (New England Biolabs) to an average length of 120 bp.

    Techniques: Binding Assay, Genome Wide, Sequencing, Immunoprecipitation

    BEN-2 shows B cell-specific nucleosome occupancy, chromatin accessibility and enrichment for the H3K27ac active enhancer histone mark across a panel of B cell lines. A. Nucleosome occupancy at BEN-2 as measured by ChART-PCR with MNase digestion. Data was normalised to the inaccessible SFTPA2 gene promoter such that a value of 1.0 represents fully compacted nucleosomes, and lower values indicate less compacted nucleosomes. B. Chromatin accessibility at BEN-2 as measured by ChART-PCR with DNase I digestion. Data have been normalised to the inaccessible SFTPA2 gene promoter. C. H3K27ac enrichment at BEN-2 as determined by ChIP-qPCR using the percent input method. Grey bars indicate H3K27ac enrichment at the target locus, and black bars show enrichment using a non-specific IgG control antibody. All data are presented as mean ± SEM from at least 3 biological replicates.

    Journal: bioRxiv

    Article Title: Regulatory architecture of the RCA gene cluster captures an intragenic TAD boundary and enhancer elements in B cells

    doi: 10.1101/2020.02.16.941070

    Figure Lengend Snippet: BEN-2 shows B cell-specific nucleosome occupancy, chromatin accessibility and enrichment for the H3K27ac active enhancer histone mark across a panel of B cell lines. A. Nucleosome occupancy at BEN-2 as measured by ChART-PCR with MNase digestion. Data was normalised to the inaccessible SFTPA2 gene promoter such that a value of 1.0 represents fully compacted nucleosomes, and lower values indicate less compacted nucleosomes. B. Chromatin accessibility at BEN-2 as measured by ChART-PCR with DNase I digestion. Data have been normalised to the inaccessible SFTPA2 gene promoter. C. H3K27ac enrichment at BEN-2 as determined by ChIP-qPCR using the percent input method. Grey bars indicate H3K27ac enrichment at the target locus, and black bars show enrichment using a non-specific IgG control antibody. All data are presented as mean ± SEM from at least 3 biological replicates.

    Article Snippet: To assess nucleosome occupancy, 1000 Gel Units MNase (New England Biolabs) was used.

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