Journal: Scientific Reports

Article Title: Cell cycle–dependent localization of the proteasome to chromatin

doi: 10.1038/s41598-020-62697-2

Figure Lengend Snippet: Proteomics analysis shows that the nuclear abundance of the proteasome is reduced in association with oncogenic transformation. ( a ) Strategy for cell transformation and subcellular fractionation. TIG-3(WT) cells were transformed by the introduction of hTert and the early region of SV40. Cellular proteins of both TIG-3(WT) cells and the transformed cells, designated TIG-3(T + SV40), were separated into three fractions corresponding to the cytoplasm (S), the nucleoplasm and proteins loosely associated with chromatin (P1), and proteins tightly associated with chromatin (P2). Each fraction was analyzed by liquid chromatography and tandem mass spectrometry (LC-MS/MS). ( b ) Validation of subcellular fractionation. Whole cell extract (WCE) and subcellular fractions of TIG-3(WT) cells were subjected to immunoblot (IB) analysis with antibodies to LDHA, HSP90, α-tubulin, and calnexin as cytoplasmic marker proteins; to c-Jun and E2F1 as nucleoplasmic marker proteins; to histone H1 as a chromatin marker protein; and to the proteasome subunits PSMA2 and PSMD1. ( c ) Label-free proteomics analysis of P1 and P2 fractions. The log 2 [fold change] for protein abundance in TIG-3(T + SV40) cells relative to TIG-3(WT) cells and the –log 10 [q-value] are shown as Volcano plots. The threshold for determining differential expression is indicated by the dashed lines (q-value of ≤0.05). Proteasomal proteins are shown in red. ( d ) KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway–based enrichment analysis of the P1 fraction with Fisher’s exact test. The top three significantly ( P < 0.05) up-regulated or down-regulated KEGG pathways in TIG-3(T + SV40) cells relative to TIG-3(WT) cells are shown. ( e ) Absolut e quantification of proteasome subunits by iMPAQT analysis. The S and P1 fractions of TIG-3(WT), TIG-3(T), and TIG-3(T + SV40) cells were analyzed for the abundance of proteasome subunits of both the 20S and 19S complexes. Data are means ± s.d. for six independent biological replicates. * P < 0.05, ** P < 0.01 compared with TIG-3(WT); # P < 0.05, ## P < 0.01 compared with TIG-3(T) (one-way ANOVA followed by Bonferroni’s post hoc test).

Article Snippet: Primary antibodies included those to PSMA2 (Cell Signaling Technology, 2455), to PSMD1 (Abcam, 140682), to LDHA (Cell Signaling Technology, 3582), to c-Jun (Cell Signaling Technology, 9165), to histone H1 (Merck Millipore, 05–457), to E2F1 (Cell Signaling Technology, 3742), to SV40 LT and ST (Santa Cruz Biotechnology, sc-148), to HSP90 (BD Transduction Laboratories, 610419), and to α-tubulin (Thermo Fisher Scientific, 13–8000), and to calnexin (Medical and Biological Laboratories, M178–3).

Techniques: Transformation Assay, Fractionation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Western Blot, Marker, Expressing

Journal: Acta biochimica et biophysica Sinica

Article Title: Nrf2 is associated with the regulation of basal transcription activity of the BRCA1 gene.

doi: 10.1093/abbs/gmt001

Figure Lengend Snippet: Figure 2 Binding status of Nrf2 complex and histone accetylation on the BRCA1 promoter in HCC38 and MCF-7 cells (A) Relative BRCA1 mRNA level was detected in HCC38 and MCF-7 cells. GAPDH was used as an internal control. (B) Relative BRCA1 protein level was detected in HCC38 and MCF-7 cells. Tubulin was used as a control. (C) Binding status of Nrf2, CBP and p300 on the BRCA1 promoter in HCC38 and MCF-7 cells. ChIP assays were performed using antibody against Nrf2, CBP, and p300. n ¼ 3, *P , 0.05 vs. control. (D) Detection of Nrf2 complex on the BRCA1 promoter in HCC38 and MCF-7 cells. ChIP-ReChIP assay was performed. n ¼ 3, *P , 0.05 vs. control. (E) Histone accetylation on the BRCA1 promoter in HCC38 and MCF-7 cells. ChIP assays were performed using antibody against acetyl-H3, acetyl-H4. n ¼ 3, *P , 0.05 vs. control.

Article Snippet: The lysates were sonicated to shear the DNA to fragments of 200–600 bp, and subjected to Acta Biochim Biophys Sin (2013) | Volume 45 | Issue 3 | Page 180 immunoprecipitation with the following antibodies, respectively, Nrf2 (Santa Cruz), acetylated histone H3 (Abcam Inc., Cambridge, USA), CBP (Chemicon, Rosemont, USA), p300 (Millipore), acetylated histone H4, (Millipore), or IgG (Santa Cruz) as negative control.

Techniques: Binding Assay, Control

Journal: Acta biochimica et biophysica Sinica

Article Title: Nrf2 is associated with the regulation of basal transcription activity of the BRCA1 gene.

doi: 10.1093/abbs/gmt001

Figure Lengend Snippet: Figure 3 Over-expression of Nrf2 activates BRCA1 expression in HCC38 cells (A and B) Over-expression of Nrf2 in HCC38 cells activated BRCA1 expression. pCMV6-XL5 or Nrf2 expression vectors was transfected into HCC38 cells for 72 h and performed reverse transcription polymerase chain reaction (RT-PCR) (A) and western blot analysis (B). 1: HCC38 cells transfected with pCMV6-XL5; 2: HCC38 cells transfected with Nrf2 vector. (C) Binding status of Nrf2, CBP, and p300 on the BRCA1 promoter in control HCC38 cells and Nrf2 over-expression HCC38 cells. ChIP assays were performed using antibody against Nrf2, CBP, and p300. n ¼ 3, *P , 0.05 vs. control. (D) Detection of Nrf2 complex on the BRCA1 promoter in control HCC38 cells and Nrf2 over-expression HCC38 cells. ChIP-ReChIP assay was performed. n ¼ 3, *P , 0.05 vs. control. (E) Histone accetylation on the BRCA1 promoter in control HCC38 cells and Nrf2 over-expression HCC38 cells. ChIP assays were performed using antibody against acetyl-H3, acetyl-H4. n ¼ 3, *P , 0.05 vs. control.

Article Snippet: The lysates were sonicated to shear the DNA to fragments of 200–600 bp, and subjected to Acta Biochim Biophys Sin (2013) | Volume 45 | Issue 3 | Page 180 immunoprecipitation with the following antibodies, respectively, Nrf2 (Santa Cruz), acetylated histone H3 (Abcam Inc., Cambridge, USA), CBP (Chemicon, Rosemont, USA), p300 (Millipore), acetylated histone H4, (Millipore), or IgG (Santa Cruz) as negative control.

Techniques: Over Expression, Expressing, Transfection, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Binding Assay, Control

Journal: Journal of Hematology & Oncology

Article Title: Small-molecule survivin inhibitor YM155 enhances radiosensitization in esophageal squamous cell carcinoma by the abrogation of G 2 checkpoint and suppression of homologous recombination repair

doi: 10.1186/s13045-014-0062-8

Figure Lengend Snippet: YM155 potentiates the activation of Cdc2 kinase and suppresses protein expression related to G 2 /M checkpoint. A. Irradiated Eca109 cells were treated with YM155 in the absence or the presence of nocodazole. Cdc2 was immunoprecipitated using Cdc2 antibody and Cdc2 kinase activity was measured by Histone H1 kinase assay described. The relative Cdc2 kinase activity was shown in the histogram (right) (*, P < 0.05; **, P < 0.01). B. Eca109 and TE13 cells were treated with YM155, X ray or the combination modality and subjected to Western blot analysis of G 2 /M checkpoint related proteins phospho-Cdc2, Cdc2, cyclin B1, phospho-Cdc25C.

Article Snippet: The protein-agarose beads and bound immune complexes were then pelleted by centrifugation and immunoprecipitates were washed twice with lysis buffer and twice with wash buffer (50 mM Hepes/NaOH, pH 7.4, 10 mM MgCl 2 , 1 mM dithio-threitol) and then subjected to a Cdc2 kinase assay using histone H1 peptide (Merck Millipore, Darmstadt) as a substrate.

Techniques: Activation Assay, Expressing, Irradiation, Immunoprecipitation, Activity Assay, Kinase Assay, Western Blot

Journal: bioRxiv

Article Title: Histone H3.3 lysine 9 and 27 control repressive chromatin states at cryptic cis -regulatory elements and bivalent promoters in mouse embryonic stem cells

doi: 10.1101/2023.05.08.539859

Figure Lengend Snippet: Heatmaps displaying the relative abundance (row z-score) of individual or combinatorial post-translational modifications of histone H3.3 (a) , H3 (b) or H4 (c) . Lysine-to-alanine mutated H3.3 peptides were used as controls in panel “a”. The peptides length was omitted for clarity from the figure and is reported here: H3K4 (3-8); H3K9/K14 (9-17); H3K18/K23 (18-26); H3K27/K36 (27-40); H3K56 (54-63); H3K79 (73-83); H4K20 (20-23); H4K5/K8/K12/K16 (4-17); H4K20 (20-23).

Article Snippet: Antibodies used with this protocol were the following: H3.3 (09-838 – Merck-Millipore); H3K9me3 (D4W1U – Cell Signalling Technology); H3K9me2 (D85B4 – Cell Signalling Technology); H3K27me3 (C36B11 – Cell Signalling Technology); H3K18ac (9675 – Cell Signalling Technology); SUZ12 (D39F6-3737 – Cell Signalling Technology).

Techniques:

Journal: bioRxiv

Article Title: Histone H3.3 lysine 9 and 27 control repressive chromatin states at cryptic cis -regulatory elements and bivalent promoters in mouse embryonic stem cells

doi: 10.1101/2023.05.08.539859

Figure Lengend Snippet: (a) Volcano plot showing differential H3K9me3 ChIP-seq peaks in K9A vs. control mESCs (consensus peakset with n=48346 broad peaks) and ChromHMM annotation of DA-H3K9me3 regions. (b) Volcano plot showing differential H3K27ac ChIP-seq peaks in K9A vs. control mESCs (consensus peakset with n=94000 peaks) and ChromHMM annotation of DA-H3K27ac regions. (c) Heatmaps of H3K9me3 and H3K27ac ChIP-seq signals at H3K9me3 regions (summit ± 5 kb). Differentially abundant regions are divided into two clusters (k-means clustering) and sorted from a high-to-low H3K9me3 signal in control. ChIP-seq signal calculated as the average of three replicates for control or mutant mESCs, in 250 bp sliding genomic windows. On the right, metaprofile plots with H3K9me3, H3K27ac, H3K9ac, and H3.3 average ChIP-seq signals are reported. (d) STRING network analysis of chromatin and transcription factors enriched at Cluster 1 (top) and Cluster 2 (bottom) DA-H3K9me3 regions. Colors indicate the chromatin complex/category (red: transcriptional co-activator; orange: chromatin remodeler/elongation complex; yellow: pluripotency TF; blue: developmental TF; purple: histone demethylase; green: heterochromatin factor; dark-blue: transcriptional repressor; light-blue: Polycomb subunit). STRING settings: physical subnetwork (the edges indicate that the proteins are part of a physical complex) – medium confidence (interaction score 0.4). (e) Nascent transcription signal at DA-H3K9me3 regions reported as log2(RPKM) values. P value: unpaired t-test (***: p-value<0.001; ns: p-value>0.05). PRO-seq experiments were performed independently for K27A and K9A. Control samples were repeated in each batch and reported in two different gray shades.

Article Snippet: Antibodies used with this protocol were the following: H3.3 (09-838 – Merck-Millipore); H3K9me3 (D4W1U – Cell Signalling Technology); H3K9me2 (D85B4 – Cell Signalling Technology); H3K27me3 (C36B11 – Cell Signalling Technology); H3K18ac (9675 – Cell Signalling Technology); SUZ12 (D39F6-3737 – Cell Signalling Technology).

Techniques: ChIP-sequencing, Mutagenesis

Journal: bioRxiv

Article Title: Histone H3.3 lysine 9 and 27 control repressive chromatin states at cryptic cis -regulatory elements and bivalent promoters in mouse embryonic stem cells

doi: 10.1101/2023.05.08.539859

Figure Lengend Snippet: (a) Stacked barplot showing the percentage of DA-H3K9me3 regions with at least one ERV. P value: a two-proportions Z-test. (b) Stacked barplot indicating the percentage of LTR elements with significant (pval<0.01) H3.3 enrichment (left), and the classification of ERV families with significant H3.3 signal (right). (c-e) Violin plot displaying (c) H3.3, (d) H3K9me3, (e) H3K27ac log2(Fold-change) signal at ERVs subfamilies (n=108), in control, K27A and K9A mESCs. Individual ERV subfamilies (data points) are colored to indicate if the ChIP enrichment is significant (gray/red/orange) or not (purple). P value: unpaired t-test. (f) Volcano plot showing differential LTRs expression in K9A vs. control mESCs. (g) Heatmap displaying H3K27ac log2(Fold-change) enrichment at four selected ERV subfamilies (complete heatmap in ). (h) Genome browser snapshot of a representative Cluster 1 DA-H3K9me3 region (highlighted), linked through the GRN to the Tlr2 gene; H3K27me3, H3K27ac, H3K9me3, and mRNA-seq tracks are shown. For ERVs annotation, only the name of the ERV located within the DA-H3K9me3 region is shown. (i) PRO-seq signal at Weak, Medium, and Strong dREG enhancers included in the GRN for K9A and control mESCs. Log2(RPKM) values are reported. P value: unpaired t-test. (j) Expression of DEGs connected to dREG enhancers through the GRN; log2(fold-change) values calculated with DESeq2 are plotted, and individual genes (data points) are colored to indicate if the differential expression is significant (orange) or not (purple). (k) Model of cis -regulatory elements rewiring in H3.3K9A mESCs. P value in a,c-e,i : ***: p-value<0.001; **: p-value<0.01; ns: p-value>0.05. Data from three biological replicates of control, K9A, or K27A clonal lines.

Article Snippet: Antibodies used with this protocol were the following: H3.3 (09-838 – Merck-Millipore); H3K9me3 (D4W1U – Cell Signalling Technology); H3K9me2 (D85B4 – Cell Signalling Technology); H3K27me3 (C36B11 – Cell Signalling Technology); H3K18ac (9675 – Cell Signalling Technology); SUZ12 (D39F6-3737 – Cell Signalling Technology).

Techniques: Expressing

Journal: bioRxiv

Article Title: Histone H3.3 lysine 9 and 27 control repressive chromatin states at cryptic cis -regulatory elements and bivalent promoters in mouse embryonic stem cells

doi: 10.1101/2023.05.08.539859

Figure Lengend Snippet: (a) Stacked barplot indicating the percentage of transposable elements with or without significant (pval<0.01) H3.3 enrichment over input in control mESCs. (b) Boxplot of H3.3 log2(Fold-change) enrichment at ERVs families and SINE elements, displaying significant H3.3 signal (from panel a and ) (c) H3K9me3 ChIP-qPCR with primers for ERVs (left) or other transposable elements (right). Rosa26 locus was used as a negative control for H3K9me3 enrichment. (d) Real-time qPCR using primers for selected transposable elements. (e) Principal component analysis of LTRs expression. (f) Volcano plot showing differential ERVs expression in K27A mESCs. (g) Heatmaps displaying H3.3, H3K9me3 and H3K27ac log2(Fold-change) enrichment at ERVs subfamilies with highest H3.3 signal (pval<0.01 & log2FC>1; n=108). ERVs subfamilies are divided in six groups, and sorted in each group by decreasing H3.3 signal in control mESCs. For each subfamily, the corresponding ERV family is reported and color-coded (ERVK in yellow; ERV1 in green and Gypsy in purple).

Article Snippet: Antibodies used with this protocol were the following: H3.3 (09-838 – Merck-Millipore); H3K9me3 (D4W1U – Cell Signalling Technology); H3K9me2 (D85B4 – Cell Signalling Technology); H3K27me3 (C36B11 – Cell Signalling Technology); H3K18ac (9675 – Cell Signalling Technology); SUZ12 (D39F6-3737 – Cell Signalling Technology).

Techniques: Negative Control, Expressing

Journal: bioRxiv

Article Title: Histone H3.3 lysine 9 and 27 control repressive chromatin states at cryptic cis -regulatory elements and bivalent promoters in mouse embryonic stem cells

doi: 10.1101/2023.05.08.539859

Figure Lengend Snippet: (a-d) Metaprofile plot of (a) H3.3, (b) H3K27me3, (c) H3K27ac, and (d) Suz12 ChIP-seq signal at TSS ± 5kb of bivalent genes (n=4117). (e) Analysis of H3K27me3 signal within SUZ12 peaks (n=3356) and in the 1.5 kb genomic region immediately upstream or downstream. (f) Metaprofile plot of H3K9me2 (left) and H3K9me3 (right) ChIP-seq signal at TSS ± 5kb of bivalent genes (n=4117). (g) Histone methyltransferase assays of PRC2 in the presence of chromatin reconstituted using different H3 variants and H3K9 modifications. In all cases, “me2” and “me3” represent the respective methyl lysine analog (MLA), and “-“ represents an unmodified lysine residue. Barplot represents the mean densitometry values as recorded from the radiograms (see ). The error bars represent the standard deviation over three replicates carried out on different days. P value: Tukey’s multiple comparisons test (****: P<0.0001, ***: P<0.001, **: P<0.005, *: P<0.05). (h) Stacked barplot indicating the percentage of genes with or without bivalent promoters (left), and details of significant differential expression (right). (i) Genome browser snapshot of a representative Cluster 2 DA-H3K9me3 region (highlighted), linked through the GRN to the Tbx20 gene; H3K27me3, H3K27ac, H3K9me3, and mRNA-seq tracks are shown. For metaprofiles in panels a-d and f , the ChIP-seq signal is calculated as the average of three biological replicates for control or mutant mESCs, in 250 bp sliding genomic windows.

Article Snippet: Antibodies used with this protocol were the following: H3.3 (09-838 – Merck-Millipore); H3K9me3 (D4W1U – Cell Signalling Technology); H3K9me2 (D85B4 – Cell Signalling Technology); H3K27me3 (C36B11 – Cell Signalling Technology); H3K18ac (9675 – Cell Signalling Technology); SUZ12 (D39F6-3737 – Cell Signalling Technology).

Techniques: ChIP-sequencing, Standard Deviation, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Histone H3.3 lysine 9 and 27 control repressive chromatin states at cryptic cis -regulatory elements and bivalent promoters in mouse embryonic stem cells

doi: 10.1101/2023.05.08.539859

Figure Lengend Snippet: (a) Volcano plots showing differential SUZ12 ChIP-seq peaks in K27A (left) or K9A (right) versus control (consensus peakset with n=3356 peaks). (b) Radiograms used to calculate mean values reported in for PRC2 methyltransferase assay. (c) Gene ontology enrichment analysis of bivalent genes upregulated only in K9A (left) or in K27A/K27A+K9A mESCs (right). (d) H3.3 ChIP-seq signal in control mESCs, at the promoter regions of bivalent genes, upregulated only in K9A or K27A/K27A+K9A. A group of comparable size with non-significantly differentially expressed bivalent genes was randomly selected for comparison. Significance was calculated with unpaired t-test (***: p-value<0.001). (e) Genome browser snapshot of a representative Cluster 2 DA-H3K9me3 region (highlighted), linked through the GRN to the Frzb gene. For ERVs annotation, only the name of the ERV located within the DA-H3K9me3 region is shown; from left to right the other elements are: RMER10A, RLTR20B2, RLTR13B1 and RMER15-int.

Article Snippet: Antibodies used with this protocol were the following: H3.3 (09-838 – Merck-Millipore); H3K9me3 (D4W1U – Cell Signalling Technology); H3K9me2 (D85B4 – Cell Signalling Technology); H3K27me3 (C36B11 – Cell Signalling Technology); H3K18ac (9675 – Cell Signalling Technology); SUZ12 (D39F6-3737 – Cell Signalling Technology).

Techniques: ChIP-sequencing

Journal: Nature Cell Biology

Article Title: Aberrant chromatin landscape following loss of the H3.3 chaperone Daxx in haematopoietic precursors leads to Pu.1-mediated neutrophilia and inflammation

doi: 10.1038/s41556-021-00774-y

Figure Lengend Snippet: a , Genotyping of Daxx +/+ ;Mx1Cre +/- and Daxx F/F ;Mx1Cre +/- mice untreated (-) or treated with pI:pC (+). Arrows indicate the wild-type, floxed and recombined alleles ( n = 5 independent experiments). b , Daxx mRNA levels upon pI:pC treatment in bone marrow. Shown is relative expression of Daxx mRNA over Tbp mRNA levels ( n = 3 mice per genotype). c , IF of Daxx in bone marrow, scale bar = 10 µm ( n = 4 mice per genotype, two independent experiments). Inlay image zoomed on representative nuclei. d , Western blot of Daxx, alpha-tubulin and H3.3 in Lineage-negative and Lineage-positive bone marrow cells ( n = 2 mice per genotype). e , Bone marrow cellularity counts before and after RBC lysis in male mice ( n = 5 mice per gender, non-parametric Mann–Whitney test, box-and-whiskers plot: min to max (whiskers), 25 th and 75 th percentile and median). f , Bone marrow cellularity counts before and after RBC lysis in female mice ( n = 5 mice per gender, non-parametric Mann–Whitney test, box-and-whiskers plot: min to max (whiskers), 25 th and 75 th percentile and median). g , Flow cytometry analysis of Ki-67 + cells in BM and HPCs ( n = 5 mice per genotype, repeated in two independent experiments, non-parametric Mann–Whitney test). h,i , Flow cytometry analysis of HSC and MPP populations ( n = 3 mice per genotype, repeated in two independent experiments, Student’s t-test). BM, bone marrow; HPC, haematopoietic progenitor cells; HSC, haematopoietic stem cell; MPP, multipotent progenitors. Data in box plots are mean and min to max. ns, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Exact p-values and numerical source data can be found in the accompanying source data. Unprocessed blots provided in Source data.

Article Snippet: We used antibodies, at a 1:20 dilution, to H3K9me3 (Active Motif), H3K27ac (Epicypher), H3K27me3 (Cell Signaling Technologies), histone H3.3 (Merck Millipore) and Pu.1 (Abcam).

Techniques: Expressing, Western Blot, Lysis, MANN-WHITNEY, Flow Cytometry

Journal: Nature Cell Biology

Article Title: Aberrant chromatin landscape following loss of the H3.3 chaperone Daxx in haematopoietic precursors leads to Pu.1-mediated neutrophilia and inflammation

doi: 10.1038/s41556-021-00774-y

Figure Lengend Snippet: a , Flow cytometry analysis of myeloid cell surface markers ( n = 6 mice per genotype, repeated in three independent experiments). b , Flow cytometry analysis of lymphoid cell surface markers ( n = 6 mice per genotype, repeated in three independent experiments). c , Frequencies of B cell progenitors ( n = 6 mice per genotype, repeated in three independent experiments. d , Western blot of Daxx, H3.3 and corresponding β-actin control in isolated B220 + cells from bone marrow ( n = 3 mice per genotype, repeated in two independent experiments). e , Bones isolated from two Daxx +/+ ;Mx1Cre and Daxx F/F ;Mx1Cre mice. f , Frequencies of erythroblast populations ( n = 6 mice per genotype, repeated in three independent experiments. Data in box plots are mean and min to max. ns, not significant; * P < 0.05,d ** P < 0.01, *** P < 0.001, **** P < 0.0001, Wilcoxon rank test. Exact p-values and numerical source data can be found in the accompanying source data. Source image file provided in Source data.

Article Snippet: We used antibodies, at a 1:20 dilution, to H3K9me3 (Active Motif), H3K27ac (Epicypher), H3K27me3 (Cell Signaling Technologies), histone H3.3 (Merck Millipore) and Pu.1 (Abcam).

Techniques: Flow Cytometry, Western Blot, Isolation

Journal: Nature Cell Biology

Article Title: Aberrant chromatin landscape following loss of the H3.3 chaperone Daxx in haematopoietic precursors leads to Pu.1-mediated neutrophilia and inflammation

doi: 10.1038/s41556-021-00774-y

Figure Lengend Snippet: a , Overview of altered H3.3 distribution and Pu.1 binding in Daxx-KO HSPCs determined by CUT&Tag assays. b , Heatmaps of ATAC-seq read distribution around the centre of H3.3-depleted enhancers. c , H3K27ac, H3K9me3 and Pu.1 CUT&Tag read distribution across H3.3-depleted enhancers (left) as well as Pu.1, ATAC-seq and H3.3 coverage around the TSS of genes close to H3.3-depleted enhancers (right). Pu.1 coverage was stratified into three clusters by k -means and ATAC-seq read distribution was plotted for the same clusters of TSS. d , Summary plot of IPA analysis for genes close to distal regions with altered Pu.1 binding. Predicted activation of the protein or biofunction is indicated in orange and predicted inhibition of the displayed protein or biofunction in blue. e , Graphical depiction of Pu.1-binding changes at distal or proximal sites close to transcription factors regulated by Pu.1. The regions with increased Pu.1 binding are shown in red and regions with decreased Pu.1 binding in green. f , Enrichment plots for ATAC-seq, H3K9me3 CUT&Tag and H3K27ac CUT&Tag at enhancers overlapping and not overlapping ERVs with increased accessibility. g , Enrichment plots for ATAC-seq, H3K9me3 CUT&Tag and H3K27ac CUT&Tag at ERVs with increased accessibility. h , Heatmaps of enhancers with increased H3K27ac showing cluster analysis with H3.3. i , Heatmaps of ERVs with increased H3K27ac showing cluster analysis with H3.3. h , i , The legend for the heatmaps is the same as b and middle graphs show cluster of enhancers or ERVs/RTEs with no difference in H3.3 while right graphs show those with reduced H3.3 loading. Daxx F/F, Daxx KO and Daxx +/+, Daxx WT. Numerical source data are provided.

Article Snippet: We used antibodies, at a 1:20 dilution, to H3K9me3 (Active Motif), H3K27ac (Epicypher), H3K27me3 (Cell Signaling Technologies), histone H3.3 (Merck Millipore) and Pu.1 (Abcam).

Techniques: Binding Assay, Activation Assay, Inhibition

Journal: Nature Cell Biology

Article Title: Aberrant chromatin landscape following loss of the H3.3 chaperone Daxx in haematopoietic precursors leads to Pu.1-mediated neutrophilia and inflammation

doi: 10.1038/s41556-021-00774-y

Figure Lengend Snippet: a , PCA of the top-500 most variable genes in KLS cells collected at 3 w.p.i. b , PCA of the top-500 most variable genes in GMP cells collected at 3 w.p.i. c , Top-five activated or inhibited haematological functions and diseases associated with KLS cells with Daxx and Pu.1 DKO- (left) and Pu.1-KO-specific (right) gene expression changes. d , Top-five activated or inhibited haematological functions and diseases associated with GMP cells with DKO- (left) and Pu.1-KO-specific (right) gene expression changes. c , d , Data are the activation Z -score from IPA Fisher’s exact tests with multiple testing-adjusted P < 0.05. An activation Z -score > 2 suggests increased activation of the indicated biofunctions and an activation Z -score < −2 suggests increased inhibition. e , Normalized read counts of ERV/RTE subtypes and satellite repeats ( n = 2 mice per genotype) in KLS cells. f , Normalized read counts of ERV/RTE subtypes and satellite repeats ( n = 2 mice per genotype) in GMP cells. e , f , Boxplots show the minimum and maximum values (box boundaries) and the mean (horizontal line). g , Number of regions that were opened or closed in Daxx-KO versus WT (left) and the number of those regions that reverted back to WT condition in DKO KLS cells (right). h , ATAC-seq read distribution around the centre of IDR-reproducible peaks identified in WT (top), Daxx-KO (middle) and DKO (bottom) KLS cells. i , Heatmap of scaled normalized RNA-seq read counts for genes upregulated in Daxx single-KO KLS and increased accessibility in nearby enhancers. j , Heatmaps of H3K9me3 enrichment over distal regions segregated in regions gaining or losing H3K9me3. k , H3.3 enrichment plots at enhancers (top) and ERVs (bottom). l , Genome browser coverage plot of the Fbp1 and Fbp2 locus. Daxx F/F, Daxx KO and Daxx +/+, Daxx WT; Pu.1 F/F, Pu.1-KO and Pu.1 +/+, Pu.1 WT. Numerical source data are provided.

Article Snippet: We used antibodies, at a 1:20 dilution, to H3K9me3 (Active Motif), H3K27ac (Epicypher), H3K27me3 (Cell Signaling Technologies), histone H3.3 (Merck Millipore) and Pu.1 (Abcam).

Techniques: Expressing, Activation Assay, Inhibition, RNA Sequencing Assay