arid1a  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc arid1a
    Cellular proliferation by AKT phosphorylation is induced by <t>ARID1A</t> knockdown. Notes: ( A ) Western blotting for the screening of ARID1A in gastric cancer cell lines. ( B – D ) After transfection of ARID1A siRNA, cell viabilities were significantly increased ( ** P <0.001; paired t -test). Knockdown of ARID1A increased the phosphorylation of AKT and the downstream S6.
    Arid1a, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    1) Product Images from "AKT inhibition is an effective treatment strategy in ARID1A-deficient gastric cancer cells"

    Article Title: AKT inhibition is an effective treatment strategy in ARID1A-deficient gastric cancer cells

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S139664

    Cellular proliferation by AKT phosphorylation is induced by ARID1A knockdown. Notes: ( A ) Western blotting for the screening of ARID1A in gastric cancer cell lines. ( B – D ) After transfection of ARID1A siRNA, cell viabilities were significantly increased ( ** P <0.001; paired t -test). Knockdown of ARID1A increased the phosphorylation of AKT and the downstream S6.
    Figure Legend Snippet: Cellular proliferation by AKT phosphorylation is induced by ARID1A knockdown. Notes: ( A ) Western blotting for the screening of ARID1A in gastric cancer cell lines. ( B – D ) After transfection of ARID1A siRNA, cell viabilities were significantly increased ( ** P <0.001; paired t -test). Knockdown of ARID1A increased the phosphorylation of AKT and the downstream S6.

    Techniques Used: Western Blot, Transfection

    ARID1A depletion leads to increased sensitivity toward AKT pathway inhibitors. Notes: Increased sensitivity of ARID1A-depleted MKN-1, MKN-28, and KATO-III cells toward GSK690693 (AKT inhibitor) was observed than that of controls. * P <0.001; paired t -test. Abbreviation: NC, normal control.
    Figure Legend Snippet: ARID1A depletion leads to increased sensitivity toward AKT pathway inhibitors. Notes: Increased sensitivity of ARID1A-depleted MKN-1, MKN-28, and KATO-III cells toward GSK690693 (AKT inhibitor) was observed than that of controls. * P <0.001; paired t -test. Abbreviation: NC, normal control.

    Techniques Used:

    Loss of ARID1A expression is associated with high sensitivity to the AKT inhibitor in gastric cancer cell lines. Notes: ARID1A-deficient MKN-45 cells showed the highest sensitivity toward GSK690693 treatment. The IC 50 value of MKN-45 was 0.043, while the IC 50 values of the ARID1A-intact MKN-1, MKN-28, and KATO-III cells were 0.132, 0.084, and 4.521, respectively ( P <0.001). The P -value indicates the divergence of the IC 50 values calculated by the F -test. Abbreviation: IC 50 , half inhibitory concentration.
    Figure Legend Snippet: Loss of ARID1A expression is associated with high sensitivity to the AKT inhibitor in gastric cancer cell lines. Notes: ARID1A-deficient MKN-45 cells showed the highest sensitivity toward GSK690693 treatment. The IC 50 value of MKN-45 was 0.043, while the IC 50 values of the ARID1A-intact MKN-1, MKN-28, and KATO-III cells were 0.132, 0.084, and 4.521, respectively ( P <0.001). The P -value indicates the divergence of the IC 50 values calculated by the F -test. Abbreviation: IC 50 , half inhibitory concentration.

    Techniques Used: Expressing, Concentration Assay

    AKT inhibition leads to increased apoptosis in ARID1A-deficient cells. Notes: ( A ) Treatment with the AKT inhibitor GSK690693 (at a concentration of 10 μmol/L) completely abrogated p-Akt induced by ARID1A knockdown in ARID1A-deficient MKN-28 cells and led to reduced p-S6, in contrast to the controls. PARP cleavage was more increased in ARID1A-knockdown cells treated with GSK690693. ( B ) Flow cytometry confirmed the increased apoptosis in ARID1A-deficient cells treated with GSK690693 (0.01 μmol/L) in contrast to the controls ( ** P <0.001; paired t -test). Abbreviations: FITC, fluorescein isothiocyanate; PARP, poly-ADP ribose polymerase.
    Figure Legend Snippet: AKT inhibition leads to increased apoptosis in ARID1A-deficient cells. Notes: ( A ) Treatment with the AKT inhibitor GSK690693 (at a concentration of 10 μmol/L) completely abrogated p-Akt induced by ARID1A knockdown in ARID1A-deficient MKN-28 cells and led to reduced p-S6, in contrast to the controls. PARP cleavage was more increased in ARID1A-knockdown cells treated with GSK690693. ( B ) Flow cytometry confirmed the increased apoptosis in ARID1A-deficient cells treated with GSK690693 (0.01 μmol/L) in contrast to the controls ( ** P <0.001; paired t -test). Abbreviations: FITC, fluorescein isothiocyanate; PARP, poly-ADP ribose polymerase.

    Techniques Used: Inhibition, Concentration Assay, Flow Cytometry

    Loss of ARID1A expression did not induce resistance to the conventional chemotherapy. Notes: To investigate the antiproliferative effect of conventional chemotherapy in ARID1A-depleted GC cells, 5-FU or cisplatin was applied at different drug concentrations (10–60 μmol/L) for 48 hours to MKN-1, MKN-28, and KATO-III cells transfected with control-shRNA and shARID1A. Drug sensitivities did not differ between these groups. Abbreviations: 5-FU, 5-fluorouracil; GC, gastric cancer.
    Figure Legend Snippet: Loss of ARID1A expression did not induce resistance to the conventional chemotherapy. Notes: To investigate the antiproliferative effect of conventional chemotherapy in ARID1A-depleted GC cells, 5-FU or cisplatin was applied at different drug concentrations (10–60 μmol/L) for 48 hours to MKN-1, MKN-28, and KATO-III cells transfected with control-shRNA and shARID1A. Drug sensitivities did not differ between these groups. Abbreviations: 5-FU, 5-fluorouracil; GC, gastric cancer.

    Techniques Used: Expressing, Transfection, shRNA

    Addition of AKT inhibitors to conventional chemotherapy increases antitumor activity in ARID1A-deficient cancer cells. Notes: ( A ) 5-FU (10 μmol/L) or cisplatin (10 μmol/L) was applied to MKN-28 and KATO-III cells in the presence of a minimal drug concentration of GSK690693 (0.01 μmol/L). Compared with single agent alone, addition of GSK690693 to the conventional chemotherapy induced more decreased cell viability in ARID1A-knockdown MKN-28 and KATO-III cells than in wild-type cells ( * P <0.01). ( B ) GSK690693 in combination with 5-FU or cisplatin induced a significant increase in apoptosis, compared with 5-FU or cisplatin alone in ARID1A-knockdown cells ( * P <0.01). ** P <0.001. Abbreviation: 5-FU, 5-fluorouracil.
    Figure Legend Snippet: Addition of AKT inhibitors to conventional chemotherapy increases antitumor activity in ARID1A-deficient cancer cells. Notes: ( A ) 5-FU (10 μmol/L) or cisplatin (10 μmol/L) was applied to MKN-28 and KATO-III cells in the presence of a minimal drug concentration of GSK690693 (0.01 μmol/L). Compared with single agent alone, addition of GSK690693 to the conventional chemotherapy induced more decreased cell viability in ARID1A-knockdown MKN-28 and KATO-III cells than in wild-type cells ( * P <0.01). ( B ) GSK690693 in combination with 5-FU or cisplatin induced a significant increase in apoptosis, compared with 5-FU or cisplatin alone in ARID1A-knockdown cells ( * P <0.01). ** P <0.001. Abbreviation: 5-FU, 5-fluorouracil.

    Techniques Used: Activity Assay, Concentration Assay

    arid1a  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc arid1a
    Cellular proliferation by AKT phosphorylation is induced by <t>ARID1A</t> knockdown. Notes: ( A ) Western blotting for the screening of ARID1A in gastric cancer cell lines. ( B – D ) After transfection of ARID1A siRNA, cell viabilities were significantly increased ( ** P <0.001; paired t -test). Knockdown of ARID1A increased the phosphorylation of AKT and the downstream S6.
    Arid1a, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/arid1a/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    arid1a - by Bioz Stars, 2023-02
    94/100 stars

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    1) Product Images from "AKT inhibition is an effective treatment strategy in ARID1A-deficient gastric cancer cells"

    Article Title: AKT inhibition is an effective treatment strategy in ARID1A-deficient gastric cancer cells

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S139664

    Cellular proliferation by AKT phosphorylation is induced by ARID1A knockdown. Notes: ( A ) Western blotting for the screening of ARID1A in gastric cancer cell lines. ( B – D ) After transfection of ARID1A siRNA, cell viabilities were significantly increased ( ** P <0.001; paired t -test). Knockdown of ARID1A increased the phosphorylation of AKT and the downstream S6.
    Figure Legend Snippet: Cellular proliferation by AKT phosphorylation is induced by ARID1A knockdown. Notes: ( A ) Western blotting for the screening of ARID1A in gastric cancer cell lines. ( B – D ) After transfection of ARID1A siRNA, cell viabilities were significantly increased ( ** P <0.001; paired t -test). Knockdown of ARID1A increased the phosphorylation of AKT and the downstream S6.

    Techniques Used: Western Blot, Transfection

    ARID1A depletion leads to increased sensitivity toward AKT pathway inhibitors. Notes: Increased sensitivity of ARID1A-depleted MKN-1, MKN-28, and KATO-III cells toward GSK690693 (AKT inhibitor) was observed than that of controls. * P <0.001; paired t -test. Abbreviation: NC, normal control.
    Figure Legend Snippet: ARID1A depletion leads to increased sensitivity toward AKT pathway inhibitors. Notes: Increased sensitivity of ARID1A-depleted MKN-1, MKN-28, and KATO-III cells toward GSK690693 (AKT inhibitor) was observed than that of controls. * P <0.001; paired t -test. Abbreviation: NC, normal control.

    Techniques Used:

    Loss of ARID1A expression is associated with high sensitivity to the AKT inhibitor in gastric cancer cell lines. Notes: ARID1A-deficient MKN-45 cells showed the highest sensitivity toward GSK690693 treatment. The IC 50 value of MKN-45 was 0.043, while the IC 50 values of the ARID1A-intact MKN-1, MKN-28, and KATO-III cells were 0.132, 0.084, and 4.521, respectively ( P <0.001). The P -value indicates the divergence of the IC 50 values calculated by the F -test. Abbreviation: IC 50 , half inhibitory concentration.
    Figure Legend Snippet: Loss of ARID1A expression is associated with high sensitivity to the AKT inhibitor in gastric cancer cell lines. Notes: ARID1A-deficient MKN-45 cells showed the highest sensitivity toward GSK690693 treatment. The IC 50 value of MKN-45 was 0.043, while the IC 50 values of the ARID1A-intact MKN-1, MKN-28, and KATO-III cells were 0.132, 0.084, and 4.521, respectively ( P <0.001). The P -value indicates the divergence of the IC 50 values calculated by the F -test. Abbreviation: IC 50 , half inhibitory concentration.

    Techniques Used: Expressing, Concentration Assay

    AKT inhibition leads to increased apoptosis in ARID1A-deficient cells. Notes: ( A ) Treatment with the AKT inhibitor GSK690693 (at a concentration of 10 μmol/L) completely abrogated p-Akt induced by ARID1A knockdown in ARID1A-deficient MKN-28 cells and led to reduced p-S6, in contrast to the controls. PARP cleavage was more increased in ARID1A-knockdown cells treated with GSK690693. ( B ) Flow cytometry confirmed the increased apoptosis in ARID1A-deficient cells treated with GSK690693 (0.01 μmol/L) in contrast to the controls ( ** P <0.001; paired t -test). Abbreviations: FITC, fluorescein isothiocyanate; PARP, poly-ADP ribose polymerase.
    Figure Legend Snippet: AKT inhibition leads to increased apoptosis in ARID1A-deficient cells. Notes: ( A ) Treatment with the AKT inhibitor GSK690693 (at a concentration of 10 μmol/L) completely abrogated p-Akt induced by ARID1A knockdown in ARID1A-deficient MKN-28 cells and led to reduced p-S6, in contrast to the controls. PARP cleavage was more increased in ARID1A-knockdown cells treated with GSK690693. ( B ) Flow cytometry confirmed the increased apoptosis in ARID1A-deficient cells treated with GSK690693 (0.01 μmol/L) in contrast to the controls ( ** P <0.001; paired t -test). Abbreviations: FITC, fluorescein isothiocyanate; PARP, poly-ADP ribose polymerase.

    Techniques Used: Inhibition, Concentration Assay, Flow Cytometry

    Loss of ARID1A expression did not induce resistance to the conventional chemotherapy. Notes: To investigate the antiproliferative effect of conventional chemotherapy in ARID1A-depleted GC cells, 5-FU or cisplatin was applied at different drug concentrations (10–60 μmol/L) for 48 hours to MKN-1, MKN-28, and KATO-III cells transfected with control-shRNA and shARID1A. Drug sensitivities did not differ between these groups. Abbreviations: 5-FU, 5-fluorouracil; GC, gastric cancer.
    Figure Legend Snippet: Loss of ARID1A expression did not induce resistance to the conventional chemotherapy. Notes: To investigate the antiproliferative effect of conventional chemotherapy in ARID1A-depleted GC cells, 5-FU or cisplatin was applied at different drug concentrations (10–60 μmol/L) for 48 hours to MKN-1, MKN-28, and KATO-III cells transfected with control-shRNA and shARID1A. Drug sensitivities did not differ between these groups. Abbreviations: 5-FU, 5-fluorouracil; GC, gastric cancer.

    Techniques Used: Expressing, Transfection, shRNA

    Addition of AKT inhibitors to conventional chemotherapy increases antitumor activity in ARID1A-deficient cancer cells. Notes: ( A ) 5-FU (10 μmol/L) or cisplatin (10 μmol/L) was applied to MKN-28 and KATO-III cells in the presence of a minimal drug concentration of GSK690693 (0.01 μmol/L). Compared with single agent alone, addition of GSK690693 to the conventional chemotherapy induced more decreased cell viability in ARID1A-knockdown MKN-28 and KATO-III cells than in wild-type cells ( * P <0.01). ( B ) GSK690693 in combination with 5-FU or cisplatin induced a significant increase in apoptosis, compared with 5-FU or cisplatin alone in ARID1A-knockdown cells ( * P <0.01). ** P <0.001. Abbreviation: 5-FU, 5-fluorouracil.
    Figure Legend Snippet: Addition of AKT inhibitors to conventional chemotherapy increases antitumor activity in ARID1A-deficient cancer cells. Notes: ( A ) 5-FU (10 μmol/L) or cisplatin (10 μmol/L) was applied to MKN-28 and KATO-III cells in the presence of a minimal drug concentration of GSK690693 (0.01 μmol/L). Compared with single agent alone, addition of GSK690693 to the conventional chemotherapy induced more decreased cell viability in ARID1A-knockdown MKN-28 and KATO-III cells than in wild-type cells ( * P <0.01). ( B ) GSK690693 in combination with 5-FU or cisplatin induced a significant increase in apoptosis, compared with 5-FU or cisplatin alone in ARID1A-knockdown cells ( * P <0.01). ** P <0.001. Abbreviation: 5-FU, 5-fluorouracil.

    Techniques Used: Activity Assay, Concentration Assay

    anti arid1a  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti arid1a
    Genome-wide analysis of <t>H3.3-ARID1A</t> chromatin co-regulation. A Genomic annotation of 40,006 genome-wide H3.3 ChIP-seq peaks in 12Z cells ( n = 2). B Distribution of H3.3 peak widths. Median H3.3 peak width is 1830 bp. C Genome-wide overlap of ARID1A and H3.3 ChIP-seq peaks. D Genome-wide association between H3.3 and other previously measured chromatin features, per genomic bp, quantified as [observed / expected]. Statistic is hypergeometric enrichment. E Enrichment for H3.3 and ARID1A co-regulation across 18 chromatin states previously modeled via ChromHMM . Left, enrichment of H3.3 peaks; center, enrichment of H3.3+ARID1A binding; right, enrichment of ARID1A binding at sites with vs. without H3.3. Statistic is hypergeometric enrichment. F Left, ARID1A binding levels (ChIP/input fold-enrichment, FE) at H3.3+ vs. H3.3− ARID1A peaks. Right, H3.3 abundance (ChIP/input fold-enrichment) at ARID1A+ vs. ARID1A− H3.3 peaks. Statistic is two-tailed, unpaired Wilcoxon’s test. G Top, enrichment of H3.3 at genes promoter-proximally bound by ARID1A. Bottom, enrichment of ARID1A+H3.3 co-binding at genes DE following ARID1A loss (siARID1A treatment). Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. H Example hg38 browser shots of genes and regulatory elements co-regulated by H3.3 and ARID1A. y -axis is log-likelihood ratio (logLR) of assay signal (compared to input chromatin for ChIP-seq or background genome for ATAC-seq). Small bars under tracks indicate significant peak detection by MACS2 (FDR < 0.05). Super-enhancers were detected by ROSE from H3K27ac ChIP-seq. * p < 0.05, ** p < 0.01, *** p < 0.001
    Anti Arid1a, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti arid1a/product/Cell Signaling Technology Inc
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti arid1a - by Bioz Stars, 2023-02
    94/100 stars

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    1) Product Images from "ARID1A-dependent maintenance of H3.3 is required for repressive CHD4-ZMYND8 chromatin interactions at super-enhancers"

    Article Title: ARID1A-dependent maintenance of H3.3 is required for repressive CHD4-ZMYND8 chromatin interactions at super-enhancers

    Journal: BMC Biology

    doi: 10.1186/s12915-022-01407-y

    Genome-wide analysis of H3.3-ARID1A chromatin co-regulation. A Genomic annotation of 40,006 genome-wide H3.3 ChIP-seq peaks in 12Z cells ( n = 2). B Distribution of H3.3 peak widths. Median H3.3 peak width is 1830 bp. C Genome-wide overlap of ARID1A and H3.3 ChIP-seq peaks. D Genome-wide association between H3.3 and other previously measured chromatin features, per genomic bp, quantified as [observed / expected]. Statistic is hypergeometric enrichment. E Enrichment for H3.3 and ARID1A co-regulation across 18 chromatin states previously modeled via ChromHMM . Left, enrichment of H3.3 peaks; center, enrichment of H3.3+ARID1A binding; right, enrichment of ARID1A binding at sites with vs. without H3.3. Statistic is hypergeometric enrichment. F Left, ARID1A binding levels (ChIP/input fold-enrichment, FE) at H3.3+ vs. H3.3− ARID1A peaks. Right, H3.3 abundance (ChIP/input fold-enrichment) at ARID1A+ vs. ARID1A− H3.3 peaks. Statistic is two-tailed, unpaired Wilcoxon’s test. G Top, enrichment of H3.3 at genes promoter-proximally bound by ARID1A. Bottom, enrichment of ARID1A+H3.3 co-binding at genes DE following ARID1A loss (siARID1A treatment). Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. H Example hg38 browser shots of genes and regulatory elements co-regulated by H3.3 and ARID1A. y -axis is log-likelihood ratio (logLR) of assay signal (compared to input chromatin for ChIP-seq or background genome for ATAC-seq). Small bars under tracks indicate significant peak detection by MACS2 (FDR < 0.05). Super-enhancers were detected by ROSE from H3K27ac ChIP-seq. * p < 0.05, ** p < 0.01, *** p < 0.001
    Figure Legend Snippet: Genome-wide analysis of H3.3-ARID1A chromatin co-regulation. A Genomic annotation of 40,006 genome-wide H3.3 ChIP-seq peaks in 12Z cells ( n = 2). B Distribution of H3.3 peak widths. Median H3.3 peak width is 1830 bp. C Genome-wide overlap of ARID1A and H3.3 ChIP-seq peaks. D Genome-wide association between H3.3 and other previously measured chromatin features, per genomic bp, quantified as [observed / expected]. Statistic is hypergeometric enrichment. E Enrichment for H3.3 and ARID1A co-regulation across 18 chromatin states previously modeled via ChromHMM . Left, enrichment of H3.3 peaks; center, enrichment of H3.3+ARID1A binding; right, enrichment of ARID1A binding at sites with vs. without H3.3. Statistic is hypergeometric enrichment. F Left, ARID1A binding levels (ChIP/input fold-enrichment, FE) at H3.3+ vs. H3.3− ARID1A peaks. Right, H3.3 abundance (ChIP/input fold-enrichment) at ARID1A+ vs. ARID1A− H3.3 peaks. Statistic is two-tailed, unpaired Wilcoxon’s test. G Top, enrichment of H3.3 at genes promoter-proximally bound by ARID1A. Bottom, enrichment of ARID1A+H3.3 co-binding at genes DE following ARID1A loss (siARID1A treatment). Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. H Example hg38 browser shots of genes and regulatory elements co-regulated by H3.3 and ARID1A. y -axis is log-likelihood ratio (logLR) of assay signal (compared to input chromatin for ChIP-seq or background genome for ATAC-seq). Small bars under tracks indicate significant peak detection by MACS2 (FDR < 0.05). Super-enhancers were detected by ROSE from H3K27ac ChIP-seq. * p < 0.05, ** p < 0.01, *** p < 0.001

    Techniques Used: Genome Wide, ChIP-sequencing, GWAS, Binding Assay, Two Tailed Test

    Genome-wide analysis of ARID1A-dependent H3.3. A MA plot of shARID1A vs. control differential H3.3 ChIP-seq ( n = 2), across 67,502 tested genomic regions. Regions are colored based on shARID1A differential H3.3 significance. Inset pie chart depicts distribution of significantly increasing and decreasing H3.3 regions ( csaw / edgeR FDR < 0.05) compared to stable H3.3 (FDR > 0.05). FDR < 0.05 was used as the significance threshold for all downstream analyses. B shARID1A differential H3.3 regions segregated by detection of ARID1A binding in wild-type cells. Left, MA plot with all genome-wide H3.3 tested regions, colored by ARID1A binding status. Right, box plot quantification of shARID1A log 2 FC H3.3 abundance, segregated by ARID1A binding status. Statistic is two-tailed, unpaired Wilcoxon’s test. C Analysis of canonical H3 (H3.1/3.2) changes (ChIP-seq, n = 2) at H3.3-marked genomic regions following ARID1A knockdown (shARID1A), segregated by ARID1A binding status as in B . Statistic is two-tailed, unpaired Wilcoxon’s test. D Enrichment of ARID1A binding detection at regions with decreasing H3.3 following ARID1A loss compared to all tested H3.3 regions. Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. E Magnitude of H3.3 change (log 2 FC) among ARID1A-bound, shARID1A significantly decreasing vs. increasing H3.3 regions. Statistic is two-tailed, unpaired Wilcoxon’s test. F Distribution of H3.3-enriched region widths among shARID1A stable vs. increasing vs. decreasing H3.3 regions. Statistic is two-tailed, unpaired Wilcoxon’s test. G Chromatin state enrichment among shARID1A increasing and decreasing H3.3 regions, calculated per 200 bp genomic interval. Statistic is hypergeometric enrichment. H Top 10 significant (FDR < 0.05) enriched Hallmark pathways (left) and GO Biological Process gene sets (right) among genes with ARID1A-bound, shARID1A decreasing promoter-proximal H3.3. I Representative hg38 locus near CCL2 displaying H3.3 maintained by ARID1A chromatin interactions. *** p < 0.001
    Figure Legend Snippet: Genome-wide analysis of ARID1A-dependent H3.3. A MA plot of shARID1A vs. control differential H3.3 ChIP-seq ( n = 2), across 67,502 tested genomic regions. Regions are colored based on shARID1A differential H3.3 significance. Inset pie chart depicts distribution of significantly increasing and decreasing H3.3 regions ( csaw / edgeR FDR < 0.05) compared to stable H3.3 (FDR > 0.05). FDR < 0.05 was used as the significance threshold for all downstream analyses. B shARID1A differential H3.3 regions segregated by detection of ARID1A binding in wild-type cells. Left, MA plot with all genome-wide H3.3 tested regions, colored by ARID1A binding status. Right, box plot quantification of shARID1A log 2 FC H3.3 abundance, segregated by ARID1A binding status. Statistic is two-tailed, unpaired Wilcoxon’s test. C Analysis of canonical H3 (H3.1/3.2) changes (ChIP-seq, n = 2) at H3.3-marked genomic regions following ARID1A knockdown (shARID1A), segregated by ARID1A binding status as in B . Statistic is two-tailed, unpaired Wilcoxon’s test. D Enrichment of ARID1A binding detection at regions with decreasing H3.3 following ARID1A loss compared to all tested H3.3 regions. Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. E Magnitude of H3.3 change (log 2 FC) among ARID1A-bound, shARID1A significantly decreasing vs. increasing H3.3 regions. Statistic is two-tailed, unpaired Wilcoxon’s test. F Distribution of H3.3-enriched region widths among shARID1A stable vs. increasing vs. decreasing H3.3 regions. Statistic is two-tailed, unpaired Wilcoxon’s test. G Chromatin state enrichment among shARID1A increasing and decreasing H3.3 regions, calculated per 200 bp genomic interval. Statistic is hypergeometric enrichment. H Top 10 significant (FDR < 0.05) enriched Hallmark pathways (left) and GO Biological Process gene sets (right) among genes with ARID1A-bound, shARID1A decreasing promoter-proximal H3.3. I Representative hg38 locus near CCL2 displaying H3.3 maintained by ARID1A chromatin interactions. *** p < 0.001

    Techniques Used: Genome Wide, ChIP-sequencing, Binding Assay, Two Tailed Test

    Transcriptional effects of H3.3 depletion and overlap with ARID1A. A Baseline relative linear expression of H3F3A ( H3-3A ) and H3F3B ( H3-3B ) gene isoforms encoding H3.3, as measured by RNA-seq ( n = 3). B Western blot for H3.3 and total H3 in control vs. siH3F3B treated cells. C Global transcriptomic effects of 24,192 genes following H3.3 knockdown via siH3F3B treatment (RNA-seq, n = 3). Red dots represent significant DE genes ( DESeq2 , FDR < 0.001). D Relative linear expression of H3F3A and H3F3B by RNA-seq in control and siH3F3B cells ( n = 3). E Volcano plot depicting siH3F3B vs. control differential gene expression (DGE). Top significant genes are labeled. F Significant overlap in DE genes following H3.3 knockdown (siH3F3B) vs. ARID1A knockdown (siARID1A). Statistic is hypergeometric enrichment. G Directional segregation of siH3F3B/siARID1A overlapping DE genes. A positive association is observed by chi-squared test, i.e., genes are more likely to be upregulated or downregulated in both conditions as opposed to antagonistic regulation. H Scatter plot of siH3F3B vs. siARID1A expression log 2 FC (with shrinkage correction) for all 19,900 transcriptome-wide commonly detected genes. Statistics are Pearson ( r ) and Spearman ( r s ) correlation coefficients. Colored dots indicate significant DE genes (FDR < 0.001) in both treatment conditions. I Association between H3.3 transcriptional repression (siH3F3B upregulation) and transcriptional co-regulation by ARID1A (siARID1A DE). Statistic is two-tailed Fisher’s exact test. J Scatter plot of 196 shared DE genes upregulated following knockdown of either H3.3 or ARID1A. These genes are mutually repressed by H3.3 and ARID1A. K Top significant (FDR < 0.05) enriched gene sets among the 196 ARID1A-H3.3 mutually repressed genes among various gene set databases
    Figure Legend Snippet: Transcriptional effects of H3.3 depletion and overlap with ARID1A. A Baseline relative linear expression of H3F3A ( H3-3A ) and H3F3B ( H3-3B ) gene isoforms encoding H3.3, as measured by RNA-seq ( n = 3). B Western blot for H3.3 and total H3 in control vs. siH3F3B treated cells. C Global transcriptomic effects of 24,192 genes following H3.3 knockdown via siH3F3B treatment (RNA-seq, n = 3). Red dots represent significant DE genes ( DESeq2 , FDR < 0.001). D Relative linear expression of H3F3A and H3F3B by RNA-seq in control and siH3F3B cells ( n = 3). E Volcano plot depicting siH3F3B vs. control differential gene expression (DGE). Top significant genes are labeled. F Significant overlap in DE genes following H3.3 knockdown (siH3F3B) vs. ARID1A knockdown (siARID1A). Statistic is hypergeometric enrichment. G Directional segregation of siH3F3B/siARID1A overlapping DE genes. A positive association is observed by chi-squared test, i.e., genes are more likely to be upregulated or downregulated in both conditions as opposed to antagonistic regulation. H Scatter plot of siH3F3B vs. siARID1A expression log 2 FC (with shrinkage correction) for all 19,900 transcriptome-wide commonly detected genes. Statistics are Pearson ( r ) and Spearman ( r s ) correlation coefficients. Colored dots indicate significant DE genes (FDR < 0.001) in both treatment conditions. I Association between H3.3 transcriptional repression (siH3F3B upregulation) and transcriptional co-regulation by ARID1A (siARID1A DE). Statistic is two-tailed Fisher’s exact test. J Scatter plot of 196 shared DE genes upregulated following knockdown of either H3.3 or ARID1A. These genes are mutually repressed by H3.3 and ARID1A. K Top significant (FDR < 0.05) enriched gene sets among the 196 ARID1A-H3.3 mutually repressed genes among various gene set databases

    Techniques Used: Expressing, RNA Sequencing Assay, Western Blot, Labeling, Two Tailed Test

    Characterization of ARID1A, CHD4, and ZMYND8 chromatin interactions co-regulating H3.3. A Genome-wide associations between ARID1A binding at H3.3+ vs. H3.3− regions for all 1135 transcriptional regulator peak sets included in the ReMap2020 peak database. Labeled factors exhibit an H3.3+ ARID1A binding association with genomic odds ratio >2 and overlap with >0.1% of ARID1A binding sites. ZMYND11 and ZMYND8 (bolded) are two of the top factors most associated with H3.3+ ARID1A binding. B Chromatin model schematic depicting hypothesized relationship between ARID1A-SWI/SNF and ZMYND8 co-regulation of H3.3, possibly mediated by co-factors. C ARID1A co-immunoprecipitation detecting physical interaction with NuRD catalytic subunit CHD4, but not ZMYND8. D CHD4 co-immunoprecipitation detecting physical interactions with both ARID1A and ZMYND8. E 10–30% glycerol gradient sedimentation and immunoblotting for SWI/SNF, NuRD, and ZMYND8. Relative fractions display native protein complexes transitioning from low molecular weight (left) to high molecular weight (right). Underlined fractions highlight potential interacting native complexes containing ZMYND8 and members of SWI/SNF (BAF) and NuRD (Mi-2β). F Genome-wide ChIP-seq ( n = 2) peak overlaps between ARID1A, CHD4, ZMYND8, and H3.3. Peak numbers within the Euler diagram are approximations and not mutually exclusive due to varying peak sizes. G Example locus on chromosome 10 displaying ARID1A, CHD4, ZMYND8, and H3.3 co-regulation. H Enrichment for ARID1A co-regulation of H3.3 peaks bound by CHD4 and/or ZMYND8. Statistic is two-tailed Fisher’s exact test. I Average ChIP-seq signal density histograms for ARID1A (left) and H3.3 (right) at H3.3 peaks bound by CHD4 and/or ZMYND8. J H3.3 abundance (ChIP FPKM) at ARID1A-bound shARID1A differential H3.3 regions co-bound by CHD4 or ZMYND8. Statistic is two-tailed, unpaired Wilcoxon’s test. K Positive association between CHD4 binding (top) and negative association between ZMYND8 binding (bottom) and ARID1A maintenance of H3.3 chromatin, genome-wide. Statistic is two-tailed Fisher’s exact test. L Average ChIP-seq signal density histograms for ARID1A, H3.3, CHD4, and ZMYND8 across ARID1A-bound H3.3 regions that decreased or were stable with shARID1A
    Figure Legend Snippet: Characterization of ARID1A, CHD4, and ZMYND8 chromatin interactions co-regulating H3.3. A Genome-wide associations between ARID1A binding at H3.3+ vs. H3.3− regions for all 1135 transcriptional regulator peak sets included in the ReMap2020 peak database. Labeled factors exhibit an H3.3+ ARID1A binding association with genomic odds ratio >2 and overlap with >0.1% of ARID1A binding sites. ZMYND11 and ZMYND8 (bolded) are two of the top factors most associated with H3.3+ ARID1A binding. B Chromatin model schematic depicting hypothesized relationship between ARID1A-SWI/SNF and ZMYND8 co-regulation of H3.3, possibly mediated by co-factors. C ARID1A co-immunoprecipitation detecting physical interaction with NuRD catalytic subunit CHD4, but not ZMYND8. D CHD4 co-immunoprecipitation detecting physical interactions with both ARID1A and ZMYND8. E 10–30% glycerol gradient sedimentation and immunoblotting for SWI/SNF, NuRD, and ZMYND8. Relative fractions display native protein complexes transitioning from low molecular weight (left) to high molecular weight (right). Underlined fractions highlight potential interacting native complexes containing ZMYND8 and members of SWI/SNF (BAF) and NuRD (Mi-2β). F Genome-wide ChIP-seq ( n = 2) peak overlaps between ARID1A, CHD4, ZMYND8, and H3.3. Peak numbers within the Euler diagram are approximations and not mutually exclusive due to varying peak sizes. G Example locus on chromosome 10 displaying ARID1A, CHD4, ZMYND8, and H3.3 co-regulation. H Enrichment for ARID1A co-regulation of H3.3 peaks bound by CHD4 and/or ZMYND8. Statistic is two-tailed Fisher’s exact test. I Average ChIP-seq signal density histograms for ARID1A (left) and H3.3 (right) at H3.3 peaks bound by CHD4 and/or ZMYND8. J H3.3 abundance (ChIP FPKM) at ARID1A-bound shARID1A differential H3.3 regions co-bound by CHD4 or ZMYND8. Statistic is two-tailed, unpaired Wilcoxon’s test. K Positive association between CHD4 binding (top) and negative association between ZMYND8 binding (bottom) and ARID1A maintenance of H3.3 chromatin, genome-wide. Statistic is two-tailed Fisher’s exact test. L Average ChIP-seq signal density histograms for ARID1A, H3.3, CHD4, and ZMYND8 across ARID1A-bound H3.3 regions that decreased or were stable with shARID1A

    Techniques Used: Genome Wide, Binding Assay, Labeling, Immunoprecipitation, Sedimentation, Western Blot, Molecular Weight, ChIP-sequencing, Two Tailed Test

    Genome-wide analysis of ARID1A-dependent CHD4 binding. A MA plot of shARID1A vs. control differential CHD4 ChIP-seq ( n = 2), across 44,567 tested genomic regions of CHD4 binding. Regions are colored based on shARID1A differential CHD4 binding significance. Inset pie chart depicts distribution of significantly increasing and decreasing CHD4 regions ( csaw / edgeR FDR < 0.05) compared to stable CHD4 (FDR > 0.05). FDR < 0.05 was used as the significance threshold for all downstream analyses. B Global analysis of ARID1A-dependent CHD4 binding based on presence of normal ARID1A binding. Box plot quantification of shARID1A log 2 FC CHD4 binding, segregated by ARID1A binding status. Statistic is two-tailed, unpaired Wilcoxon’s test. C Chromatin state enrichment among shARID1A increasing and decreasing CHD4 binding regions, calculated as observed/expected genomic fold-enrichment per genomic bp. Statistic is hypergeometric enrichment. D Enrichment of ARID1A binding detection at regions with decreasing CHD4 binding following ARID1A loss compared to all tested CHD4 binding sites. Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. E Enrichment of ARID1A-dependent H3.3 maintenance (shARID1A decreasing H3.3 abundance) at regions with decreasing CHD4 binding following ARID1A loss compared to all tested CHD4 binding sites. Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. F Associations between presence of H3.3 (wild-type peak) and ARID1A-bound, ARID1A-dependent CHD4 binding. Statistic is hypergeometric enrichment. G Associations between ZMYND8 co-binding (wild-type peak) and ARID1A-bound, ARID1A-dependent CHD4 binding. Statistic is hypergeometric enrichment. *** p < 0.001
    Figure Legend Snippet: Genome-wide analysis of ARID1A-dependent CHD4 binding. A MA plot of shARID1A vs. control differential CHD4 ChIP-seq ( n = 2), across 44,567 tested genomic regions of CHD4 binding. Regions are colored based on shARID1A differential CHD4 binding significance. Inset pie chart depicts distribution of significantly increasing and decreasing CHD4 regions ( csaw / edgeR FDR < 0.05) compared to stable CHD4 (FDR > 0.05). FDR < 0.05 was used as the significance threshold for all downstream analyses. B Global analysis of ARID1A-dependent CHD4 binding based on presence of normal ARID1A binding. Box plot quantification of shARID1A log 2 FC CHD4 binding, segregated by ARID1A binding status. Statistic is two-tailed, unpaired Wilcoxon’s test. C Chromatin state enrichment among shARID1A increasing and decreasing CHD4 binding regions, calculated as observed/expected genomic fold-enrichment per genomic bp. Statistic is hypergeometric enrichment. D Enrichment of ARID1A binding detection at regions with decreasing CHD4 binding following ARID1A loss compared to all tested CHD4 binding sites. Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. E Enrichment of ARID1A-dependent H3.3 maintenance (shARID1A decreasing H3.3 abundance) at regions with decreasing CHD4 binding following ARID1A loss compared to all tested CHD4 binding sites. Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. F Associations between presence of H3.3 (wild-type peak) and ARID1A-bound, ARID1A-dependent CHD4 binding. Statistic is hypergeometric enrichment. G Associations between ZMYND8 co-binding (wild-type peak) and ARID1A-bound, ARID1A-dependent CHD4 binding. Statistic is hypergeometric enrichment. *** p < 0.001

    Techniques Used: Genome Wide, Binding Assay, ChIP-sequencing, Two Tailed Test

    H3.3 enhancer regulation by ARID1A, CHD4, and ZMYND8. A Heatmap of chromatin features at 15,925 active typical enhancers and 1374 distal super-enhancer constituents (H3K27ac peaks co-marked by ATAC) segregated by ARID1A ± CHD4 ± ZMYND8 binding. Enhancers are centered on the H3K27ac peak, and signal is displayed as indicated for the flanking 5 kb in either direction. B ZMYND8 binding detection at ARID1A-bound typical and super-enhancers with or without CHD4 co-binding. Statistic is two-tailed Fisher’s exact test. C Association between ARID1A+CHD4+ZMYND8 co-binding at enhancers and presence of H3.3. H3.3+ super-enhancers show the most frequent co-binding. Statistic is two-tailed Fisher’s exact test. D Chromatin features at active super-enhancer constituents segregated by ARID1A loss-driven H3K27-acetylation dynamics: hyperacetylated, de-acetylated, or stably acetylated. Left, average ChIP-seq signal density histograms across enhancer classes. Right, violin plots quantifying signal (ChIP/input fold-enrichment) across enhancer classes. Statistic is two-tailed, unpaired Wilcoxon’s test
    Figure Legend Snippet: H3.3 enhancer regulation by ARID1A, CHD4, and ZMYND8. A Heatmap of chromatin features at 15,925 active typical enhancers and 1374 distal super-enhancer constituents (H3K27ac peaks co-marked by ATAC) segregated by ARID1A ± CHD4 ± ZMYND8 binding. Enhancers are centered on the H3K27ac peak, and signal is displayed as indicated for the flanking 5 kb in either direction. B ZMYND8 binding detection at ARID1A-bound typical and super-enhancers with or without CHD4 co-binding. Statistic is two-tailed Fisher’s exact test. C Association between ARID1A+CHD4+ZMYND8 co-binding at enhancers and presence of H3.3. H3.3+ super-enhancers show the most frequent co-binding. Statistic is two-tailed Fisher’s exact test. D Chromatin features at active super-enhancer constituents segregated by ARID1A loss-driven H3K27-acetylation dynamics: hyperacetylated, de-acetylated, or stably acetylated. Left, average ChIP-seq signal density histograms across enhancer classes. Right, violin plots quantifying signal (ChIP/input fold-enrichment) across enhancer classes. Statistic is two-tailed, unpaired Wilcoxon’s test

    Techniques Used: Binding Assay, Two Tailed Test, Stable Transfection, ChIP-sequencing

    ZMYND8-mediated chromatin repression is associated with H4(K16)ac. A Heatmap of clustered, normalized feature emission probabilities, and associated functional annotation of the new 12-feature, genome-wide chromatin 25-state model. States (S_) are labeled based on order of normalized emission probability clustering. See “ ” for details on optimal model selection. SE: super-enhancer. B Genomic fold-enrichment (FE) for ARID1A, CHD4, ZMYND8, co-binding, and shARID1A decreasing H3.3 among the 25 chromatin states. Statistic is hypergeometric enrichment test. C Modeled chromatin states among reference gene promoter-proximal regions (±3 kb around annotated TSS). Left, proportion of promoter-proximal chromatin for siARID1A DE genes ( DESeq2 , FDR < 0.0001) belonging to each of the 25 states. Center, ratio of promoter-proximal chromatin states associated with siARID1A DE genes (FDR < 0.0001) compared to stable genes (FDR > 0.05). Right, ratio of promoter-proximal chromatin states associated with ARID1A transcriptional repression (i.e., siARID1A upregulation) compared to activation (i.e., siARID1A downregulation). D Violin plots quantifying chromatin feature signal at H4K16ac+ (purple) vs. H4K16ac− (gray) promoter-proximal super-enhancer constituent H3K27ac peaks. Statistic is two-tailed, unpaired Wilcoxon’s test. E Principal component analysis (PCA) of RNA-seq expression log 2 FC (shrinkage-corrected) values for siCHD4, siZMYND8, siH3F3B, and siARID1A treatment conditions vs. controls ( n = 3). In total, 1974 genes with s log 2 FC variance >0.1 were used for PCA. F Schematic of identifying mechanistic genes co-repressed by ARID1A, H3.3, CHD4, and ZMYND8, i.e., upregulated ( DESeq2 , FDR < 0.05) with siARID1A, siH3F3B, siCHD4, and siZMYND8 treatments. G Clustered heatmap of expression log 2 FC values for 60 co-repressed genes upregulated in all 4 knockdown conditions. Rightmost column demarcates presence of H4K16ac peaks over promoter-proximal region or gene body. H Top gene sets enriched (hypergeometric enrichment test, FDR < 0.05) among the 60 ARID1A-CHD4-ZMYND8-H3.3 co-repressed genes from various gene set databases. I Example target gene loci, PLAU and TRIO , marked by nearby H3.3+ super-enhancers within H4(K16)ac+ domains that are co-bound by ARID1A, CHD4, and ZMYND8, where ARID1A loss leads to significant depletion of H3.3 (ChIP-seq FDR < 0.05), and knockdown of ARID1A, H3.3, CHD4, or ZMYND8 leads to significant expression upregulation (RNA-seq FDR < 0.05)
    Figure Legend Snippet: ZMYND8-mediated chromatin repression is associated with H4(K16)ac. A Heatmap of clustered, normalized feature emission probabilities, and associated functional annotation of the new 12-feature, genome-wide chromatin 25-state model. States (S_) are labeled based on order of normalized emission probability clustering. See “ ” for details on optimal model selection. SE: super-enhancer. B Genomic fold-enrichment (FE) for ARID1A, CHD4, ZMYND8, co-binding, and shARID1A decreasing H3.3 among the 25 chromatin states. Statistic is hypergeometric enrichment test. C Modeled chromatin states among reference gene promoter-proximal regions (±3 kb around annotated TSS). Left, proportion of promoter-proximal chromatin for siARID1A DE genes ( DESeq2 , FDR < 0.0001) belonging to each of the 25 states. Center, ratio of promoter-proximal chromatin states associated with siARID1A DE genes (FDR < 0.0001) compared to stable genes (FDR > 0.05). Right, ratio of promoter-proximal chromatin states associated with ARID1A transcriptional repression (i.e., siARID1A upregulation) compared to activation (i.e., siARID1A downregulation). D Violin plots quantifying chromatin feature signal at H4K16ac+ (purple) vs. H4K16ac− (gray) promoter-proximal super-enhancer constituent H3K27ac peaks. Statistic is two-tailed, unpaired Wilcoxon’s test. E Principal component analysis (PCA) of RNA-seq expression log 2 FC (shrinkage-corrected) values for siCHD4, siZMYND8, siH3F3B, and siARID1A treatment conditions vs. controls ( n = 3). In total, 1974 genes with s log 2 FC variance >0.1 were used for PCA. F Schematic of identifying mechanistic genes co-repressed by ARID1A, H3.3, CHD4, and ZMYND8, i.e., upregulated ( DESeq2 , FDR < 0.05) with siARID1A, siH3F3B, siCHD4, and siZMYND8 treatments. G Clustered heatmap of expression log 2 FC values for 60 co-repressed genes upregulated in all 4 knockdown conditions. Rightmost column demarcates presence of H4K16ac peaks over promoter-proximal region or gene body. H Top gene sets enriched (hypergeometric enrichment test, FDR < 0.05) among the 60 ARID1A-CHD4-ZMYND8-H3.3 co-repressed genes from various gene set databases. I Example target gene loci, PLAU and TRIO , marked by nearby H3.3+ super-enhancers within H4(K16)ac+ domains that are co-bound by ARID1A, CHD4, and ZMYND8, where ARID1A loss leads to significant depletion of H3.3 (ChIP-seq FDR < 0.05), and knockdown of ARID1A, H3.3, CHD4, or ZMYND8 leads to significant expression upregulation (RNA-seq FDR < 0.05)

    Techniques Used: Functional Assay, Genome Wide, Labeling, Selection, Binding Assay, Activation Assay, Two Tailed Test, RNA Sequencing Assay, Expressing, ChIP-sequencing

    Mechanistic gene expression alterations in human endometriomas. A Left, enrichment for ARID1A-H3.3 co-repressive chromatin mechanistic gene sets among human endometrioma (ovarian endometriosis) vs. control endometrium DE genes reported by Hawkins et al. , compared to all unique measured genes. Right, proportion of overlapping DE genes that are upregulated vs. downregulated in endometriomas, compared to all unique measured genes. Statistic is hypergeometric enrichment. B Box plots displaying endometrioma expression log 2 FC values for probes annotated to genes within mechanistic gene sets, compared to all measured probes. Statistic is two-tailed, unpaired Wilcoxon’s test. C Relative expression box-dot plots of 6 genes upregulated in endometriomas vs. control endometrium that are co-repressed by ARID1A, H3.3, CHD4, and ZMYND8. Statistic is limma FDR-adjusted p . * p < 0.05, ** p < 0.01, *** p < 0.001
    Figure Legend Snippet: Mechanistic gene expression alterations in human endometriomas. A Left, enrichment for ARID1A-H3.3 co-repressive chromatin mechanistic gene sets among human endometrioma (ovarian endometriosis) vs. control endometrium DE genes reported by Hawkins et al. , compared to all unique measured genes. Right, proportion of overlapping DE genes that are upregulated vs. downregulated in endometriomas, compared to all unique measured genes. Statistic is hypergeometric enrichment. B Box plots displaying endometrioma expression log 2 FC values for probes annotated to genes within mechanistic gene sets, compared to all measured probes. Statistic is two-tailed, unpaired Wilcoxon’s test. C Relative expression box-dot plots of 6 genes upregulated in endometriomas vs. control endometrium that are co-repressed by ARID1A, H3.3, CHD4, and ZMYND8. Statistic is limma FDR-adjusted p . * p < 0.05, ** p < 0.01, *** p < 0.001

    Techniques Used: Expressing, Two Tailed Test

    Proposed model of H3.3 chromatin regulation by ARID1A-SWI/SNF and co-regulators. ARID1A and SWI/SNF chromatin remodeling activities are required for H3.3 incorporation or maintenance at certain active regulatory elements across the genome, such as super-enhancers. When ARID1A is mutated or lost, H3.3 maintenance is disrupted, and nucleosome composition shifts toward canonical H3.1/3.2 at ARID1A-bound sites. Consequential to local H3.3 depletion, H3.3 reader factor occupancy is reduced—such as the CHD4-containing NuRD complex—leading to impaired chromatin regulation and aberrant target gene expression. At H3.3+ H4K16ac+ super-enhancer-like elements located promoter-proximally upstream of genes, H3.3 maintenance by ARID1A-SWI/SNF is associated with repression of transcriptional hyperactivation and the NuRD cofactor ZMYND8
    Figure Legend Snippet: Proposed model of H3.3 chromatin regulation by ARID1A-SWI/SNF and co-regulators. ARID1A and SWI/SNF chromatin remodeling activities are required for H3.3 incorporation or maintenance at certain active regulatory elements across the genome, such as super-enhancers. When ARID1A is mutated or lost, H3.3 maintenance is disrupted, and nucleosome composition shifts toward canonical H3.1/3.2 at ARID1A-bound sites. Consequential to local H3.3 depletion, H3.3 reader factor occupancy is reduced—such as the CHD4-containing NuRD complex—leading to impaired chromatin regulation and aberrant target gene expression. At H3.3+ H4K16ac+ super-enhancer-like elements located promoter-proximally upstream of genes, H3.3 maintenance by ARID1A-SWI/SNF is associated with repression of transcriptional hyperactivation and the NuRD cofactor ZMYND8

    Techniques Used: Expressing

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    Cell Signaling Technology Inc anti arid1a
    A , Representation of 42 distinct histone H3 peptide measurements by mass spectrometry in public CCLE global chromatin profiling data, specific ally for endometrial cancer cell lines stratified by <t>ARID1A</t> mutation status. Heatmap values are relative peptide abundance Z-scores, where 0 (white) is the mean across all pan-cancer cell lines. H3 peptides were then ranked ( y -axis) by differential abundance between ARID1A mutant vs. wild-type lines. B , Summary of ARID1A mutant peptide associations across all cancer cell lines ( n = 896, y -axis) or specifically endometrial cancer lines ( n = 27, x -axis). C - D , Box dot plots showing relative abundance of H3.3 vs. canonical H3.1 peptides in ARID1A wild-type and mutant lines: C , pan-cancer lines; D , endometrial lines. Statistic is two-tailed, unpaired Welch’s t -test.
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    1) Product Images from "ARID1A maintains transcriptionally repressive H3.3 associated with CHD4-ZMYND8 chromatin interactions"

    Article Title: ARID1A maintains transcriptionally repressive H3.3 associated with CHD4-ZMYND8 chromatin interactions

    Journal: bioRxiv

    doi: 10.1101/2022.02.27.482165

    A , Representation of 42 distinct histone H3 peptide measurements by mass spectrometry in public CCLE global chromatin profiling data, specific ally for endometrial cancer cell lines stratified by ARID1A mutation status. Heatmap values are relative peptide abundance Z-scores, where 0 (white) is the mean across all pan-cancer cell lines. H3 peptides were then ranked ( y -axis) by differential abundance between ARID1A mutant vs. wild-type lines. B , Summary of ARID1A mutant peptide associations across all cancer cell lines ( n = 896, y -axis) or specifically endometrial cancer lines ( n = 27, x -axis). C - D , Box dot plots showing relative abundance of H3.3 vs. canonical H3.1 peptides in ARID1A wild-type and mutant lines: C , pan-cancer lines; D , endometrial lines. Statistic is two-tailed, unpaired Welch’s t -test.
    Figure Legend Snippet: A , Representation of 42 distinct histone H3 peptide measurements by mass spectrometry in public CCLE global chromatin profiling data, specific ally for endometrial cancer cell lines stratified by ARID1A mutation status. Heatmap values are relative peptide abundance Z-scores, where 0 (white) is the mean across all pan-cancer cell lines. H3 peptides were then ranked ( y -axis) by differential abundance between ARID1A mutant vs. wild-type lines. B , Summary of ARID1A mutant peptide associations across all cancer cell lines ( n = 896, y -axis) or specifically endometrial cancer lines ( n = 27, x -axis). C - D , Box dot plots showing relative abundance of H3.3 vs. canonical H3.1 peptides in ARID1A wild-type and mutant lines: C , pan-cancer lines; D , endometrial lines. Statistic is two-tailed, unpaired Welch’s t -test.

    Techniques Used: Mass Spectrometry, Mutagenesis, Two Tailed Test

    Repeated analysis of H3.3 and H3.1 abundance in CCLE lines that are wild-type for all 74 human histone genes annotated by Nacev et al. Statistic is two-tailed, unpaired Welch’s t -test. A , H3.3 (left) and H3.1 (right) peptide abundances in ARID1A mutant vs. wild-type pan-cancer lines, as in . B , H3.3 (left) and H3.1 (right) peptide abundances in ARID1A mutant vs. wild-type endometrial cancer lines, as in .
    Figure Legend Snippet: Repeated analysis of H3.3 and H3.1 abundance in CCLE lines that are wild-type for all 74 human histone genes annotated by Nacev et al. Statistic is two-tailed, unpaired Welch’s t -test. A , H3.3 (left) and H3.1 (right) peptide abundances in ARID1A mutant vs. wild-type pan-cancer lines, as in . B , H3.3 (left) and H3.1 (right) peptide abundances in ARID1A mutant vs. wild-type endometrial cancer lines, as in .

    Techniques Used: Two Tailed Test, Mutagenesis

    A , Genomic annotation of 40,006 genome-wide H3.3 ChIP-seq peaks in 12Z cells. B , Distribution of H3.3 peak widths. Median H3.3 peak width is 1830 bp. C , Genome-wide overlap of ARID1A and H3.3 ChIP-seq peaks. D , Genome-wide association between H3.3 and other previously measured chromatin features, per genomic bp. Statistic is hypergeometric enrichment. E , Enrichment for H3.3 and ARID1A co-regulation across 18 chromatin states previously modeled via ChromHMM . Left, enrichment of H3.3 peaks; center, enrichment of H3.3+ARID1A binding; right, enrichment of ARID1A binding at sites with vs. without H3.3. Statistic is hypergeometric enrichment. F , Left, ARID1 A binding levels (ChIP/input fold-enrichment, FE) at H3.3+ vs. H3.3-ARID1A peaks. Right, H3.3 abundance (ChIP/input fold-enrichment) at ARID1A+ vs. ARID1A-H3.3 peaks. Statistic is two-tailed, unpaired Wilcoxon’s test. G , Association between ARID1A and H3.3 co-binding at promoter proximal (<3 kb from a TSS) vs. distal (>3 kb from a TSS) peaks. Statistic is two-tailed Fisher’s exact test. H , Left, ARID1A binding levels at promoter vs. distal peaks with or without H3.3. Right, H3.3 abundance at promoter vs. distal peaks with or without ARID1A. Statistic is two-tailed, unpaired Wilcoxon’s test. I , Top, enrichment of H3.3 at genes promoter-bound by ARID1A. Bottom, enrichment of ARID1A+H3.3 co-binding at genes DE following ARID1A loss (siARID1A treatment). Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. J , Example hg38 browser shots of genes and regulatory elements co-regulated by H3.3 and ARID1A. y -axis is log-likelihood ratio (logLR) of assay signal (compared to input chromatin for ChIP-seq or background genome for ATAC-seq). Small bars under tracks indicate significant peak detection by MACS2 (FDR < 0.05). Super-enhancers were detected by ROSE from H3K27ac ChIP-seq. * p < 0.05, ** p < 0.01, *** p < 0.001.
    Figure Legend Snippet: A , Genomic annotation of 40,006 genome-wide H3.3 ChIP-seq peaks in 12Z cells. B , Distribution of H3.3 peak widths. Median H3.3 peak width is 1830 bp. C , Genome-wide overlap of ARID1A and H3.3 ChIP-seq peaks. D , Genome-wide association between H3.3 and other previously measured chromatin features, per genomic bp. Statistic is hypergeometric enrichment. E , Enrichment for H3.3 and ARID1A co-regulation across 18 chromatin states previously modeled via ChromHMM . Left, enrichment of H3.3 peaks; center, enrichment of H3.3+ARID1A binding; right, enrichment of ARID1A binding at sites with vs. without H3.3. Statistic is hypergeometric enrichment. F , Left, ARID1 A binding levels (ChIP/input fold-enrichment, FE) at H3.3+ vs. H3.3-ARID1A peaks. Right, H3.3 abundance (ChIP/input fold-enrichment) at ARID1A+ vs. ARID1A-H3.3 peaks. Statistic is two-tailed, unpaired Wilcoxon’s test. G , Association between ARID1A and H3.3 co-binding at promoter proximal (<3 kb from a TSS) vs. distal (>3 kb from a TSS) peaks. Statistic is two-tailed Fisher’s exact test. H , Left, ARID1A binding levels at promoter vs. distal peaks with or without H3.3. Right, H3.3 abundance at promoter vs. distal peaks with or without ARID1A. Statistic is two-tailed, unpaired Wilcoxon’s test. I , Top, enrichment of H3.3 at genes promoter-bound by ARID1A. Bottom, enrichment of ARID1A+H3.3 co-binding at genes DE following ARID1A loss (siARID1A treatment). Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. J , Example hg38 browser shots of genes and regulatory elements co-regulated by H3.3 and ARID1A. y -axis is log-likelihood ratio (logLR) of assay signal (compared to input chromatin for ChIP-seq or background genome for ATAC-seq). Small bars under tracks indicate significant peak detection by MACS2 (FDR < 0.05). Super-enhancers were detected by ROSE from H3K27ac ChIP-seq. * p < 0.05, ** p < 0.01, *** p < 0.001.

    Techniques Used: Genome Wide, ChIP-sequencing, GWAS, Binding Assay, Two Tailed Test

    A , MA plot of shARID1A vs. control differential H3.3 ChIP-seq, n = 67,502 tested genomic regions. Regions are colored based on shARID1A differential H3.3 significance. FDR < 0.05 was used as the significance threshold for all downstream analyses. B , Baseline H3.3 abundance (ChIP FPKM) at regions with stable H3.3 following ARID1A knockdown, regions that display increasing H3.3, and regions that display decreasing H3.3. Statistic is two-tailed, unpaired Wilcoxon’s test. C , shARID1A differential H3.3 regions segregated by detection of ARID1A binding in wild-type cells. Left, MA plot with all genome-wide H3.3 tested regions, colored by ARID1A binding status. Right, box plot quantification of shARID1A log 2 FC H3.3 abundance, segregated by ARID1A binding status. Statistic is two-tailed, unpaired Wilcoxon’s test. D , Enrichment of ARID1A binding detection at regions with decreasing H3.3 following ARID1A loss compared to all tested H3.3 regions. Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. E , Magnitude of H3.3 change (log 2 FC) among ARID1A-bound (direct) shARID1A significantly decreasing vs. increasing H3.3 regions. Statistic is two-tailed, unpaired Wilcoxon’s test. F , Distribution of H3.3-enriched region widths among shARID1A stable vs. increasing vs. decreasing H3.3 regions. Statistic is two-tailed, unpaired Wilcoxon’s test. G , Chromatin state enrichment among shARID1A increasing and decreasing H3.3 regions, calculated per 200 bp genomic interval. Statistic is hypergeometric enrichment. H , Top 10 significant (FDR < 0.05) enriched Hallmark pathways (left) and GO Biological Process gene sets (right) among genes with shARID1A direct (ARID1A-bound) decreasing promoter H3.3. I , Representative hg38 locus near CCL2 displaying H3.3 maintained by ARID1A chromatin interactions. *** p < 0.001.
    Figure Legend Snippet: A , MA plot of shARID1A vs. control differential H3.3 ChIP-seq, n = 67,502 tested genomic regions. Regions are colored based on shARID1A differential H3.3 significance. FDR < 0.05 was used as the significance threshold for all downstream analyses. B , Baseline H3.3 abundance (ChIP FPKM) at regions with stable H3.3 following ARID1A knockdown, regions that display increasing H3.3, and regions that display decreasing H3.3. Statistic is two-tailed, unpaired Wilcoxon’s test. C , shARID1A differential H3.3 regions segregated by detection of ARID1A binding in wild-type cells. Left, MA plot with all genome-wide H3.3 tested regions, colored by ARID1A binding status. Right, box plot quantification of shARID1A log 2 FC H3.3 abundance, segregated by ARID1A binding status. Statistic is two-tailed, unpaired Wilcoxon’s test. D , Enrichment of ARID1A binding detection at regions with decreasing H3.3 following ARID1A loss compared to all tested H3.3 regions. Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. E , Magnitude of H3.3 change (log 2 FC) among ARID1A-bound (direct) shARID1A significantly decreasing vs. increasing H3.3 regions. Statistic is two-tailed, unpaired Wilcoxon’s test. F , Distribution of H3.3-enriched region widths among shARID1A stable vs. increasing vs. decreasing H3.3 regions. Statistic is two-tailed, unpaired Wilcoxon’s test. G , Chromatin state enrichment among shARID1A increasing and decreasing H3.3 regions, calculated per 200 bp genomic interval. Statistic is hypergeometric enrichment. H , Top 10 significant (FDR < 0.05) enriched Hallmark pathways (left) and GO Biological Process gene sets (right) among genes with shARID1A direct (ARID1A-bound) decreasing promoter H3.3. I , Representative hg38 locus near CCL2 displaying H3.3 maintained by ARID1A chromatin interactions. *** p < 0.001.

    Techniques Used: ChIP-sequencing, Two Tailed Test, Binding Assay, Genome Wide

    A , ARID1A immunoblot, compared to β-actin loading control, in 12Z cells treated with lentiviral shRNA particles to acutely knockdown ARID1A compared to non-targeting control shRNA. Wild-type condition shown as reference. B , Immunoblot for H3.3 and ARID1A, compared to total H3 and β-actin loading controls, respectively, in 12Z cells treated with siRNA to acutely knockdown ARID1A (siARID1A). C , RNA-seq linear gene expression data (normalized counts) for H3.3-encoding gene isoforms, H3F3A and H3F3B , following ARID1A knockdown by siRNA (siARID1A). D , Enrichment of regions displaying shARID1A significant differential H3.3 abundance among promoters, typical enhancers, and super-enhancers compared to all tested H3.3 regions. Gene promoters are defined as within 3 kb of a TSS. Enhancers are defined as ATAC+ H3K27ac peaks located >3 kb from a TSS. Super-enhancers were further distinguished from typical enhancers by ROSE . Statistics are hypergeometric enrichment and pairwise two-tailed Fisher’s exact test. E , Distribution of significantly increasing vs. decreasing genomic H3.3 with ARID1A knockdown among the classes described in C . Statistics are hypergeometric enrichment and pairwise two-tailed Fisher’s exact test.
    Figure Legend Snippet: A , ARID1A immunoblot, compared to β-actin loading control, in 12Z cells treated with lentiviral shRNA particles to acutely knockdown ARID1A compared to non-targeting control shRNA. Wild-type condition shown as reference. B , Immunoblot for H3.3 and ARID1A, compared to total H3 and β-actin loading controls, respectively, in 12Z cells treated with siRNA to acutely knockdown ARID1A (siARID1A). C , RNA-seq linear gene expression data (normalized counts) for H3.3-encoding gene isoforms, H3F3A and H3F3B , following ARID1A knockdown by siRNA (siARID1A). D , Enrichment of regions displaying shARID1A significant differential H3.3 abundance among promoters, typical enhancers, and super-enhancers compared to all tested H3.3 regions. Gene promoters are defined as within 3 kb of a TSS. Enhancers are defined as ATAC+ H3K27ac peaks located >3 kb from a TSS. Super-enhancers were further distinguished from typical enhancers by ROSE . Statistics are hypergeometric enrichment and pairwise two-tailed Fisher’s exact test. E , Distribution of significantly increasing vs. decreasing genomic H3.3 with ARID1A knockdown among the classes described in C . Statistics are hypergeometric enrichment and pairwise two-tailed Fisher’s exact test.

    Techniques Used: Western Blot, shRNA, RNA Sequencing Assay, Expressing, Two Tailed Test

    A , Baseline relative linear expression of H3F3A ( H3-3A ) and H3F3B ( H3-3B ) gene isoforms encoding H3.3, as measured by RNA-seq. B , Western blot for H3.3 and total H3 in control vs. siH3F3B treated cells. C , Global transcriptomic effects of 24,192 genes following H3.3 knockdown via siH3F3B treatment. Red dots represent significant DE genes ( DESeq2 , FDR < 0.001). D , Relative linear expression of H3F3A and H3F3B by RNA-seq in control and siH3F3B cells. E , Volcano plot depicting siH3F3B vs. control differential gene expression (DGE). Top significant genes are labeled. F , Significant overlap in DE genes following H3.3 knockdown (siH3F3B) vs. ARID1A knockdown (siARID1A). Statistic is hypergeometric enrichment. G , Directional segregation of siH3F3B/siARID1A overlapping DE genes. A positive association is observed by Chi-squared test, i.e., genes are more likely to be upregulated or downregulated in both conditions as opposed to antagonistic regulation. H , Scatter plot of siH3F3B vs. siARID1A expression log 2 FC (with shrinkage correction) for all 19,900 transcriptome-wide commonly detected genes. Statistic is pearson ( r ) and Spearman ( r s ) correlation coefficients. Colored dots indicate significant DE genes (FDR < 0.001) in both treatment conditions. I , Enrichment for H3.3 repression (siH3F3B upregulation) among siH3F3B genes which are also affected by ARID1A loss (siARID1A). Statistic is hypergeometric enrichment test. J , Scatter plot of 196 shared DE genes upregulated following knockdown of either H3.3 and ARID1A. These genes are mutually repressed by H3.3 and ARID1A. K , Top significant (FDR < 0.05) enriched gene sets among the 196 ARID1A-H3.3 mutually repressed genes among various gene set databases.
    Figure Legend Snippet: A , Baseline relative linear expression of H3F3A ( H3-3A ) and H3F3B ( H3-3B ) gene isoforms encoding H3.3, as measured by RNA-seq. B , Western blot for H3.3 and total H3 in control vs. siH3F3B treated cells. C , Global transcriptomic effects of 24,192 genes following H3.3 knockdown via siH3F3B treatment. Red dots represent significant DE genes ( DESeq2 , FDR < 0.001). D , Relative linear expression of H3F3A and H3F3B by RNA-seq in control and siH3F3B cells. E , Volcano plot depicting siH3F3B vs. control differential gene expression (DGE). Top significant genes are labeled. F , Significant overlap in DE genes following H3.3 knockdown (siH3F3B) vs. ARID1A knockdown (siARID1A). Statistic is hypergeometric enrichment. G , Directional segregation of siH3F3B/siARID1A overlapping DE genes. A positive association is observed by Chi-squared test, i.e., genes are more likely to be upregulated or downregulated in both conditions as opposed to antagonistic regulation. H , Scatter plot of siH3F3B vs. siARID1A expression log 2 FC (with shrinkage correction) for all 19,900 transcriptome-wide commonly detected genes. Statistic is pearson ( r ) and Spearman ( r s ) correlation coefficients. Colored dots indicate significant DE genes (FDR < 0.001) in both treatment conditions. I , Enrichment for H3.3 repression (siH3F3B upregulation) among siH3F3B genes which are also affected by ARID1A loss (siARID1A). Statistic is hypergeometric enrichment test. J , Scatter plot of 196 shared DE genes upregulated following knockdown of either H3.3 and ARID1A. These genes are mutually repressed by H3.3 and ARID1A. K , Top significant (FDR < 0.05) enriched gene sets among the 196 ARID1A-H3.3 mutually repressed genes among various gene set databases.

    Techniques Used: Expressing, RNA Sequencing Assay, Western Blot, Labeling

    A , Genome-wide associations between ARID1A binding at H3.3+ vs. H3.3-regions for all 1135 transcriptional regulator peak sets included in the ReMap2020 peak database. Labeled factors exhibit an H3.3+ ARID1A binding association with genomic odds ratio >2 and overlap with >0.1% of ARID1A binding sites. ZMYND11 and ZMYND8 (bolded) are two of the top factors most associated with H3.3+ ARID1A binding. B , Chromatin model schematic depicting hypothesized relationship between ARID1A-SWI/SNF and ZMYND8 co-regulation of H3.3, possibly mediated by co-factors. C , ARID1A co-immunoprecipitation detecting physical interaction with NuRD catalytic subunit CHD4, but not ZMYND8. D , CHD4 co-immunoprecipitation detecting physical interactions with both ARID1A and ZMYND8. E , 10-30% glycerol gradient sedimentation and immunoblotting for SWI/SNF, NuRD, and ZMYND8. Relative fractions display native protein complexes transitioning from low molecular weight (left) to high molecular weight (right). Underlined fractions highlight potential interacting native complexes containing ZMYND8 and members of SWI/SNF (BAF) and NuRD (Mi-2β). F , Genome-wide ChIP-seq peak overlaps between ARID1A, CHD4, ZMYND8, and H3.3. Peak numbers within the Euler diagram are approximations and not mutually exclusive due to varying peak sizes. G , Example locus on chromosome 10 displaying ARID1A, CHD4, ZMYND8, and H3.3 co-regulation. H , Enrichment for ARID1A co-regulation of H3.3 peaks bound by CHD4 and/or ZMYND8. Statistic is two-tailed Fisher’s exact test. I , Average ChIP-seq signal density histograms for ARID1A (left) and H3.3 (right) at H3.3 peaks bound by CHD4 and/or ZMYND8. J , H3.3 abundance (ChIP FPKM) at ARID1A-bound shARID1A differential H3.3 regions co-bound by CHD4 or ZMYND8. Statistic is two-tailed, unpaired Wilcoxon’s test. K , Positive association between CHD4 binding (top) and negative association between ZMYND8 binding (bottom) and ARID1A direct maintenance of H3.3 chromatin, genome-wide. Statistic is two-tailed Fisher’s exact test. L , Average ChIP-seq signal density histograms for ARID1A, H3.3, CHD4, and ZMYND8 across ARID1A-bound H3.3 regions that decreased or were stable with shARID1A.
    Figure Legend Snippet: A , Genome-wide associations between ARID1A binding at H3.3+ vs. H3.3-regions for all 1135 transcriptional regulator peak sets included in the ReMap2020 peak database. Labeled factors exhibit an H3.3+ ARID1A binding association with genomic odds ratio >2 and overlap with >0.1% of ARID1A binding sites. ZMYND11 and ZMYND8 (bolded) are two of the top factors most associated with H3.3+ ARID1A binding. B , Chromatin model schematic depicting hypothesized relationship between ARID1A-SWI/SNF and ZMYND8 co-regulation of H3.3, possibly mediated by co-factors. C , ARID1A co-immunoprecipitation detecting physical interaction with NuRD catalytic subunit CHD4, but not ZMYND8. D , CHD4 co-immunoprecipitation detecting physical interactions with both ARID1A and ZMYND8. E , 10-30% glycerol gradient sedimentation and immunoblotting for SWI/SNF, NuRD, and ZMYND8. Relative fractions display native protein complexes transitioning from low molecular weight (left) to high molecular weight (right). Underlined fractions highlight potential interacting native complexes containing ZMYND8 and members of SWI/SNF (BAF) and NuRD (Mi-2β). F , Genome-wide ChIP-seq peak overlaps between ARID1A, CHD4, ZMYND8, and H3.3. Peak numbers within the Euler diagram are approximations and not mutually exclusive due to varying peak sizes. G , Example locus on chromosome 10 displaying ARID1A, CHD4, ZMYND8, and H3.3 co-regulation. H , Enrichment for ARID1A co-regulation of H3.3 peaks bound by CHD4 and/or ZMYND8. Statistic is two-tailed Fisher’s exact test. I , Average ChIP-seq signal density histograms for ARID1A (left) and H3.3 (right) at H3.3 peaks bound by CHD4 and/or ZMYND8. J , H3.3 abundance (ChIP FPKM) at ARID1A-bound shARID1A differential H3.3 regions co-bound by CHD4 or ZMYND8. Statistic is two-tailed, unpaired Wilcoxon’s test. K , Positive association between CHD4 binding (top) and negative association between ZMYND8 binding (bottom) and ARID1A direct maintenance of H3.3 chromatin, genome-wide. Statistic is two-tailed Fisher’s exact test. L , Average ChIP-seq signal density histograms for ARID1A, H3.3, CHD4, and ZMYND8 across ARID1A-bound H3.3 regions that decreased or were stable with shARID1A.

    Techniques Used: Genome Wide, Binding Assay, Labeling, Immunoprecipitation, Sedimentation, Western Blot, Molecular Weight, ChIP-sequencing, Two Tailed Test

    A , Heatmap of chromatin features at 15,925 active typical enhancers and 1374 distal super-enhancer constituents (H3K27ac peaks co-marked by ATAC) segregated by ARID1A ± CHD4 ± ZMYND8 binding. Enhancers are centered on the H3K27ac peak, and signal is displayed as indicated for the flanking 5 kb in either direction. B , ZMYND8 binding detection at ARID1A-bound typical and super-enhancers with or without CHD4 co-binding. Statistic is two-tailed Fisher’s exact test. C , Association between ARID1A+CHD4+ZMYND8 co-binding at enhancers and presence of H3.3. H3.3+ super-enhancers show the most frequent co-binding. Statistic is two-tailed Fisher’s exact test. D , Chromatin features at active super-enhancer constituents segregated by ARID1A loss-driven H3K27-acetylation dynamics: hyper-acetylated, de-acetylated, or stably acetylated. Left, average ChIP-seq signal density histograms across enhancer classes. Right, violin plots quantifying signal (ChIP/input fold-enrichment) across enhancer classes. Statistic is two-tailed, unpaired Wilcoxon’s test.
    Figure Legend Snippet: A , Heatmap of chromatin features at 15,925 active typical enhancers and 1374 distal super-enhancer constituents (H3K27ac peaks co-marked by ATAC) segregated by ARID1A ± CHD4 ± ZMYND8 binding. Enhancers are centered on the H3K27ac peak, and signal is displayed as indicated for the flanking 5 kb in either direction. B , ZMYND8 binding detection at ARID1A-bound typical and super-enhancers with or without CHD4 co-binding. Statistic is two-tailed Fisher’s exact test. C , Association between ARID1A+CHD4+ZMYND8 co-binding at enhancers and presence of H3.3. H3.3+ super-enhancers show the most frequent co-binding. Statistic is two-tailed Fisher’s exact test. D , Chromatin features at active super-enhancer constituents segregated by ARID1A loss-driven H3K27-acetylation dynamics: hyper-acetylated, de-acetylated, or stably acetylated. Left, average ChIP-seq signal density histograms across enhancer classes. Right, violin plots quantifying signal (ChIP/input fold-enrichment) across enhancer classes. Statistic is two-tailed, unpaired Wilcoxon’s test.

    Techniques Used: Binding Assay, Two Tailed Test, Stable Transfection, ChIP-sequencing

    A , Heatmap of clustered, normalized feature emission probabilities and associated functional annotation of the new 12 feature, genome-wide chromatin 25-state model. States (S_) are labeled based on order of normalized emission probability clustering. See methods for details on optimal model selection. B , Genomic fold-enrichment (FE) for ARID1A, CHD4, ZMYND8, co-binding, and shARID1A direct decreasing H3.3 among the 25 chromatin states. Statistic is hypergeometric enrichment test. C , Modeled chromatin states among reference gene promoter regions (±3 kb around annotated TSS). Left, proportion of promoter chromatin for siARID1A DE genes ( DESeq2 , FDR < 0.0001) belonging to each of the 25 states. Center, ratio of promoter chromatin states associated with siARID1A DE genes (FDR < 0.0001) compared to stable genes (FDR > 0.05). Right, ratio of promoter chromatin states associated with ARID1A transcriptional repression (i.e. siARID1A upregulation) compared to activation (i.e. siARID1A downregulation). D , Violin plots quantifying chromatin feature signal at H4K16ac+ (purple) vs. H4K16ac- (gray) promoter super-enhancer constituent H3K27ac peaks. Statistic is two-tailed, unpaired Wilcoxon’s test. E , Principal component analysis (PCA) of RNA-seq expression log 2 FC (shrinkage-corrected) values for siCHD4, siZMYND8, siH3F3B, and siARID1A treatment conditions vs. controls. 1974 genes with s log 2 FC variance >0.1 were used for PCA. F , Schematic of identifying mechanistic genes co-repressed by ARID1A, H3.3, CHD4, and ZMYND8, i.e., upregulated ( DESeq2 , FDR < 0.05) with siARID1A, siH3F3B, siCHD4, and siZMYND8 treatments. G , Clustered heatmap of expression log 2 FC values for 60 co-repressed genes upregulated in all 4 knockdown conditions. Rightmost column demarcates presence of H4K16ac peaks over promoter or gene body. H , Top gene sets enriched (hypergeometric enrichment test, FDR < 0.05) among the 60 ARID1A-CHD4-ZMYND8-H3.3 co-repressed genes from various gene set databases. I , Example target gene loci, PLAU and TRIO , marked by nearby H3.3+ super-enhancers within H4(K16)ac+ domains that are co-bound by ARID1A, CHD4, ZMYND8, where ARID1A loss leads to significant depletion of H3.3 (ChIP-seq FDR < 0.05), and knockdown of ARID1A, H3.3, CHD4, or ZMYND8 leads to significant expression upregulation (RNA-seq FDR < 0.05).
    Figure Legend Snippet: A , Heatmap of clustered, normalized feature emission probabilities and associated functional annotation of the new 12 feature, genome-wide chromatin 25-state model. States (S_) are labeled based on order of normalized emission probability clustering. See methods for details on optimal model selection. B , Genomic fold-enrichment (FE) for ARID1A, CHD4, ZMYND8, co-binding, and shARID1A direct decreasing H3.3 among the 25 chromatin states. Statistic is hypergeometric enrichment test. C , Modeled chromatin states among reference gene promoter regions (±3 kb around annotated TSS). Left, proportion of promoter chromatin for siARID1A DE genes ( DESeq2 , FDR < 0.0001) belonging to each of the 25 states. Center, ratio of promoter chromatin states associated with siARID1A DE genes (FDR < 0.0001) compared to stable genes (FDR > 0.05). Right, ratio of promoter chromatin states associated with ARID1A transcriptional repression (i.e. siARID1A upregulation) compared to activation (i.e. siARID1A downregulation). D , Violin plots quantifying chromatin feature signal at H4K16ac+ (purple) vs. H4K16ac- (gray) promoter super-enhancer constituent H3K27ac peaks. Statistic is two-tailed, unpaired Wilcoxon’s test. E , Principal component analysis (PCA) of RNA-seq expression log 2 FC (shrinkage-corrected) values for siCHD4, siZMYND8, siH3F3B, and siARID1A treatment conditions vs. controls. 1974 genes with s log 2 FC variance >0.1 were used for PCA. F , Schematic of identifying mechanistic genes co-repressed by ARID1A, H3.3, CHD4, and ZMYND8, i.e., upregulated ( DESeq2 , FDR < 0.05) with siARID1A, siH3F3B, siCHD4, and siZMYND8 treatments. G , Clustered heatmap of expression log 2 FC values for 60 co-repressed genes upregulated in all 4 knockdown conditions. Rightmost column demarcates presence of H4K16ac peaks over promoter or gene body. H , Top gene sets enriched (hypergeometric enrichment test, FDR < 0.05) among the 60 ARID1A-CHD4-ZMYND8-H3.3 co-repressed genes from various gene set databases. I , Example target gene loci, PLAU and TRIO , marked by nearby H3.3+ super-enhancers within H4(K16)ac+ domains that are co-bound by ARID1A, CHD4, ZMYND8, where ARID1A loss leads to significant depletion of H3.3 (ChIP-seq FDR < 0.05), and knockdown of ARID1A, H3.3, CHD4, or ZMYND8 leads to significant expression upregulation (RNA-seq FDR < 0.05).

    Techniques Used: Functional Assay, Genome Wide, Labeling, Selection, Binding Assay, Activation Assay, Two Tailed Test, RNA Sequencing Assay, Expressing, ChIP-sequencing

    A , Euler diagram displaying overlapping genome-wide ChIP-seq peaks (called by MACS2 ) for pan-H4ac compared to ARID1A. 48.6% of pan-H4ac peaks are co-marked by ARID1A binding. B , Euler diagram displaying overlapping gene promoters marked by pan-H4ac (H4ac+) and ARID1A binding. Statistic is hypergeometric enrichment test. C , Heatmap displaying ChIP-seq signal across 10,558 genome-wide pan-H4ac ChIP-seq peaks. Signal is quantified as ChIP – Input. Peaks are ranked by overall pan-H4ac signal and stratified by ARID1A binding and promoter (<3 kb from a TSS) vs. distal (>3 kb from a TSS). D , Annotation and directional breakdown of 6205 significant (FDR < 0.05) differentially accessible genomic regions, measured by ATAC-seq, following ARID1A depletion via siRNA (siARID1A). Regions are further segregated based on ARID1A binding status. E , Association of pan-H4ac with siARID1A differential ATAC regions directly bound by ARID1A and separated by direction of accessibility change genome-wide (left), at promoters (center), and at distal elements (right). Statistic is hypergeometric enrichment test and two-tailed Fisher’s exact test. # p < 0.10, *** p < 0.001.
    Figure Legend Snippet: A , Euler diagram displaying overlapping genome-wide ChIP-seq peaks (called by MACS2 ) for pan-H4ac compared to ARID1A. 48.6% of pan-H4ac peaks are co-marked by ARID1A binding. B , Euler diagram displaying overlapping gene promoters marked by pan-H4ac (H4ac+) and ARID1A binding. Statistic is hypergeometric enrichment test. C , Heatmap displaying ChIP-seq signal across 10,558 genome-wide pan-H4ac ChIP-seq peaks. Signal is quantified as ChIP – Input. Peaks are ranked by overall pan-H4ac signal and stratified by ARID1A binding and promoter (<3 kb from a TSS) vs. distal (>3 kb from a TSS). D , Annotation and directional breakdown of 6205 significant (FDR < 0.05) differentially accessible genomic regions, measured by ATAC-seq, following ARID1A depletion via siRNA (siARID1A). Regions are further segregated based on ARID1A binding status. E , Association of pan-H4ac with siARID1A differential ATAC regions directly bound by ARID1A and separated by direction of accessibility change genome-wide (left), at promoters (center), and at distal elements (right). Statistic is hypergeometric enrichment test and two-tailed Fisher’s exact test. # p < 0.10, *** p < 0.001.

    Techniques Used: Genome Wide, ChIP-sequencing, Binding Assay, Two Tailed Test

    A , Immunoblot for (top) ZMYND8 compared to (bottom) β-actin loading control in 12Z cells treated with non-targeting control siRNA or siZMYND8 (ZMYND8 knockdown). B , RNA-seq expression of ZMYND8 in control and siZMYND8 cells. Statistic is FDR-adjusted DESeq2 Wald test. C , Volcano plot for differential gene expression between siZMYND8 and control cells. FDR < 0.001 was used as a significance threshold. Top significant ZMYND8-dependent genes are labeled. D - F , CHD4 siRNA knockdown (siCHD4) framework and RNA-seq analysis as in A - C . G , Euler diagram displaying overlap between siZMYND8 differential gene expression (DGE) and ZMYND8 promoter-bound genes. Statistic is hypergeometric enrichment test. H , Euler diagram displaying overlap between siCHD4 DGE and CHD4 promoter-bound genes. Statistic is hypergeometric enrichment test. I , Left, association of siZMYND8 DGE among ARID1A-H3.3 co-regulated gene classes described in . Right, distribution of significantly upregulated (ZMYND8 repressed) vs. downregulated (ZMYND8 activated) siZMYND8 DE genes among ARID1A-H3.3 co-regulated gene classes. Statistic is two-tailed Fisher’s exact test. J , RNA-seq DGE (FDR < 0.001) overlap and enrichment across the four analyzed knockdown conditions: siARID1A, siH3F3B, siCHD4, and siZMYND8. The black cell diagonal represents the number of total significant DE genes in that condition. The bottom-left triangle displays the number of overlapping DE genes between pairwise knockdowns. The upper-right triangle displays the overlap enrichment significance by hypergeometric enrichment test. A reduced 18,077 gene set universe was used that contains genes with detected expression in all analyzed conditions. K , Overlap of FDR < 0.001 DGE sets across the four analyzed knockdown conditions. L , Overlap of FDR < 0.05 DGE sets across the four analyzed knockdown conditions, corresponding with .
    Figure Legend Snippet: A , Immunoblot for (top) ZMYND8 compared to (bottom) β-actin loading control in 12Z cells treated with non-targeting control siRNA or siZMYND8 (ZMYND8 knockdown). B , RNA-seq expression of ZMYND8 in control and siZMYND8 cells. Statistic is FDR-adjusted DESeq2 Wald test. C , Volcano plot for differential gene expression between siZMYND8 and control cells. FDR < 0.001 was used as a significance threshold. Top significant ZMYND8-dependent genes are labeled. D - F , CHD4 siRNA knockdown (siCHD4) framework and RNA-seq analysis as in A - C . G , Euler diagram displaying overlap between siZMYND8 differential gene expression (DGE) and ZMYND8 promoter-bound genes. Statistic is hypergeometric enrichment test. H , Euler diagram displaying overlap between siCHD4 DGE and CHD4 promoter-bound genes. Statistic is hypergeometric enrichment test. I , Left, association of siZMYND8 DGE among ARID1A-H3.3 co-regulated gene classes described in . Right, distribution of significantly upregulated (ZMYND8 repressed) vs. downregulated (ZMYND8 activated) siZMYND8 DE genes among ARID1A-H3.3 co-regulated gene classes. Statistic is two-tailed Fisher’s exact test. J , RNA-seq DGE (FDR < 0.001) overlap and enrichment across the four analyzed knockdown conditions: siARID1A, siH3F3B, siCHD4, and siZMYND8. The black cell diagonal represents the number of total significant DE genes in that condition. The bottom-left triangle displays the number of overlapping DE genes between pairwise knockdowns. The upper-right triangle displays the overlap enrichment significance by hypergeometric enrichment test. A reduced 18,077 gene set universe was used that contains genes with detected expression in all analyzed conditions. K , Overlap of FDR < 0.001 DGE sets across the four analyzed knockdown conditions. L , Overlap of FDR < 0.05 DGE sets across the four analyzed knockdown conditions, corresponding with .

    Techniques Used: Western Blot, RNA Sequencing Assay, Expressing, Labeling, Two Tailed Test

    Enrichment of H4K16ac at gene promoters or gene bodies of ARID1A-H3.3-ZMYND8-CHD4 co-repressed genes—i.e. genes that are upregulated (FDR < 0.05) following treatment with siARID1A, siH3F3B, siZMYND8, and siCHD4—compared to all expressed genes with chromatin data. Statistic is hypergeometric enrichment test.
    Figure Legend Snippet: Enrichment of H4K16ac at gene promoters or gene bodies of ARID1A-H3.3-ZMYND8-CHD4 co-repressed genes—i.e. genes that are upregulated (FDR < 0.05) following treatment with siARID1A, siH3F3B, siZMYND8, and siCHD4—compared to all expressed genes with chromatin data. Statistic is hypergeometric enrichment test.

    Techniques Used:

    RNA-seq analysis of an ARID1A ± CHD4 co-knockdown experiment ( n = 3 per condition). A , Relative linear RNA-seq expression for (left) CHD4 and (right) ARID1A among siRNA treated conditions. Statistic is DESeq2 FDR-adjusted Wald test. B , Euler diagram displaying DGE (FDR < 0.001) overlap of siCHD4, siARID1A, and siARID1A+siCHD4 conditions compared to control cells. C , Clustered heatmap of expression alterations (rlog) at 22 genes displaying additive repression by ARID1A and CHD4. These genes are significantly upregulated in siCHD4, siARID1A, and siARID1A+siCHD4 conditions compared to control cells and siARID1A+siCHD4 vs. single-knockdown conditions. *** FDR < 0.001
    Figure Legend Snippet: RNA-seq analysis of an ARID1A ± CHD4 co-knockdown experiment ( n = 3 per condition). A , Relative linear RNA-seq expression for (left) CHD4 and (right) ARID1A among siRNA treated conditions. Statistic is DESeq2 FDR-adjusted Wald test. B , Euler diagram displaying DGE (FDR < 0.001) overlap of siCHD4, siARID1A, and siARID1A+siCHD4 conditions compared to control cells. C , Clustered heatmap of expression alterations (rlog) at 22 genes displaying additive repression by ARID1A and CHD4. These genes are significantly upregulated in siCHD4, siARID1A, and siARID1A+siCHD4 conditions compared to control cells and siARID1A+siCHD4 vs. single-knockdown conditions. *** FDR < 0.001

    Techniques Used: RNA Sequencing Assay, Expressing

    A , Left, enrichment for ARID1A-H3.3 co-repressive chromatin mechanistic gene sets among human endometrioma (ovarian endometriosis) vs. control endometrium DE genes reported by Hawkins et al. , compared to all unique measured genes. Right, proportion of overlapping DE genes that are upregulated vs. downregulated in endometriomas, compared to all unique measured genes. Statistic is hypergeometric enrichment. B , Box plots displaying endometrioma expression log 2 FC values for probes annotated to genes within mechanistic gene sets, compared to all measured probes. Statistic is two-tailed, unpaired Wilcoxon’s test. C , Relative expression box-dot plots of 6 genes upregulated in endometriomas vs. control endometrium that are co-repressed by ARID1A, H3.3, CHD4, and ZMYND8. Statistic is limma FDR-adjusted p . * p < 0.05, ** p < 0.01, *** p < 0.001.
    Figure Legend Snippet: A , Left, enrichment for ARID1A-H3.3 co-repressive chromatin mechanistic gene sets among human endometrioma (ovarian endometriosis) vs. control endometrium DE genes reported by Hawkins et al. , compared to all unique measured genes. Right, proportion of overlapping DE genes that are upregulated vs. downregulated in endometriomas, compared to all unique measured genes. Statistic is hypergeometric enrichment. B , Box plots displaying endometrioma expression log 2 FC values for probes annotated to genes within mechanistic gene sets, compared to all measured probes. Statistic is two-tailed, unpaired Wilcoxon’s test. C , Relative expression box-dot plots of 6 genes upregulated in endometriomas vs. control endometrium that are co-repressed by ARID1A, H3.3, CHD4, and ZMYND8. Statistic is limma FDR-adjusted p . * p < 0.05, ** p < 0.01, *** p < 0.001.

    Techniques Used: Expressing, Two Tailed Test

    complex ii  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc complex ii
    Complex Ii, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    arid1a  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc arid1a
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    Cell Signaling Technology Inc arid1a
    Somatic recurrent mutations in all the 3 thymic squamous cell carcinoma (TSCC) nodules
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    1) Product Images from "A mutational profile in multiple thymic squamous cell carcinoma"

    Article Title: A mutational profile in multiple thymic squamous cell carcinoma

    Journal: Gland Surgery

    doi: 10.21037/gs.2019.11.08

    Somatic recurrent mutations in all the 3 thymic squamous cell carcinoma (TSCC) nodules
    Figure Legend Snippet: Somatic recurrent mutations in all the 3 thymic squamous cell carcinoma (TSCC) nodules

    Techniques Used: Mutagenesis

    Immunohistochemistry of TP53 and ARID1A in multiple thymic squamous cell carcinoma (TSCC) nodules.
    Figure Legend Snippet: Immunohistochemistry of TP53 and ARID1A in multiple thymic squamous cell carcinoma (TSCC) nodules.

    Techniques Used: Immunohistochemistry

    anti arid1a  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti arid1a
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    Cell Signaling Technology Inc anti arid1a antibody
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    Cell Signaling Technology Inc arid1a
    ( A ) SWI/SNF subunit gene alterations including <t>ARID1A</t> are present in 290 cases (71%) of a TCGA cohort comprising 408 muscle-invasive urothelial bladder carcinomas with 106 patients (26%) showing ARID1A gene alterations. The 25 depicted subunit genes are categorized into „core“, „BAF-specific (BAF)“, „PBAF-specific (PBAF)”and „variant/accessory (variant)”subunit genes. Note: nBAF (neuronal BAF)-specific subunit genes have been excluded. US: unknown significance, PD: putative driver, Amp: amplification, Deep del: deep deletion, Non-pap: non-papillary, pap: papillary. ( B ) Frequencies of ARID1A gene alteration types in the TCGA cohort. ( C ) ARID1A mRNA expression levels in patient samples harboring ARID1A truncating mutations (trunc, n = 81) compared to specimens without these alterations (non-trunc, n = 327). * P < 0.05 (unpaired t test). Horizotal line (red): median expression. ( D ) Univariate Kaplan-Meier survival curves displaying overall survival of patients harboring ARID1A truncating mutations (trunc) and deep deletions (del) (n = 83; black curve) in relation to patients with ARID1A gene amplification (amp), gene gain (gain) and wildtype (WT) ARID1A gene sequence (n = 233; grey curve). Cases with ARID1A missense mutations of unknown significance and shallow ARID1A gene deletions have been excluded from the analysis.
    Arid1a, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "ARID1A-deficiency in urothelial bladder cancer: No predictive biomarker for EZH2-inhibitor treatment response?"

    Article Title: ARID1A-deficiency in urothelial bladder cancer: No predictive biomarker for EZH2-inhibitor treatment response?

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202965

    ( A ) SWI/SNF subunit gene alterations including ARID1A are present in 290 cases (71%) of a TCGA cohort comprising 408 muscle-invasive urothelial bladder carcinomas with 106 patients (26%) showing ARID1A gene alterations. The 25 depicted subunit genes are categorized into „core“, „BAF-specific (BAF)“, „PBAF-specific (PBAF)”and „variant/accessory (variant)”subunit genes. Note: nBAF (neuronal BAF)-specific subunit genes have been excluded. US: unknown significance, PD: putative driver, Amp: amplification, Deep del: deep deletion, Non-pap: non-papillary, pap: papillary. ( B ) Frequencies of ARID1A gene alteration types in the TCGA cohort. ( C ) ARID1A mRNA expression levels in patient samples harboring ARID1A truncating mutations (trunc, n = 81) compared to specimens without these alterations (non-trunc, n = 327). * P < 0.05 (unpaired t test). Horizotal line (red): median expression. ( D ) Univariate Kaplan-Meier survival curves displaying overall survival of patients harboring ARID1A truncating mutations (trunc) and deep deletions (del) (n = 83; black curve) in relation to patients with ARID1A gene amplification (amp), gene gain (gain) and wildtype (WT) ARID1A gene sequence (n = 233; grey curve). Cases with ARID1A missense mutations of unknown significance and shallow ARID1A gene deletions have been excluded from the analysis.
    Figure Legend Snippet: ( A ) SWI/SNF subunit gene alterations including ARID1A are present in 290 cases (71%) of a TCGA cohort comprising 408 muscle-invasive urothelial bladder carcinomas with 106 patients (26%) showing ARID1A gene alterations. The 25 depicted subunit genes are categorized into „core“, „BAF-specific (BAF)“, „PBAF-specific (PBAF)”and „variant/accessory (variant)”subunit genes. Note: nBAF (neuronal BAF)-specific subunit genes have been excluded. US: unknown significance, PD: putative driver, Amp: amplification, Deep del: deep deletion, Non-pap: non-papillary, pap: papillary. ( B ) Frequencies of ARID1A gene alteration types in the TCGA cohort. ( C ) ARID1A mRNA expression levels in patient samples harboring ARID1A truncating mutations (trunc, n = 81) compared to specimens without these alterations (non-trunc, n = 327). * P < 0.05 (unpaired t test). Horizotal line (red): median expression. ( D ) Univariate Kaplan-Meier survival curves displaying overall survival of patients harboring ARID1A truncating mutations (trunc) and deep deletions (del) (n = 83; black curve) in relation to patients with ARID1A gene amplification (amp), gene gain (gain) and wildtype (WT) ARID1A gene sequence (n = 233; grey curve). Cases with ARID1A missense mutations of unknown significance and shallow ARID1A gene deletions have been excluded from the analysis.

    Techniques Used: Variant Assay, Amplification, Expressing, Sequencing

    (A) ARID1A expression in J82 wildtype (WT) cells and J82 single-cell clones, either transfected with an ARID1A -specific shRNA or a scrambled shRNA control, on protein (upper panel) and mRNA level (bottom panel). ACTB served as a loading control for western blot analysis. ARID1A mRNA expression levels are normalized to median mRNA expression of J82 control cells. ( B - C ) ARID1A protein detection in fixed, hematoxylin-stained J82 cells transfected with a vector expressing a scrambled shRNA (J82 shNeg 3) ( B ) and an ARID1A -specific shRNA sequence (J82 shARID1A 13) ( C ). Bar: 100μm. ( D ) ARID1A expression in urothelial bladder cell lines on protein (upper panel) and mRNA level (bottom panel). HT1376 and JMSU-1 cells, both harboring a homozygous ARID1A frameshift mutation (HT1376: p.S186fs*209, JMSU-1: p.R911fs) show strongly reduced ARID1A mRNA and protein expression. ACTB served as a loading control for western blot analysis. ARID1A mRNA levels are expressed relative to ARID1A mRNA expression in TERT-NHUC cells. ( E ) Muscle-invasive urothelial bladder carcinoma exhibiting an ARID1A frameshift deletion (p.P1314Lfs*14) lacks ARID1A protein expression in cancer cells. Arrow: stromal cells expressing ARID1A protein. Bar: 100μm.
    Figure Legend Snippet: (A) ARID1A expression in J82 wildtype (WT) cells and J82 single-cell clones, either transfected with an ARID1A -specific shRNA or a scrambled shRNA control, on protein (upper panel) and mRNA level (bottom panel). ACTB served as a loading control for western blot analysis. ARID1A mRNA expression levels are normalized to median mRNA expression of J82 control cells. ( B - C ) ARID1A protein detection in fixed, hematoxylin-stained J82 cells transfected with a vector expressing a scrambled shRNA (J82 shNeg 3) ( B ) and an ARID1A -specific shRNA sequence (J82 shARID1A 13) ( C ). Bar: 100μm. ( D ) ARID1A expression in urothelial bladder cell lines on protein (upper panel) and mRNA level (bottom panel). HT1376 and JMSU-1 cells, both harboring a homozygous ARID1A frameshift mutation (HT1376: p.S186fs*209, JMSU-1: p.R911fs) show strongly reduced ARID1A mRNA and protein expression. ACTB served as a loading control for western blot analysis. ARID1A mRNA levels are expressed relative to ARID1A mRNA expression in TERT-NHUC cells. ( E ) Muscle-invasive urothelial bladder carcinoma exhibiting an ARID1A frameshift deletion (p.P1314Lfs*14) lacks ARID1A protein expression in cancer cells. Arrow: stromal cells expressing ARID1A protein. Bar: 100μm.

    Techniques Used: Expressing, Clone Assay, Transfection, shRNA, Western Blot, Staining, Plasmid Preparation, Sequencing, Mutagenesis

    (A-F) Immunohistochemical images representative of observed median ARID1A protein expression in normal urothelium of the urinary bladder (NU, n = 21) (A), pTa low-grade (pTa LG, n = 62) (B) and pTa high-grade (pTa HG, n = 30) tumors (C), carcinoma in situ (CIS, n = 175) (D), pT1 high-grade (pT1 HG, n = 31) (E) and muscle-invasive bladder cancers (MIBC, n = 64) (F). Arrows in A: CIS. Bar: 100μm. (G) Quantification of ARID1A protein expression by box plot analysis. Horizontal lines/numbers: grouped medians. Boxes: 25–75% quartiles. Vertical lines: range, minimum and maximum. Only significant differences are shown. * P < 0.05, ** P <0.01, *** P < 0.001 (Kruskal-Wallis-test, Dunn’s multiple comparison post test), IRS: immunoreactive score. (H) Quantification of ARID1A protein expression loss (IRS = 0–2).
    Figure Legend Snippet: (A-F) Immunohistochemical images representative of observed median ARID1A protein expression in normal urothelium of the urinary bladder (NU, n = 21) (A), pTa low-grade (pTa LG, n = 62) (B) and pTa high-grade (pTa HG, n = 30) tumors (C), carcinoma in situ (CIS, n = 175) (D), pT1 high-grade (pT1 HG, n = 31) (E) and muscle-invasive bladder cancers (MIBC, n = 64) (F). Arrows in A: CIS. Bar: 100μm. (G) Quantification of ARID1A protein expression by box plot analysis. Horizontal lines/numbers: grouped medians. Boxes: 25–75% quartiles. Vertical lines: range, minimum and maximum. Only significant differences are shown. * P < 0.05, ** P <0.01, *** P < 0.001 (Kruskal-Wallis-test, Dunn’s multiple comparison post test), IRS: immunoreactive score. (H) Quantification of ARID1A protein expression loss (IRS = 0–2).

    Techniques Used: Immunohistochemical staining, Expressing, In Situ

    ARID1A, EZH2 protein expression and amount of tri-methylated H3K27 (H3K27me3) in stably transfected J82 single-cell clones ( A ) and transiently transfected UROtsa cells ( B ). ACTB served as a loading control for western blot analysis. WT: wildtype. Densitometrical evaluation of the western blot results shown in A and B are depicted in ( C ) and ( D ), respectively. ( E ) EZH2 mRNA expression in urothelial carcinomas of the TCGA 2017 data set exhibiting “low” (n = 204) and “high” ARID1A (n = 204) mRNA expression. Dichotomization into both groups is based on median ARID1A mRNA levels in all tumor samples. ns: not significant (unpaired t test). ( F ) EZH2 protein expression and amount of tri-methylated H3K27 (H3K27me3) in ARID1A-positive (median ARID1A Remmele score: 12, n = 84 for EZH2 cohort and n = 105 for H3K27me3 cohort) as well as ARID1A-deficient (median ARID1A Remmele score: 0, n = 18) urothelial bladder carcinoma cases. Immunohistochemical images are representative of observed median expression of EZH2 and H3K27me3 in the respective group. Bar: 100μm. ( G ) Quantification of EZH2 and H3K27me3 levels by box plot analysis. Horizontal lines: grouped medians. Boxes: 25–75% quartiles. Vertical lines: range, minimum and maximum. IRS: immunoreactive score (Remmele score). ns: not significant (Mann-Whitney-U test).
    Figure Legend Snippet: ARID1A, EZH2 protein expression and amount of tri-methylated H3K27 (H3K27me3) in stably transfected J82 single-cell clones ( A ) and transiently transfected UROtsa cells ( B ). ACTB served as a loading control for western blot analysis. WT: wildtype. Densitometrical evaluation of the western blot results shown in A and B are depicted in ( C ) and ( D ), respectively. ( E ) EZH2 mRNA expression in urothelial carcinomas of the TCGA 2017 data set exhibiting “low” (n = 204) and “high” ARID1A (n = 204) mRNA expression. Dichotomization into both groups is based on median ARID1A mRNA levels in all tumor samples. ns: not significant (unpaired t test). ( F ) EZH2 protein expression and amount of tri-methylated H3K27 (H3K27me3) in ARID1A-positive (median ARID1A Remmele score: 12, n = 84 for EZH2 cohort and n = 105 for H3K27me3 cohort) as well as ARID1A-deficient (median ARID1A Remmele score: 0, n = 18) urothelial bladder carcinoma cases. Immunohistochemical images are representative of observed median expression of EZH2 and H3K27me3 in the respective group. Bar: 100μm. ( G ) Quantification of EZH2 and H3K27me3 levels by box plot analysis. Horizontal lines: grouped medians. Boxes: 25–75% quartiles. Vertical lines: range, minimum and maximum. IRS: immunoreactive score (Remmele score). ns: not significant (Mann-Whitney-U test).

    Techniques Used: Expressing, Methylation, Stable Transfection, Transfection, Clone Assay, Western Blot, Immunohistochemical staining, MANN-WHITNEY

    Amount of tri-methylated H3K27 (H3K27me3) and EZH2 protein expression in J82 ( A ) and UROtsa ( B ) wildtype cells following GSK126 treatment for 24/48/72 hours in the indicated concentrations in comparison to DMSO control. Densitometrical evaluation of the western blot results shown in A and B are depicted in ( C ) and ( D ), respectively. ( E ) Dose-response curves for J82 ARID1A-depleted single-cell clones and controls treated with the indicated GSK126 concentrations for 72h. Error bars (n = 3): SEM. ( F ) Dose-response curves for UROtsa ARID1A-depleted cells and controls treated with the indicated GSK126 concentrations for 72h. Error bars (n = 3): SEM. ( G ) Dose-response curves for bladder cancer cell lines without genetic SWI/SNF alterations (RT112, SCaBER, J82) and ARID1A -mutated cells (HT1376, JMSU-1, VM-CUB-1) treated with the indicated GSK126 concentrations for 72h. Error bars (n = 3): SEM. WT: wildtype. ( H ) Determined growth IC50 (GI50) values for all cell lines treated with GSK126 for 72h. Green label: normal urothelial model UROtsa, black label: cell lines wihout genetic SWI/SNF alterations, red label: ARID1A -mutated cell lines.
    Figure Legend Snippet: Amount of tri-methylated H3K27 (H3K27me3) and EZH2 protein expression in J82 ( A ) and UROtsa ( B ) wildtype cells following GSK126 treatment for 24/48/72 hours in the indicated concentrations in comparison to DMSO control. Densitometrical evaluation of the western blot results shown in A and B are depicted in ( C ) and ( D ), respectively. ( E ) Dose-response curves for J82 ARID1A-depleted single-cell clones and controls treated with the indicated GSK126 concentrations for 72h. Error bars (n = 3): SEM. ( F ) Dose-response curves for UROtsa ARID1A-depleted cells and controls treated with the indicated GSK126 concentrations for 72h. Error bars (n = 3): SEM. ( G ) Dose-response curves for bladder cancer cell lines without genetic SWI/SNF alterations (RT112, SCaBER, J82) and ARID1A -mutated cells (HT1376, JMSU-1, VM-CUB-1) treated with the indicated GSK126 concentrations for 72h. Error bars (n = 3): SEM. WT: wildtype. ( H ) Determined growth IC50 (GI50) values for all cell lines treated with GSK126 for 72h. Green label: normal urothelial model UROtsa, black label: cell lines wihout genetic SWI/SNF alterations, red label: ARID1A -mutated cell lines.

    Techniques Used: Methylation, Expressing, Western Blot, Clone Assay

    ( A ) Representative western blot analysis of TERT-immortalized normal human urothelial cells (TERT-NHUC) treated with two different ARID1A -specific siRNAs (si ARID1A_4 and 6 ) and a negative control (siNeg). ACTB was used as a loading control. Experiments have been performed in triplicate. ( B ) Densitometrical evaluation of the western blot results shown in A. Protein expression of control cells (siNeg) was set to 1. ( C ) Representative western blot analysis of SV40 large T-antigen-immortalized UROtsa cells treated with two different ARID1A -specific siRNAs (si ARID1A_4 and 6 ) and a negative control (siNeg). ACTB was used as a loading control. Experiments have been performed in triplicate. ( D ) Densitometrical evaluation of the western blot results shown in C. Protein expression of control cells (siNeg) was set to 1. ( E ) Cell growth analysis of UROtsa cells treated with two different ARID1A -specific siRNAs (si ARID1A_4 and 6 ) in comparison to the siRNA negative control (siNeg) and UROtsa wildtype cells (untreated). The mean cell number after 96 hours cell growth of independent experiments (n = 3) was calculated. The values depicted were normalized to cellular growth of the siNeg control. Vertical lines: standard deviation of triplicates. * P <0.05, ** P <0.01, ns: not significant (repeated measures ANOVA, Tukey’s multiple comparison test). ( F ) Left: representative colony formation assay in six-well plates containing UROtsa cells treated with two different ARID1A -specific siRNAs (si ARID1A_4 and 6 ) in comparison to a siRNA negative control (siNeg) and wildtype cells without treatment (untreated) two weeks after cell seeding. Right: Densitometrical evaluation of 2D colony growth of triplicate experiments. The mean colony growth was calculated and the data were normalized to colony growth of the control (siNeg). Horizontal lines: mean values of triplicate experiments. Vertical lines: standard deviation of triplicates. * P <0.05, ns: not significant (repeated measures ANOVA, Tukey’s multiple comparison test).
    Figure Legend Snippet: ( A ) Representative western blot analysis of TERT-immortalized normal human urothelial cells (TERT-NHUC) treated with two different ARID1A -specific siRNAs (si ARID1A_4 and 6 ) and a negative control (siNeg). ACTB was used as a loading control. Experiments have been performed in triplicate. ( B ) Densitometrical evaluation of the western blot results shown in A. Protein expression of control cells (siNeg) was set to 1. ( C ) Representative western blot analysis of SV40 large T-antigen-immortalized UROtsa cells treated with two different ARID1A -specific siRNAs (si ARID1A_4 and 6 ) and a negative control (siNeg). ACTB was used as a loading control. Experiments have been performed in triplicate. ( D ) Densitometrical evaluation of the western blot results shown in C. Protein expression of control cells (siNeg) was set to 1. ( E ) Cell growth analysis of UROtsa cells treated with two different ARID1A -specific siRNAs (si ARID1A_4 and 6 ) in comparison to the siRNA negative control (siNeg) and UROtsa wildtype cells (untreated). The mean cell number after 96 hours cell growth of independent experiments (n = 3) was calculated. The values depicted were normalized to cellular growth of the siNeg control. Vertical lines: standard deviation of triplicates. * P <0.05, ** P <0.01, ns: not significant (repeated measures ANOVA, Tukey’s multiple comparison test). ( F ) Left: representative colony formation assay in six-well plates containing UROtsa cells treated with two different ARID1A -specific siRNAs (si ARID1A_4 and 6 ) in comparison to a siRNA negative control (siNeg) and wildtype cells without treatment (untreated) two weeks after cell seeding. Right: Densitometrical evaluation of 2D colony growth of triplicate experiments. The mean colony growth was calculated and the data were normalized to colony growth of the control (siNeg). Horizontal lines: mean values of triplicate experiments. Vertical lines: standard deviation of triplicates. * P <0.05, ns: not significant (repeated measures ANOVA, Tukey’s multiple comparison test).

    Techniques Used: Western Blot, Negative Control, Expressing, Standard Deviation, Colony Assay

    arid1a  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc arid1a
    PDGFRβ forms ligand-dependent complex with Fer and TMF-1, affecting composition of the SWI–SNF remodeling complex. (a) Immunofluorescence staining of colocalization of PDGFRβ and Fer kinase. PDGFRβ was immunostained with extra PDGFRβ (red), chromatin with DAPI (blue), and the Fer kinase was stained in green. Yellow indicates colocalization between PDGFRβ and Fer. Bars, 10 µm. (b) Coimmunoprecipitation of PDGFRβ and Fer kinase in the nucleus and in the cytoplasm. Immunoprecipitated complexes were immunoblotted for PDGFRβ (top) or Fer (second panel). 10% of the input material was analyzed by immunoblotting for PDGFRβ and Fer (two bottom panels). (c) PDGFRβ forms PDGF-BB–inducible complexes with TMF-1 and Fer in the nucleus. TMF-1, Fer, and <t>ARID1A</t> immunocomplexes were immunoblotted for PDGFRβ, TMF-1, and Fer. 10% of the input lysate was loaded on the last two lanes on each blot; nuclear TMF-1 was poorly detected by direct immunoblotting as opposite to immunoblotting after IP. The membrane was reblotted for lamin A/C as a loading control. (d) Dissociation of TMF-1–Brg-1 and Brg-1–BAF170 nuclear complexes upon PDGF-BB stimulation. TMF-1, BAF170 and ARID1A immunocomplexes were immunoblotted for Brg-1 and reblotted for BAF170 or blotted for ARID1A and reblotted for TMF-1. 10% of the input lysate was loaded on each gel. Arrows indicate ARID1A protein. (e) Dissociation of TMF-1–Brg-1 and Brg-1–BAF155 nuclear complexes upon PDGF-BB stimulation. This experiment reproduces the result from d, but an antibody against the BAF155 core subunit of SWI–SNF chromatin remodeling complex was used instead of BAF170 for IP. Arrows indicate ARID1A protein. (f) Kinase-inactive PDGFRβ binds TMF-1 and prevents PDGF-BB–inducible dissociation of the SWI–SNF complex. Cells were pretreated with AG1296 and stimulated with PDGF-BB. TMF-1 immunocomplexes from nuclear extracts were blotted for PDGFRβ (top) and reblotted for TMF-1 (second) or blotted for Brg-1 (third) and reblotted for TMF-1 (bottom). Molecular mass was measured in kilodaltons. IB, immunoblotting; IP, immunoprecipitation.
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    1) Product Images from "PDGFRβ translocates to the nucleus and regulates chromatin remodeling via TATA element–modifying factor 1"

    Article Title: PDGFRβ translocates to the nucleus and regulates chromatin remodeling via TATA element–modifying factor 1

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201706118

    PDGFRβ forms ligand-dependent complex with Fer and TMF-1, affecting composition of the SWI–SNF remodeling complex. (a) Immunofluorescence staining of colocalization of PDGFRβ and Fer kinase. PDGFRβ was immunostained with extra PDGFRβ (red), chromatin with DAPI (blue), and the Fer kinase was stained in green. Yellow indicates colocalization between PDGFRβ and Fer. Bars, 10 µm. (b) Coimmunoprecipitation of PDGFRβ and Fer kinase in the nucleus and in the cytoplasm. Immunoprecipitated complexes were immunoblotted for PDGFRβ (top) or Fer (second panel). 10% of the input material was analyzed by immunoblotting for PDGFRβ and Fer (two bottom panels). (c) PDGFRβ forms PDGF-BB–inducible complexes with TMF-1 and Fer in the nucleus. TMF-1, Fer, and ARID1A immunocomplexes were immunoblotted for PDGFRβ, TMF-1, and Fer. 10% of the input lysate was loaded on the last two lanes on each blot; nuclear TMF-1 was poorly detected by direct immunoblotting as opposite to immunoblotting after IP. The membrane was reblotted for lamin A/C as a loading control. (d) Dissociation of TMF-1–Brg-1 and Brg-1–BAF170 nuclear complexes upon PDGF-BB stimulation. TMF-1, BAF170 and ARID1A immunocomplexes were immunoblotted for Brg-1 and reblotted for BAF170 or blotted for ARID1A and reblotted for TMF-1. 10% of the input lysate was loaded on each gel. Arrows indicate ARID1A protein. (e) Dissociation of TMF-1–Brg-1 and Brg-1–BAF155 nuclear complexes upon PDGF-BB stimulation. This experiment reproduces the result from d, but an antibody against the BAF155 core subunit of SWI–SNF chromatin remodeling complex was used instead of BAF170 for IP. Arrows indicate ARID1A protein. (f) Kinase-inactive PDGFRβ binds TMF-1 and prevents PDGF-BB–inducible dissociation of the SWI–SNF complex. Cells were pretreated with AG1296 and stimulated with PDGF-BB. TMF-1 immunocomplexes from nuclear extracts were blotted for PDGFRβ (top) and reblotted for TMF-1 (second) or blotted for Brg-1 (third) and reblotted for TMF-1 (bottom). Molecular mass was measured in kilodaltons. IB, immunoblotting; IP, immunoprecipitation.
    Figure Legend Snippet: PDGFRβ forms ligand-dependent complex with Fer and TMF-1, affecting composition of the SWI–SNF remodeling complex. (a) Immunofluorescence staining of colocalization of PDGFRβ and Fer kinase. PDGFRβ was immunostained with extra PDGFRβ (red), chromatin with DAPI (blue), and the Fer kinase was stained in green. Yellow indicates colocalization between PDGFRβ and Fer. Bars, 10 µm. (b) Coimmunoprecipitation of PDGFRβ and Fer kinase in the nucleus and in the cytoplasm. Immunoprecipitated complexes were immunoblotted for PDGFRβ (top) or Fer (second panel). 10% of the input material was analyzed by immunoblotting for PDGFRβ and Fer (two bottom panels). (c) PDGFRβ forms PDGF-BB–inducible complexes with TMF-1 and Fer in the nucleus. TMF-1, Fer, and ARID1A immunocomplexes were immunoblotted for PDGFRβ, TMF-1, and Fer. 10% of the input lysate was loaded on the last two lanes on each blot; nuclear TMF-1 was poorly detected by direct immunoblotting as opposite to immunoblotting after IP. The membrane was reblotted for lamin A/C as a loading control. (d) Dissociation of TMF-1–Brg-1 and Brg-1–BAF170 nuclear complexes upon PDGF-BB stimulation. TMF-1, BAF170 and ARID1A immunocomplexes were immunoblotted for Brg-1 and reblotted for BAF170 or blotted for ARID1A and reblotted for TMF-1. 10% of the input lysate was loaded on each gel. Arrows indicate ARID1A protein. (e) Dissociation of TMF-1–Brg-1 and Brg-1–BAF155 nuclear complexes upon PDGF-BB stimulation. This experiment reproduces the result from d, but an antibody against the BAF155 core subunit of SWI–SNF chromatin remodeling complex was used instead of BAF170 for IP. Arrows indicate ARID1A protein. (f) Kinase-inactive PDGFRβ binds TMF-1 and prevents PDGF-BB–inducible dissociation of the SWI–SNF complex. Cells were pretreated with AG1296 and stimulated with PDGF-BB. TMF-1 immunocomplexes from nuclear extracts were blotted for PDGFRβ (top) and reblotted for TMF-1 (second) or blotted for Brg-1 (third) and reblotted for TMF-1 (bottom). Molecular mass was measured in kilodaltons. IB, immunoblotting; IP, immunoprecipitation.

    Techniques Used: Immunofluorescence, Staining, Immunoprecipitation, Western Blot

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    Cell Signaling Technology Inc arid1a
    Cellular proliferation by AKT phosphorylation is induced by <t>ARID1A</t> knockdown. Notes: ( A ) Western blotting for the screening of ARID1A in gastric cancer cell lines. ( B – D ) After transfection of ARID1A siRNA, cell viabilities were significantly increased ( ** P <0.001; paired t -test). Knockdown of ARID1A increased the phosphorylation of AKT and the downstream S6.
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    Genome-wide analysis of <t>H3.3-ARID1A</t> chromatin co-regulation. A Genomic annotation of 40,006 genome-wide H3.3 ChIP-seq peaks in 12Z cells ( n = 2). B Distribution of H3.3 peak widths. Median H3.3 peak width is 1830 bp. C Genome-wide overlap of ARID1A and H3.3 ChIP-seq peaks. D Genome-wide association between H3.3 and other previously measured chromatin features, per genomic bp, quantified as [observed / expected]. Statistic is hypergeometric enrichment. E Enrichment for H3.3 and ARID1A co-regulation across 18 chromatin states previously modeled via ChromHMM . Left, enrichment of H3.3 peaks; center, enrichment of H3.3+ARID1A binding; right, enrichment of ARID1A binding at sites with vs. without H3.3. Statistic is hypergeometric enrichment. F Left, ARID1A binding levels (ChIP/input fold-enrichment, FE) at H3.3+ vs. H3.3− ARID1A peaks. Right, H3.3 abundance (ChIP/input fold-enrichment) at ARID1A+ vs. ARID1A− H3.3 peaks. Statistic is two-tailed, unpaired Wilcoxon’s test. G Top, enrichment of H3.3 at genes promoter-proximally bound by ARID1A. Bottom, enrichment of ARID1A+H3.3 co-binding at genes DE following ARID1A loss (siARID1A treatment). Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. H Example hg38 browser shots of genes and regulatory elements co-regulated by H3.3 and ARID1A. y -axis is log-likelihood ratio (logLR) of assay signal (compared to input chromatin for ChIP-seq or background genome for ATAC-seq). Small bars under tracks indicate significant peak detection by MACS2 (FDR < 0.05). Super-enhancers were detected by ROSE from H3K27ac ChIP-seq. * p < 0.05, ** p < 0.01, *** p < 0.001
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    Cell Signaling Technology Inc complex ii
    Genome-wide analysis of <t>H3.3-ARID1A</t> chromatin co-regulation. A Genomic annotation of 40,006 genome-wide H3.3 ChIP-seq peaks in 12Z cells ( n = 2). B Distribution of H3.3 peak widths. Median H3.3 peak width is 1830 bp. C Genome-wide overlap of ARID1A and H3.3 ChIP-seq peaks. D Genome-wide association between H3.3 and other previously measured chromatin features, per genomic bp, quantified as [observed / expected]. Statistic is hypergeometric enrichment. E Enrichment for H3.3 and ARID1A co-regulation across 18 chromatin states previously modeled via ChromHMM . Left, enrichment of H3.3 peaks; center, enrichment of H3.3+ARID1A binding; right, enrichment of ARID1A binding at sites with vs. without H3.3. Statistic is hypergeometric enrichment. F Left, ARID1A binding levels (ChIP/input fold-enrichment, FE) at H3.3+ vs. H3.3− ARID1A peaks. Right, H3.3 abundance (ChIP/input fold-enrichment) at ARID1A+ vs. ARID1A− H3.3 peaks. Statistic is two-tailed, unpaired Wilcoxon’s test. G Top, enrichment of H3.3 at genes promoter-proximally bound by ARID1A. Bottom, enrichment of ARID1A+H3.3 co-binding at genes DE following ARID1A loss (siARID1A treatment). Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. H Example hg38 browser shots of genes and regulatory elements co-regulated by H3.3 and ARID1A. y -axis is log-likelihood ratio (logLR) of assay signal (compared to input chromatin for ChIP-seq or background genome for ATAC-seq). Small bars under tracks indicate significant peak detection by MACS2 (FDR < 0.05). Super-enhancers were detected by ROSE from H3K27ac ChIP-seq. * p < 0.05, ** p < 0.01, *** p < 0.001
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    Cell Signaling Technology Inc anti arid1a antibody
    Genome-wide analysis of <t>H3.3-ARID1A</t> chromatin co-regulation. A Genomic annotation of 40,006 genome-wide H3.3 ChIP-seq peaks in 12Z cells ( n = 2). B Distribution of H3.3 peak widths. Median H3.3 peak width is 1830 bp. C Genome-wide overlap of ARID1A and H3.3 ChIP-seq peaks. D Genome-wide association between H3.3 and other previously measured chromatin features, per genomic bp, quantified as [observed / expected]. Statistic is hypergeometric enrichment. E Enrichment for H3.3 and ARID1A co-regulation across 18 chromatin states previously modeled via ChromHMM . Left, enrichment of H3.3 peaks; center, enrichment of H3.3+ARID1A binding; right, enrichment of ARID1A binding at sites with vs. without H3.3. Statistic is hypergeometric enrichment. F Left, ARID1A binding levels (ChIP/input fold-enrichment, FE) at H3.3+ vs. H3.3− ARID1A peaks. Right, H3.3 abundance (ChIP/input fold-enrichment) at ARID1A+ vs. ARID1A− H3.3 peaks. Statistic is two-tailed, unpaired Wilcoxon’s test. G Top, enrichment of H3.3 at genes promoter-proximally bound by ARID1A. Bottom, enrichment of ARID1A+H3.3 co-binding at genes DE following ARID1A loss (siARID1A treatment). Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. H Example hg38 browser shots of genes and regulatory elements co-regulated by H3.3 and ARID1A. y -axis is log-likelihood ratio (logLR) of assay signal (compared to input chromatin for ChIP-seq or background genome for ATAC-seq). Small bars under tracks indicate significant peak detection by MACS2 (FDR < 0.05). Super-enhancers were detected by ROSE from H3K27ac ChIP-seq. * p < 0.05, ** p < 0.01, *** p < 0.001
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    Cellular proliferation by AKT phosphorylation is induced by ARID1A knockdown. Notes: ( A ) Western blotting for the screening of ARID1A in gastric cancer cell lines. ( B – D ) After transfection of ARID1A siRNA, cell viabilities were significantly increased ( ** P <0.001; paired t -test). Knockdown of ARID1A increased the phosphorylation of AKT and the downstream S6.

    Journal: OncoTargets and therapy

    Article Title: AKT inhibition is an effective treatment strategy in ARID1A-deficient gastric cancer cells

    doi: 10.2147/OTT.S139664

    Figure Lengend Snippet: Cellular proliferation by AKT phosphorylation is induced by ARID1A knockdown. Notes: ( A ) Western blotting for the screening of ARID1A in gastric cancer cell lines. ( B – D ) After transfection of ARID1A siRNA, cell viabilities were significantly increased ( ** P <0.001; paired t -test). Knockdown of ARID1A increased the phosphorylation of AKT and the downstream S6.

    Article Snippet: The following reagents and antibodies were used: GSK690693 (Cell Signalling Technology, Danvers, MA, USA), 5-fluorouracil (5-FU) and cisplatin (both from Sigma-Aldrich, St Louis, MO, USA), ARID1A, p-AKT, AKT, p-S6, poly-ADP ribose polymerase (1:1,000; Cell Signalling Technology), and β-actin (1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

    Techniques: Western Blot, Transfection

    ARID1A depletion leads to increased sensitivity toward AKT pathway inhibitors. Notes: Increased sensitivity of ARID1A-depleted MKN-1, MKN-28, and KATO-III cells toward GSK690693 (AKT inhibitor) was observed than that of controls. * P <0.001; paired t -test. Abbreviation: NC, normal control.

    Journal: OncoTargets and therapy

    Article Title: AKT inhibition is an effective treatment strategy in ARID1A-deficient gastric cancer cells

    doi: 10.2147/OTT.S139664

    Figure Lengend Snippet: ARID1A depletion leads to increased sensitivity toward AKT pathway inhibitors. Notes: Increased sensitivity of ARID1A-depleted MKN-1, MKN-28, and KATO-III cells toward GSK690693 (AKT inhibitor) was observed than that of controls. * P <0.001; paired t -test. Abbreviation: NC, normal control.

    Article Snippet: The following reagents and antibodies were used: GSK690693 (Cell Signalling Technology, Danvers, MA, USA), 5-fluorouracil (5-FU) and cisplatin (both from Sigma-Aldrich, St Louis, MO, USA), ARID1A, p-AKT, AKT, p-S6, poly-ADP ribose polymerase (1:1,000; Cell Signalling Technology), and β-actin (1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

    Techniques:

    Loss of ARID1A expression is associated with high sensitivity to the AKT inhibitor in gastric cancer cell lines. Notes: ARID1A-deficient MKN-45 cells showed the highest sensitivity toward GSK690693 treatment. The IC 50 value of MKN-45 was 0.043, while the IC 50 values of the ARID1A-intact MKN-1, MKN-28, and KATO-III cells were 0.132, 0.084, and 4.521, respectively ( P <0.001). The P -value indicates the divergence of the IC 50 values calculated by the F -test. Abbreviation: IC 50 , half inhibitory concentration.

    Journal: OncoTargets and therapy

    Article Title: AKT inhibition is an effective treatment strategy in ARID1A-deficient gastric cancer cells

    doi: 10.2147/OTT.S139664

    Figure Lengend Snippet: Loss of ARID1A expression is associated with high sensitivity to the AKT inhibitor in gastric cancer cell lines. Notes: ARID1A-deficient MKN-45 cells showed the highest sensitivity toward GSK690693 treatment. The IC 50 value of MKN-45 was 0.043, while the IC 50 values of the ARID1A-intact MKN-1, MKN-28, and KATO-III cells were 0.132, 0.084, and 4.521, respectively ( P <0.001). The P -value indicates the divergence of the IC 50 values calculated by the F -test. Abbreviation: IC 50 , half inhibitory concentration.

    Article Snippet: The following reagents and antibodies were used: GSK690693 (Cell Signalling Technology, Danvers, MA, USA), 5-fluorouracil (5-FU) and cisplatin (both from Sigma-Aldrich, St Louis, MO, USA), ARID1A, p-AKT, AKT, p-S6, poly-ADP ribose polymerase (1:1,000; Cell Signalling Technology), and β-actin (1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

    Techniques: Expressing, Concentration Assay

    AKT inhibition leads to increased apoptosis in ARID1A-deficient cells. Notes: ( A ) Treatment with the AKT inhibitor GSK690693 (at a concentration of 10 μmol/L) completely abrogated p-Akt induced by ARID1A knockdown in ARID1A-deficient MKN-28 cells and led to reduced p-S6, in contrast to the controls. PARP cleavage was more increased in ARID1A-knockdown cells treated with GSK690693. ( B ) Flow cytometry confirmed the increased apoptosis in ARID1A-deficient cells treated with GSK690693 (0.01 μmol/L) in contrast to the controls ( ** P <0.001; paired t -test). Abbreviations: FITC, fluorescein isothiocyanate; PARP, poly-ADP ribose polymerase.

    Journal: OncoTargets and therapy

    Article Title: AKT inhibition is an effective treatment strategy in ARID1A-deficient gastric cancer cells

    doi: 10.2147/OTT.S139664

    Figure Lengend Snippet: AKT inhibition leads to increased apoptosis in ARID1A-deficient cells. Notes: ( A ) Treatment with the AKT inhibitor GSK690693 (at a concentration of 10 μmol/L) completely abrogated p-Akt induced by ARID1A knockdown in ARID1A-deficient MKN-28 cells and led to reduced p-S6, in contrast to the controls. PARP cleavage was more increased in ARID1A-knockdown cells treated with GSK690693. ( B ) Flow cytometry confirmed the increased apoptosis in ARID1A-deficient cells treated with GSK690693 (0.01 μmol/L) in contrast to the controls ( ** P <0.001; paired t -test). Abbreviations: FITC, fluorescein isothiocyanate; PARP, poly-ADP ribose polymerase.

    Article Snippet: The following reagents and antibodies were used: GSK690693 (Cell Signalling Technology, Danvers, MA, USA), 5-fluorouracil (5-FU) and cisplatin (both from Sigma-Aldrich, St Louis, MO, USA), ARID1A, p-AKT, AKT, p-S6, poly-ADP ribose polymerase (1:1,000; Cell Signalling Technology), and β-actin (1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

    Techniques: Inhibition, Concentration Assay, Flow Cytometry

    Loss of ARID1A expression did not induce resistance to the conventional chemotherapy. Notes: To investigate the antiproliferative effect of conventional chemotherapy in ARID1A-depleted GC cells, 5-FU or cisplatin was applied at different drug concentrations (10–60 μmol/L) for 48 hours to MKN-1, MKN-28, and KATO-III cells transfected with control-shRNA and shARID1A. Drug sensitivities did not differ between these groups. Abbreviations: 5-FU, 5-fluorouracil; GC, gastric cancer.

    Journal: OncoTargets and therapy

    Article Title: AKT inhibition is an effective treatment strategy in ARID1A-deficient gastric cancer cells

    doi: 10.2147/OTT.S139664

    Figure Lengend Snippet: Loss of ARID1A expression did not induce resistance to the conventional chemotherapy. Notes: To investigate the antiproliferative effect of conventional chemotherapy in ARID1A-depleted GC cells, 5-FU or cisplatin was applied at different drug concentrations (10–60 μmol/L) for 48 hours to MKN-1, MKN-28, and KATO-III cells transfected with control-shRNA and shARID1A. Drug sensitivities did not differ between these groups. Abbreviations: 5-FU, 5-fluorouracil; GC, gastric cancer.

    Article Snippet: The following reagents and antibodies were used: GSK690693 (Cell Signalling Technology, Danvers, MA, USA), 5-fluorouracil (5-FU) and cisplatin (both from Sigma-Aldrich, St Louis, MO, USA), ARID1A, p-AKT, AKT, p-S6, poly-ADP ribose polymerase (1:1,000; Cell Signalling Technology), and β-actin (1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

    Techniques: Expressing, Transfection, shRNA

    Addition of AKT inhibitors to conventional chemotherapy increases antitumor activity in ARID1A-deficient cancer cells. Notes: ( A ) 5-FU (10 μmol/L) or cisplatin (10 μmol/L) was applied to MKN-28 and KATO-III cells in the presence of a minimal drug concentration of GSK690693 (0.01 μmol/L). Compared with single agent alone, addition of GSK690693 to the conventional chemotherapy induced more decreased cell viability in ARID1A-knockdown MKN-28 and KATO-III cells than in wild-type cells ( * P <0.01). ( B ) GSK690693 in combination with 5-FU or cisplatin induced a significant increase in apoptosis, compared with 5-FU or cisplatin alone in ARID1A-knockdown cells ( * P <0.01). ** P <0.001. Abbreviation: 5-FU, 5-fluorouracil.

    Journal: OncoTargets and therapy

    Article Title: AKT inhibition is an effective treatment strategy in ARID1A-deficient gastric cancer cells

    doi: 10.2147/OTT.S139664

    Figure Lengend Snippet: Addition of AKT inhibitors to conventional chemotherapy increases antitumor activity in ARID1A-deficient cancer cells. Notes: ( A ) 5-FU (10 μmol/L) or cisplatin (10 μmol/L) was applied to MKN-28 and KATO-III cells in the presence of a minimal drug concentration of GSK690693 (0.01 μmol/L). Compared with single agent alone, addition of GSK690693 to the conventional chemotherapy induced more decreased cell viability in ARID1A-knockdown MKN-28 and KATO-III cells than in wild-type cells ( * P <0.01). ( B ) GSK690693 in combination with 5-FU or cisplatin induced a significant increase in apoptosis, compared with 5-FU or cisplatin alone in ARID1A-knockdown cells ( * P <0.01). ** P <0.001. Abbreviation: 5-FU, 5-fluorouracil.

    Article Snippet: The following reagents and antibodies were used: GSK690693 (Cell Signalling Technology, Danvers, MA, USA), 5-fluorouracil (5-FU) and cisplatin (both from Sigma-Aldrich, St Louis, MO, USA), ARID1A, p-AKT, AKT, p-S6, poly-ADP ribose polymerase (1:1,000; Cell Signalling Technology), and β-actin (1:5,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA).

    Techniques: Activity Assay, Concentration Assay

    Genome-wide analysis of H3.3-ARID1A chromatin co-regulation. A Genomic annotation of 40,006 genome-wide H3.3 ChIP-seq peaks in 12Z cells ( n = 2). B Distribution of H3.3 peak widths. Median H3.3 peak width is 1830 bp. C Genome-wide overlap of ARID1A and H3.3 ChIP-seq peaks. D Genome-wide association between H3.3 and other previously measured chromatin features, per genomic bp, quantified as [observed / expected]. Statistic is hypergeometric enrichment. E Enrichment for H3.3 and ARID1A co-regulation across 18 chromatin states previously modeled via ChromHMM . Left, enrichment of H3.3 peaks; center, enrichment of H3.3+ARID1A binding; right, enrichment of ARID1A binding at sites with vs. without H3.3. Statistic is hypergeometric enrichment. F Left, ARID1A binding levels (ChIP/input fold-enrichment, FE) at H3.3+ vs. H3.3− ARID1A peaks. Right, H3.3 abundance (ChIP/input fold-enrichment) at ARID1A+ vs. ARID1A− H3.3 peaks. Statistic is two-tailed, unpaired Wilcoxon’s test. G Top, enrichment of H3.3 at genes promoter-proximally bound by ARID1A. Bottom, enrichment of ARID1A+H3.3 co-binding at genes DE following ARID1A loss (siARID1A treatment). Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. H Example hg38 browser shots of genes and regulatory elements co-regulated by H3.3 and ARID1A. y -axis is log-likelihood ratio (logLR) of assay signal (compared to input chromatin for ChIP-seq or background genome for ATAC-seq). Small bars under tracks indicate significant peak detection by MACS2 (FDR < 0.05). Super-enhancers were detected by ROSE from H3K27ac ChIP-seq. * p < 0.05, ** p < 0.01, *** p < 0.001

    Journal: BMC Biology

    Article Title: ARID1A-dependent maintenance of H3.3 is required for repressive CHD4-ZMYND8 chromatin interactions at super-enhancers

    doi: 10.1186/s12915-022-01407-y

    Figure Lengend Snippet: Genome-wide analysis of H3.3-ARID1A chromatin co-regulation. A Genomic annotation of 40,006 genome-wide H3.3 ChIP-seq peaks in 12Z cells ( n = 2). B Distribution of H3.3 peak widths. Median H3.3 peak width is 1830 bp. C Genome-wide overlap of ARID1A and H3.3 ChIP-seq peaks. D Genome-wide association between H3.3 and other previously measured chromatin features, per genomic bp, quantified as [observed / expected]. Statistic is hypergeometric enrichment. E Enrichment for H3.3 and ARID1A co-regulation across 18 chromatin states previously modeled via ChromHMM . Left, enrichment of H3.3 peaks; center, enrichment of H3.3+ARID1A binding; right, enrichment of ARID1A binding at sites with vs. without H3.3. Statistic is hypergeometric enrichment. F Left, ARID1A binding levels (ChIP/input fold-enrichment, FE) at H3.3+ vs. H3.3− ARID1A peaks. Right, H3.3 abundance (ChIP/input fold-enrichment) at ARID1A+ vs. ARID1A− H3.3 peaks. Statistic is two-tailed, unpaired Wilcoxon’s test. G Top, enrichment of H3.3 at genes promoter-proximally bound by ARID1A. Bottom, enrichment of ARID1A+H3.3 co-binding at genes DE following ARID1A loss (siARID1A treatment). Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. H Example hg38 browser shots of genes and regulatory elements co-regulated by H3.3 and ARID1A. y -axis is log-likelihood ratio (logLR) of assay signal (compared to input chromatin for ChIP-seq or background genome for ATAC-seq). Small bars under tracks indicate significant peak detection by MACS2 (FDR < 0.05). Super-enhancers were detected by ROSE from H3K27ac ChIP-seq. * p < 0.05, ** p < 0.01, *** p < 0.001

    Article Snippet: The following primary antibodies were used: anti-ARID1A (D2A8U, Cell Signaling), anti-CHD4 (D4B7, Cell Signaling), anti-ZMYND8 (A302-089, Bethyl), anti-ZMYND8 (Atlas), anti-BRG1 (ab110641, abcam), anti-BAF155 (D7F8S, Cell Signaling), anti-HDAC1 (10E2, Cell Signaling), anti-histone H3.3 (ab176840, abcam), anti-histone H3.3 (2D7-H1, abnova), and anti-histone H3 (D1H2, Cell Signaling).

    Techniques: Genome Wide, ChIP-sequencing, GWAS, Binding Assay, Two Tailed Test

    Genome-wide analysis of ARID1A-dependent H3.3. A MA plot of shARID1A vs. control differential H3.3 ChIP-seq ( n = 2), across 67,502 tested genomic regions. Regions are colored based on shARID1A differential H3.3 significance. Inset pie chart depicts distribution of significantly increasing and decreasing H3.3 regions ( csaw / edgeR FDR < 0.05) compared to stable H3.3 (FDR > 0.05). FDR < 0.05 was used as the significance threshold for all downstream analyses. B shARID1A differential H3.3 regions segregated by detection of ARID1A binding in wild-type cells. Left, MA plot with all genome-wide H3.3 tested regions, colored by ARID1A binding status. Right, box plot quantification of shARID1A log 2 FC H3.3 abundance, segregated by ARID1A binding status. Statistic is two-tailed, unpaired Wilcoxon’s test. C Analysis of canonical H3 (H3.1/3.2) changes (ChIP-seq, n = 2) at H3.3-marked genomic regions following ARID1A knockdown (shARID1A), segregated by ARID1A binding status as in B . Statistic is two-tailed, unpaired Wilcoxon’s test. D Enrichment of ARID1A binding detection at regions with decreasing H3.3 following ARID1A loss compared to all tested H3.3 regions. Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. E Magnitude of H3.3 change (log 2 FC) among ARID1A-bound, shARID1A significantly decreasing vs. increasing H3.3 regions. Statistic is two-tailed, unpaired Wilcoxon’s test. F Distribution of H3.3-enriched region widths among shARID1A stable vs. increasing vs. decreasing H3.3 regions. Statistic is two-tailed, unpaired Wilcoxon’s test. G Chromatin state enrichment among shARID1A increasing and decreasing H3.3 regions, calculated per 200 bp genomic interval. Statistic is hypergeometric enrichment. H Top 10 significant (FDR < 0.05) enriched Hallmark pathways (left) and GO Biological Process gene sets (right) among genes with ARID1A-bound, shARID1A decreasing promoter-proximal H3.3. I Representative hg38 locus near CCL2 displaying H3.3 maintained by ARID1A chromatin interactions. *** p < 0.001

    Journal: BMC Biology

    Article Title: ARID1A-dependent maintenance of H3.3 is required for repressive CHD4-ZMYND8 chromatin interactions at super-enhancers

    doi: 10.1186/s12915-022-01407-y

    Figure Lengend Snippet: Genome-wide analysis of ARID1A-dependent H3.3. A MA plot of shARID1A vs. control differential H3.3 ChIP-seq ( n = 2), across 67,502 tested genomic regions. Regions are colored based on shARID1A differential H3.3 significance. Inset pie chart depicts distribution of significantly increasing and decreasing H3.3 regions ( csaw / edgeR FDR < 0.05) compared to stable H3.3 (FDR > 0.05). FDR < 0.05 was used as the significance threshold for all downstream analyses. B shARID1A differential H3.3 regions segregated by detection of ARID1A binding in wild-type cells. Left, MA plot with all genome-wide H3.3 tested regions, colored by ARID1A binding status. Right, box plot quantification of shARID1A log 2 FC H3.3 abundance, segregated by ARID1A binding status. Statistic is two-tailed, unpaired Wilcoxon’s test. C Analysis of canonical H3 (H3.1/3.2) changes (ChIP-seq, n = 2) at H3.3-marked genomic regions following ARID1A knockdown (shARID1A), segregated by ARID1A binding status as in B . Statistic is two-tailed, unpaired Wilcoxon’s test. D Enrichment of ARID1A binding detection at regions with decreasing H3.3 following ARID1A loss compared to all tested H3.3 regions. Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. E Magnitude of H3.3 change (log 2 FC) among ARID1A-bound, shARID1A significantly decreasing vs. increasing H3.3 regions. Statistic is two-tailed, unpaired Wilcoxon’s test. F Distribution of H3.3-enriched region widths among shARID1A stable vs. increasing vs. decreasing H3.3 regions. Statistic is two-tailed, unpaired Wilcoxon’s test. G Chromatin state enrichment among shARID1A increasing and decreasing H3.3 regions, calculated per 200 bp genomic interval. Statistic is hypergeometric enrichment. H Top 10 significant (FDR < 0.05) enriched Hallmark pathways (left) and GO Biological Process gene sets (right) among genes with ARID1A-bound, shARID1A decreasing promoter-proximal H3.3. I Representative hg38 locus near CCL2 displaying H3.3 maintained by ARID1A chromatin interactions. *** p < 0.001

    Article Snippet: The following primary antibodies were used: anti-ARID1A (D2A8U, Cell Signaling), anti-CHD4 (D4B7, Cell Signaling), anti-ZMYND8 (A302-089, Bethyl), anti-ZMYND8 (Atlas), anti-BRG1 (ab110641, abcam), anti-BAF155 (D7F8S, Cell Signaling), anti-HDAC1 (10E2, Cell Signaling), anti-histone H3.3 (ab176840, abcam), anti-histone H3.3 (2D7-H1, abnova), and anti-histone H3 (D1H2, Cell Signaling).

    Techniques: Genome Wide, ChIP-sequencing, Binding Assay, Two Tailed Test

    Transcriptional effects of H3.3 depletion and overlap with ARID1A. A Baseline relative linear expression of H3F3A ( H3-3A ) and H3F3B ( H3-3B ) gene isoforms encoding H3.3, as measured by RNA-seq ( n = 3). B Western blot for H3.3 and total H3 in control vs. siH3F3B treated cells. C Global transcriptomic effects of 24,192 genes following H3.3 knockdown via siH3F3B treatment (RNA-seq, n = 3). Red dots represent significant DE genes ( DESeq2 , FDR < 0.001). D Relative linear expression of H3F3A and H3F3B by RNA-seq in control and siH3F3B cells ( n = 3). E Volcano plot depicting siH3F3B vs. control differential gene expression (DGE). Top significant genes are labeled. F Significant overlap in DE genes following H3.3 knockdown (siH3F3B) vs. ARID1A knockdown (siARID1A). Statistic is hypergeometric enrichment. G Directional segregation of siH3F3B/siARID1A overlapping DE genes. A positive association is observed by chi-squared test, i.e., genes are more likely to be upregulated or downregulated in both conditions as opposed to antagonistic regulation. H Scatter plot of siH3F3B vs. siARID1A expression log 2 FC (with shrinkage correction) for all 19,900 transcriptome-wide commonly detected genes. Statistics are Pearson ( r ) and Spearman ( r s ) correlation coefficients. Colored dots indicate significant DE genes (FDR < 0.001) in both treatment conditions. I Association between H3.3 transcriptional repression (siH3F3B upregulation) and transcriptional co-regulation by ARID1A (siARID1A DE). Statistic is two-tailed Fisher’s exact test. J Scatter plot of 196 shared DE genes upregulated following knockdown of either H3.3 or ARID1A. These genes are mutually repressed by H3.3 and ARID1A. K Top significant (FDR < 0.05) enriched gene sets among the 196 ARID1A-H3.3 mutually repressed genes among various gene set databases

    Journal: BMC Biology

    Article Title: ARID1A-dependent maintenance of H3.3 is required for repressive CHD4-ZMYND8 chromatin interactions at super-enhancers

    doi: 10.1186/s12915-022-01407-y

    Figure Lengend Snippet: Transcriptional effects of H3.3 depletion and overlap with ARID1A. A Baseline relative linear expression of H3F3A ( H3-3A ) and H3F3B ( H3-3B ) gene isoforms encoding H3.3, as measured by RNA-seq ( n = 3). B Western blot for H3.3 and total H3 in control vs. siH3F3B treated cells. C Global transcriptomic effects of 24,192 genes following H3.3 knockdown via siH3F3B treatment (RNA-seq, n = 3). Red dots represent significant DE genes ( DESeq2 , FDR < 0.001). D Relative linear expression of H3F3A and H3F3B by RNA-seq in control and siH3F3B cells ( n = 3). E Volcano plot depicting siH3F3B vs. control differential gene expression (DGE). Top significant genes are labeled. F Significant overlap in DE genes following H3.3 knockdown (siH3F3B) vs. ARID1A knockdown (siARID1A). Statistic is hypergeometric enrichment. G Directional segregation of siH3F3B/siARID1A overlapping DE genes. A positive association is observed by chi-squared test, i.e., genes are more likely to be upregulated or downregulated in both conditions as opposed to antagonistic regulation. H Scatter plot of siH3F3B vs. siARID1A expression log 2 FC (with shrinkage correction) for all 19,900 transcriptome-wide commonly detected genes. Statistics are Pearson ( r ) and Spearman ( r s ) correlation coefficients. Colored dots indicate significant DE genes (FDR < 0.001) in both treatment conditions. I Association between H3.3 transcriptional repression (siH3F3B upregulation) and transcriptional co-regulation by ARID1A (siARID1A DE). Statistic is two-tailed Fisher’s exact test. J Scatter plot of 196 shared DE genes upregulated following knockdown of either H3.3 or ARID1A. These genes are mutually repressed by H3.3 and ARID1A. K Top significant (FDR < 0.05) enriched gene sets among the 196 ARID1A-H3.3 mutually repressed genes among various gene set databases

    Article Snippet: The following primary antibodies were used: anti-ARID1A (D2A8U, Cell Signaling), anti-CHD4 (D4B7, Cell Signaling), anti-ZMYND8 (A302-089, Bethyl), anti-ZMYND8 (Atlas), anti-BRG1 (ab110641, abcam), anti-BAF155 (D7F8S, Cell Signaling), anti-HDAC1 (10E2, Cell Signaling), anti-histone H3.3 (ab176840, abcam), anti-histone H3.3 (2D7-H1, abnova), and anti-histone H3 (D1H2, Cell Signaling).

    Techniques: Expressing, RNA Sequencing Assay, Western Blot, Labeling, Two Tailed Test

    Characterization of ARID1A, CHD4, and ZMYND8 chromatin interactions co-regulating H3.3. A Genome-wide associations between ARID1A binding at H3.3+ vs. H3.3− regions for all 1135 transcriptional regulator peak sets included in the ReMap2020 peak database. Labeled factors exhibit an H3.3+ ARID1A binding association with genomic odds ratio >2 and overlap with >0.1% of ARID1A binding sites. ZMYND11 and ZMYND8 (bolded) are two of the top factors most associated with H3.3+ ARID1A binding. B Chromatin model schematic depicting hypothesized relationship between ARID1A-SWI/SNF and ZMYND8 co-regulation of H3.3, possibly mediated by co-factors. C ARID1A co-immunoprecipitation detecting physical interaction with NuRD catalytic subunit CHD4, but not ZMYND8. D CHD4 co-immunoprecipitation detecting physical interactions with both ARID1A and ZMYND8. E 10–30% glycerol gradient sedimentation and immunoblotting for SWI/SNF, NuRD, and ZMYND8. Relative fractions display native protein complexes transitioning from low molecular weight (left) to high molecular weight (right). Underlined fractions highlight potential interacting native complexes containing ZMYND8 and members of SWI/SNF (BAF) and NuRD (Mi-2β). F Genome-wide ChIP-seq ( n = 2) peak overlaps between ARID1A, CHD4, ZMYND8, and H3.3. Peak numbers within the Euler diagram are approximations and not mutually exclusive due to varying peak sizes. G Example locus on chromosome 10 displaying ARID1A, CHD4, ZMYND8, and H3.3 co-regulation. H Enrichment for ARID1A co-regulation of H3.3 peaks bound by CHD4 and/or ZMYND8. Statistic is two-tailed Fisher’s exact test. I Average ChIP-seq signal density histograms for ARID1A (left) and H3.3 (right) at H3.3 peaks bound by CHD4 and/or ZMYND8. J H3.3 abundance (ChIP FPKM) at ARID1A-bound shARID1A differential H3.3 regions co-bound by CHD4 or ZMYND8. Statistic is two-tailed, unpaired Wilcoxon’s test. K Positive association between CHD4 binding (top) and negative association between ZMYND8 binding (bottom) and ARID1A maintenance of H3.3 chromatin, genome-wide. Statistic is two-tailed Fisher’s exact test. L Average ChIP-seq signal density histograms for ARID1A, H3.3, CHD4, and ZMYND8 across ARID1A-bound H3.3 regions that decreased or were stable with shARID1A

    Journal: BMC Biology

    Article Title: ARID1A-dependent maintenance of H3.3 is required for repressive CHD4-ZMYND8 chromatin interactions at super-enhancers

    doi: 10.1186/s12915-022-01407-y

    Figure Lengend Snippet: Characterization of ARID1A, CHD4, and ZMYND8 chromatin interactions co-regulating H3.3. A Genome-wide associations between ARID1A binding at H3.3+ vs. H3.3− regions for all 1135 transcriptional regulator peak sets included in the ReMap2020 peak database. Labeled factors exhibit an H3.3+ ARID1A binding association with genomic odds ratio >2 and overlap with >0.1% of ARID1A binding sites. ZMYND11 and ZMYND8 (bolded) are two of the top factors most associated with H3.3+ ARID1A binding. B Chromatin model schematic depicting hypothesized relationship between ARID1A-SWI/SNF and ZMYND8 co-regulation of H3.3, possibly mediated by co-factors. C ARID1A co-immunoprecipitation detecting physical interaction with NuRD catalytic subunit CHD4, but not ZMYND8. D CHD4 co-immunoprecipitation detecting physical interactions with both ARID1A and ZMYND8. E 10–30% glycerol gradient sedimentation and immunoblotting for SWI/SNF, NuRD, and ZMYND8. Relative fractions display native protein complexes transitioning from low molecular weight (left) to high molecular weight (right). Underlined fractions highlight potential interacting native complexes containing ZMYND8 and members of SWI/SNF (BAF) and NuRD (Mi-2β). F Genome-wide ChIP-seq ( n = 2) peak overlaps between ARID1A, CHD4, ZMYND8, and H3.3. Peak numbers within the Euler diagram are approximations and not mutually exclusive due to varying peak sizes. G Example locus on chromosome 10 displaying ARID1A, CHD4, ZMYND8, and H3.3 co-regulation. H Enrichment for ARID1A co-regulation of H3.3 peaks bound by CHD4 and/or ZMYND8. Statistic is two-tailed Fisher’s exact test. I Average ChIP-seq signal density histograms for ARID1A (left) and H3.3 (right) at H3.3 peaks bound by CHD4 and/or ZMYND8. J H3.3 abundance (ChIP FPKM) at ARID1A-bound shARID1A differential H3.3 regions co-bound by CHD4 or ZMYND8. Statistic is two-tailed, unpaired Wilcoxon’s test. K Positive association between CHD4 binding (top) and negative association between ZMYND8 binding (bottom) and ARID1A maintenance of H3.3 chromatin, genome-wide. Statistic is two-tailed Fisher’s exact test. L Average ChIP-seq signal density histograms for ARID1A, H3.3, CHD4, and ZMYND8 across ARID1A-bound H3.3 regions that decreased or were stable with shARID1A

    Article Snippet: The following primary antibodies were used: anti-ARID1A (D2A8U, Cell Signaling), anti-CHD4 (D4B7, Cell Signaling), anti-ZMYND8 (A302-089, Bethyl), anti-ZMYND8 (Atlas), anti-BRG1 (ab110641, abcam), anti-BAF155 (D7F8S, Cell Signaling), anti-HDAC1 (10E2, Cell Signaling), anti-histone H3.3 (ab176840, abcam), anti-histone H3.3 (2D7-H1, abnova), and anti-histone H3 (D1H2, Cell Signaling).

    Techniques: Genome Wide, Binding Assay, Labeling, Immunoprecipitation, Sedimentation, Western Blot, Molecular Weight, ChIP-sequencing, Two Tailed Test

    Genome-wide analysis of ARID1A-dependent CHD4 binding. A MA plot of shARID1A vs. control differential CHD4 ChIP-seq ( n = 2), across 44,567 tested genomic regions of CHD4 binding. Regions are colored based on shARID1A differential CHD4 binding significance. Inset pie chart depicts distribution of significantly increasing and decreasing CHD4 regions ( csaw / edgeR FDR < 0.05) compared to stable CHD4 (FDR > 0.05). FDR < 0.05 was used as the significance threshold for all downstream analyses. B Global analysis of ARID1A-dependent CHD4 binding based on presence of normal ARID1A binding. Box plot quantification of shARID1A log 2 FC CHD4 binding, segregated by ARID1A binding status. Statistic is two-tailed, unpaired Wilcoxon’s test. C Chromatin state enrichment among shARID1A increasing and decreasing CHD4 binding regions, calculated as observed/expected genomic fold-enrichment per genomic bp. Statistic is hypergeometric enrichment. D Enrichment of ARID1A binding detection at regions with decreasing CHD4 binding following ARID1A loss compared to all tested CHD4 binding sites. Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. E Enrichment of ARID1A-dependent H3.3 maintenance (shARID1A decreasing H3.3 abundance) at regions with decreasing CHD4 binding following ARID1A loss compared to all tested CHD4 binding sites. Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. F Associations between presence of H3.3 (wild-type peak) and ARID1A-bound, ARID1A-dependent CHD4 binding. Statistic is hypergeometric enrichment. G Associations between ZMYND8 co-binding (wild-type peak) and ARID1A-bound, ARID1A-dependent CHD4 binding. Statistic is hypergeometric enrichment. *** p < 0.001

    Journal: BMC Biology

    Article Title: ARID1A-dependent maintenance of H3.3 is required for repressive CHD4-ZMYND8 chromatin interactions at super-enhancers

    doi: 10.1186/s12915-022-01407-y

    Figure Lengend Snippet: Genome-wide analysis of ARID1A-dependent CHD4 binding. A MA plot of shARID1A vs. control differential CHD4 ChIP-seq ( n = 2), across 44,567 tested genomic regions of CHD4 binding. Regions are colored based on shARID1A differential CHD4 binding significance. Inset pie chart depicts distribution of significantly increasing and decreasing CHD4 regions ( csaw / edgeR FDR < 0.05) compared to stable CHD4 (FDR > 0.05). FDR < 0.05 was used as the significance threshold for all downstream analyses. B Global analysis of ARID1A-dependent CHD4 binding based on presence of normal ARID1A binding. Box plot quantification of shARID1A log 2 FC CHD4 binding, segregated by ARID1A binding status. Statistic is two-tailed, unpaired Wilcoxon’s test. C Chromatin state enrichment among shARID1A increasing and decreasing CHD4 binding regions, calculated as observed/expected genomic fold-enrichment per genomic bp. Statistic is hypergeometric enrichment. D Enrichment of ARID1A binding detection at regions with decreasing CHD4 binding following ARID1A loss compared to all tested CHD4 binding sites. Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. E Enrichment of ARID1A-dependent H3.3 maintenance (shARID1A decreasing H3.3 abundance) at regions with decreasing CHD4 binding following ARID1A loss compared to all tested CHD4 binding sites. Statistics are hypergeometric enrichment test and pairwise two-tailed Fisher’s exact test. F Associations between presence of H3.3 (wild-type peak) and ARID1A-bound, ARID1A-dependent CHD4 binding. Statistic is hypergeometric enrichment. G Associations between ZMYND8 co-binding (wild-type peak) and ARID1A-bound, ARID1A-dependent CHD4 binding. Statistic is hypergeometric enrichment. *** p < 0.001

    Article Snippet: The following primary antibodies were used: anti-ARID1A (D2A8U, Cell Signaling), anti-CHD4 (D4B7, Cell Signaling), anti-ZMYND8 (A302-089, Bethyl), anti-ZMYND8 (Atlas), anti-BRG1 (ab110641, abcam), anti-BAF155 (D7F8S, Cell Signaling), anti-HDAC1 (10E2, Cell Signaling), anti-histone H3.3 (ab176840, abcam), anti-histone H3.3 (2D7-H1, abnova), and anti-histone H3 (D1H2, Cell Signaling).

    Techniques: Genome Wide, Binding Assay, ChIP-sequencing, Two Tailed Test

    H3.3 enhancer regulation by ARID1A, CHD4, and ZMYND8. A Heatmap of chromatin features at 15,925 active typical enhancers and 1374 distal super-enhancer constituents (H3K27ac peaks co-marked by ATAC) segregated by ARID1A ± CHD4 ± ZMYND8 binding. Enhancers are centered on the H3K27ac peak, and signal is displayed as indicated for the flanking 5 kb in either direction. B ZMYND8 binding detection at ARID1A-bound typical and super-enhancers with or without CHD4 co-binding. Statistic is two-tailed Fisher’s exact test. C Association between ARID1A+CHD4+ZMYND8 co-binding at enhancers and presence of H3.3. H3.3+ super-enhancers show the most frequent co-binding. Statistic is two-tailed Fisher’s exact test. D Chromatin features at active super-enhancer constituents segregated by ARID1A loss-driven H3K27-acetylation dynamics: hyperacetylated, de-acetylated, or stably acetylated. Left, average ChIP-seq signal density histograms across enhancer classes. Right, violin plots quantifying signal (ChIP/input fold-enrichment) across enhancer classes. Statistic is two-tailed, unpaired Wilcoxon’s test

    Journal: BMC Biology

    Article Title: ARID1A-dependent maintenance of H3.3 is required for repressive CHD4-ZMYND8 chromatin interactions at super-enhancers

    doi: 10.1186/s12915-022-01407-y

    Figure Lengend Snippet: H3.3 enhancer regulation by ARID1A, CHD4, and ZMYND8. A Heatmap of chromatin features at 15,925 active typical enhancers and 1374 distal super-enhancer constituents (H3K27ac peaks co-marked by ATAC) segregated by ARID1A ± CHD4 ± ZMYND8 binding. Enhancers are centered on the H3K27ac peak, and signal is displayed as indicated for the flanking 5 kb in either direction. B ZMYND8 binding detection at ARID1A-bound typical and super-enhancers with or without CHD4 co-binding. Statistic is two-tailed Fisher’s exact test. C Association between ARID1A+CHD4+ZMYND8 co-binding at enhancers and presence of H3.3. H3.3+ super-enhancers show the most frequent co-binding. Statistic is two-tailed Fisher’s exact test. D Chromatin features at active super-enhancer constituents segregated by ARID1A loss-driven H3K27-acetylation dynamics: hyperacetylated, de-acetylated, or stably acetylated. Left, average ChIP-seq signal density histograms across enhancer classes. Right, violin plots quantifying signal (ChIP/input fold-enrichment) across enhancer classes. Statistic is two-tailed, unpaired Wilcoxon’s test

    Article Snippet: The following primary antibodies were used: anti-ARID1A (D2A8U, Cell Signaling), anti-CHD4 (D4B7, Cell Signaling), anti-ZMYND8 (A302-089, Bethyl), anti-ZMYND8 (Atlas), anti-BRG1 (ab110641, abcam), anti-BAF155 (D7F8S, Cell Signaling), anti-HDAC1 (10E2, Cell Signaling), anti-histone H3.3 (ab176840, abcam), anti-histone H3.3 (2D7-H1, abnova), and anti-histone H3 (D1H2, Cell Signaling).

    Techniques: Binding Assay, Two Tailed Test, Stable Transfection, ChIP-sequencing

    ZMYND8-mediated chromatin repression is associated with H4(K16)ac. A Heatmap of clustered, normalized feature emission probabilities, and associated functional annotation of the new 12-feature, genome-wide chromatin 25-state model. States (S_) are labeled based on order of normalized emission probability clustering. See “ ” for details on optimal model selection. SE: super-enhancer. B Genomic fold-enrichment (FE) for ARID1A, CHD4, ZMYND8, co-binding, and shARID1A decreasing H3.3 among the 25 chromatin states. Statistic is hypergeometric enrichment test. C Modeled chromatin states among reference gene promoter-proximal regions (±3 kb around annotated TSS). Left, proportion of promoter-proximal chromatin for siARID1A DE genes ( DESeq2 , FDR < 0.0001) belonging to each of the 25 states. Center, ratio of promoter-proximal chromatin states associated with siARID1A DE genes (FDR < 0.0001) compared to stable genes (FDR > 0.05). Right, ratio of promoter-proximal chromatin states associated with ARID1A transcriptional repression (i.e., siARID1A upregulation) compared to activation (i.e., siARID1A downregulation). D Violin plots quantifying chromatin feature signal at H4K16ac+ (purple) vs. H4K16ac− (gray) promoter-proximal super-enhancer constituent H3K27ac peaks. Statistic is two-tailed, unpaired Wilcoxon’s test. E Principal component analysis (PCA) of RNA-seq expression log 2 FC (shrinkage-corrected) values for siCHD4, siZMYND8, siH3F3B, and siARID1A treatment conditions vs. controls ( n = 3). In total, 1974 genes with s log 2 FC variance >0.1 were used for PCA. F Schematic of identifying mechanistic genes co-repressed by ARID1A, H3.3, CHD4, and ZMYND8, i.e., upregulated ( DESeq2 , FDR < 0.05) with siARID1A, siH3F3B, siCHD4, and siZMYND8 treatments. G Clustered heatmap of expression log 2 FC values for 60 co-repressed genes upregulated in all 4 knockdown conditions. Rightmost column demarcates presence of H4K16ac peaks over promoter-proximal region or gene body. H Top gene sets enriched (hypergeometric enrichment test, FDR < 0.05) among the 60 ARID1A-CHD4-ZMYND8-H3.3 co-repressed genes from various gene set databases. I Example target gene loci, PLAU and TRIO , marked by nearby H3.3+ super-enhancers within H4(K16)ac+ domains that are co-bound by ARID1A, CHD4, and ZMYND8, where ARID1A loss leads to significant depletion of H3.3 (ChIP-seq FDR < 0.05), and knockdown of ARID1A, H3.3, CHD4, or ZMYND8 leads to significant expression upregulation (RNA-seq FDR < 0.05)

    Journal: BMC Biology

    Article Title: ARID1A-dependent maintenance of H3.3 is required for repressive CHD4-ZMYND8 chromatin interactions at super-enhancers

    doi: 10.1186/s12915-022-01407-y

    Figure Lengend Snippet: ZMYND8-mediated chromatin repression is associated with H4(K16)ac. A Heatmap of clustered, normalized feature emission probabilities, and associated functional annotation of the new 12-feature, genome-wide chromatin 25-state model. States (S_) are labeled based on order of normalized emission probability clustering. See “ ” for details on optimal model selection. SE: super-enhancer. B Genomic fold-enrichment (FE) for ARID1A, CHD4, ZMYND8, co-binding, and shARID1A decreasing H3.3 among the 25 chromatin states. Statistic is hypergeometric enrichment test. C Modeled chromatin states among reference gene promoter-proximal regions (±3 kb around annotated TSS). Left, proportion of promoter-proximal chromatin for siARID1A DE genes ( DESeq2 , FDR < 0.0001) belonging to each of the 25 states. Center, ratio of promoter-proximal chromatin states associated with siARID1A DE genes (FDR < 0.0001) compared to stable genes (FDR > 0.05). Right, ratio of promoter-proximal chromatin states associated with ARID1A transcriptional repression (i.e., siARID1A upregulation) compared to activation (i.e., siARID1A downregulation). D Violin plots quantifying chromatin feature signal at H4K16ac+ (purple) vs. H4K16ac− (gray) promoter-proximal super-enhancer constituent H3K27ac peaks. Statistic is two-tailed, unpaired Wilcoxon’s test. E Principal component analysis (PCA) of RNA-seq expression log 2 FC (shrinkage-corrected) values for siCHD4, siZMYND8, siH3F3B, and siARID1A treatment conditions vs. controls ( n = 3). In total, 1974 genes with s log 2 FC variance >0.1 were used for PCA. F Schematic of identifying mechanistic genes co-repressed by ARID1A, H3.3, CHD4, and ZMYND8, i.e., upregulated ( DESeq2 , FDR < 0.05) with siARID1A, siH3F3B, siCHD4, and siZMYND8 treatments. G Clustered heatmap of expression log 2 FC values for 60 co-repressed genes upregulated in all 4 knockdown conditions. Rightmost column demarcates presence of H4K16ac peaks over promoter-proximal region or gene body. H Top gene sets enriched (hypergeometric enrichment test, FDR < 0.05) among the 60 ARID1A-CHD4-ZMYND8-H3.3 co-repressed genes from various gene set databases. I Example target gene loci, PLAU and TRIO , marked by nearby H3.3+ super-enhancers within H4(K16)ac+ domains that are co-bound by ARID1A, CHD4, and ZMYND8, where ARID1A loss leads to significant depletion of H3.3 (ChIP-seq FDR < 0.05), and knockdown of ARID1A, H3.3, CHD4, or ZMYND8 leads to significant expression upregulation (RNA-seq FDR < 0.05)

    Article Snippet: The following primary antibodies were used: anti-ARID1A (D2A8U, Cell Signaling), anti-CHD4 (D4B7, Cell Signaling), anti-ZMYND8 (A302-089, Bethyl), anti-ZMYND8 (Atlas), anti-BRG1 (ab110641, abcam), anti-BAF155 (D7F8S, Cell Signaling), anti-HDAC1 (10E2, Cell Signaling), anti-histone H3.3 (ab176840, abcam), anti-histone H3.3 (2D7-H1, abnova), and anti-histone H3 (D1H2, Cell Signaling).

    Techniques: Functional Assay, Genome Wide, Labeling, Selection, Binding Assay, Activation Assay, Two Tailed Test, RNA Sequencing Assay, Expressing, ChIP-sequencing

    Mechanistic gene expression alterations in human endometriomas. A Left, enrichment for ARID1A-H3.3 co-repressive chromatin mechanistic gene sets among human endometrioma (ovarian endometriosis) vs. control endometrium DE genes reported by Hawkins et al. , compared to all unique measured genes. Right, proportion of overlapping DE genes that are upregulated vs. downregulated in endometriomas, compared to all unique measured genes. Statistic is hypergeometric enrichment. B Box plots displaying endometrioma expression log 2 FC values for probes annotated to genes within mechanistic gene sets, compared to all measured probes. Statistic is two-tailed, unpaired Wilcoxon’s test. C Relative expression box-dot plots of 6 genes upregulated in endometriomas vs. control endometrium that are co-repressed by ARID1A, H3.3, CHD4, and ZMYND8. Statistic is limma FDR-adjusted p . * p < 0.05, ** p < 0.01, *** p < 0.001

    Journal: BMC Biology

    Article Title: ARID1A-dependent maintenance of H3.3 is required for repressive CHD4-ZMYND8 chromatin interactions at super-enhancers

    doi: 10.1186/s12915-022-01407-y

    Figure Lengend Snippet: Mechanistic gene expression alterations in human endometriomas. A Left, enrichment for ARID1A-H3.3 co-repressive chromatin mechanistic gene sets among human endometrioma (ovarian endometriosis) vs. control endometrium DE genes reported by Hawkins et al. , compared to all unique measured genes. Right, proportion of overlapping DE genes that are upregulated vs. downregulated in endometriomas, compared to all unique measured genes. Statistic is hypergeometric enrichment. B Box plots displaying endometrioma expression log 2 FC values for probes annotated to genes within mechanistic gene sets, compared to all measured probes. Statistic is two-tailed, unpaired Wilcoxon’s test. C Relative expression box-dot plots of 6 genes upregulated in endometriomas vs. control endometrium that are co-repressed by ARID1A, H3.3, CHD4, and ZMYND8. Statistic is limma FDR-adjusted p . * p < 0.05, ** p < 0.01, *** p < 0.001

    Article Snippet: The following primary antibodies were used: anti-ARID1A (D2A8U, Cell Signaling), anti-CHD4 (D4B7, Cell Signaling), anti-ZMYND8 (A302-089, Bethyl), anti-ZMYND8 (Atlas), anti-BRG1 (ab110641, abcam), anti-BAF155 (D7F8S, Cell Signaling), anti-HDAC1 (10E2, Cell Signaling), anti-histone H3.3 (ab176840, abcam), anti-histone H3.3 (2D7-H1, abnova), and anti-histone H3 (D1H2, Cell Signaling).

    Techniques: Expressing, Two Tailed Test

    Proposed model of H3.3 chromatin regulation by ARID1A-SWI/SNF and co-regulators. ARID1A and SWI/SNF chromatin remodeling activities are required for H3.3 incorporation or maintenance at certain active regulatory elements across the genome, such as super-enhancers. When ARID1A is mutated or lost, H3.3 maintenance is disrupted, and nucleosome composition shifts toward canonical H3.1/3.2 at ARID1A-bound sites. Consequential to local H3.3 depletion, H3.3 reader factor occupancy is reduced—such as the CHD4-containing NuRD complex—leading to impaired chromatin regulation and aberrant target gene expression. At H3.3+ H4K16ac+ super-enhancer-like elements located promoter-proximally upstream of genes, H3.3 maintenance by ARID1A-SWI/SNF is associated with repression of transcriptional hyperactivation and the NuRD cofactor ZMYND8

    Journal: BMC Biology

    Article Title: ARID1A-dependent maintenance of H3.3 is required for repressive CHD4-ZMYND8 chromatin interactions at super-enhancers

    doi: 10.1186/s12915-022-01407-y

    Figure Lengend Snippet: Proposed model of H3.3 chromatin regulation by ARID1A-SWI/SNF and co-regulators. ARID1A and SWI/SNF chromatin remodeling activities are required for H3.3 incorporation or maintenance at certain active regulatory elements across the genome, such as super-enhancers. When ARID1A is mutated or lost, H3.3 maintenance is disrupted, and nucleosome composition shifts toward canonical H3.1/3.2 at ARID1A-bound sites. Consequential to local H3.3 depletion, H3.3 reader factor occupancy is reduced—such as the CHD4-containing NuRD complex—leading to impaired chromatin regulation and aberrant target gene expression. At H3.3+ H4K16ac+ super-enhancer-like elements located promoter-proximally upstream of genes, H3.3 maintenance by ARID1A-SWI/SNF is associated with repression of transcriptional hyperactivation and the NuRD cofactor ZMYND8

    Article Snippet: The following primary antibodies were used: anti-ARID1A (D2A8U, Cell Signaling), anti-CHD4 (D4B7, Cell Signaling), anti-ZMYND8 (A302-089, Bethyl), anti-ZMYND8 (Atlas), anti-BRG1 (ab110641, abcam), anti-BAF155 (D7F8S, Cell Signaling), anti-HDAC1 (10E2, Cell Signaling), anti-histone H3.3 (ab176840, abcam), anti-histone H3.3 (2D7-H1, abnova), and anti-histone H3 (D1H2, Cell Signaling).

    Techniques: Expressing