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. 2023 Oct 23;51(21):11634–11651. doi: 10.1093/nar/gkad907

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© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 5. — BRD9 collaborates with BRD4, SMAD2/3 and β-CATENIN to regulate gene expression in hESCs. (A) Distribution of BRD9 target sites relative to TSSs in hESCs, determined by ChIP-seq. (B) Chromatin state enrichment of BRD9 target sites in hESCs. ESC chromatin states were defined in Ernst et al. (20) using ChromHMM. Rows represent chromatin states and their mnemonics. Columns give the frequency of the indicated histone marks for each chromatin state (ChromHMM emission probabilities), color-coded from blue (highest) to white (lowest). Enrichment of BRD9 in each chromatin state is shown. (C) KEGG pathway enrichment analysis with BRD9 target sites within 10 kb of their TSS. Significantly enriched KEGG pathways (P < 0.05) are presented. (Dand E) Genome browser view of ChIP-seq tracks for the histone mark H3K27ac at the NODAL (D) and NANOG (E) loci in hESCs cultured with and without 10 μM I-BRD9 for 2 days. Regions highlighted in brown signify ESC promoter and enhancer regions. The values on the y-axis represent fold enrichment over control. (F) H3K27ac levels at TGF-β-related genes in hESCs, treated or not with 100 nM dBRD9A for 2 days, determined by ChIP-qPCR. (G) Co-IP experiment with α-BRD4 antibody showing the interaction of BRD4 with P300, BRD9, RNAP II, SMAD2 and β-CATENIN. (H) Venn diagram depicting the number of H3K27ac peaks that are bound by BRD4, BRD9 and SMAD2 from H3K27ac, BRD4, BRD9 and SMAD2 ChIP-seq analyses. (Iand J) Genome browser view of BRD4, BRD9, SMAD2/3, β-CATENIN binding and H3K27ac modification at the NODAL (I) and NANOG (J) loci in hESCs. (K) BRD4 binding at the indicated genes in hESCs, treated or not with 100 nM dBRD9A for 48 h, determined by ChIP-qPCR, (L and M) SMAD2 binding at TGF-β-related genes (L) and NANOG, OCT4, WNT3 and WNT3A genes (M) in hESCs cultured in E8 medium, treated or not with 10 μM I-BRD9 for 2 days, determined by ChIP-qPCR, (N–P) BRD9 binding at TGF-β-related genes (N), and pluripotency genes NANOG, OCT4 and SOX2 (O), WNT3 and WNT3A genes (P) in hESCs cultured in E8 medium, treated or not with 50 nM JQ1 for 2 days, determined by ChIP-qPCR. (Q–S) β-CATENIN binding at TGF-β-related genes (Q), and pluripotency genes NANOG, OCT4 and SOX2 (R), WNT3 and WNT3A genes (S) in hESCs cultured in E8 medium, treated or not with 50 nM JQ1 for 2 days, determined by ChIP-qPCR.