Fig. 6: mH2A2 is a negative regulator of BRD4.

a Scatter plot of input-corrected ChIP-seq signals for mH2A2, H3K4me1, H3K27ac, p300 and BRD4, and ATAC-seq after over-expression of mH2A2 against control over-expression (GFP-H2A) at all CRE (Active, n = 16341; APL, 5194; ATAC-only, 13921; Inactive, 14875 peaks) in MDA-MB-231L cells. APL, Active Promoter-Like. The linear regression line (solid line) is shown along with the line y = x for reference. b Boxplots showing the log₂ fold change of input-corrected ChIP-seq signals of each histone mark, histone variant or DNA binding protein in a in mH2A2 over-expression over control over-expression. c Scatter plot of input-corrected ChIP-seq signals for BRD4 after over-expression of mH2A2 against control over-expression (GFP-H2A) at CUT&RUN peaks that are specific to short (n = 2151) and long (n = 27684) isoforms of BRD4, and those that were common to both (n = 21836) (CUT&RUN peaks from Wu et al.⁴⁶) in MDA-MB-231L cells. The linear regression lines for each set of peaks are shown as solid lines along with the line y = x for reference. d Top 10 significantly enriched DNA-binding molecules whose binding sites as defined by ChIP-seq peaks from ReMap are enriched in peaks that lost BRD4 (fold change < 0.5) on over-expression of mH2A2 in MDA-MB-231L cells (enrichment statistics computed using ReMapEnrich). e Immunoblots from chromatin extracts in MDA-MB-231L cells with over-expression of mH2A1-GFP and mH2A2-GFP constructs (and H2A-GFP as control) probed for BRD4, GFP and histone H3 (loading control). Fold change quantification over control (H2A-GFP) after H3 normalization (right). Data represented are mean with SE, n = 3 (t-test). f Representative immunoblots from chromatin extracts in MDA-MB-231L cells with over-expression of mH2A2-GFP and H2A-GFP after MNase immunoprecipitation. Extracts were probed for GFP, H4 and H4K12ac. Quantification of IP over input ration (right). Data represented are mean with SE, n = 3 (t-test). g Boxplots showing the Z scores of the log-normalized input-corrected ChIP-seq signal of histone mark H4K12ac in mammary epithelial cells, melanocytes and MCF7 breast cancer cells. The number of datapoints, n, equals the number of CRE per class shown in Fig. 1c. In all boxplots, the middle line represents the median, the lower and upper edges of the rectangle represent the first and third quartiles and the lower and upper whiskers represent the interquartile range (IQR) × 1.5.