Supplementary Figure 4: MacroH2A acts as a redundant layer of epigenetic repression (related to Fig. 3).

(a) MA plot showing log₂ fold change (y axis) of gene expression during macroH2A2-GFP induction in dKO iDFs, 6 h versus 0 h (left panel) and 24 h versus 0 h (right panel), against the ranked expression level at 0 h (x axis). Red and blue dots represent genes significantly up or downregulated, respectively (FDR < 0.05, log₂[fold change] > 1 or < −1, n = 3 biological replicates). (b) RT-qPCR analysis of indicated genes at different time points after dox induction. Error bars represent s.d. from n = 2 independent experiments. (c) Experimental scheme showing treatment of WT and dKO iDFs with a combination of GSK126 (EZH2 inhibitor), 5AzaC (DNA methyltransferase inhibitor), TSA (histone deacetylase inhibitor). (d) Genome browser tracks showing occupancy of indicated histone variant/modifications. Expression levels in FPKM in dKO iDFs are showing in the boxes below. (e) RT-qPCR anaylsis showing activation of repressed genes in WT and dKO cells after simultaneous alleviation of multiple layers of epigenetic repression. Runx1 used as a negative control. Dotted line highlighted value 1 representing no change between drug treated and untreated. Error bars represent s.d. (n = 2). P values are calculated from two-tailed t test (*P < 0.05). (f) Percentage of inactive or active genes overlapping with transient macroH2A2 peaks. (g) Metagene plot showing the occupancy of elongating RNA polymerase II (Pol II S2P) in MEFs²⁷ at active and inactive genes in iDFs. (h) Expression levels (FPKM) in inducible macroH2A2-GFP dKO iDFs (0 hour) and their promoter association with CpG islands (CGI) for the panel of genes selected for qPCR validations. (i) nChIP-qPCR analysis of macroH2A2-GFP occupancy 6 h and 24 h after induction. Same data was used as in Fig. 3e, but macroH2A-GFP ChIP was normalized to input. Three negative control regions (chr2, chr6 and chr14) were used for normalization to calculate relative enrichment. Error bars represent s.d. from n = 3 independent experiments. (j) nChIP-qPCR analysis of H2A occupancy 6 hour and 24 hour after macroH2A2-GFP induction. H2A ChIP was normalized to H2B. Error bars represent s.d. from n = 2 independent experiments.