Supplementary Figure 7: Transcriptional inhibition by FP leads to accumulation of macroH2A2 at steady state (related to Fig. 6).
From: Transcription-associated histone pruning demarcates macroH2A chromatin domains
(a) Genotyping genomic PCR analysis of iDF clones with WT (+/+), heterozygous KO (±) or homozygous KO (-/-) TSS of Tks4. (b) Western blots of whole cell extract from WT iDFs after 4-hour FP treatment; NT, no treatment. S2P, serine-2 phosphorylation. S5P, serine-5 phosphorylation. β-tubulin used as loading control. (c-d and g-h) RT-qPCR analysis of WT iDFs (c and g) and iDFs constitutively expressing macroH2A2-GFP (d and h) treated with FP for 18 h. 18 S rRNA used as internal reference. Error bars represent s.d. from n = 2 independent experiments. P values are calculated from a two-tailed t test comparing FP treated with untreated (*P < 0.05, ** P < 0.01, *** P < 0.001). (e-f and i-j) nChIP-qPCR analysis of WT iDFs (e and i) and iDFs constitutively expressing macroH2A2-GFP (f and j) showing the occupancy of endogenous macroH2A2 or macroH2A2-GFP after indicated time of FP treatment. MacroH2A2 ChIP was normalized to H2B. Primers target non-CGI gene body regions. Error bars represent s.d. from n = 2 independent experiments. Insets in (e and f) shows macroH2A2 (H2afy2) gene expression by RT-qPCR. Error bars represent s.d. from n = 2 independent experiments.
