GSE109740
Transcription-associated histone pruning demarcates macroH2A chromatin domains
Here, we characterized de novo chromatin deposition of macroH2A2 using temporal genomic profiling in cells devoid of all macroH2A isoforms. We find that macroH2A2 is first pervasively deposited genome-wide and subsequently pruned to establish mature domains. This transient incorporation occurs preferentially at transcribed regions adjacent to future mature macroH2A2 domains and transcriptional inhibition prevents the clearing of promiscuously incorporated macroH2A2. Furthermore, CRISPR/Cas9-based manipulation of the transcriptional activity at a given locus reveals that transcriptional silencing triggers ectopic macroH2A2 accumulation, while activation depletes pre-existing macroH2A2. We demonstrate that the FACT (facilitates chromatin transcription) complex is required for macroH2A2 removal from transcribed chromatin. Taken together, we have identified a transcription-associated pruning mechanism that establishes and maintains the faithful genomic localization of macroH2A variants. Overall design: Native ChIP-seq (nChIP-seq) for macroH2A2-GFP was performed at 0, 6, 24 hours and 5 days after dox addtion in macroH2A1/2 dKO iDFs with inducible macroH2A2-GFP transgene. Additionally, nChIP-seq for macroH2A2-GFP was done 24 hours post dox induction with the last 18 hours under flavopiridol (CDK9 inhibitor) treatment. Further, inducible iDFs were treated with Ezh2 inhibitor GSK126 and native ChIP-seq for macroH2A2-GFP was performed 6 and 24 hours post dox addition. Input sample was sequenced as control. PolyA mRNA-seq was performed at 0, 6 and 24 hours after dox induction in biological triplicates in the same cell line.