Inactivation of the histone H3 K36 methyltransferase NSD1 confers resistance to EZH2 inhibition [ChIP-Seq]

Disruption of antagonism between SWI/SNF chromatin remodelers and Polycomb repressor complexes drives the formation of numerous cancer types. Recently, an inhibitor of Polycomb protein EZH2 was approved for treatment of sarcomas mutant in SWI/SNF subunit SMARCB1, but resistance occurs. Here we sought to identify additional contributors to SWI/SNF-Polycomb antagonism and potential resistance mechanisms. We performed CRISPR screens and found that NSD1 loss caused resistance to EZH2 inhibition. NSD1 loss reduced H3K36me2 and impaired transcriptional activation of targets inhibited by EZH2 or activated by SMARCB1. Further, inhibiting the H3K36me2 demethylase KDM2A restored EZH2 inhibition in cells lacking NSD1. Our results expand the mechanistic understanding of SWI/SNF and Polycomb interplay and identify NSD1 as key for coordinating this transcriptional control. G401 cells were engineered to express either GFP or SMARCB1 (B1) under doxycycline treatment and further transfected with either control (sgC) or NSD1 (sgN) targeting sgRNAs. Cells were then treated with 1ug/mL dox for 24h. For EZH2 inhibition studies, G401 cells stably expressing shCTRL or shNSD1 RNAs were treated for 72h with 1uM GSK126. In brief, cells were cross-linked directly on the plate using 1% paraformaldehyde for 10 min at room temperature and quenched with glycine. Nuclei were isolated using the Covaris TruCHIP kit and Chromatin extracts were sonicated for 8 min using a Covaris E220 focused ultrasonicator at a peak power of 140, a duty factor of 5 and 200 cycles per burst. Chromatin immunoprecipitation was carried overnight with antibodies bound to dynabeads. Chromatin was de-crosslinked, digested with RNase and Proteinase K and DNA was ourified using a Zymo ChIP DNA kit.