Citation
- Authors: Drosos Y. et al.
- Year: 2022
- Journal: Mol Cell 82 2472-2489.e8
- Applications: in vitro / shRNA and DNA cotransfection / PEIpro
- Cell type: 293-RTV
Method
To generate lentiviral particles, 293-RTV (ATCC) cells were co-transfected with an envelope plasmid (pVSVG), packaging vector (psPAX2), and shRNA, TET3G or SMARCB1 expression vectors with PEI Pro (Polysciences Inc.). The medium containing the lentiviral particles was harvested 48 and 72 h after transfection and centrifuged at 20,000 r.p.m. for 2h at 4 °C. Virus-containing pellets were resuspended in PBS and the viral titer was determined. 48 h after transduction, transduced cells were selected in the presence of 1 μg/mL puromycin.
Abstract
Disruption of antagonism between SWI/SNF chromatin remodelers and polycomb repressor complexes drives the formation of numerous cancer types. Recently, an inhibitor of the polycomb protein EZH2 was approved for the treatment of a sarcoma mutant in the SWI/SNF subunit SMARCB1, but resistance occurs. Here, we performed CRISPR screens in SMARCB1-mutant rhabdoid tumor cells to identify genetic contributors to SWI/SNF-polycomb antagonism and potential resistance mechanisms. We found that loss of the H3K36 methyltransferase NSD1 caused resistance to EZH2 inhibition. We show that NSD1 antagonizes polycomb via cooperation with SWI/SNF and identify co-occurrence of NSD1 inactivation in SWI/SNF-defective cancers, indicating in vivo relevance. We demonstrate that H3K36me2 itself has an essential role in the activation of polycomb target genes as inhibition of the H3K36me2 demethylase KDM2A restores the efficacy of EZH2 inhibition in SWI/SNF-deficient cells lacking NSD1. Together our data expand the mechanistic understanding of SWI/SNF and polycomb interplay and identify NSD1 as the key for coordinating this transcriptional control.