Abstract 2995: CDK9 phosphorylates BRG1 chromatin remodeler

Abstract

Abstract Cyclin-dependent kinase 9 (CDK9) belongs to the class of CDKs involved in transcription regulation along with CDK7, 8, 10-13. Earlier works have established that CDK9 is the catalytic subunit of P-TEFb, and a transcriptional activator. CDK9 inhibition holds promise for patients belonging to Chronic Lymphocytic Leukemia (CLL) and BRD4-NUT-rearranged NUT midline carcinoma (NMC) groups. It is therefore fundamentally important to understand CDK9 mediated gene regulation. CDK9 in complex with its regulatory subunit, Cyclin T1 or T2, is known to promote RNAPII promoter-proximal pause release by phosphorylating negative elongation factors. Additionally, phosphorylation of the C-terminal domain (CTD) of RNAPII on Serine-2 allows recruitment of RNA processing factors, which work on the nascent RNA as it emerges from RNAPII. It is however not known whether CDK9 has any transcriptional repression activity. Earlier work from our lab demonstrated that long-term CDK9 inhibition leads to the activation of BRG1, an ATP-dependent nucleosome-remodeling complex from the SWI/SNF family of proteins. We therefore hypothesized that CDK9 directly phosphorylates BRG1 and regulates its activity. Using Co-immunoprecipitation (Co-IP) assay we discovered that CDK9 associates with BRG1 endogenously in colon cancer cells. We tested the association of these proteins more rigorously by transiently over-expressing either FLAG-tagged or GFP-tagged CDK9 in HEK293T cells and performed immunoprecipitation using BRG1 antibody. CDK9 was found to co-precipitate with BRG1. We validated this via reciprocal Co-IPs. We performed an in vitro kinase assay using purified CDK9 and BRG1 proteins to demonstrate that CDK9 directly phosphorylates BRG1 and that inhibition of CDK9 leads to reduced phosphorylation of BRG1. We validated these results in vivo in colon cancer cells. Furthermore, our LC-MS/MS data lead to the identification of novel phosphorylation sites in BRG1 following CDK9 inhibition. These findings provide a potential mechanism for the transcriptional repressor activity associated with CDK9 and may be relevant for developing therapies targeting this transcriptional regulator. Citation Format: Somnath Pandey, Hanghang Zhang, Carlos A. Barrero, Salim Merali, Xavier Graña, Jean-Pierre Issa. CDK9 phosphorylates BRG1 chromatin remodeler [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2995.