Abstract LB-A29: Divergent mechanisms of transcriptional regulation by SAGA member and epigenetic modifier USP22

SAGA is a 2 MDa multi-subunit complex containing two assembly modules (TAF and SPT) and two enzymatic modules (HAT and DUB) which exhibit acetyltransferase activity via GCN5 and deubiquitylase activity via USP22. Multiple studies in yeast, flies, mice, and human systems have shown the SAGA DUB module to participate in a concerted series of biochemical steps during transcription, removing monoubiquitin from histone H2B to mediate Pol II phosphorylation and elongation downstream. High USP22 expression has been reported as a biomarker of late-stage aggressive tumors across multiple cancer types, and our previous work demonstrated that USP22 is necessary for this phenotype, as depletion of USP22 severely impairs tumor cell growth and proliferation. We utilized the ER stress response as an activator-driven transcriptional program to clarify a number of details surrounding the role of hSAGA in transcriptional regulation. ChIP-seq of endogenous human USP22 reveals a tightly regulated USP22-containing DUB module that occupies the promoters of only a handful of activated genes, despite hundreds of ER stress response genes altered within 2 hours of induction. In contrast, promoter occupancy of the HAT module subunit GCN5 is widespread and does not require activation of a transcriptional program. USP22 depletion in this system elicits significant effects on GTF recruitment and assembly of the PIC. Notably, USP22 occupancy appears entirely exclusive of chromatin-associated Ub-H2B, and shRNA-mediated depletion of USP22 fails to elicit an increase in either global or local Ub-H2B levels. These data imply that removal of monoubiquitin from histone H2B is not regulated by USP22 in higher eukaryotes. Given that several non-histone substrates of USP22 have been characterized, we performed a proteome-wide screen to measure changes in endogenous ubiquitylation levels following USP22 depletion from cells. Our data indicate that USP22-mediated deubiquitylation of Mediator tail subunits as well as the large subunit of the Pol II holoenzyme may be significant steps in efficient transcriptional activation. ChIP of core and tail Mediator subunits reveals that USP22 depletion impairs their recruitment to USP22-bound ER stress response gene promoters. Given the specialized role of USP22 in activator-driven transcriptional events and its prominence as a driver of tumor progression, we predict USP22 to be a suitable enzymatic substrate for targeted therapy in cancer. Our current efforts include high-throughput screening of compound libraries against USP22 enzymatic activity in vitro as a preliminary drug discovery campaign. Citation Format: Sabrina Butt, Timothy J. Stanek, Victoria J. Gennaro, Chris McNair, Kristen L. Pauley, Karen Knudsen, Steven B. McMahon. Divergent mechanisms of transcriptional regulation by SAGA member and epigenetic modifier USP22 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A29.