ATXN7L3 and ENY2 Coordinate Activity of Multiple H2B Deubiquitinases Important for Cellular Proliferation and Tumor Growth

Abstract

SUMMARY Histone H2B monoubiquitination (H2Bub1) is centrally involved in gene regulation. The deubiquitination module (DUBm) of the SAGA complex is a major regulator of global H2Bub1 levels, and components of this DUBm are linked to both neurodegenerative diseases and cancer. Unexpectedly, we find that ablation of USP22, the enzymatic center of the DUBm, leads to a reduction, rather than an increase, in global H2bub1 levels. In contrast, depletion of non-enzymatic components, ATXN7L3 or ENY2, results in increased H2Bub1. These observations led us to discover two new H2Bub1 DUBs, USP27X and USP51, which function independently of SAGA and which compete with USP22 for ATXN7L3 and ENY2 for activity. Like USP22, USP51 and USP27X are required for normal cell proliferation, and their depletion suppresses tumor growth. Our results reveal that ATXN7L3 and ENY2 orchestrate activities of multiple deubiquitinating enzymes and that imbalances in these activities likely potentiate human diseases including cancer.

Cytoplasmic ATXN7L3B Interferes with Nuclear Functions of the SAGA Deubiquitinase Module

“…Antibodies used in this study include anti-ATXN7L3B (4331-1; homemade), anti-ATXN7L3 (catalog number A301-800A; Bethyl Labs), anti-ENY2 (catalog number GTX128034; GeneTex), anti-USP22 (3933-1; homemade [17]), anti-GCN5 (catalog number 3305; Cell Signaling), anti-␤-actin (catalog number Sc-47778; Santa Cruz), anti-␤-tubulin (catalog number 2146S; Millipore), anti-H2Bub1 (catalog number 05-1312; Millipore), anti-H2B (catalog number 07-371; Millipore), anti-Flag (M2; catalog number F3165; Sigma), antihemagglutinin (anti-HA; catalog number 3724; Cell Signaling), and anti-V5 (catalog number 46-0705; Invitrogen).…”

“…Sample preparation for multidimensional protein identification technology (MudPIT) analyses was performed as described previously (17). Briefly, 293T cells, stably expressing pINTO-N-FH-ATXN7L3, pINTO-N-FH-ATXN7L3B, or empty vector, were harvested by trypsinization, washed twice in ice-cold PBS, pelleted by centrifugation, and resuspended in 5 pellet volumes of buffer C (20 mM Tris-HCl [pH 7.9], 20% glycerol, 420 mM NaCl, 1.5 mM MgCl 2 , 0.1% NP-40, 0.2 mM EDTA, 1 mM DTT, 1 mM PMSF, protease inhibitors).…”

Mechanisms of regulation and diversification of deubiquitylating enzyme function

“…The crystal structures of yeast mDUB provided a rationale for this by revealing that all subunits form extensive contacts with each other in a manner that could stabilize and activate Ubp8 (Köhler et al, 2010;Samara et al, 2010). Interestingly, ATXN7L3 and ENY2 of mammalian mDUB also bind to and activate two additional DUBs, USP27X (also known as USP27) and USP51, although not as part of the SAGA complex (Atanassov et al, 2016). This suggests that these two noncatalytic subunits of mDUB could act as master regulators of histone H2B deubiquitylation by multiple DUBs.…”

Study of the SAGA deubiquitination module: identification of new modulators and its implication on Spinocerebellar Ataxia Type 7

“…The DUBm contains yUbp8/hUSP22, ySus1/hENY2, ySgf73/hATXN7 and ySgf11/hATXN7L3 and is responsible for the deubiquitination of histone H2B (Atanassov et al, 2016;Galán and Rodríguez-Navarro, 2012;Helmlinger et al, 2004).…”

“…Our observed unchanged levels of H2Bub 1 might be due to the existence of compensatory DUBs enzymes. For instance, a recent study has discovered that two H2Bub 1 DUB enzymes function independently of SAGA and compete with USP22 for ATXNL3 and ENY2 for DUB activity (Atanassov et al, 2016). Previous research has attributed the existence of polyQ-ATXN7 with transcriptional dysregulation (Chou et al, 2010), thus, another possibility is that transcriptional dysregulation caused by an impairment of the DUB activity might be found in those tissues affected by the disease, such as cone-rode receptors and cerebellum.…”