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.

SUMMARY Histone acetyltransferases (HATs) play important roles in gene regulation and DNA repair by influencing the accessibility of chromatin to transcription factors and repair proteins. Here we show that deletion of Gcn5 leads to telomere dysfunction in mouse and human cells. Biochemical studies reveal that depletion of Gcn5 or ubiquitin specific protease 22 (Usp22), which is another bona fide component of the Gcn5-containing SAGA complex, increases ubiquitination and turnover of TRF1, a primary component of the telomeric shelterin complex. Inhibition of the proteasome or over expression of USP22 opposes this effect. The USP22 deubiquitinating module requires association with SAGA complexes for activity, and we find that depletion of Gcn5 compromises this association in mammalian cells. Thus, our results indicate that Gcn5 regulates TRF1 levels through effects on Usp22 activity and SAGA integrity.

Ubiquitin-specific protease 22 (USP22) edits the histone code by deubiquitinating H2A and H2B as part of the mammalian SAGA (Spt-Ada-Gcn5) complex, and is required for transcriptional regulation and normal cell-cycle progression. Here, we show that USP22 affects the expression of p21 by altering far upstream element (FUSE)-binding protein 1 (FBP1) ubiquitination, as ablation of USP22 leads to increased FBP1 ubiquitination and decreased FBP1 protein occupancy at the p21 gene. Surprisingly, increased polyubiquitination of FBP1 does not alter its protein stability, but instead modulates the stable recruitment of FBP1 to target loci. Our results indicate a mechanism by which USP22 regulates cell proliferation and tumorigenesis.

In cancer cells, epithelial-to-mesenchymal transition (EMT) is controlled by Snail1, a transcriptional factor also required for the activation of cancer-associated fibroblasts (CAF). Snail1 is short-lived in normal epithelial cells as a consequence of its coordinated and continuous ubiquitination by several F-box-specific E3 ligases, but its degradation is prevented in cancer cells and in activated fibroblasts. Here, we performed an siRNA screen and identified USP27X as a deubiquitinase that increases Snail1 stability. Expression of USP27X in breast and pancreatic cancer cell lines and tumors positively correlated with Snail1 expression levels. Accordingly, downregulation of USP27X decreased Snail1 protein in several tumor cell lines. USP27X depletion impaired Snail1-dependent cell migration and invasion and metastasis formation and increased cellular sensitivity to cisplatin. USP27X was upregulated by TGFb during EMT and was required for TGFb-induced expression of Snail1 and other mesenchymal markers in epithelial cells and CAF. In agreement with this, depletion of USP27X prevented TGFb-induced EMT and fibroblast activation. Collectively, these results indicate that USP27X is an essential protein controlling Snail1 expression and function and may serve as a target for inhibition of Snail1-dependent tumoral invasion and chemoresistance. Significance: These findings show that inhibition of USP27X destabilizes Snail1 to impair EMT and renders tumor cells sensitive to chemotherapy, thus opening new strategies for the inhibition of Snail1 expression and its protumoral actions.

a b s t r a c tPost-translational modifications of the histones are centrally involved in the regulation of all DNAtemplated processes, including gene transcription, DNA replication, recombination, and repair. These modifications are often dynamic, and their removal is just as important as their addition in proper regulation of cellular functions. Although histone acetylation/deacetylation and histone methylation/demethylation are highly studied, the functions and regulation of histone ubiquitination and deubiquitination are less well understood. This review highlights our current understanding of how histone ubiquitination impacts gene transcription, DNA repair, and cell cycle progression, and stresses the importance of deubiquitinases to normal cellular functions as well as to disease states such as cancer.