Un nouveau mécanisme de régulation des complexes épigénétiques BAP1/ASXLs par ubiquitination

Ubiquitination is a post-translational modification of proteins that involves covalently attaching the ubiquitin moiety to the lysine residues of the target protein. This modification has been reported to have a significant impact on the function, localization and stability of these targets. Once established by enzymes called E3 ligases, ubiquitination can be removed by specific enzymes called deubiquitinases, thus modulating the effects caused by this modification. BAP1 (or BRCA1-Associated Protein1) is a deubiquitinase, from the UCH (Ubiquitin C-terminal Hydrolases) family, that was originally identified as a partner of the BRCA1 (BReast Cancer Associated gene 1) tumor suppressor. We and other research groups have shown that BAP1 is associated with other co-factors forming a multi-protein complex involved in several cellular processes such as gene transcription, chromatin regulation, cell cycle regulation and DNA damage response. The major target of BAP1 is ubiquitinated histone H2A, a histone mark that has been frequently associated with a repressive chromatin conformation. What are the mechanisms regulating the BAP1 complex allowing it to perform its biological functions? Does this involve post-translational modifications affecting BAP1 partners? These questions are still incompletely answered. Thus, the objectives of our studies are to characterize the mechanism and the function of the BAP1 complex by studying the post-translational modifications that could affect its obligate partners including ASXLs.  To address these questions, we studied the post-translational modifications affecting BAP1 and its two mutually exclusive co-factors ASXL1 and ASXL2 (Additional Sex Comb-like 1,2). We demonstrated that ASXL1 and ASXL2 are mono-ubiquitinated only when associated with BAP1. Taking into account that the BAP1/ASXLs complexes are highly conserved during evolution, we also demonstrated that the mono-ubiquitination of ASXLs is important for Drosophila development. Using RNAi and CRISPR/Cas9 gene depletion methods and loss-of-function mutants of BAP1 and ASXL2, we identified the precise site of ASXLs ubiquitination, the enzymes responsible for establishing this mono-ubiquitination as well as its effect on catalytic activity of BAP1. On the other hand, we investigated Drosophila development as well as human cell cycle progression to identify the biological function of ASXLs mono-ubiquitination. Our results indicate that the mono-ubiquitination of ASXL2 on lysine 370 in the presence of BAP1 is a conserved post-translational modification catalyzed directly by the UBE2E family of ubiquitin-conjugating enzymes (UBE2E1, 2, 3 in mammals and UbcD2 in Drosophila). This mono-ubiquitination event stimulates the catalytic activity of BAP1 in mammals and its Drosophila ortholog Calypso towards H2Aub in vivo and in vitro. Blocking the mono-ubiquitination of ASXLs, by mutations targeting lysine K370, induces an inhibition of BAP1 catalytic activity causing a deregulation of human cell cycle progression and a haltere-to-wing homeotic transformation in Drosophila. In addition, we were able to assess the importance of ASXLs mono-ubiquitination in cancer using the mesothelioma tumor model, demonstrating a strong correlation between the expression of BAP1/ASXL2 and UBE2Es.  Our results indicate the importance of post-translational modifications, including mono-ubiquitination, in the regulation of the function and stability of the BAP1 complex. Moreover, we describe a novel mechanism of activation of a deubiquitinase by the mono-ubiquitination of its co-factor. Further studies will be needed to shed more light on the link between BAP1/ASXLs activation by mono-ubiquitination and the tumor suppressor function of BAP1 via H2Aub deubiquitination. On the other hand, we have noticed that the depletion of PSMD14, a deubiquitinase associated with the proteasome regulatory particle, induces not only a drastic reduction of H2Aub in the cell, but also rapid cell death. This prompted us initially to investigate the involvement of the catalytic activity of the proteasome in the regulation of H2Aub in connection with cell death. Although we did not find a direct link between PSMD14 and H2Aub deubiquitination, we identified several candidates (DUBs and E2s) involved in the induction of cell death while overcoming acquired resistance against proteasome catalytic inhibitors. These candidates may represent attractive targets for developing specific inhibitors to counteract resistance to proteasome inhibitors.

Keyphrases: histone H2A K118/K119 monoubiquitination, complexe PR-DUB, ubiquitination, deubiquitination, BAP1, ASXL1, ASXL2, Chromatin, Histones post-translational modifications, Polycomb proteins, PR-DUB complex, Cell proliferation, Tumor suppressor