Régulation du complexe suppresseur de tumeurs BAP1/ASXL2 par ubiquitination

Introduction: Monobiquitination of histone H2A on lysine 119 (H2AK119ub) is an epigenomic modification catalyzed by the Polycomb complex PRC1 via its ubiquitin ligase Ring1B/BMI1. This modification plays an important role in the regulation of gene expression, and is required for the control of several cellular processes associated with cell development and proliferation. This modification is reversed by the deubiquitinase (DUB) BAP1, a tumor suppressor considered as the most frequently mutated DUB in human cancers. In Drosophila, the deubiquitination of H2AK118ub, the analog of H2AK119ub, is catalyzed by the Polycomb protein Calypso, the ortholog of BAP1, which is associated with its partner ASX, and forms the Polycomb repressor complex (PR-DUB). We have previously shown that BAP1 forms, in mammals, two mutually exclusive complexes with two factors of the Polycomb proteins family, ASXL1 and ASXL2 (ASXL1/2), which are two ASX orthologs. The association of BAP1 with ASXL1/2 stimulates its DUB activity and is necessary for the normal coordination of the cell cycle. But how BAP1 acts as a tumor suppressor, and how these associated proteins are regulated at the molecular level remain important questions to be addressed. Interestingly, we identified a constitutive monoubiquitination that appears on ASXL1/2 only in the presence of BAP1, and we hypothesized that this monoubiquitination event plays an essential role in the protein stability of the BAP1/ASXL1/2 complex and the coordination of its DUB function.                                                                                                                                                           Methods and results: In our study, we were interested in the ASXL2 transcriptional regulator, which interacts, via its ASXM2 domain, with the C-terminal domain (CTD) of BAP1. This interaction results in a monoubiquitination event on lysine 370 of the ASXM2 domain and this regulates the function of the BAP1/ASXL2 complex. Interestingly, monoubiquitination of lysine K370 stabilizes ASXL2, but can also initiate polyubiquitination and degradation by the proteasome. In addition, we demonstrated that monoubiquitination of ASXL2 stimulates DUB activity of BAP1 on H2AK119ub. By conducting siRNA screening of ubiquitin conjugating enzymes (E2) and DUBs of all the human genome, we demonstrated that monoubiquitination of ASXM2 is directly catalyzed by members of the UBE2E family (UBE2E1, UBE2E2 and UBE2E3) and is reversed by the proteasomal DUB PSMD14. At the cellular level, we have observed that the expression of a mutated form of ASXL2 on its ubiquitination site, K370R, delays cell proliferation. The same delay of cell proliferation is also observed following the depletion of UBE2E3 by the CRISPR/Cas9 approach. Finally, we have identified cancer mutations of BAP1, which abolish the monoubiquitination of ASXM2 when expressed in human cells, suggesting the importance of this post-translational modification for tumor suppression.  Conclusion and relevance: Our results indicate that BAP1 and ASXL2 are tightly co-regulated by ubiquitination. This allows the BAP1/ASXL2 complex to control its DUB activity and function, promoting normal cell proliferation and preventing malignant transformation. In conclusion, understanding how the BAP1 complex is regulated by ASXL1/2 cofactors will allow us to better establish its tumor suppressor role, and this might help in the development of new therapies against cancer.

Keyphrases: BAP1, ASXL2, Ubiquitination, H2AK119ub, Cancer, BAP1, ASXL2, Ubiquitination, Deubiquitination, H2AK119ub, Cancer