O-GlcNAcylation of FOXK1 orchestrates the E2F pathway and promotes oncogenesis.

Gene transcription is a highly regulated process, and deregulation of transcription factors activity underlies numerous pathologies including cancer. Albeit near four decades of studies have established that the E2F pathway is a core transcriptional network that govern cell division in multi-cellular organisms1,2, the molecular mechanisms that underlie the functions of E2F transcription factors remain incompletely understood. FOXK1 and FOXK2 transcription factors have recently emerged as important regulators of cell metabolism, autophagy and cell differentiation3-6. While both FOXK1 and FOXK2 interact with the histone H2AK119ub deubiquitinase BAP1 and possess many overlapping functions in normal biology, their specific functions as well as deregulation of their transcriptional activity in cancer is less clear and sometimes contradictory7-13. Here, we show that elevated expression of FOXK1, but not FOXK2, in primary normal cells promotes transcription of E2F target genes associated with increased proliferation and delayed entry into cellular senescence. FOXK1 expressing cells are highly prone to cellular transformation revealing important oncogenic properties of FOXK1 in tumor initiation. High expression of FOXK1 in patient tumors is also highly correlated with E2F gene expression. Mechanistically, we demonstrate that FOXK1, but not FOXK2, is specifically modified by O-GlcNAcylation. FOXK1 O-GlcNAcylation is modulated during the cell cycle with the highest levels occurring during the time of E2F pathway activation at G1/S. Moreover, loss of FOXK1 O-GlcNAcylation impairs FOXK1 ability to promote cell proliferation, cellular transformation and tumor growth. Mechanistically, expression of FOXK1 O-GlcNAcylation-defective mutants results in reduced recruitment of BAP1 to gene regulatory regions. This event is associated with a concomitant increase in the levels of histone H2AK119ub and a decrease in the levels of H3K4me1, resulting in a transcriptional repressive chromatin environment. Our results define an essential role of O-GlcNAcylation in modulating the functions of FOXK1 in controlling the cell cycle of normal and cancer cells through orchestration of the E2F pathway.

Persistent Acetylation of Histone H3 Lysine 56 Compromises the Activity of DNA Replication Origins.

In Saccharomyces cerevisiae, newly synthesized histones H3 are acetylated on lysine 56 (H3 K56ac) by the Rtt109 acetyltransferase prior to their deposition on nascent DNA behind replication forks. Two deacetylases of the sirtuin family, Hst3 and Hst4, remove H3 K56ac from chromatin after S phase. hst3Δ hst4Δ cells present constitutive H3 K56ac, which sensitizes cells to replicative stress via unclear mechanisms. A chemogenomic screen revealed that DBF4 heterozygosity sensitizes cells to NAM-induced inhibition of sirtuins. DBF4 and CDC7 encode subunits of the Dbf4-dependent kinase (DDK), which activates origins of DNA replication during S phase. We show that (i) cells harboring the dbf4-1 or cdc7-4 hypomorphic alleles are sensitized to NAM, and that (ii) the sirtuins Sir2, Hst1, Hst3, and Hst4 promote DNA replication in cdc7-4 cells. We further demonstrate that Rif1, an inhibitor of DDK-dependent activation of origins, causes DNA damage and replication defects in NAM-treated cells and hst3Δ hst4Δ mutants. cdc7-4 hst3Δ hst4Δ cells are shown to display delayed initiation of DNA replication, which is not due to intra-S checkpoint activation but requires Rtt109-dependent H3 K56ac. Our results suggest that constitutive H3 K56ac sensitizes cells to replicative stress in part by negatively influencing the activation of origins of DNA replication.

Immunoprecipitation and Western blot-based detection of protein O-GlcNAcylation in cells.

Detection of protein O-GlcNAcylation could be challenging. By using the host-cell factor 1 (HCF-1), a known O-GlcNAcylated protein, we immunoprecipitated HCF-1 from transfected HEK293T cells or endogenous HCF-1 from HeLa cells to detect its O-GlcNAc levels by Western blotting. We also take advantage of RNAi or chemical inhibitors to modulate OGT and OGA activities before HCF-1 immunoprecipitation. For complete details on the use and execution of this protocol, please refer to Daou et al. (2011).

Starvation-induced proteasome assemblies in the nucleus link amino acid supply to apoptosis.

Eukaryotic cells have evolved highly orchestrated protein catabolic machineries responsible for the timely and selective disposal of proteins and organelles, thereby ensuring amino acid recycling. However, how protein degradation is coordinated with amino acid supply and protein synthesis has remained largely elusive. Here we show that the mammalian proteasome undergoes liquid-liquid phase separation in the nucleus upon amino acid deprivation. We termed these proteasome condensates SIPAN (Starvation-Induced Proteasome Assemblies in the Nucleus) and show that these are a common response of mammalian cells to amino acid deprivation. SIPAN undergo fusion events, rapidly exchange proteasome particles with the surrounding milieu and quickly dissolve following amino acid replenishment. We further show that: (i) SIPAN contain K48-conjugated ubiquitin, (ii) proteasome inhibition accelerates SIPAN formation, (iii) deubiquitinase inhibition prevents SIPAN resolution and (iv) RAD23B proteasome shuttling factor is required for SIPAN formation. Finally, SIPAN formation is associated with decreased cell survival and p53-mediated apoptosis, which might contribute to tissue fitness in diverse pathophysiological conditions.

Roles and mechanisms of BAP1 deubiquitinase in tumor suppression.

The BAP1 gene has emerged as a major tumor suppressor mutated with various frequencies in numerous human malignancies, including uveal melanoma, malignant pleural mesothelioma, clear cell renal cell carcinoma, intrahepatic cholangiocarcinoma, hepatocellular carcinoma, and thymic epithelial tumors. BAP1 mutations are also observed at low frequency in other malignancies including breast, colorectal, pancreatic, and bladder cancers. BAP1 germline mutations are associated with high incidence of mesothelioma, uveal melanoma, and other cancers, defining the "BAP1 cancer syndrome." Interestingly, germline BAP1 mutations constitute an important paradigm for gene-environment interactions, as loss of BAP1 predisposes to carcinogen-induced tumorigenesis. Inactivating mutations of BAP1 are also identified in sporadic cancers, denoting the importance of this gene for normal tissue homeostasis and tumor suppression, although some oncogenic properties have also been attributed to BAP1. BAP1 belongs to the deubiquitinase superfamily of enzymes, which are responsible for the maturation and turnover of ubiquitin as well as the reversal of substrate ubiquitination, thus regulating ubiquitin signaling. BAP1 is predominantly nuclear and interacts with several chromatin-associated factors, assembling multi-protein complexes with mutually exclusive partners. BAP1 exerts its function through highly regulated deubiquitination of its substrates. As such, BAP1 orchestrates chromatin-associated processes including gene expression, DNA replication, and DNA repair. BAP1 also exerts cytoplasmic functions, notably in regulating Ca2+ signaling at the endoplasmic reticulum. This DUB is also subjected to multiple post-translational modifications, notably phosphorylation and ubiquitination, indicating that several signaling pathways tightly regulate its function. Recent progress indicated that BAP1 plays essential roles in multiple cellular processes including cell proliferation and differentiation, cell metabolism, as well as cell survival and death. In this review, we summarize the biological and molecular functions of BAP1 and explain how the inactivation of this DUB might cause human cancers. We also highlight some of the unresolved questions and suggest potential new directions.

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones.

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate.  Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Monoubiquitination of ASXLs controls the deubiquitinase activity of the tumor suppressor BAP1.

The tumor suppressor and deubiquitinase (DUB) BAP1 and its Drosophila ortholog Calypso assemble DUB complexes with the transcription regulators Additional sex combs-like (ASXL1, ASXL2, ASXL3) and Asx respectively. ASXLs and Asx use their DEUBiquitinase ADaptor (DEUBAD) domain to stimulate BAP1/Calypso DUB activity. Here we report that monoubiquitination of the DEUBAD is a general feature of ASXLs and Asx. BAP1 promotes DEUBAD monoubiquitination resulting in an increased stability of ASXL2, which in turn stimulates BAP1 DUB activity. ASXL2 monoubiquitination is directly catalyzed by UBE2E family of Ubiquitin-conjugating enzymes and regulates mammalian cell proliferation. Remarkably, Calypso also regulates Asx monoubiquitination and transgenic flies expressing monoubiquitination-defective Asx mutant exhibit developmental defects. Finally, the protein levels of ASXL2, BAP1 and UBE2E enzymes are highly correlated in mesothelioma tumors suggesting the importance of this signaling axis for tumor suppression. We propose that monoubiquitination orchestrates a molecular symbiosis relationship between ASXLs and BAP1.