The forkhead transcription factor FOXK2 acts as a chromatin targeting factor for the BAP1-containing histone deubiquitinase complex.
ABSTRACT: There are numerous forkhead transcription factors in mammalian cells but we know little about the molecular functions of the majority of these. FOXK2 is a ubiquitously expressed family member suggesting an important function across multiple cell types. Here, we show that FOXK2 binds to the SIN3A and PR-DUB complexes. The PR-DUB complex contains the important tumour suppressor protein, the deubiquitinase BAP1. FOXK2 recruits BAP1 to DNA, promotes local histone deubiquitination and causes changes in target gene activity. Our results therefore provide an important link between BAP1 and the transcription factor FOXK2 and demonstrate how BAP1 can be recruited to specific regulatory loci.
Project description: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.
Project description:The deubiquitinase (DUB) and tumor suppressor BAP1 catalyzes ubiquitin removal from histone H2A Lys-119 and coordinates cell proliferation, but how BAP1 partners modulate its function remains poorly understood. Here, we report that BAP1 forms two mutually exclusive complexes with the transcriptional regulators ASXL1 and ASXL2, which are necessary for maintaining proper protein levels of this DUB. Conversely, BAP1 is essential for maintaining ASXL2, but not ASXL1, protein stability. Notably, cancer-associated loss of BAP1 expression results in ASXL2 destabilization and hence loss of its function. ASXL1 and ASXL2 use their ASXM domains to interact with the C-terminal domain (CTD) of BAP1, and these interactions are required for ubiquitin binding and H2A deubiquitination. The deubiquitination-promoting effect of ASXM requires intramolecular interactions between catalytic and non-catalytic domains of BAP1, which generate a composite ubiquitin-binding interface (CUBI). Notably, the CUBI engages multiple interactions with ubiquitin involving (i) the ubiquitin carboxyl hydrolase catalytic domain of BAP1, which interacts with the hydrophobic patch of ubiquitin, and (ii) the CTD domain, which interacts with a charged patch of ubiquitin. Significantly, we identified cancer-associated mutations of BAP1 that disrupt the CUBI and notably an in-frame deletion in the CTD that inhibits its interaction with ASXL1/2 and DUB activity and deregulates cell proliferation. Moreover, we demonstrated that BAP1 interaction with ASXL2 regulates cell senescence and that ASXL2 cancer-associated mutations disrupt BAP1 DUB activity. Thus, inactivation of the BAP1/ASXL2 axis might contribute to cancer development.
Project description:The transcriptional control circuitry in eukaryotic cells is complex and is orchestrated by combinatorially acting transcription factors. Forkhead transcription factors often function in concert with heterotypic transcription factors to specify distinct transcriptional programs. Here, we demonstrate that FOXK2 participates in combinatorial transcriptional control with the AP-1 transcription factor. FOXK2 binding regions are widespread throughout the genome and are often coassociated with AP-1 binding motifs. FOXK2 acts to promote AP-1-dependent gene expression changes in response to activation of the AP-1 pathway. In this context, FOXK2 is required for the efficient recruitment of AP-1 to chromatin. Thus, we have uncovered an important new molecular mechanism that controls AP-1-dependent gene expression.
Project description:Several mammalian forkhead transcription factors have been shown to impact on cell cycle regulation and are themselves linked to cell cycle control systems. Here we have investigated the little studied mammalian forkhead transcription factor FOXK2 and demonstrate that it is subject to control by cell cycle-regulated protein kinases. FOXK2 exhibits a periodic rise in its phosphorylation levels during the cell cycle, with hyperphosphorylation occurring in mitotic cells. Hyperphosphorylation occurs in a cyclin-dependent kinase (CDK)·cyclin-dependent manner with CDK1·cyclin B as the major kinase complex, although CDK2 and cyclin A also appear to be important. We have mapped two CDK phosphorylation sites, serines 368 and 423, which play a role in defining FOXK2 function through regulating its stability and its activity as a transcriptional repressor protein. These two CDK sites appear vital for FOXK2 function because expression of a mutant lacking these sites cannot be tolerated and causes apoptosis.
Project description:Attachment of ubiquitin to lysine 119 of Histone 2A (H2AK119Ub) is an epigenetic mark characteristic of repressed developmental genes, which is removed by the Polycomb Repressive-Deubiquitinase (PR-DUB) complex. Here we report the crystal structure of the Drosophila PR-DUB, revealing that the deubiquitinase Calypso and its activating partner ASX form a 2:2 complex. The bidentate Calypso-ASX complex is generated by dimerisation of two activated Calypso proteins through their coiled-coil regions. Disrupting the Calypso dimer interface does not affect inherent catalytic activity, but inhibits removal of H2AK119Ub as a consequence of impaired recruitment to nucleosomes. Mutating the equivalent surface on the human counterpart, BAP1, also compromises activity on nucleosomes. Together, this suggests that high local concentrations drive assembly of bidentate PR-DUB complexes on chromatin-providing a mechanistic basis for enhanced PR-DUB activity at specific genomic foci, and the impact of distinct classes of PR-DUB mutations in tumorigenesis.
Project description:Polycomb group (PcG) proteins are transcriptional repressors that control processes ranging from the maintenance of cell fate decisions and stem cell pluripotency in animals to the control of flowering time in plants. In Drosophila, genetic studies identified more than 15 different PcG proteins that are required to repress homeotic (HOX) and other developmental regulator genes in cells where they must stay inactive. Biochemical analyses established that these PcG proteins exist in distinct multiprotein complexes that bind to and modify chromatin of target genes. Among those, Polycomb repressive complex 1 (PRC1) and the related dRing-associated factors (dRAF) complex contain an E3 ligase activity for monoubiquitination of histone H2A (refs 1-4). Here we show that the uncharacterized Drosophila PcG gene calypso encodes the ubiquitin carboxy-terminal hydrolase BAP1. Biochemically purified Calypso exists in a complex with the PcG protein ASX, and this complex, named Polycomb repressive deubiquitinase (PR-DUB), is bound at PcG target genes in Drosophila. Reconstituted recombinant Drosophila and human PR-DUB complexes remove monoubiquitin from H2A but not from H2B in nucleosomes. Drosophila mutants lacking PR-DUB show a strong increase in the levels of monoubiquitinated H2A. A mutation that disrupts the catalytic activity of Calypso, or absence of the ASX subunit abolishes H2A deubiquitination in vitro and HOX gene repression in vivo. Polycomb gene silencing may thus entail a dynamic balance between H2A ubiquitination by PRC1 and dRAF, and H2A deubiquitination by PR-DUB.
Project description:The tumor suppressor BAP1 associates with ASXL1/2 to form the core Polycomb complex PR-DUB, which catalyzes the removal of mono-ubiquitin from several substrates including histone H2A. This complex also mediates the poly-deubiquitination of HCFC1, OGT and PCG1-α, preventing them from proteasomal degradation. Surprisingly, considering its role in a Polycomb complex, no transcriptional signature was consistently found among BAP1-inactivated tumor types. It was hypothesized that BAP1 tumor suppressor activity could reside, at least in part, in stabilizing proteins through its poly-deubiquitinase activity. Quantitative mass spectrometry and gene expression arrays were used to investigate the consequences of BAP1 expression modulation in the NCI-H226 mesothelioma cell line. Analysis of differentially expressed proteins revealed enrichment in cytoskeleton organization, mitochondrial activity and ROS management, while gene expression analysis revealed enrichment in the epithelial-to-mesenchymal transition pathway. Functional assessments in BAP1 inactivated, BAP1 wild-type and BAP1 catalytically dead-expressing NCI-H226 and QR mesothelioma cell lines confirmed alteration of these pathways and demonstrated that BAP1 deubiquitinase activity was mandatory to maintain these phenotypes. Interestingly, monitoring intracellular ROS levels partly restored the morphology and the mitochondrial activity. Finally, the study suggests new tumorigenic and cellular functions of BAP1 and shows for the first time the interest of studying the proteome as readout of BAP1 inactivation.
Project description:The transcription factor KLF5 is highly expressed in basal-like breast cancer and promotes breast cancer cell proliferation, survival, migration and tumour growth. Here we show that, in breast cancer cells, KLF5 is stabilized by the deubiquitinase (DUB) BAP1. With a genome-wide siRNA library screen of DUBs, we identify BAP1 as a bona fide KLF5 DUB. BAP1 interacts directly with KLF5 and stabilizes KLF5 via deubiquitination. KLF5 is in the BAP1/HCF-1 complex, and this newly identified complex promotes cell cycle progression partially by inhibiting p27 gene expression. Furthermore, BAP1 knockdown inhibits tumorigenicity and lung metastasis, which can be rescued partially by ectopic expression of KLF5. Collectively, our findings not only identify BAP1 as the DUB for KLF5, but also reveal a critical mechanism that regulates KLF5 expression in breast cancer. Our findings indicate that BAP1 could be a potential therapeutic target for breast and other cancers.
Project description:Malignant mesothelioma (MM) is poorly responsive to systemic cytotoxic chemotherapy and invariably fatal. Here we describe a screen of 94 drugs in 15 exome-sequenced MM lines and the discovery of a subset defined by loss of function of the nuclear deubiquitinase BRCA associated protein-1 (BAP1) that demonstrate heightened sensitivity to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand). This association is observed across human early passage MM cultures, mouse xenografts and human tumour explants. We demonstrate that BAP1 deubiquitinase activity and its association with ASXL1 to form the Polycomb repressive deubiquitinase complex (PR-DUB) impacts TRAIL sensitivity implicating transcriptional modulation as an underlying mechanism. Death receptor agonists are well-tolerated anti-cancer agents demonstrating limited therapeutic benefit in trials without a targeting biomarker. We identify BAP1 loss-of-function mutations, which are frequent in MM, as a potential genomic stratification tool for TRAIL sensitivity with immediate and actionable therapeutic implications.
Project description:Post-translational modification of histone proteins plays a major role in histone-DNA packaging and ultimately gene expression. Attachment of ubiquitin to the C-terminal tail of histone H2A (H2AK119Ub in mammals) is particularly relevant to the repression of gene transcription, and is removed by the Polycomb Repressive-Deubiquitinase (PR-DUB) complex. Here, we outline recent advances in the understanding of PR-DUB regulation, which have come through structural studies of the Drosophila melanogaster PR-DUB, biochemical investigation of the human PR-DUB, and functional studies of proteins that associate with the PR-DUB. In humans, mutations in components of the PR-DUB frequently give rise to malignant mesothelioma, melanomas, and renal cell carcinoma, and increase disease risk from carcinogens. Diverse mechanisms may underlie disruption of the PR-DUB across this spectrum of disease. Comparing and contrasting the PR-DUB in mammals and Drosophila reiterates the importance of H2AK119Ub through evolution, provides clues as to how the PR-DUB is dysregulated in disease, and may enable new treatment approaches in cancers where the PR-DUB is disrupted.