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 ...

University of Montréal

Sinai Health System

The candidate tumor suppressor BAP1 is a deubiquitinating enzyme (DUB) involved in the regulation of cell proliferation, although the molecular mechanisms governing its function remain poorly defined. BAP1 was recently shown to interact with and deubiquitinate the transcriptional regulator host cell factor 1 (HCF-1). Here we show that BAP1 assembles multiprotein complexes containing numerous transcription factors and cofactors, including HCF-1 and the transcription factor Yin Yang 1 (YY1). Through its coiled-coil motif, BAP1 directly interacts with the zinc fingers of YY1. Moreover, HCF-1 interacts with the middle region of YY1 encompassing the glycine-lysine-rich domain and is essential for the formation of a ternary complex with YY1 and BAP1 in vivo. BAP1 activates transcription in an enzymatic-activity-dependent manner and regulates the expression of a variety of genes involved in numerous cellular processes. We further show that BAP1 and HCF-1 are recruited by YY1 to the promoter of the cox7c gene, which encodes a mitochondrial protein used here as a model of BAP1-activated gene expression. Our findings (i) establish a direct link between BAP1 and the transcriptional control of genes regulating cell growth and proliferation and (ii) shed light on a novel mechanism of transcription regulation involving ubiquitin signaling.

The ubiquitin carboxyl hydrolase BAP1 forms a ternary complex with YY1 and HCF-1 and is a critical regulator of gene expression.

Host Cell Factor 1 (HCF-1) plays critical roles in regulating gene expression in a plethora of physiological processes. HCF-1 is first synthesized as a precursor, and subsequently specifically proteolytically cleaved within a large middle region termed the proteolytic processing domain (PPD). Although the underlying mechanism remains enigmatic, proteolysis of HCF-1 regulates its transcriptional activity and is important for cell cycle progression. Here we report that HCF-1 proteolysis is a regulated process. We demonstrate that a large proportion of the signaling enzyme O-linked-N-acetylglucosaminyl transferase (OGT) is complexed with HCF-1 and this interaction is essential for HCF-1 cleavage. Moreover, HCF-1 is, in turn, required for stabilizing OGT in the nucleus. We provide evidence indicating that OGT regulates HCF-1 cleavage via interaction with and O-GlcNAcylation of the HCF-1 PPD. In contrast, although OGT also interacts with the basic domain in the HCF-1 amino-terminal subunit, neither the interaction nor the O-GlcNAcylation of this region are required for proteolysis. Moreover, we show that OGT-mediated modulation of HCF-1 impacts the expression of the herpes simplex virus immediate-early genes, targets of HCF-1 during the initiation of viral infection. Together the data indicate that O-GlcNAcylation of HCF-1 is a signal for its proteolytic processing and reveal a unique crosstalk between these posttranslational modifications. Additionally, interactions of OGT with multiple HCF-1 domains may indicate that OGT has several functions in association with HCF-1.

Crosstalk between O-GlcNAcylation and proteolytic cleavage regulates the host cell factor-1 maturation pathway.

The development of electron-based, unimolecular dissociation MS, i.e. electron capture and electron transfer dissociation (ECD and ETD, respectively), has greatly increased the speed and reliability of labile PTM site assignment. The field of intracellular O-GlcNAc (O-linked N-acetylglucosamine) signaling has especially advanced with the advent of ETD MS. Only within the last five years have proteomic-scale experiments utilizing ETD allowed the assignment of hundreds of O-GlcNAc sites within cells and subcellular structures. Our ability to identify and unambiguously assign the site of O-GlcNAc modifications using ETD is rapidly increasing our understanding of this regulatory glycosylation and its potential interaction with other PTMs. Here, we discuss the advantages of using ETD, complimented with collisional-activation MS, in a study of the extensively O-GlcNAcylated protein Host Cell Factor C1 (HCF-1). HCF-1 is a transcriptional coregulator that forms a stable complex with O-GlcNAc transferase and controls cell cycle progression. ETD, along with higher energy collisional dissociation (HCD) MS, was employed to assign the PTMs of the HCF-1 protein isolated from HEK293T cells. These include 19 sites of O-GlcNAcylation, two sites of phosphorylation, and two sites bearing dimethylarginine, and showcase the residue-specific, PTM complexity of this regulator of cell proliferation.

Electron transfer dissociation (ETD): the mass spectrometric breakthrough essential for O-GlcNAc protein site assignments-a study of the O-GlcNAcylated protein host cell factor C1.

The tumor suppressor BAP1 interacts with chromatin-associated proteins and regulates cell proliferation, but its mechanism of action and regulation remain poorly defined. We show that the ubiquitin-conjugating enzyme UBE2O multi-monoubiquitinates the nuclear localization signal of BAP1, thereby inducing its cytoplasmic sequestration. This activity is counteracted by BAP1 autodeubiquitination through intramolecular interactions. Significantly, we identified cancer-derived BAP1 mutations that abrogate autodeubiquitination and promote its cytoplasmic retention, indicating that BAP1 autodeubiquitination ensures tumor suppression. The antagonistic relationship between UBE2O and BAP1 is also observed during adipogenesis, whereby UBE2O promotes differentiation and cytoplasmic localization of BAP1. Finally, we established a putative targeting consensus sequence of UBE2O and identified numerous chromatin remodeling factors as potential targets, several of which tested positive for UBE2O-mediated ubiquitination. Thus, UBE2O defines an atypical ubiquitin-signaling pathway that coordinates the function of BAP1 and establishes a paradigm for regulation of nuclear trafficking of chromatin-associated proteins. 

Autodeubiquitination protects the tumor suppressor BAP1 from cytoplasmic sequestration mediated by the atypical ubiquitin ligase UBE2O.

IKAROS is a critical regulator of hematopoietic cell fate and its dynamic expression pattern is required for proper hematopoiesis. In collaboration with the Nucleosome Remodeling and Deacetylase (NuRD) complex, it promotes gene repression and activation. It remains to be clarified how IKAROS can support transcription activation while being associated with the HDAC-containing complex NuRD. IKAROS also binds to the Positive-Transcription Elongation Factor b (P-TEFb) at gene promoters. Here, we demonstrate that NuRD and P-TEFb are assembled in a complex that can be recruited to specific genes by IKAROS. The expression level of IKAROS influences the recruitment of the NuRD-P-TEFb complex to gene regulatory regions and facilitates transcription elongation by transferring the Protein Phosphatase 1α (PP1α), an IKAROS-binding protein and P-TEFb activator, to CDK9. We show that an IKAROS mutant that is unable to bind PP1α cannot sustain gene expression and impedes normal differentiation of Ik(NULL) hematopoietic progenitors. Finally, the knock-down of the NuRD subunit Mi2 reveals that the occupancy of the NuRD complex at transcribed regions of genes favors the relief of POL II promoter-proximal pausing and thereby, promotes transcription elongation.

The IKAROS interaction with a complex including chromatin remodeling and transcription elongation activities is required for hematopoiesis.

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. 

The BAP1/ASXL2 Histone H2A Deubiquitinase Complex Regulates Cell Proliferation and Is Disrupted in Cancer.

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.

Salima Daou

Maisonneuve-Rosemont Hospital Research Center and Department of Medicine

University of Montréal

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Salima Daou

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University of Montréal

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Maisonneuve-Rosemont Hospital Research Center and Department of Medicine