Dissecting OGT's TPR domain to identify determinants of cellular function.

O-GlcNAc transferase (OGT) is an essential mammalian enzyme that glycosylates myriad intracellular proteins and cleaves the transcriptional coregulator Host Cell Factor 1 to regulate cell cycle processes. Via these catalytic activities as well as noncatalytic protein-protein interactions, OGT maintains cell homeostasis. OGT's tetratricopeptide repeat (TPR) domain is important in substrate recognition, but there is little information on how changing the TPR domain impacts its cellular functions. Here, we investigate how altering OGT's TPR domain impacts cell growth after the endogenous enzyme is deleted. We find that disrupting the TPR residues required for OGT dimerization leads to faster cell growth, whereas truncating the TPR domain slows cell growth. We also find that OGT requires eight of its 13 TPRs to sustain cell viability. OGT-8, like the nonviable shorter OGT variants, is mislocalized and has reduced Ser/Thr glycosylation activity; moreover, its interactions with most of wild-type OGT's binding partners are broadly attenuated. Therefore, although OGT's five N-terminal TPRs are not essential for cell viability, they are required for proper subcellular localization and for mediating many of OGT's protein-protein interactions. Because the viable OGT truncation variant we have identified preserves OGT's essential functions, it may facilitate their identification.

Associations between Extracellular Matrix Protein 1 Gene Polymorphism and Progression of Liver Disease.

Background: Our study aimed to investigate the relationship between extracellular matrix 1 (ECM1) gene polymorphism and progression of liver fibrosis in the Chinese population. Methods: A total 656 patients with hepatitis B virus (HBV) infection and 298 healthy individuals of the Chinese Han population were recruited for a retrospective case-control study. Of the disease group, 104 cases had chronic hepatitis B (CHB), 266 had LC, and 286 had hepatocellular carcinoma (HCC). Subjects were frequency-matched according to age and gender. Polymorphisms of the ECM1 gene were examined using the MassARRAY SNP genotyping method. Results: There were no associations between genotype and allele frequencies of ECM1 rs3737240 and rs13294 loci with the risk of CHB and CHB-related HCC. After adjustment for age, sex, smoking status, and drinking habits, the GT genotype was dramatically related to a reduced risk of chronic HBV infection in both non-HCC (OR = 0.68, 95% CI: 0.49-0.94) and total chronic HBV infection patients (OR = 0.75, 95% CI: 0.56-1.00). Haplotype analyses revealed twelve protective haplotypes against total chronic HBV infection and four against non-HCC chronic HBV infection. Conclusion: ECM1 gene polymorphism in rs3834087 and rs3754217 loci is associated with a reduced risk of chronic HBV infection but not with liver fibrosis development and the occurrence of HCC.

Rare germline heterozygous missense variants in BRCA1-associated protein 1, BAP1, cause a syndromic neurodevelopmental disorder.

Nuclear deubiquitinase BAP1 (BRCA1-associated protein 1) is a core component of multiprotein complexes that promote transcription by reversing the ubiquitination of histone 2A (H2A). BAP1 is a tumor suppressor whose germline loss-of-function variants predispose to cancer. To our knowledge, there are very rare examples of different germline variants in the same gene causing either a neurodevelopmental disorder (NDD) or a tumor predisposition syndrome. Here, we report a series of 11 de novo germline heterozygous missense BAP1 variants associated with a rare syndromic NDD. Functional analysis showed that most of the variants cannot rescue the consequences of BAP1 inactivation, suggesting a loss-of-function mechanism. In T cells isolated from two affected children, H2A deubiquitination was impaired. In matching peripheral blood mononuclear cells, histone H3 K27 acetylation ChIP-seq indicated that these BAP1 variants induced genome-wide chromatin state alterations, with enrichment for regulatory regions surrounding genes of the ubiquitin-proteasome system (UPS). Altogether, these results define a clinical syndrome caused by rare germline missense BAP1 variants that alter chromatin remodeling through abnormal histone ubiquitination and lead to transcriptional dysregulation of developmental genes.

Mutations in Hcfc1 and Ronin result in an inborn error of cobalamin metabolism and ribosomopathy.

Combined methylmalonic acidemia and homocystinuria (cblC) is the most common inborn error of intracellular cobalamin metabolism and due to mutations in Methylmalonic Aciduria type C and Homocystinuria (MMACHC). Recently, mutations in the transcriptional regulators HCFC1 and RONIN (THAP11) were shown to result in cellular phenocopies of cblC. Since HCFC1/RONIN jointly regulate MMACHC, patients with mutations in these factors suffer from reduced MMACHC expression and exhibit a cblC-like disease. However, additional de-regulated genes and the resulting pathophysiology is unknown. Therefore, we have generated mouse models of this disease. In addition to exhibiting loss of Mmachc, metabolic perturbations, and developmental defects previously observed in cblC, we uncovered reduced expression of target genes that encode ribosome protein subunits. We also identified specific phenotypes that we ascribe to deregulation of ribosome biogenesis impacting normal translation during development. These findings identify HCFC1/RONIN as transcriptional regulators of ribosome biogenesis during development and their mutation results in complex syndromes exhibiting aspects of both cblC and ribosomopathies.

Mammalian cell proliferation requires noncatalytic functions of O-GlcNAc transferase.

O-GlcNAc transferase (OGT), found in the nucleus and cytoplasm of all mammalian cell types, is essential for cell proliferation. Why OGT is required for cell growth is not known. OGT performs two enzymatic reactions in the same active site. In one, it glycosylates thousands of different proteins, and in the other, it proteolytically cleaves another essential protein involved in gene expression. Deconvoluting OGT's myriad cellular roles has been challenging because genetic deletion is lethal; complementation methods have not been established. Here, we developed approaches to replace endogenous OGT with separation-of-function variants to investigate the importance of OGT's enzymatic activities for cell viability. Using genetic complementation, we found that OGT's glycosyltransferase function is required for cell growth but its protease function is dispensable. We next used complementation to construct a cell line with degron-tagged wild-type OGT. When OGT was degraded to very low levels, cells stopped proliferating but remained viable. Adding back catalytically inactive OGT rescued growth. Therefore, OGT has an essential noncatalytic role that is necessary for cell proliferation. By developing a method to quantify how OGT's catalytic and noncatalytic activities affect protein abundance, we found that OGT's noncatalytic functions often affect different proteins from its catalytic functions. Proteins involved in oxidative phosphorylation and the actin cytoskeleton were especially impacted by the noncatalytic functions. We conclude that OGT integrates both catalytic and noncatalytic functions to control cell physiology.

MYC regulates ribosome biogenesis and mitochondrial gene expression programs through its interaction with host cell factor-1.

The oncoprotein transcription factor MYC is a major driver of malignancy and a highly validated but challenging target for the development of anticancer therapies. Novel strategies to inhibit MYC may come from understanding the co-factors it uses to drive pro-tumorigenic gene expression programs, providing their role in MYC activity is understood. Here we interrogate how one MYC co-factor, host cell factor (HCF)-1, contributes to MYC activity in a human Burkitt lymphoma setting. We identify genes connected to mitochondrial function and ribosome biogenesis as direct MYC/HCF-1 targets and demonstrate how modulation of the MYC-HCF-1 interaction influences cell growth, metabolite profiles, global gene expression patterns, and tumor growth in vivo. This work defines HCF-1 as a critical MYC co-factor, places the MYC-HCF-1 interaction in biological context, and highlights HCF-1 as a focal point for development of novel anti-MYC therapies.

Tumours form when cells lose control of their growth. Usually, cells produce signals that control how much and how often they divide. But if these signals become faulty, cells may grow too quickly or multiply too often. For example, a group of proteins known as MYC proteins activate growth genes in a cell, but too much of these proteins causes cells to grow uncontrollably. With one third of all cancer deaths linked to excess MYC proteins, these molecules could be key targets for anti-cancer drugs. However, current treatments fail to target these proteins. One option for treating cancers linked to MYC proteins could be to target proteins that work alongside MYC proteins, such as the protein HCF-1, which can attach to MYC proteins. To test if HCF-1 could be a potential drug target, Popay et al. first studied how HCF-1 and MYC proteins interacted using specific cancer cells grown in the laboratory. This revealed that when the two proteins connected, they activated genes that trigger rapid cell growth. When these cancer cells were then injected into mice, tumours quickly grew. However, when the MYC and HCF-1 attachments in the cancer cells were disrupted, the tumours shrunk. This suggests that if anti-cancer drugs were able to target HCF-1 proteins, they could potentially reduce or even reverse the growth of tumours. While further research is needed to identify drug candidates, these findings reveal a promising target for treating tumours that stem from over-abundant MYC proteins.

Novel exon-skipping variant disrupting the basic domain of HCFC1 causes intellectual disability without metabolic abnormalities in both male and female patients.

HCFC1, a global transcriptional regulator, has been shown to associate with MMACHC expression. Pathogenic variants in HCFC1 cause X-linked combined methylmalonic acidemia and hyperhomocysteinemia, CblX type (MIM# 309541). Recent studies showed that certain variants in HCFC1 are associated with X-linked intellectual disability with mild or absent metabolic abnormalities. Here, we report five subjects (three males, two females) from the same family with a novel predicted loss of function HCFC1 variant. All five patients exhibit developmental delay or intellectual disability/learning difficulty and some dysmorphic features; findings were milder in the female as compared to male subjects. Biochemical studies in all patients did not show methylmalonic acidemia or hyperhomocysteinemia but revealed elevated vitamin B12 levels. Trio exome sequencing of the proband and his parents revealed a maternally inherited novel variant in HCFC1 designated as c.1781_1803 + 3del26insCA (NM_005334). Targeted testing confirmed the presence of the same variant in two half-siblings and maternal great uncle. In silico analysis showed that the variant is expected to reduce the quality of the splice donor site in intron 10 and causes abnormal splicing. Sequencing of proband's cDNA revealed exon 10 skipping. Further molecular studies in the two manifesting females revealed moderate and high skewing of X inactivation. Our results support previous observation that HCFC1 variants located outside the Kelch domain exhibit dissociation of the clinical and biochemical phenotype and cause milder or no metabolic changes. We also show that this novel variant can be associated with a phenotype in females, although with milder severity, but further studies are needed to understand the role of skewed X inactivation among females in this rare disorder. Our work expands the genotypes and phenotypes associated with HCFC1-related disorder.

HCF-1 promotes cell cycle progression by regulating the expression of CDC42.

The eukaryotic cell cycle involves a highly orchestrated series of events in which the cellular genome is replicated during a synthesis (S) phase and each of the two resulting copies are segregated properly during mitosis (M). Host cell factor-1 (HCF-1) is a transcriptional co-regulator that is essential for and has been implicated in basic cellular processes, such as transcriptional regulation and cell cycle progression. Although a series of HCF-1 transcriptional targets have been identified, few functional clues have been provided, especially for chromosome segregation. Our results showed that HCF-1 activated CDC42 expression by binding to the -881 to -575 region upstream of the CDC42 transcription start site, and the regulation of CDC42 expression by HCF-1 was correlated with cell cycle progression. The overexpression of a spontaneously cycling and constitutively active CDC42 mutant (CDC42F28L) rescued G1 phase delay and multinucleate defects in mitosis upon the loss of HCF-1. Therefore, these results establish that HCF-1 ensures proper cell cycle progression by regulating the expression of CDC42, which indicates a possible mechanism of cell cycle coordination and the regulation mode of typical Rho GTPases.

Hcfc1a regulates neural precursor proliferation and asxl1 expression in the developing brain.

BACKGROUND: Precise regulation of neural precursor cell (NPC) proliferation and differentiation is essential to ensure proper brain development and function. The HCFC1 gene encodes a transcriptional co-factor that regulates cell proliferation, and previous studies suggest that HCFC1 regulates NPC number and differentiation. However, the molecular mechanism underlying these cellular deficits has not been completely characterized. METHODS: Here we created a zebrafish harboring mutations in the hcfc1a gene (the hcfc1aco60/+ allele), one ortholog of HCFC1, and utilized immunohistochemistry and RNA-sequencing technology to understand the function of hcfc1a during neural development. RESULTS: The hcfc1aco60/+ allele results in an increased number of NPCs and increased expression of neuronal and glial markers. These neural developmental deficits are associated with larval hypomotility and the abnormal expression of asxl1, a polycomb transcription factor, which we identified as a downstream effector of hcfc1a. Inhibition of asxl1 activity and/or expression in larvae harboring the hcfc1aco60/+ allele completely restored the number of NPCs to normal levels. CONCLUSION: Collectively, our data demonstrate that hcfc1a regulates NPC number, NPC proliferation, motor behavior, and brain development.

THAP11F80L cobalamin disorder-associated mutation reveals normal and pathogenic THAP11 functions in gene expression and cell proliferation.

Twelve human THAP proteins share the THAP domain, an evolutionary conserved zinc-finger DNA-binding domain. Studies of different THAP proteins have indicated roles in gene transcription, cell proliferation and development. We have analyzed this protein family, focusing on THAP7 and THAP11. We show that human THAP proteins possess differing homo- and heterodimer formation properties and interaction abilities with the transcriptional co-regulator HCF-1. HEK-293 cells lacking THAP7 were viable but proliferated more slowly. In contrast, HEK-293 cells were very sensitive to THAP11 alteration. Nevertheless, HEK-293 cells bearing a THAP11 mutation identified in a patient suffering from cobalamin disorder (THAP11F80L) were viable although proliferated more slowly. Cobalamin disorder is an inborn vitamin deficiency characterized by neurodevelopmental abnormalities, most often owing to biallelic mutations in the MMACHC gene, whose gene product MMACHC is a key enzyme in the cobalamin (vitamin B12) metabolic pathway. We show that THAP11F80L selectively affected promoter binding by THAP11, having more deleterious effects on a subset of THAP11 targets, and resulting in altered patterns of gene expression. In particular, THAP11F80L exhibited a strong effect on association with the MMACHC promoter and led to a decrease in MMACHC gene transcription, suggesting that the THAP11F80L mutation is directly responsible for the observed cobalamin disorder.

Heat-Shock Protein 90 Controls the Expression of Cell-Cycle Genes by Stabilizing Metazoan-Specific Host-Cell Factor HCFC1.

Molecular chaperones such as heat-shock proteins (HSPs) help in protein folding. Their function in the cytosol has been well studied. Notably, chaperones are also present in the nucleus, a compartment where proteins enter after completing de novo folding in the cytosol, and this raises an important question about chaperone function in the nucleus. We performed a systematic analysis of the nuclear pool of heat-shock protein 90. Three orthogonal and independent analyses led us to the core functional interactome of HSP90. Computational and biochemical analyses identify host cell factor C1 (HCFC1) as a transcriptional regulator that depends on HSP90 for its stability. HSP90 was required to maintain the expression of HCFC1-targeted cell-cycle genes. The regulatory nexus between HSP90 and the HCFC1 module identified in this study sheds light on the relevance of chaperones in the transcription of cell-cycle genes. Our study also suggests a therapeutic avenue of combining chaperone and transcription inhibitors for cancer treatment.

HCF-1 Regulates De Novo Lipogenesis through a Nutrient-Sensitive Complex with ChREBP.

Carbohydrate response element binding protein (ChREBP) is a key transcriptional regulator of de novo lipogenesis (DNL) in response to carbohydrates and in hepatic steatosis. Mechanisms underlying nutrient modulation of ChREBP are under active investigation. Here we identify host cell factor 1 (HCF-1) as a previously unknown ChREBP-interacting protein that is enriched in liver biopsies of nonalcoholic steatohepatitis (NASH) patients. Biochemical and genetic studies show that HCF-1 is O-GlcNAcylated in response to glucose as a prerequisite for its binding to ChREBP and subsequent recruitment of OGT, ChREBP O-GlcNAcylation, and activation. The HCF-1:ChREBP complex resides at lipogenic gene promoters, where HCF-1 regulates H3K4 trimethylation to prime recruitment of the Jumonji C domain-containing histone demethylase PHF2 for epigenetic activation of these promoters. Overall, these findings define HCF-1's interaction with ChREBP as a previously unappreciated mechanism whereby glucose signals are both relayed to ChREBP and transmitted for epigenetic regulation of lipogenic genes.

High OGT activity is essential for MYC-driven proliferation of prostate cancer cells.

O-GlcNAc transferase (OGT) is overexpressed in aggressive prostate cancer. OGT modifies intra-cellular proteins via single sugar conjugation (O-GlcNAcylation) to alter their activity. We recently discovered the first fast-acting OGT inhibitor OSMI-2. Here, we probe the stability and function of the chromatin O-GlcNAc and identify transcription factors that coordinate with OGT to promote proliferation of prostate cancer cells. Methods: Chromatin immunoprecipitation (ChIP) coupled to sequencing (seq), formaldehyde-assisted isolation of regulatory elements, RNA-seq and reverse-phase protein arrays (RPPA) were used to study the importance of OGT for chromatin structure and transcription. Mass spectrometry, western blot, RT-qPCR, cell cycle analysis and viability assays were used to establish the role of OGT for MYC-related processes. Prostate cancer patient data profiled for both mRNA and protein levels were used to validate findings. Results: We show for the first time that OGT inhibition leads to a rapid loss of O-GlcNAc chromatin mark. O-GlcNAc ChIP-seq regions overlap with super-enhancers (SE) and MYC binding sites. OGT inhibition leads to down-regulation of SE-dependent genes. We establish the first O-GlcNAc chromatin consensus motif, which we use as a bait for mass spectrometry. By combining the proteomic data from oligonucleotide enrichment with O-GlcNAc and MYC ChIP-mass spectrometry, we identify host cell factor 1 (HCF-1) as an interaction partner of MYC. Inhibition of OGT disrupts this interaction and compromises MYC's ability to confer androgen-independent proliferation to prostate cancer cells. We show that OGT is required for MYC-mediated stabilization of mitotic proteins, including Cyclin B1, and/or the increased translation of their coding transcripts. This implies that increased expression of mRNA is not always required to achieve increased protein expression and confer aggressive phenotype. Indeed, high expression of Cyclin B1 protein has strong predictive value in prostate cancer patients (p=0.000014) while mRNA does not. Conclusions: OGT promotes SE-dependent gene expression. OGT activity is required for the interaction between MYC and HCF-1 and expression of MYC-regulated mitotic proteins. These features render OGT essential for the androgen-independent, MYC-driven proliferation of prostate cancer cells. Androgen-independency is the major mechanism of prostate cancer progression, and our study identifies OGT as an essential mediator in this process.

Insulin Receptor Associates with Promoters Genome-wide and Regulates Gene Expression.

Insulin receptor (IR) signaling is central to normal metabolic control and dysregulated in prevalent chronic diseases. IR binds insulin at the cell surface and transduces rapid signaling via cytoplasmic kinases. However, mechanisms mediating long-term effects of insulin remain unclear. Here, we show that IR associates with RNA polymerase II in the nucleus, with striking enrichment at promoters genome-wide. The target genes were highly enriched for insulin-related functions including lipid metabolism and protein synthesis and diseases including diabetes, neurodegeneration, and cancer. IR chromatin binding was increased by insulin and impaired in an insulin-resistant disease model. Promoter binding by IR was mediated by coregulator host cell factor-1 (HCF-1) and transcription factors, revealing an HCF-1-dependent pathway for gene regulation by insulin. These results show that IR interacts with transcriptional machinery at promoters and identify a pathway regulating genes linked to insulin's effects in physiology and disease.

Rapid Recapitulation of Nonalcoholic Steatohepatitis upon Loss of Host Cell Factor 1 Function in Mouse Hepatocytes

Host-cell factor 1 (HCF-1), encoded by the ubiquitously expressed X-linked gene Hcfc1, is an epigenetic coregulator important for mouse development and cell proliferation, including during liver regeneration. We used a hepatocyte-specific inducible Hcfc1 knock-out allele (called Hcfc1hepKO), to induce HCF-1 loss in hepatocytes of hemizygous Hcfc1hepKO/Y males by four days. In heterozygous Hcfc1hepKO/+ females, owing to random X-chromosome inactivation, upon Hcfc1hepKO allele induction, a 50/50 mix of HCF-1 positive and negative hepatocyte clusters is engineered. The livers with Hcfc1hepKO/Y hepatocytes displayed a 21-24-day terminal non-alcoholic fatty liver (NAFL) followed by non-alcoholic steatohepatitis (NASH) disease progression typical of severe NAFL disease (NAFLD). In contrast, in livers with heterozygous Hcfc1hepKO/+ hepatocytes, HCF-1-positive hepatocytes replaced HCF-1-negative hepatocytes and revealed only mild-NAFL development. Loss of HCF-1 led to loss of PGC1α protein, probably owing to its destabilization, and deregulation of gene expression particularly of genes involved in mitochondrial structure and function, likely explaining the severe Hcfc1 hepKO/Y liver pathology. Thus, HCF-1 is essential for hepatocyte function, likely playing both transcriptional and non-transcriptional roles. These genetically-engineered loss-of-HCF-1 mice can be used to study NASH as well as NAFLD resolution.

Asxl1 exerts an antiproliferative effect on mouse lung maturation via epigenetic repression of the E2f1-Nmyc axis.

Although additional sex combs-like 1 (ASXL1) has been extensively described in hematologic malignancies, little is known about the molecular role of ASXL1 in organ development. Here, we show that Asxl1 ablation in mice results in postnatal lethality due to cyanosis, a respiratory failure. This lung defect is likely caused by higher proliferative potential and reduced expression of surfactant proteins, leading to reduced air space and defective lung maturation. By microarray analysis, we identified E2F1-responsive genes, including Nmyc, as targets repressed by Asxl1. Nmyc and Asxl1 are reciprocally expressed during the fetal development of normal mouse lungs, whereas Nmyc downregulation is impaired in Asxl1-deficient lungs. Together with E2F1 and ASXL1, host cell factor 1 (HCF-1), purified as an Asxl1-bound protein, is recruited to the E2F1-binding site of the Nmyc promoter. The interaction occurs between the C-terminal region of Asxl1 and the N-terminal Kelch domain of HCF-1. Trimethylation (me3) of histone H3 lysine 27 (H3K27) is enriched in the Nmyc promoter upon Asxl1 overexpression, whereas it is downregulated in Asxl1-deleted lung and -depleted A549 cells, similar to H3K9me3, another repressive histone marker. Overall, these findings suggest that Asxl1 modulates proliferation of lung epithelial cells via the epigenetic repression of Nmyc expression, deficiency of which may cause hyperplasia, leading to dyspnea.

Endoplasmic Reticulum Homeostasis Is Modulated by the Forkhead Transcription Factor FKH-9 During Infection of Caenorhabditis elegans.

Animals have evolved critical mechanisms to maintain cellular and organismal proteostasis during development, disease, and exposure to environmental stressors. The Unfolded Protein Response (UPR) is a conserved pathway that senses and responds to the accumulation of misfolded proteins in the endoplasmic reticulum (ER) lumen. We have previously demonstrated that the IRE-1-XBP-1 branch of the UPR is required to maintain Caenorhabditis elegans ER homeostasis during larval development in the presence of pathogenic Pseudomonas aeruginosa In this study, we identify loss-of-function mutations in four conserved transcriptional regulators that suppress the larval lethality of xbp-1 mutant animals caused by immune activation in response to infection by pathogenic bacteria: FKH-9, a forkhead family transcription factor; ARID-1, an ARID/Bright domain-containing transcription factor; HCF-1, a transcriptional regulator that associates with histone modifying enzymes; and SIN-3, a subunit of a histone deacetylase complex. Further characterization of FKH-9 suggests that loss of FKH-9 enhances resistance to the ER toxin tunicamycin and results in enhanced ER-associated degradation (ERAD). Increased ERAD activity of fkh-9 loss-of-function mutants is accompanied by a diminished capacity to degrade cytosolic proteasomal substrates and a corresponding increased sensitivity to the proteasomal inhibitor bortezomib. Our data underscore how the balance between ER and cytosolic proteostasis can be influenced by compensatory activation of ERAD during the physiological ER stress of infection and immune activation.

The conserved threonine-rich region of the HCF-1PRO repeat activates promiscuous OGT:UDP-GlcNAc glycosylation and proteolysis activities.

O-Linked GlcNAc transferase (OGT) possesses dual glycosyltransferase-protease activities. OGT thereby stably glycosylates serines and threonines of numerous proteins and, via a transient glutamate glycosylation, cleaves a single known substrate-the so-called HCF-1PRO repeat of the transcriptional co-regulator host-cell factor 1 (HCF-1). Here, we probed the relationship between these distinct glycosylation and proteolytic activities. For proteolysis, the HCF-1PRO repeat possesses an important extended threonine-rich region that is tightly bound by the OGT tetratricopeptide-repeat (TPR) region. We report that linkage of this HCF-1PRO-repeat, threonine-rich region to heterologous substrate sequences also potentiates robust serine glycosylation with the otherwise poor Rp-αS-UDP-GlcNAc diastereomer phosphorothioate and UDP-5S-GlcNAc OGT co-substrates. Furthermore, it potentiated proteolysis of a non-HCF-1PRO-repeat cleavage sequence, provided it contained an appropriately positioned glutamate residue. Using serine- or glutamate-containing HCF-1PRO-repeat sequences, we show that proposed OGT-based or UDP-GlcNAc-based serine-acceptor residue activation mechanisms can be circumvented independently, but not when disrupted together. In contrast, disruption of both proposed activation mechanisms even in combination did not inhibit OGT-mediated proteolysis. These results reveal a multiplicity of OGT glycosylation strategies, some leading to proteolysis, which could be targets of alternative molecular regulatory strategies.

O-GlcNAcylation Signal Mediates Proteasome Inhibitor Resistance in Cancer Cells by Stabilizing NRF1.

Cancer cells often heavily depend on the ubiquitin-proteasome system (UPS) for their growth and survival. Irrespective of their strong dependence on the proteasome activity, cancer cells, except for multiple myeloma, are mostly resistant to proteasome inhibitors. A major cause of this resistance is the proteasome bounce-back response mediated by NRF1, a transcription factor that coordinately activates proteasome subunit genes. To identify new targets for efficient suppression of UPS, we explored, using immunoprecipitation and mass spectrometry, the possible existence of nuclear proteins that cooperate with NRF1 and identified O-linked N-acetylglucosamine transferase (OGT) and host cell factor C1 (HCF-1) as two proteins capable of forming a complex with NRF1. O-GlcNAcylation catalyzed by OGT was essential for NRF1 stabilization and consequent upregulation of proteasome subunit genes. Meta-analysis of breast and colorectal cancers revealed positive correlations in the relative protein abundance of OGT and proteasome subunits. OGT inhibition was effective at sensitizing cancer cells to a proteasome inhibitor both in culture cells and a xenograft mouse model. Since active O-GlcNAcylation is a feature of cancer metabolism, our study has clarified a novel linkage between cancer metabolism and UPS function and added a new regulatory axis to the regulation of the proteasome activity.

The Cellular Coactivator HCF-1 Is Required for Glucocorticoid Receptor-Mediated Transcription of Bovine Herpesvirus 1 Immediate Early Genes.

Following productive infection, bovine herpesvirus 1 (BoHV-1) establishes latency in sensory neurons. As in other alphaherpesviruses, expression of BoHV-1 immediate early (IE) genes is regulated by an enhancer complex containing the viral IE activator VP16, the cellular transcription factor Oct-1, and transcriptional coactivator HCF-1, which is assembled on an IE enhancer core element (TAATGARAT). Expression of the IE transcription unit that encodes the viral IE activators bICP0 and bICP4 may also be induced by the activated glucocorticoid receptor (GR) via two glucocorticoid response elements (GREs) located upstream of the enhancer core. Strikingly, lytic infection and reactivation from latency are consistently enhanced by glucocorticoid treatment in vivo As the coactivator HCF-1 is essential for IE gene expression of alphaherpesviruses and recruited by multiple transcription factors, we tested whether HCF-1 is required for glucocorticoid-induced IE gene expression. Depletion of HCF-1 reduced GR-mediated activation of the IE promoter in mouse neuroblastoma cells (Neuro-2A). More importantly, HCF-1-mediated GR activation of the promoter was dependent on the presence of GRE sites but independent of the TAATGARAT enhancer core element. HCF-1 was also recruited to the GRE region of a promoter lacking the enhancer core, consistent with a direct role of the coactivator in mediating GR-induced transcription. Similarly, during productive lytic infection, HCF-1 and GR occupied the IE region containing the GREs. These studies indicate HCF-1 is critical for GR activation of the viral IE genes and suggests that glucocorticoid induction of viral reactivation proceeds via an HCF-1-GR mechanism in the absence of the viral IE activator VP16.IMPORTANCE BoHV-1 transcription is rapidly activated during stress-induced reactivation from latency. The immediate early transcription unit 1 (IEtu1) promoter is regulated by the GR via two GREs. The IEtu1 promoter regulates expression of two viral transcriptional regulatory proteins, infected cell proteins 0 and 4 (bICP0 and bICP4), and thus must be stimulated during reactivation. This study demonstrates that activation of the IEtu1 promoter by the synthetic corticosteroid dexamethasone requires HCF-1. Interestingly, the GRE sites, but not the IE enhancer core element (TAATGARAT), were required for HCF-1-mediated GR promoter activation. The GR and HCF-1 were recruited to the IEtu1 promoter in transfected and infected cells. Collectively, these studies indicate that HCF-1 is critical for GR activation of the viral IE genes and suggest that an HCF-1-GR complex can stimulate the IEtu1 promoter in the absence of the viral IE activator VP16.