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IMPORTANCE: Kaposi's sarcoma-associated herpesvirus (KSHV) is a cancer-causing human herpesvirus that establishes a persistent infection in humans. The lytic viral cycle plays a crucial part in lifelong infection as it is involved in the viral dissemination. The master regulator of the KSHV lytic replication cycle is the viral replication and transcription activator (RTA) protein, which is necessary and sufficient to push the virus from latency into the lytic phase. Thus, the identification of host factors utilized by RTA for controlling the lytic cycle can help to find novel targets that could be used for the development of antiviral therapies against KSHV. Using a proteomics approach, we have identified a novel interaction between RTA and the cellular E3 ubiquitin ligase complex RNF20/40, which we have shown to be necessary for promoting RTA-induced KSHV lytic cycle.

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Histone H2B monoubiquitylation plays essential roles in chromatin-based transcriptional processes. A RING-type E3 ligase (yeast Bre1 or human RNF20/RNF40) and an E2 ubiquitin-conjugating enzyme (yeast Rad6 or human hRAD6A), together, precisely deposit ubiquitin on H2B K123 in yeast or K120 in humans. Here, we developed a chemical trapping strategy and successfully captured the transient structures of Bre1- or RNF20/RNF40-mediated ubiquitin transfer from Rad6 or hRAD6A to nucleosomal H2B. Our structures show that Bre1 and RNF40 directly bind nucleosomal DNA, exhibiting a conserved E3/E2/nucleosome interaction pattern from yeast to humans for H2B monoubiquitylation. We also find an uncanonical non-hydrophobic contact in the Bre1 RING-Rad6 interface, which positions Rad6 directly above the target H2B lysine residue. Our study provides mechanistic insights into the site-specific monoubiquitylation of H2B, reveals a critical role of nucleosomal DNA in mediating E3 ligase recognition, and provides a framework for understanding the cancer-driving mutations of RNF20/RNF40.

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BACKGROUND: Type 1 diabetes is one of the chronic autoimmune diseases. Its features include the immune-triggered pancreatic beta-cells destruction. Ubiquitin ligases RNF20 and RNF40 have been discovered to participate into beta cells gene expression, insulin secretion, and expression of vitamin D receptors (VDRs). However, no reports about the role of RNF20/RNF40 in type 1 diabetes are known till now. The aim of this study was to clarify the role of RNF20/RNF40 in type 1 diabetes and explore the mechanism. METHODS: In this study, streptozotocin (STZ)-induced mice type 1 diabetes model was used. The protein expressions of genes were examined through Western blot analysis. Fasting blood glucose was detected through glucose meter. The plasma insulin was tested through the commercial kit. Hematoxylin and eosin staining was utilized to observe pathological changes of pancreatic tissues. Immunofluorescence assay was performed to evaluate the level of insulin. The levels of pro-inflammatory cytokines in serum were assessed by enzyme-linked-immunosorbent serologic assay. The cell apoptosis was measured through terminal deoxynucleotidyl transferase dUTP nick end labelling assay. RESULTS: STZ was used to stimulate mice model for type 1 diabetes. At first, both RNF20 and RNF40 expressions were down-regulated in STZ-mediated type 1 diabetes. Additionally, RNF20/RNF40 improved hyperglycemia in STZ-stimulated mice. Moreover, RNF20/RNF40 relieved pancreatic tissue injury in STZ-induced mice. Further experiments found that RNF20/RNF40 rescued the strengthened inflammation mediated by STZ treatment. The cell apoptosis was enhanced in the pancreatic tissues of STZ-triggered mice, but this effect was weakened by overexpression of RNF20/RNF40. Besides, the VDR expression was positively regulated by RNF20/RNF40. Finally, VDR knockdown reversed improved hyperglycemia, inflammation, and cell apoptosis stimulated by overexpression of RNF20/RNF40. CONCLUSION: Our findings proved that RNF20/RNF40 activated VDR to relieve type 1 diabetes. This work might highlight the functioning of RNF20/RNF40 in the treatment of type 1 diabetes.

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Background: Type 1 diabetes is one of the chronic autoimmune diseases. Its features include the immune-triggered pancreatic beta-cells destruction. Ubiquitin ligases RNF20 and RNF40 have been discovered to participate into beta cells gene expression, insulin secretion, and expression of vitamin D receptors (VDRs). However, no reports about the role of RNF20/RNF40 in type 1 diabetes are known till now. The aim of this study was to clarify the role of RNF20/RNF40 in type 1 diabetes and explore the mechanism. Methods: In this study, streptozotocin (STZ)-induced mice type 1 diabetes model was used. The protein expressions of genes were examined through Western blot analysis. Fasting blood glucose was detected through glucose meter. The plasma insulin was tested through the commercial kit. Hematoxylin and eosin staining was utilized to observe pathological changes of pancreatic tissues. Immunofluorescence assay was performed to evaluate the level of insulin. The levels of pro-inflammatory cytokines in serum were assessed by enzyme-linked-immunosorbent serologic assay. The cell apoptosis was measured through terminal deoxynucleotidyl transferase dUTP nick end labelling assay. Results: STZ was used to stimulate mice model for type 1 diabetes. At first, both RNF20 and RNF40 expressions were down-regulated in STZ-mediated type 1 diabetes. Additionally, RNF20/RNF40 improved hyperglycemia in STZ-stimulated mice. Moreover, RNF20/RNF40 relieved pancreatic tissue injury in STZ-induced mice. Further experiments found that RNF20/RNF40 rescued the strengthened inflammation mediated by STZ treatment. The cell apoptosis was enhanced in the pancreatic tissues of STZ-triggered mice, but this effect was weakened by overexpression of RNF20/RNF40 (AU)

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Epigenetic regulation plays an essential role in driving precise transcriptional programs during development and homeostasis. Among epigenetic mechanisms, histone mono-ubiquitination has emerged as an important post-transcriptional modification. Two major histone mono-ubiquitination events are the mono-ubiquitination of histone H2A at lysine 119 (H2AK119ub), placed by Polycomb repressive complex 1 (PRC1), and histone H2B lysine 120 mono-ubiquitination (H2BK120ub), placed by the heteromeric RNF20/RNF40 complex. Both of these events play fundamental roles in shaping the chromatin epigenetic landscape and cellular identity. In this review we summarize the current understandings of molecular concepts behind histone mono-ubiquitination, focusing on their recently identified roles in tissue development and pathologies.

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COVID-19, caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has presented a serious risk to global public health. The viral main protease Mpro (also called 3Clpro) encoded by NSP5 is an enzyme essential for viral replication. However, very few host proteins have been experimentally validated as targets of 3Clpro. Here, through bioinformatics analysis of 300 interferon stimulatory genes (ISGs) based on the prediction method NetCorona, we identify RNF20 (Ring Finger Protein 20) as a novel target of 3Clpro. We have also provided evidence that 3Clpro, but not the mutant 3ClproC145A without catalytic activity, cleaves RNF20 at a conserved Gln521 across species, which subsequently prevents SREBP1 from RNF20-mediated degradation and promotes SARS-CoV-2 replication. We show that RNA interference (RNAi)-mediated depletion of either RNF20 or RNF40 significantly enhances viral replication, indicating the antiviral role of RNF20/RNF40 complex against SARS-CoV-2. The involvement of SREBP1 in SARS-CoV-2 infection is evidenced by a decrease of viral replication in the cells with SREBP1 knockdown and inhibitor AM580. Taken together, our findings reveal RNF20 as a novel host target for SARS-CoV-2 main protease and indicate that 3Clpro inhibitors may treat COVID-19 through not only blocking viral polyprotein cleavage but also enhancing host antiviral response.

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Numerous epigenetic alterations are observed in cancer cells, and dysregulation of mono-ubiquitination of histone H2B (H2Bub1) has often been linked to tumorigenesis. H2Bub1 is a dynamic post-translational histone modification associated with transcriptional elongation and DNA damage response. Histone H2B monoubiquitination occurs in the site of lysine 120, written predominantly by E3 ubiquitin ligases RNF20/RNF40 and deubiquitinated by ubiquitin specific peptidase 22 (USP22). RNF20/40 is often altered in the primary tumors including colorectal cancer, breast cancer, ovarian cancer, prostate cancer, and lung cancer, and the loss of H2Bub1 is usually associated with poor prognosis in tumor patients. The purpose of this review is to summarize the current knowledge of H2Bub1 in transcription, DNA damage response and primary tumors. This review also provides novel options for exploiting the potential therapeutic target H2Bub1 in personalized cancer therapy.

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p53 is a key transcription factor to regulate gene transcription. However, the molecular mechanism of chromatin-associated p53 on gene transcription remains elusive. Here, using unbiased protein affinity purification, we found that the RNF20/40 complex associated with p53 on the chromatin. Further analyses indicated that p53 mediated the recruitment of the RNF20/40 complex to p53 target gene loci including p21 and PUMA loci and regulated the transcription of p21 and PUMA via the RNF20/40 complex-dependent histone H2B ubiquitination (ubH2B). Lacking the RNF20/40 complex suppressed not only ubH2B but also the generation of the mature mRNA of p21 and PUMA. Moreover, ubH2B was recognized by the ubiquitin-binding motif of pre-mRNA processing splicing factor 8 (PRPF8), a subunit in the spliceosome, and PRPF8 was required for the maturation of the mRNA of p21 and PUMA. Our study unveils a novel p53-dependent pathway that regulates mRNA splicing for tumor suppression.

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BACKGROUND: Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths worldwide, and deciphering underlying molecular mechanism is essential. The loss of monoubiquitinated histone H2B (H2Bub1) was correlated with poor prognosis of CRC patients and, accordingly, H2Bub1 was suggested as a tumor-suppressive mark. Surprisingly, our previous work revealed that the H2B ubiquitin ligase RING finger protein 40 (RNF40) might exert tumor-promoting functions. Here, we investigated the effect of RNF40 loss on tumorigenic features of CRC cells and their survival in vitro. METHODS: We evaluated the effects of RNF40 depletion in several human CRC cell lines in vitro. To evaluate cell cycle progression, cells were stained with propidium iodide and analyzed by flow cytometry. In addition, to assess apoptosis rates, caspase 3/7 activity was assessed in a Celigo® S-based measurement and, additionally, an Annexin V assay was performed. Genomic occupancy of H2Bub1, H3K79me3, and H3K27ac was determined by chromatin immunoprecipitation. Transcriptome-wide effects of RNF40 loss were evaluated based on mRNA-seq results, qRT-PCR, and Western blot. To rescue apoptosis-related effects, cells were treated with Z-VAD-FMK. RESULTS: Human CRC cell lines displayed decreased cell numbers in vitro after RNF40 depletion. While the differences in confluence were not mediated by changes in cell cycle progression, we discovered highly increased apoptosis rates after RNF40 knockdown due to elevated caspase 3/7 activity. This effect can be explained by reduced mRNA levels of anti-apoptotic and upregulation of pro-apoptotic BCL2 family members. Moreover, the direct occupancy of the RNF40-mediated H2B monoubiquitination was observed in the transcribed region of anti-apoptotic genes. Caspase inhibition by Z-VAD-FMK treatment rescued apoptosis in RNF40-depleted cells. However, knockdown cells still displayed decreased tumorigenic features despite the absence of apoptosis. CONCLUSIONS: Our findings reveal that RNF40 is essential for maintaining tumorigenic features of CRC cells in vitro by controlling the expression of genes encoding central apoptotic regulators.

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There is growing evidence highlighting the importance of monoubiquitination as part of the histone code. Monoubiquitination, the covalent attachment of a single ubiquitin molecule at specific lysines of histone tails, has been associated with transcriptional elongation and the DNA damage response. Sites function as scaffolds or docking platforms for proteins involved in transcription or DNA repair; however, not all sites are equal, with some sites resulting in actively transcribed chromatin and others associated with gene silencing. All events are written by E3 ubiquitin ligases, predominantly of the RING (really interesting new gene) finger type. One of the most well-studied events is monoubiquitination of histone H2B at lysine 120 (H2Bub1), written predominantly by the RING finger complex RNF20-RNF40 and generally associated with active transcription. Monoubiquitination of histone H2A at lysine 119 (H2AK119ub1) is also well-studied, its E3 ubiquitin ligase constituting part of thePolycomb Repressor Complex 1 (PRC1), RING1B-BMI1, associated with transcriptional silencing. Both modifications are activated as part of the DNA damage response. Histone monoubiquitination is a key epigenomic event shaping the chromatin landscape of malignancy and influencing how cells respond to DNA damage. This review discusses a number of these sites and the E3 RING finger ubiquitin ligases that write them.

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Histone posttranslational modifications play fundamental roles in the regulation of double-stranded DNA break (DSB) repair. RNF20/RNF40-mediated monoubiquitination of histone H2B on lysine 120 (H2Bub) has been suggested as a potential mediator of DSB repair, although the nature and function of this posttranslational modification remain enigmatic. In this report, we demonstrate that RNF20 and RNF40 are required for DSB repair leading to homologous recombination (HR) and class switch recombination, a process driven by nonhomologous end joining (NHEJ), in mouse B cells. These findings suggest a role for RNF20 and RNF40 in DSB repair proximal to NHEJ/HR pathway choice and likely in the signaling of DSBs. We found that DSBs led to a global increase in H2Bub but not the transcription-associated posttranslational modifications H3K4me3 and H3K79me2. We also found that H2AX phosphorylation was dispensable for H2Bub and that ATM and ATR jointly regulate ionizing radiation (IR)-induced H2Bub. Together, our results suggest that RNF20, RNF40, and H2Bub may represent a novel pathway for DSB sensing and repair.

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BACKGROUND AND AIMS: Inflammatory bowel diseases are linked to an increased risk of developing colorectal cancer [CRC]. Previous studies suggested that the H2B ubiquitin ligase RING finger protein-20 [RNF20] inhibited inflammatory signaling mediated by the nuclear factor kappa-light-chain-enhancer of activated B cells [NF-κB]. However, the role of RNF40, the obligate heterodimeric partner of RNF20, in the context of inflammation and CRC has not been addressed. Here, we examined the effect of RNF40 loss on CRC cells in vitro and on inflammation and inflammatory signaling in vitro and in vivo. METHODS: We evaluated H2Bub1 levels in human and murine colorectal tumors by immunohistochemistry. Moreover, we correlated H2Bub1 and RNF40 levels in vivo and assessed the consequences of RNF40 depletion on cellular phenotype and gene expression in CRC cells in vitro. Finally, we examined the effect of a colon-specific loss of Rnf40 in a murine model of colitis, and assessed both local and systemic inflammation-associated consequences. RESULTS: In vitro studies revealed that the tumorigenic phenotype of CRC cells decreased after RNF40 depletion and displayed gene expression changes related to chromosome segregation and DNA replication, as well as decreased induction of several NF-κB-associated cytokines. This effect was associated with decreased nuclear localization of NF-κB following tumor necrosis factor alpha treatment. Consistently, the colon-specific loss of Rnf40 exerted a protective local, as well as systemic, effect following acute colitis. CONCLUSIONS: Our findings suggest that RNF40 plays a central role in the maintenance of tumorigenic features and inflammatory signaling by promoting nuclear NF-κB activity.

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Cullin-RING-type E3 ligases (CRLs) control a broad range of biological processes by ubiquitylating numerous cellular substrates. However, the role of CRL E3 ligases in chromatid cohesion is unknown. In this study, we identified a new CRL-type E3 ligase (designated as CRL7SMU1 complex) that has an essential role in the maintenance of chromatid cohesion. We demonstrate that SMU1, DDB1, CUL7 and RNF40 are integral components of this complex. SMU1, by acting as a substrate recognition module, binds to H2B and mediates monoubiquitylation at the lysine (K) residue K120 through CRL7SMU1 E3 ligase complex. Depletion of CRL7SMU1 leads to loss of H2B ubiquitylation at the SMC1a locus and, thus, subsequently compromised SMC1a expression in cells. Knockdown of CRL7SMU1 components or loss of H2B ubiquitylation leads to defective sister chromatid cohesion, which is rescued by restoration of SMC1a expression. Together, our results unveil an important role of CRL7SMU1 E3 ligase in promoting H2B ubiquitylation for maintenance of sister chromatid cohesion during mitosis.This article has an associated First Person interview with the first author of the paper.

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Human RING-finger protein 40 (RNF40) is reported as an E3 ligase of H2B ubiquitination. RNF40 needs to couple with its homolog RNF20 to format a complex to regulate DNA double strand break (DSB) response and chromatin stability. Deficient expression of RNF40 might cause incorrect DNA repair and contribute to genomic instability, leading to an abnormal transcriptional program. Incorrect DSB repair and aberrant gene transcription play important roles in tumorigenesis. The role in primary hepatocellular carcinoma (HCC), however, remains unclear. In this study, we selected 103 cases of HCC for immunohistochemistry to explore the role of RNF40 in HCC. The relationship between RNF40 expression and clinicopathological features of HCC was evaluated. RNF40 was mainly localized in the nucleus, where the percentage of low and high staining of RNF40 in tumor tissues was 50.4% (53/103) and 49.6% (50/103), respectively. By contrast, in para-normal tissues the percentage was 92.2% (95/103) and 7.8% (8/103) respectively. Expression of RNF40 in tumor tissues was significantly higher than that in para-normal tissues (P>0.01). Expression of RNF40 had significant association with AFP and TNM tumor stage (both P>0.01). However, age, gender, Hepatitis B Virus infection, liver cirrhosis, tumor size, tumor number, differential stage, and tumor thrombosis were not associated with RNF40 expression. Meanwhile, HCC patients with high expression of RNF40 had lower 5 year overall survival rates and disease-free survival rates (P>0.05). RNF40 is, potentially, an independent prognostic factor for survival in HCC.

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BACKGROUND: Monoubiquitination of H2B (H2Bub1) is a largely enigmatic histone modification that has been linked to transcriptional elongation. Because of this association, it has been commonly assumed that H2Bub1 is an exclusively positively acting histone modification and that increased H2Bub1 occupancy correlates with increased gene expression. In contrast, depletion of the H2B ubiquitin ligases RNF20 or RNF40 alters the expression of only a subset of genes. RESULTS: Using conditional Rnf40 knockout mouse embryo fibroblasts, we show that genes occupied by low to moderate amounts of H2Bub1 are selectively regulated in response to Rnf40 deletion, whereas genes marked by high levels of H2Bub1 are mostly unaffected by Rnf40 loss. Furthermore, we find that decreased expression of RNF40-dependent genes is highly associated with widespread narrowing of H3K4me3 peaks. H2Bub1 promotes the broadening of H3K4me3 to increase transcriptional elongation, which together lead to increased tissue-specific gene transcription. Notably, genes upregulated following Rnf40 deletion, including Foxl2, are enriched for H3K27me3, which is decreased following Rnf40 deletion due to decreased expression of the Ezh2 gene. As a consequence, increased expression of some RNF40-"suppressed" genes is associated with enhancer activation via FOXL2. CONCLUSION: Together these findings reveal the complexity and context-dependency whereby one histone modification can have divergent effects on gene transcription. Furthermore, we show that these effects are dependent upon the activity of other epigenetic regulatory proteins and histone modifications.

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The Mediator complex orchestrates multiple transcription factors with the Pol II apparatus for precise transcriptional control. However, its interplay with the surrounding chromatin remains poorly understood. Here, we analyze differential histone modifications between WT and MED23(-/-) (KO) cells and identify H2B mono-ubiquitination at lysine 120 (H2Bub) as a MED23-dependent histone modification. Using tandem affinity purification and mass spectrometry, we find that MED23 associates with the RNF20/40 complex, the enzyme for H2Bub, and show that this association is critical for the recruitment of RNF20/40 to chromatin. In a cell-free system, Mediator directly and substantially increases H2Bub on recombinant chromatin through its cooperation with RNF20/40 and the PAF complex. Integrative genome-wide analyses show that MED23 depletion specifically reduces H2Bub on a subset of MED23-controlled genes. Importantly, MED23-coupled H2Bub levels are oppositely regulated during myogenesis and lung carcinogenesis. In sum, these results establish a mechanistic link between the Mediator complex and a critical chromatin modification in coordinating transcription with cell growth and differentiation.

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Ubiquitination has traditionally been viewed in the context of polyubiquitination that is essential for marking proteins for degradation via the proteasome. Recent discoveries have shed light on key cellular roles for monoubiquitination, including as a post-translational modification (PTM) of histones such as histone H2B. Monoubiquitination plays a significant role as one of the largest histone PTMs, alongside smaller, better-studied modifications such as methylation, acetylation and phosphorylation. Monoubiquitination of histone H2B at lysine 120 (H2Bub1) has been shown to have key roles in transcription, the DNA damage response and stem cell differentiation. The H2Bub1 enzymatic cascade involves E3 RING finger ubiquitin ligases, with the main E3 generally accepted to be the RNF20-RNF40 complex, and deubiquitinases including ubiquitin-specific protease 7 (USP7), USP22 and USP44. H2Bub1 has been shown to physically disrupt chromatin strands, fostering a more open chromatin structure accessible to transcription factors and DNA repair proteins. It also acts as a recruiting signal, actively attracting proteins with roles in transcription and DNA damage. H2Bub1 also appears to play central roles in histone cross-talk, influencing methylation events on histone H3, including H3K4 and H3K79. Most significantly, global levels of H2Bub1 are low to absent in advanced cancers including breast, colorectal, lung and parathyroid, marking H2Bub1 and the enzymes that regulate it as key molecules of interest as possible new therapeutic targets for the treatment of cancer. This review offers an overview of current knowledge regarding H2Bub1 and highlights links between dysregulation of H2Bub1-associated enzymes, stem cells and malignancy.

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