RNF8-mediated multi-ubiquitination of MCM7: Linking disassembly of the CMG helicase with DNA damage response in human cells.

DNA damage causes genomic instability. To maintain genome integrity, cells have evolved DNA damage response, which is involved in replication fork disassembly and DNA replication termination. However, the mechanism underlying the regulation of replication fork disassembly and its connection with DNA damage repair remain elusive. The CMG-MCM7 subunit ubiquitination functions on the eukaryotic replication fork disassembly at replication termination. Until now, only ubiquitin ligases CUL2LRR1 have been reported catalyzing MCM7 ubiquitination in human cells. This study discovered that in human cells, the ubiquitin ligase RNF8 catalyzes K63-linked multi-ubiquitination of MCM7 at K145 both in vivo and in vitro. The multi-ubiquitination of MCM7 is dynamically regulated during the cell cycle, primarily presenting on chromatin during the late S phase. Additionally, MCM7 polyubiquitylation is promoted by RNF168 and BRCA1 during DNA replication termination. Upon DNA damage, the RNF8-mediated polyubiquitination of MCM7 decreased significantly during the late S phase. This study highlights the novel role of RNF8-catalyzed polyubiquitination of MCM7 in the regulation of replication fork disassembly in human cells and linking it to DNA damage response.

Key chromatin regulator-related genes associated with the risk of coronary artery disease regulate the expression of HCFC1, RNF8, TNP1 and SET.

Chromatin regulators are indispensable upstream epigenetic regulators.The emergence and progression of atherosclerosis has been demonstrated to be influenced by smooth muscle-related chromatin regulators, such as ZEB2 and MAFF. However, specific chromatin regulators and their possible roles have not been clarified. Information was gathered from 51 patients diagnosed with coronary artery disease (CAD) and 50 individuals in good health from the GEO database. 440 genes were identified as having differential expression across the two datasets, and these genes were linked to cellular reactions. Enrichment of pathways related to histone modification and transcriptional regulatory factors was observed in GO and KEGG analyses. Four machine learning models (RF, SVM, GLM, and XGB) were developed using the expression profiles of 440 chromatin-associated genes in the CAD cohort to pinpoint genes with significant diagnostic potential. After evaluating residuals, root mean square errors, receiver operating characteristic curves, and immune-infiltration, four key genes (HCFC1, RNF8, TNP1, and SET) were identified. Gene expression in different blood vessel levels in atherosclerotic plaques in a mouse model of coronary artery disease showed significant variations. The gene expression levels in macrophages aligned with clinical data from the GEO database as expected. This discovery is crucial for future analysis and the prediction of drug and miRNA targets. In conclusion, we found that the four hub genes are important in the mechanism of CAD. These findings provide new ideas for the study of potential epigenetic predictive markers and therapeutic targets to be used in determining a treatment strategy for CAD.

SiRNF8 Delivered by DNA Framework Nucleic Acid Effectively Sensitizes Chemotherapy in Colon Cancer.

Background: The evident side effects and decreased drug sensitivity significantly restrict the use of chemotherapy. However, nanoparticles based on biomaterials are anticipated to address this challenge. Methods: Through bioinformatics analysis and colon cancer samples, we initially investigated the expression level of RNF8 in colon cancer. Next, we constructed nanocarrier for delivering siRNF8 based on DNA tetrahedron (si-Tet), and Doxorubicin (DOX) was further intercalated into the DNA structure (si-DOX-Tet) for combination therapy. Further, the effects and mechanism of RNF8 inhibition on the sensitivity of colon cancer cells to DOX chemotherapy have also been studied. Results: RNF8 expression was increased in colon cancer. Agarose gel electrophoresis, transmission electron microscopy, and size distribution and potential analysis confirmed the successful preparation of the two nanoparticles, with particle sizes of 10.29 and 37.29 nm, respectively. Fluorescence imaging reveals that the carriers can be internalized into colon cancer cells and escape from lysosomes after 12 hours of treatment, effectively delivering siRNF8 and DOX. Importantly, Western blot analysis verified treatment with 50nM si-Tet silenced RNF8 expression by approximately 50% in colon cancer cells, and combined treatment significantly inhibited cell proliferation. Furthermore, the CCK-8 assay demonstrated that si-Tet treatment enhanced the sensitivity of colon cancer cells to the three chemotherapeutic drugs. Significant more DNA damage was detected after treatment with both si-Tet or si-DOX-Tet. Further flow cytometry analysis revealed that si-DOX-Tet treatment led to significantly more apoptosis, approximately 1.6-fold higher than treatment with DOX alone. Mechanistically, inhibiting RNF8 led to decreased ABCG2 expression and DOX efflux, but increased DNA damage, thereby enhancing the chemotherapeutic effect of DOX. Conclusion: We have successfully constructed si-DOX-Tet. By inhibiting the expression of RNF8, it enhances the chemotherapy sensitivity of DOX. Therefore, this tetrahedral FNA nanocarrier offers a new approach for the combined treatment of colon cancer.

Decabromodiphenyl ether induces the chromosome association disorders of spermatocytes and deformation failures of spermatids in mice.

The environmental presence of decabromodiphenyl ether (BDE-209), which is toxic to the male reproductive system, is widespread. The current study investigated its mechanism of toxicity in mice. The results showed, that BDE-209 induced DNA damage, decreased the expression of the promoter of meiosis spermatogenesis- and oogenesis-specific basic helix-loop-helix 1 (Sohlh1), meiosis related-factors Lethal (3) malignant brain tumor like 2 (L3MBTL2), PIWI-like protein 2 (MILI), Cyclin-dependent kinase 2 (CDK2), Cyclin A, synaptonemal complex protein 1 (SYCP1) and synaptonemal complex protein 3 (SYCP3), and caused spermatogenic cell apoptosis, resulting in a decrease in sperm quantity and quality. Furthermore, BDE-209 downregulated the levels of anaphase-promoting complex/cyclosome (APC/C), increased the expression of PIWI-like protein 1 (MIWI) in the cytoplasm of elongating spermatids, and decreased the nuclear levels of RING finger protein 8 (RNF8), ubiquitinated (ub)-H2A/ub-H2B, and Protamine 1 (PRM1)/Protamine 2 (PRM2), while increasing H2A/H2B nuclear levels in spermatids. The reproductive toxicity was persistent for 50 days following the withdrawal of BDE-209 exposure. The results suggested that BDE-209 inhibits the initiation of meiosis by decreasing the expression of Sohlh1. Furthermore, the reduced expression of L3MBTL2 inhibited the formation of chromosomal synaptonemal complexes by depressing the expression of meiosis regulators affecting the meiotic progression and also inhibited histone ubiquitination preventing the replacement of histones by protamines, by preventing RNF8 from entering nuclei, which affected the evolution of spermatids into mature sperm.

ZEB1 promotes DNA homologous recombination repair and contributes to the 5-Fluorouracil resistance in colorectal cancer.

Chemotherapy resistance represents a significant obstacle in clinical practice of colorectal cancer (CRC). In this study we aim to clarify the underlying mechanism of chemotherapy resistance mediated by ZEB1 in CRC. shRNA-mediated repression of ZEB1 induced DNA damage in SW480 and RKO cells. Ectopic expression of ZEB1 suppressed the DNA damage caused by ZEB1 knocking down in SW480 and RKO cells. In addition, ZEB1 directly targeted several DNA damage response (DDR) factors including NBS1, RNF8 and RNF168, and thereby the homologous recombination (HR) repair is mediated by ZEB1 via NBS1, RNF8 and RNF168 in CRC cells. Furthermore, ZEB1 maintained chromosome stability in CRC cells. By inducing NBS1, RNF8 and RNF168, ZEB1 is capable of promoting the 5-Fluorouracil (5-FU) resistance in CRC cells via enhancing the DDR signaling and DNA repair. The high expression of ZEB1, NBS1, RNF8 and RNF168 is associated with chemotherapy resistance in primary CRC patients. In conclusion, ZEB1 directly induces the expression of NBS1, RNF8 and RNF168, and thereby enhances DNA HR repair in CRC. The ZEB1-mediated DNA repair contributes to the 5-FU resistance in CRC.

miR-146a-5p regulates autophagy and NLRP3 inflammasome activation in epithelial barrier damage in the in vitro cell model of ulcerative colitis through the RNF8/Notch1/mTORC1 pathway.

Ulcerative colitis (UC) is a chronic inflammatory disease affecting the colon that can be influenced by microRNAs (miRNAs). This study aims to investigate the impact of miR-146a-5p on lipopolysaccharide (LPS)-induced Caco-2/HT-29 cell autophagy and NLRP3 inflammasome activation and the underlying mechanism, with the aim of identifying potential therapeutic targets. We used LPS to establish Caco-2/HT-29 cell models and measured cell viability by CCK-8. The levels of miR-146a-5p, RNF8, markers of NLRP3 inflammasome activation and autophagy, proteins involved in the Notch1/mTORC1 pathway, and inflammatory factors were assessed by RT-qPCR, Western blot, and ELISA. Intestinal epithelial barrier function was evaluated by measuring transepithelial electrical resistance. Autophagic flux was measured using tandem fluorescent-labeled LC3. miR-146a-5p was highly-expressed in LPS-induced Caco-2/HT-29 cells, and autophagy flux was blocked at the autolysosomal stage after LPS induction. Inhibition of miR-146a-5p suppressed NLRP3 inflammasome activation, reduced intestinal epithelial barrier damage, and facilitated autophagy inhibition in LPS-induced Caco-2/HT-29 cells. The autophagy inhibitor NH4Cl partially nullified the inhibitory effects of miR-146a-5p inhibition on NLRP3 inflammation activation. miR-146a-5p targeted RNF8, and silencing RNF8 partly abrogated the action of miR-146a-5p inhibition on promoting autophagy and inhibiting NLRP3 inflammasome activation. miR-146a-5p inhibition suppressed the Notch1/mTORC1 pathway activation by upregulating RNF8. Inhibition of the Notch1/mTORC1 pathway partially nullified the function of silencing RNF8 on inhibiting autophagy and bolstering NLRP3 inflammasome activation. In conclusion, miR-146a-5p inhibition may be a potential therapeutic approach for UC, as it facilitates autophagy of LPS-stimulated Caco-2/HT-29 cells, inhibits NLRP3 inflammasome activation, and reduces intestinal epithelial barrier damage by upregulating RNF8 and suppressing the Notch1/mTORC1 pathway.

RNF8 depletion attenuates hepatocellular carcinoma progression by inhibiting epithelial-mesenchymal transition and enhancing drug sensitivity.

Despite substantial advances that have been made in understanding the etiology of hepatocellular carcinoma (HCC), the early-stage diagnosis and treatment of advanced-stage HCC remain a major challenge. RNF8, an E3 ligase important for the DNA damage response, has been proven to facilitate the progression of breast and lung cancer, but its role in HCC remains unclear. In this study, we find that the expression of RNF8 is up-regulated in HCC tissues and positively correlated with poor prognosis of HCC. Furthermore, silencing RNF8 by siRNAs attenuates the migration of HCC cells and inhibits epithelial-mesenchymal transition (EMT) by regulating the expressions of proteins including N-cadherin, ß-catenin, snail, and ZO-1. Moreover, Kaplan‒Meier survival analysis shows that high RNF8 expression predicts poor survival benefits from sorafenib. Finally, cell viability assay demonstrates that RNF8 depletion enhances the sensitivity of HCC cells to sorafenib and lenvatinib treatment. We hypothesize that the inhibitory role of RNF8 in EMT and its enhancing effects on anti-cancer drugs orchestrate the protective effects of RNF8 deficiency in HCC, which indicates its potential in clinical application.

A functional reference map of the RNF8 interactome in cancer.

BACKGROUND: RNF8 is an E3 ligase identified as a critical DNA damage-responsive protein. Recently, multiple reports have shown that RNF8 could be used as an important therapeutic target for cancer chemo/radiotherapy. However, the understanding of RNF8 remains limited due to the lack of its interactome reference map and comprehensive analysis of RNF8 in diverse cancers, which underscores the need to map the interactome of RNF8 via high-throughput methods. RESULTS: A two-way identification method based on LC-MS was designed for the identification of the RNF8 interactome with high-specificity. By in silico analysis and in vitro validation, we identified a new reference map of the RNF8 interactome network containing many new targets, such as YBX1, DNMT1, and HDCA1, new biological functions and the gene-disease associations of RNF8. Our results revealed a close relationship between RNF8 and neurodegenerative diseases or tumor-infiltrating immune cells using bulk RNA-seq and scRNA-seq datasets. As a proof of concept of our interactome map, we validated the direct binding between RNF8 and YBX1 and showed that RNF8 catalyzed the ubiquitination of YBX1. These results demonstrated that RNF8 might be a crucial regulator of YBX1. CONCLUSIONS: Our work provides a unique framework for researchers and clinicians who seek to better explore or understand RNF8-regulated biological functions in cancers. This study will hopefully facilitate the rational design and further development of anti-RNF8 therapy in cancers.

Neuroprotective Effect of E3 Ubiquitin Ligase RNF8 Against Ischemic Stroke via HDAC2 Stability Reduction and Reelin-Dependent GSK3ß Inhibition.

Loss of E3 ubiquitin ligase RING finger protein 8 (RNF8) may lead to neuronal DNA damage and apoptosis. In order to expand on our knowledge on the mechanistic basis underlying neuronal death in ischemic stroke, the present study sought to investigate the potential role of E3 ubiquitin ligase RNF8 on ischemic stroke and explore the underlying downstream mechanism. Middle cerebral artery occlusion (MCAO) in mice and oxygen-glucose deprivation/reoxygenation (OGD/R) in neurons were induced to simulate an ischemic stroke environment. It was found that downregulation of RNF8 and Reelin occurred in MCAO mice and OGD/R-exposed neurons. Silencing of RNF8 enhanced the MCAO-induced neuronal apoptosis and oxidative stress. Mechanistically, RNF8 enhanced the ubiquitination and degradation of HDAC2, thus attenuating OGD/R-induced neuronal apoptosis and oxidative stress. Moreover, HDAC2 inhibited Reelin expression through deacetylation of H3K27me3 in its promoter, causing reduced glycogen synthase kinase-3beta (GSK3ß)-Ser9 phosphorylation and the resultant elevated GSK3ß activity. By this mechanism, RNF8 alleviated ischemic stroke. Coherently, this study suggests that RNF8 plays a neuroprotective effect against ischemic stroke by downregulating HDAC2 expression and inducing Reelin-induced GSK3ß inhibition.

New Synthetic Lethality Re-Sensitizing Platinum-Refractory Cancer Cells to Cisplatin In Vitro: The Rationale to Co-Use PARP and ATM Inhibitors.

The pro-apoptotic tumor suppressor BIN1 inhibits the activities of the neoplastic transcription factor MYC, poly (ADP-ribose) polymerase-1 (PARP1), and ATM Ser/Thr kinase (ATM) by separate mechanisms. Although BIN1 deficits increase cancer-cell resistance to DNA-damaging chemotherapeutics, such as cisplatin, it is not fully understood when BIN1 deficiency occurs and how it provokes cisplatin resistance. Here, we report that the coordinated actions of MYC, PARP1, and ATM assist cancer cells in acquiring cisplatin resistance by BIN1 deficits. Forced BIN1 depletion compromised cisplatin sensitivity irrespective of Ser15-phosphorylated, pro-apoptotic TP53 tumor suppressor. The BIN1 deficit facilitated ATM to phosphorylate the DNA-damage-response (DDR) effectors, including MDC1. Consequently, another DDR protein, RNF8, bound to ATM-phosphorylated MDC1 and protected MDC1 from caspase-3-dependent proteolytic cleavage to hinder cisplatin sensitivity. Of note, long-term and repeated exposure to cisplatin naturally recapitulated the BIN1 loss and accompanying RNF8-dependent cisplatin resistance. Simultaneously, endogenous MYC was remarkably activated by PARP1, thereby repressing the BIN1 promoter, whereas PARP inhibition abolished the hyperactivated MYC-dependent BIN1 suppression and restored cisplatin sensitivity. Since the BIN1 gene rarely mutates in human cancers, our results suggest that simultaneous inhibition of PARP1 and ATM provokes a new BRCAness-independent synthetic lethal effect and ultimately re-establishes cisplatin sensitivity even in platinum-refractory cancer cells.

RNF8-mediated regulation of Akt promotes lung cancer cell survival and resistance to DNA damage.

Despite the tremendous success of targeted and conventional therapies for lung cancer, therapeutic resistance is a common and major clinical challenge. RNF8 is a ubiquitin E3 ligase that plays essential roles in the DNA damage response; however, its role in the pathogenesis of lung cancer is unclear. Here, we report that RNF8 is overexpressed in lung cancer and positively correlates with the expression of p-Akt and poor survival of patients with non-small-cell lung cancer. In addition, we identify RNF8 as the E3 ligase for regulating the activation of Akt by K63-linked ubiquitination under physiological and genotoxic conditions, which leads to lung cancer cell proliferation and resistance to chemotherapy. Together, our study suggests that RNF8 could be a very promising target in precision medicine for lung cancer.

Protective Effects of Sesamin on Cytoxan-Induced Spermatogenesis Dysfunction by Regulating RNF8-ubH2A/ubH2B Pathways in Male Mice.

Most of the clinically infertile patients show spermatogenesis dysfunction. Cyclophosphamide, as an anticancer drug, can induce spermatogenesis dysfunction. Sesamin is the main bioactive component of natural lignans in sesame. It is abundant in sesame oil and has strong biological activities such as antioxidant, antibacterial, and hypoglycemic properties. By establishing the model of spermatogenic dysfunction induced by cyclophosphamide in male mice and then feeding sesamin (50, 100, and 200 mg/kg) for 2 weeks, we proved that sesamin can improve the reproductive organ damage induced by cyclophosphamide and increase the number and activity of sperms. Sesamin can resist cyclophosphamide-induced sperm nuclear maturity and DNA damage by increasing the expression levels of histones H2A and H2B in the testis. In addition, sesamin can improve the ubiquitination of histones regulated by RNF8 to protect the testis. In conclusion, these results suggest that sesamin can improve spermatogenic dysfunction induced by cyclophosphamide, which may be mediated by ubiquitination of histones.

The RNF8 and RNF168 Ubiquitin Ligases Regulate Pro- and Anti-Resection Activities at Broken DNA Ends During Non-Homologous End Joining.

The RING-type E3 ubiquitin ligases RNF8 and RNF168 recruit DNA damage response (DDR) factors to chromatin flanking DNA double strand breaks (DSBs) including 53BP1, which protects DNA ends from resection during DNA DSB repair by non-homologous end joining (NHEJ). Deficiency of RNF8 or RNF168 does not lead to demonstrable NHEJ defects, but like deficiency of 53BP1, the combined deficiency of XLF and RNF8 or RNF168 leads to diminished NHEJ in lymphocytes arrested in G0/G1 phase. The function of RNF8 in NHEJ depends on its E3 ubiquitin ligase activity. Loss of RNF8 or RNF168 in G0/G1-phase lymphocytes leads to the resection of broken DNA ends, demonstrating that RNF8 and RNF168 function to protect DNA ends from nucleases, pos sibly through the recruitment of 53BP1. However, the loss of 53BP1 leads to more severe resection than the loss of RNF8 or RNF168. Moreover, in 53BP1-deficient cells, the loss of RNF8 or RNF168 leads to diminished DNA end resection. We conclude that RNF8 and RNF168 regulate pathways that both prevent and promote DNA end resection in cells arrested in G0/G1 phase.

Feedback repression of PPARα signaling by Let-7 microRNA.

Peroxisome proliferator-activated receptor α (PPARα) controls hepatic lipid homeostasis and is the target of lipid-lowering fibrate drugs. PPARα activation represses expression of let-7 microRNA (miRNA), but the function of let-7 in PPARα signaling and lipid metabolism is unknown. In the current study, a hepatocyte-specific let-7b/c2 knockout (let7b/c2ΔHep) mouse line is generated, and these mice are found to exhibit pronounced resistance to diet-induced obesity and fatty liver. Let-7 inhibition by hepatocyte-specific let-7 sponge expression shows similar phenotypes as let7b/c2ΔHep mice. RNA sequencing (RNA-seq) analysis reveals that hepatic PPARα signaling is repressed in let7b/c2ΔHep mice. Protein expression of the obligate PPARα heterodimer partner retinoid X receptor α (RXRα) is reduced in the livers of let7b/c2ΔHep mice. Ring finger protein 8 (Rnf8), which is a direct target of let-7, is elevated in let7b/c2ΔHep mouse liver and identified as a E3 ubiquitin ligase for RXRα. This study highlights a let-7-RNF8-RXRα regulatory axis that modulates hepatic lipid catabolism.

RNF8-CDH1 Co-Expression Predicts Clinical Benefit of Chemoradiotherapy in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and exhibits an overall poor outcome. Due to the lack of targeted therapy, conventional systemic chemotherapy has been the main strategy for the treatment of TNBC. Further evidence has shown that combining radiation with chemotherapy is also a suitable treatment based on DNA repair deficiencies in patients with TNBC. However, the preferred treatment for metastatic TNBC remains unclear. Therefore, identification of biomarkers is an unmet need in personalized therapy for TNBC. RNF8 (ring finger protein 8) is a ubiquitin ligase implicated in TNBC metastasis; however, its role in TNBC pathogenesis is unclear. The purpose of the present study was to investigate the roles of the RNF8-CDH1(Cadherin 1) axis in node-positive TNBC patients. We found that the RNF8high/CDH1low index was significantly higher in patients with TNBC than in patients without TNBC. Furthermore, patients with an RNF8high/CDH1low index displayed poorer outcomes than those with an RNF8low-medium/CDH1medium-high index. Notably, as compared to patients with an RNF8low-medium/CDH1medium-high index, those with an RNF8high/CDH1low index had a poorer survival rate with chemotherapy treatment alone. The combination of radiation and chemotherapy resulted in a better survival rate than chemotherapy alone in patients with an RNF8high/CDH1low index. Taken together, the RNF8high/CDH1low index not only functions as a prognostic and therapeutic marker but may also act as a target in the development of anti-cancer agents for patients with TNBC.

CDX2 inducible microRNAs sustain colon cancer by targeting multiple DNA damage response pathway factors.

Meta-analysis of transcripts in colon adenocarcinoma patient tissues led to the identification of a DNA damage responsive miR signature called DNA damage sensitive miRs (DDSMs). DDSMs were experimentally validated in the cancerous colon tissues obtained from an independent cohort of colon cancer patients and in multiple cellular systems with high levels of endogenous DNA damage. All the tested DDSMs were transcriptionally upregulated by a common intestine-specific transcription factor, CDX2. Reciprocally, DDSMs were repressed via the recruitment of HDAC1/2-containing complexes onto the CDX2 promoter. These miRs downregulated multiple key targets in the DNA damage response (DDR) pathway, namely BRCA1, ATM, Chk1 (also known as CHEK1) and RNF8. CDX2 directly regulated the DDSMs, which led to increased tumor volume and metastasis in multiple preclinical models. In colon cancer patient tissues, the DDSMs negatively correlated with BRCA1 levels, were associated with decreased probability of survival and thereby could be used as a prognostic biomarker. This article has an associated First Person interview with the first author of the paper.

RNF8 is not required for histone-to-protamine exchange in spermiogenesis†.

While an E3 ubiquitin ligase, RNF8, was initially reported to be required for histone-to-protamine exchange in spermiogenesis, we subsequently demonstrated that RNF8 is not involved in this process. Nevertheless, reflecting a lingering misunderstanding in the field, a growing number of studies have continued to postulate a requirement for RNF8 in the histone-to-protamine exchange. For example, a recent study claimed that a mouse PIWI protein, MIWI, controls RNF8-mediated histone-to-protamine exchange. Here, confirming our earlier conclusions, we show that RNF8 is required neither for the establishment of histone H4K16 acetylation, which is an initial step in histone removal during spermiogenesis, nor for the incorporation of two protamine proteins, PRM1 and PRM2. Thus, whereas RNF8 mediates ubiquitination of H2A on the sex chromosomes in meiosis, during the prior stage of spermatogenesis, our genetic evidence underscores that RNF8 is not involved in histone-to-protamine exchange.

RNF8-ubiquitinated KMT5A is required for RNF168-induced H2A ubiquitination in response to DNA damage.

Histone modifications play critical roles in DNA damage repair to safeguard genome integrity. However, how different histone modifiers coordinate to build appropriate chromatin context for DNA damage repair is largely unknown. Here, we report a novel interplay between the histone methyltransferase KMT5A and two E3 ligases RNF8 and RNF168 in establishing the histone modification status for DNA damage repair. KMT5A is a newly identified substrate of RNF8 in vitro and in vivo. In response to DNA double-strand breaks (DSBs), RNF8 promotes KMT5A recruitment onto damaged chromatin in a ubiquitination-dependent manner. RNF8-induced KMT5A ubiquitination increases the binding capacity of KMT5A to RNF168. Interestingly, KMT5A not only drives a local increase in H4K20 monomethylation at DSBs, but also promotes RNF168's activity in catalyzing H2A ubiquitination. We proved that the interaction between the H2A acidic patch and KMT5A R188/R189 residues is critical for KMT5A-mediated regulation of H2A ubiquitination. Taken together, our results highlight a new role for KMT5A in linking H4K20 methylation and H2A ubiquitination and provide insight into the histone modification network during DNA damage repair.

Targeting RNF8 effectively reverses cisplatin and doxorubicin resistance in endometrial cancer.

BACKGROUND: Endometrial cancer (EC) is one of the most frequent gynecological malignancy worldwide. However, resistance to chemotherapy remains one of the major difficulties in the treatment of EC. Thus, there is an urgent requirement to understand mechanisms of chemoresistance and identify novel regimens for patients with EC. We found that protein and mRNA expression levels of RNF8 were significantly increased in both cisplatin and doxorubicin resistant EC cells. Cell survival assay showed that RNF deficiency significantly enhanced the sensitivity of resistant EC cells to cisplatin and doxorubicin (P < 0.01). In addition, chemoresistant EC cells exhibited increased NHEJ efficiency. Knockout of RNF8 in chemoresistant EC cells significantly reduced NHEJ efficiency and prolonged Ku80 retention on DSB. Moreover, cisplatin resistant AN3CA xenograft showed that RNF8 deficiency overcame cisplatin resistance. Our in vitro and in vivo assays provide evidence for RNF8, which is a NHEJ factor, serving as a promising, novel target in EC chemotherapy.

Histone Ubiquitination: An Integrative Signaling Platform in Genome Stability.

Complex mechanisms are in place to maintain genome stability. Ubiquitination of chromatin plays a central role in these mechanisms. The ever-growing complexity of the ubiquitin (Ub) code and of chromatin modifications and dynamics challenges our ability to fully understand how histone ubiquitination regulates genome stability. Here we review the current knowledge on specific, low-abundant histone ubiquitination events that are highly regulated within the cellular DNA damage response (DDR), with particular emphasis on the latest discovery of Ub phosphorylation as a novel regulator of the DDR signaling pathway. We discuss players involved and potential implications of histone (phospho)ubiquitination on chromatin structure, and we highlight exciting open questions for future research.