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The RNA recognition motif (RRM) binds to nucleic acids as well as proteins. More than one such domain is found in the pre-mRNA processing hnRNP proteins. While the mode of RNA recognition by RRMs is known, the molecular basis of their protein interaction remains obscure. Here we describe the mode of interaction between hnRNP L and LL with the methyltransferase SETD2. We demonstrate that for the interaction to occur, a leucine pair within a highly conserved stretch of SETD2 insert their side chains in hydrophobic pockets formed by hnRNP L RRM2. Notably, the structure also highlights that RRM2 can form a ternary complex with SETD2 and RNA. Remarkably, mutating the leucine pair in SETD2 also results in its reduced interaction with other hnRNPs. Importantly, the similarity that the mode of SETD2-hnRNP L interaction shares with other related protein-protein interactions reveals a conserved design by which splicing regulators interact with one another., Interaction between SETD2 and hnRNP L has previously been shown to be implicated in coupling gene transcription and mRNA processing. Here the authors elucidate the molecular basis of this functional interaction, showing that the RRM domain of hnRNP L possesses non-overlapping binding interfaces for engaging RNA and SETD2.

Human SETD2 is the unique histone methyltransferase that generates H3K36 trimethylation (H3K36me3), an epigenetic mark that plays a key role in normal hematopoiesis. Interestingly, recurrent inactivating mutations of SETD2 and aberrant H3K36me3 are increasingly reported to be involved in hematopoietic malignancies. Benzene (BZ) is a ubiquitous environmental pollutant and carcinogen that causes leukemia. The leukemogenic properties of BZ depend on its biotransformation in the bone marrow into oxidative metabolites, in particular 1,4-benzoquinone (BQ). This hematotoxic metabolite can form DNA and protein adducts that result in the damage and the alteration of cellular processes. Recent studies suggest that BZ-dependent leukemogenesis could depend on epigenetic perturbations, notably aberrant histone methylation. We investigated whether H3K36 trimethylation by SETD2 could be impacted by BZ and its hematotoxic metabolites. Herein, we show that BQ, the major leukemogenic metabolite of BZ, inhibits irreversibly the human histone methyltransferase SETD2, resulting in decreased H3K36me3. Our mechanistic studies further indicate that the BQ-dependent inactivation of SETD2 is due to covalent binding of BQ to reactive Zn-finger cysteines within the catalytic domain of the enzyme. The formation of these quinoprotein adducts results in loss of enzyme activity and protein crosslinks/oligomers. Experiments conducted in hematopoietic cells confirm that exposure to BQ results in the formation of SETD2 crosslinks/oligomers and concomitant loss of H3K36me3 in cells. Taken together, our data indicate that BQ, a major hematotoxic metabolite of BZ, could contribute to BZ-dependent leukemogenesis by perturbing the functions of SETD2, a histone lysine methyltransferase of hematopoietic relevance. SIGNIFICANCE STATEMENT: Benzoquinone is a major leukemogenic metabolite of benzene. Dysregulation of histone methyltransferase is involved in hematopoietic malignancies. This study found that benzoquinone irreversibly impairs SET domain containing 2, a histone H3K36 methyltransferase that plays a key role in hematopoiesis. Benzoquinone forms covalent adducts on Zn-finger cysteines within the catalytic site, leading to loss of activity, protein crosslinks/oligomers, and concomitant decrease of H3K36me3 histone mark. These data provide evidence that a leukemogenic metabolite of benzene can impair a key epigenetic enzyme.

Alterations in global histone methylation regulate gene expression and participate in cancer onset and progression. The profile of histone methylation marks in pediatric astrocytomas is currently understudied with limited data on their distribution among grades. The global expression patterns of repressive histone marks H3K9me3, H3K27me3, and H4K20me3 and active H3K4me3 and H3K36me3 along with their writers SUV39H1, SETDB1, EZH2, MLL2, and SETD2 were investigated in 46 pediatric astrocytomas and normal brain tissues. Associations between histone marks and modifying enzymes with clinicopathological characteristics and disease-specific survival were studied along with their functional impact in proliferation and migration of pediatric astrocytoma cell lines using selective inhibitors in vitro. Upregulation of histone methyltransferase gene expression and deregulation of histone code were detected in astrocytomas compared to normal brain tissues, with higher levels of SUV39H1, SETDB1, and SETD2 as well as H4K20me3 and H3K4me3 histone marks. Pilocytic astrocytomas exhibited lower MLL2 levels compared to diffusely infiltrating tumors indicating a differential pattern of epigenetic regulator expression between the two types of astrocytic neoplasms. Moreover, higher H3K9me3, H3K36me3, and SETDB1 expression was detected in grade IIΙ/IV compared to grade II astrocytomas. In univariate analysis, elevated H3K9me3 and MLL2 and diminished SUV39H1 expression adversely affected survival. Upon multivariate survival analysis, only SUV39H1 expression was revealed as an independent prognostic factor of adverse significance. Treatment of pediatric astrocytoma cell lines with SUV39H1 inhibitor reduced proliferation and cell migration. Our data implicate H3K9me3 and SUV39H1 in the pathobiology of pediatric astrocytomas, with SUV39H1 yielding prognostic information independent of other clinicopathologic variables. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13311-021-01090-x.

Gene discovery efforts in autism spectrum disorder have identified heterozygous defects in chromatin remodeller genes, the ‘readers, writers and erasers’ of methyl marks on chromatin, as major contributors to this disease. Despite this advance, a convergent aetiology between these defects and aberrant chromatin architecture or gene expression has remained elusive. Recently, data have begun to emerge that chromatin remodellers also function directly on the cytoskeleton. Strongly associated with autism spectrum disorder, the SETD2 histone methyltransferase for example, has now been shown to directly methylate microtubules of the mitotic spindle. However, whether microtubule methylation occurs in post-mitotic cells, for example on the neuronal cytoskeleton, is not known. We found the SETD2 α-tubulin lysine 40 trimethyl mark occurs on microtubules in the brain and in primary neurons in culture, and that the SETD2 C-terminal SRI domain is required for binding and methylation of α-tubulin. A CRISPR knock-in of a pathogenic SRI domain mutation (Setd2SRI) that disables microtubule methylation revealed at least one wild-type allele was required in mice for survival, and while viable, heterozygous Setd2SRI/wtmice exhibited an anxiety-like phenotype. Finally, whereas RNA-sequencing (RNA-seq) and chromatin immunoprecipitation-sequencing (ChIP-seq) showed no concomitant changes in chromatin methylation or gene expression in Setd2SRI/wtmice, primary neurons exhibited structural deficits in axon length and dendritic arborization. These data provide the first demonstration that microtubules of neurons are methylated, and reveals a heterozygous chromatin remodeller defect that specifically disables microtubule methylation is sufficient to drive an autism-associated phenotype.

Patients with polycystic kidney disease (PKD) are at a high risk of developing renal cell carcinoma (RCC). However, little is known about genetic alterations or changes in signaling pathways during the transition from PKD to RCC. SET domain–containing 2 (SETD2) is a histone methyltransferase, which catalyzes tri-methylation of H3K36 (H3K36me3) and has been identified as a tumor suppressor in clear cell renal cell carcinoma (ccRCC), but the underlying mechanism remains largely unexplored. Here we report that knockout of SETD2 in a c-MYC–driven PKD mouse model drove the transition to ccRCC. SETD2 inhibited β-catenin activity at transcriptional and posttranscriptional levels by competing with β-catenin for binding promoters of target genes and maintaining transcript levels of members of the β-catenin destruction complex. Thus, SETD2 deficiency enhanced the epithelial-to-mesenchymal transition and tumorigenesis through the hyperactivation of Wnt/β-catenin signaling. Our findings reveal previously unrecognized roles of SETD2-mediated competitive DNA binding and H3K36me3 modification in regulating Wnt/β-catenin signaling during the transition from PKD to ccRCC. The novel autochthonous mouse models of PKD and ccRCC will be useful for preclinical research into disease progression. Significance: These findings characterize multiple mechanisms by which SETD2 inhibits β-catenin activity during the transition of polycystic kidney disease to renal cell carcinoma, providing a potential therapeutic strategy for high-risk patients.

The histone H3 lysine 36 methyltransferase SET-domain-containing 2 (SETD2) has been reported to be frequently mutated or deleted in many types of human cancer. However, the role of SETD2 in lung adenocarcinoma (LUAD) has not been well documented. In the present study, we found that SETD2 was significantly down-regulated both in LUAD tissues and cell lines. Functionally, the increased expression of SETD2 significantly attenuated the proliferation of cancer cells by affecting the cell cycle, whereas SETD2 deficiency dramatically improved these proliferative abilities of cancer cells. Through conjoint analysis of RNA-seq and ChIP data, we identified a functional target gene of SETD2, CXCL1, and its expression was negatively correlated with that of SETD2. Moreover, SETD2 deletion stimulated cell cycle-related proteins to promote LUAD. Further mechanistic studies demonstrated that histone H3 lysine 36 trimethylation (H3K36me3) catalyzed by SETD2 interacted with the promoter of CXCL1 to regulate its transcription and downstream signaling pathways, contributing to tumorigenesis in vitro and in vivo. Our findings suggested that SETD2 inhibited tumor growth via suppressing CXCL1-mediated activation of cell cycle, indicating that the regulation of H3K36me3 level by targeting SETD2 and/or the administration of downstream CXCL1 might represent a potential therapeutic way for new treatment in LUAD.

The SET domain containing 2, histone lysine methyltransferase encoded by SETD2 is a dual-function methyltransferase for histones and microtubules and plays an important role for transcriptional regulation, genomic stability, and cytoskeletal functions. Specifically, SETD2 is associated with trimethylation of histone H3 at lysine 36 (H3K36me3) and methylation of α-tubulin at lysine 40. Heterozygous loss of function and missense variants have previously been described with Luscan-Lumish syndrome (LLS), which is characterized by overgrowth, neurodevelopmental features, and absence of overt congenital anomalies. We have identified 15 individuals with de novo variants in codon 1740 of SETD2 whose features differ from those with LLS. Group 1 consists of 12 individuals with heterozygous variant c.5218C>T p.(Arg1740Trp) and Group 2 consists of 3 individuals with heterozygous variant c.5219G>A p.(Arg1740Gln). The phenotype of Group 1 includes microcephaly, profound intellectual disability, congenital anomalies affecting several organ systems, and similar facial features. Individuals in Group 2 had moderate to severe intellectual disability, low normal head circumference, and absence of additional major congenital anomalies. While LLS is likely due to loss of function of SETD2, the clinical features seen in individuals with variants affecting codon 1740 are more severe suggesting an alternative mechanism, such as gain of function, effects on epigenetic regulation, or posttranslational modification of the cytoskeleton. Our report is a prime example of different mutations in the same gene causing diverging phenotypes and the features observed in Group 1 suggest a new clinically recognizable syndrome uniquely associated with the heterozygous variant c.5218C>T p.(Arg1740Trp) in SETD2.

To define the role of SETD2 in the WNT5a signaling in the context of osteoclastogenesis, we exploited two different models: in vitro osteoclast differentiation, and K/BxN serum-induced arthritis model. We found that SETD2 and WNT5a were upregulated during osteoclast differentiation and after induction of arthritis. Using gain- and loss-of-function approaches in the myeloid cell, we confirmed that SETD2 regulated the osteoclast markers, and WNT5a via modulating active histone marks by enriching H3K36me3, and by reducing repressive H3K27me3 mark. Additionally, during osteoclastic differentiation, the transcription of Wnt5a was also associated with the active histone H3K9 and H4K8 acetylations. Mechanistically, SETD2 directed induction of NF-κβ expression facilitated the recruitment of H3K9Ac and H4K8Ac around the TSS region of the Wnt5a gene, thereby, assisting osteoclast differentiation. Together these findings for the first time revealed that SETD2 mediated epigenetic regulation of Wnt5a plays a critical role in osteoclastogenesis and induced arthritis. Graphic abstract Model for the Role of SETD2 dependent regulation of osteoclastic differentiation. A In monocyte cells SETD2-dependent H3K36 trimethylation help to create open chromatin region along with active enhancer mark, H3K27Ac. This chromatin state facilitated the loss of a suppressive H3K27me3 mark. B Additionally, SETD2 mediated induction of NF-κβ expression leads to the recruitment of histone acetyl transferases, P300/PCAF, to the Wnt5a gene and establish H3K9Ac and H4K8Ac marks. Along with other activation marks, these acetylation marks help in Wnt5a transcription which leads to osteoclastogenesis.

Epigenetic regulation disorder is important in the onset and pathogenesis of inflammatory bowel disease (IBD). SETD2, a trimethyltransferase of histone H3K36, is frequently mutated in IBD samples with a high risk of developing colorectal cancer (CRC). However, functions of SETD2 in IBD and colitis-associated CRC remain largely undefined. Here, we found that SETD2 modulates oxidative stress to attenuate colonic inflammation and tumorigenesis in mice. SETD2 expression became decreased in IBD patients and dextran sodium sulfate (DSS)-induced colitic mice. Setd2Vil-KO mice showed increased susceptibility to DSS-induced colitis, accompanied by more severe epithelial barrier disruption and markedly increased intestinal permeability that subsequently facilitated inflammation-associated CRC. Mechanistically, we found that Setd2 depletion resulted in excess reactive oxygen species (ROS) by directly down-regulating antioxidant genes, which led to defects in barrier integrity and subsequently inflammatory damage. Moreover, overexpression of antioxidant PRDX6 in Setd2Vil-KO intestinal epithelial cells (IECs) largely alleviated the overproductions of ROS and improved the cellular survival. Together, our findings highlight an epigenetic mechanism by which SETD2 modulates oxidative stress to regulate intestinal epithelial homeostasis and attenuate colonic inflammation and tumorigenesis. SETD2 might therefore be a pivotal regulator that maintains the homeostasis of the intestinal mucosal barrier.

Post-translational modifications to tubulin are important for many microtubule-based functions inside cells. It was recently shown that methylation of tubulin by the histone methyltransferase SETD2 occurs on mitotic spindle microtubules during cell division, with its absence resulting in mitotic defects. However, the catalytic mechanism of methyl addition to tubulin is unclear. We used a truncated version of human wild type SETD2 (tSETD2) containing the catalytic SET and C-terminal Set2–Rpb1–interacting (SRI) domains to investigate the biochemical mechanism of tubulin methylation. We found that recombinant tSETD2 had a higher activity toward tubulin dimers than polymerized microtubules. Using recombinant single-isotype tubulin, we demonstrated that methylation was restricted to lysine 40 of α-tubulin. We then introduced pathogenic mutations into tSETD2 to probe the recognition of histone and tubulin substrates. A mutation in the catalytic domain (R1625C) allowed tSETD2 to bind to tubulin but not methylate it, whereas a mutation in the SRI domain (R2510H) caused loss of both tubulin binding and methylation. Further investigation of the role of the SRI domain in substrate binding found that mutations within this region had differential effects on the ability of tSETD2 to bind to tubulin versus the binding partner RNA polymerase II for methylating histones in vivo, suggesting distinct mechanisms for tubulin and histone methylation by SETD2. Finally, we found that substrate recognition also requires the negatively charged C-terminal tail of α-tubulin. Together, this study provides a framework for understanding how SETD2 serves as a dual methyltransferase for both histone and tubulin methylation.

Objectives Cutaneous wound healing is one of the major medical problems worldwide. Epigenetic modifiers have been identified as important players in skin development, homeostasis and wound repair. SET domain–containing 2 (SETD2) is the only known histone H3K36 tri‐methylase; however, its role in skin wound healing remains unclear. Materials and Methods To elucidate the biological role of SETD2 in wound healing, conditional gene targeting was used to generate epidermis‐specific Setd2‐deficient mice. Wound‐healing experiments were performed on the backs of mice, and injured skin tissues were collected and analysed by haematoxylin and eosin (H&E) and immunohistochemical staining. In vitro, CCK8 and scratch wound‐healing assays were performed on Setd2‐knockdown and Setd2‐overexpression human immortalized keratinocyte cell line (HaCaT). In addition, RNA‐seq and H3K36me3 ChIP‐seq analyses were performed to identify the dysregulated genes modulated by SETD2. Finally, the results were validated in functional rescue experiments using AKT and mTOR inhibitors (MK2206 and rapamycin). Results Epidermis‐specific Setd2‐deficient mice were successfully established, and SETD2 deficiency resulted in accelerated re‐epithelialization during cutaneous wound healing by promoting keratinocyte proliferation and migration. Furthermore, the loss of SETD2 enhanced the scratch closure and proliferation of keratinocytes in vitro. Mechanistically, the deletion of Setd2 resulted in the activation of AKT/mTOR signalling pathway, while the pharmacological inhibition of AKT and mTOR with MK2206 and rapamycin, respectively, delayed wound closure. Conclusions Our results showed that SETD2 loss promoted cutaneous wound healing via the activation of AKT/mTOR signalling., The epidermis‐specific Setd2‐deficient mice showed accelerated re‐epithelialization during cutaneous wound healing by promoting keratinocytes proliferation and migration. Mechanistically, deletion of Setd2 resulted in the activation the AKT/mTOR signalling pathway and pharmacological inhibitions of AKT and mTOR with MK2206 and rapamycin delayed wound closure, respectively.

Hotspot histone H3 mutations have emerged as drivers of oncogenesis in cancers of multiple lineages. Specifically, H3 lysine 36 to methionine (H3K36M) mutations are recurrently identified in chondroblastomas, undifferentiated sarcomas, and head and neck cancers. While the mutation reduces global levels of both H3K36 dimethylation (H3K36me2) and trimethylation (H3K36me3) by dominantly inhibiting their respective specific methyltransferases, the relative contribution of these methylation states to the chromatin and phenotypic changes associated with H3K36M remains unclear. Here, we specifically deplete H3K36me2 or H3K36me3 in mesenchymal cells, using CRISPR-Cas9 to separately knock out the corresponding methyltransferases NSD1/2 or SETD2. By profiling and comparing the epigenomic and transcriptomic landscapes of these cells with cells expressing the H3.3K36M oncohistone, we find that the loss of H3K36me2 could largely recapitulate H3.3K36M's effect on redistribution of H3K27 trimethylation (H3K27me3) and gene expression. Consistently, knockout of Nsd1/2, but not Setd2, phenocopies the differentiation blockade and hypersensitivity to the DNA-hypomethylating agent induced by H3K36M. Together, our results support a functional divergence between H3K36me2 and H3K36me3 and their nonredundant roles in H3K36M-driven oncogenesis.

Patients with idiopathic pulmonary fibrosis (IPF) are at higher risk of developing lung cancers including squamous cell lung carcinoma (SCC), which typically carries a poor prognosis. Although the molecular basis of cancer development subsequent to IPF has not been fully investigated, we recently reported two epigenetic phenotypes characterized by frequent and infrequent DNA hypermethylation in SCC, and an association of the infrequent hypermethylation phenotype with IPF-associated SCCs. Here, we conducted targeted exon sequencing in SCCs with and without IPF using the Human Lung Cancer Panel to investigate the genetic basis of IPF-associated SCC. SCCs with and without IPF displayed comparable numbers of total mutations (137 ± 22 vs 131 ± 27, P = .5), nonsynonymous mutations (72 ± 14 vs 69 ± 16, P = .5), indels (3.0 ± 3.5 vs 3.0 ± 3.9, P = 1) and synonymous mutations (62 ± 9.1 vs 60 ± 12, P = .5). Signature 1 was the predominant signature in SCCs with and without IPF. SETD2 and NFE2L2 mutations were significantly associated with IPF (44% vs 13%, P = .03 for SETD2; 38% vs 10%, P = .04 for NFE2L2). MYC amplification, assessed by copy number variant analysis, was also significantly associated with IPF (18.8% vs 0%, P = .04). Mutations in TP53 and CDKN2A were observed relatively frequently in SCCs with frequent hypermethylation (P = .02 for TP53 and P = .06 for CDKN2A). Survival analysis revealed that the SETD2 mutation was significantly associated with worse prognosis (P = .04). Collectively, we found frequent involvement of SETD2 and NFE2L2 mutations and MYC amplification in SCCs with IPF, and an association of a SETD2 mutation with poorer prognosis.

Both SETD2-mediated H3K36me3 and miRNAs play critical epigenetic roles in inflammatory bowel disease (IBD) and involve in the dysfunctional intestinal barrier. However, little is known about cross-talk between these two types of regulators in IBD progression. We performed small RNA sequencing of Setd2 epithelium-specific knockout mice (Setd2Vil-KO) and wild-type controls, both with DSS-induced colitis, and designed a framework for integrative analysis. Firstly, we integrated the downloaded ChIP-seq data with miRNA expression profiles and identified a significant intersection of pre-miRNA expression and H3K36me3 modification. A significant inverse correlation was detected between changes of H3K36me3 modification and expression of the 171 peak-covered miRNAs. We further integrated RNA-seq data with predicted miRNA targets to screen negatively regulated miRNA-mRNA pairs and found the H3K36me3-associated differentially expressed microRNAs significantly enriched in cell-cell junction and signaling pathways. Using network analysis, we identified ten hub miRNAs, among which six are H3K36me3-associated, suggesting therapeutic targets for IBD patients with SETD2-deficiency.

A high percentage of pediatric gliomas and bone tumors reportedly harbor missense mutations at glycine 34 in genes encoding histone variant H3.3. We find that these H3.3 G34 mutations directly alter the enhancer chromatin landscape of mesenchymal stem cells by impeding methylation at lysine 36 on histone H3 (H3K36) by SETD2, but not by the NSD1/2 enzymes. The reduction of H3K36 methylation by G34 mutations promotes an aberrant gain of PRC2-mediated H3K27me2/3 and loss of H3K27ac at active enhancers containing SETD2 activity. This altered histone modification profile promotes a unique gene expression profile that supports enhanced tumor development in vivo. Our findings are mirrored in G34W-containing giant cell tumors of bone where patient-derived stromal cells exhibit gene expression profiles associated with early osteoblastic differentiation. Overall, we demonstrate that H3.3 G34 oncohistones selectively promote PRC2 activity by interfering with SETD2-mediated H3K36 methylation. We propose that PRC2-mediated silencing of enhancers involved in cell differentiation represents a potential mechanism by which H3.3 G34 mutations drive these tumors.

SETD2 regulates actin dynamics and cell migration via methylation of actin at Lys68 in a cellular complex containing Huntingtin., The methyltransferase SET domain–containing 2 (SETD2) was originally identified as Huntingtin (HTT) yeast partner B. However, a SETD2 function associated with the HTT scaffolding protein has not been elucidated, and no linkage between HTT and methylation has yet been uncovered. Here, we show that SETD2 is an actin methyltransferase that trimethylates lysine-68 (ActK68me3) in cells via its interaction with HTT and the actin-binding adapter HIP1R. ActK68me3 localizes primarily to the insoluble F-actin cytoskeleton in cells and regulates actin polymerization/depolymerization dynamics. Disruption of the SETD2-HTT-HIP1R axis inhibits actin methylation, causes defects in actin polymerization, and impairs cell migration. Together, these data identify SETD2 as a previously unknown HTT effector regulating methylation and polymerization of actin filaments and provide new avenues for understanding how defects in SETD2 and HTT drive disease via aberrant cytoskeletal methylation.

Background and aims Trimethylation of Lys36 on histone 3 (H3K36me3) catalyzed by histone methyltransferase SET domain-containing 2 (SETD2) is one of the most conserved epigenetic marks from yeast to mammals. SETD2 is frequently mutated in multiple cancers and acts as a tumor suppressor. Approach and results Here, using a liver-specific Setd2 depletion model, we found that Setd2 deficiency is sufficient to trigger spontaneous HCC. Meanwhile, Setd2 depletion significantly increased tumor and tumor size of a diethylnitrosamine-induced HCC model. The mechanistic study showed that Setd2 suppresses HCC not only through modulating DNA damage response, but also by regulating lipid metabolism in the liver. Setd2 deficiency down-regulated H3K36me3 enrichment and expression of cholesterol efflux genes and caused lipid accumulation. High-fat diet enhanced lipid accumulation and promoted the development of HCC in Setd2-deficient mice. Chromatin immunoprecipitation sequencing analysis further revealed that Setd2 depletion induced c-Jun/activator protein 1 (AP-1) activation in the liver, which was trigged by accumulated lipid. c-Jun acts as an oncogene in HCC and functions through inhibiting p53 in Setd2-deficient cells. Conclusions We revealed the roles of Setd2 in HCC and the underlying mechanisms in regulating cholesterol homeostasis and c-Jun/AP-1 signaling.

The process of autophagy is dysregulated in many cancers including clear cell renal cell carcinoma (ccRCC). Autophagy involves the coordination of numerous autophagy-related (ATG) genes, as well as processes involving the actin cytoskeleton. The histone methyltransferase SETD2, frequently inactivated in ccRCC, has recently been shown to also methylate cytoskeletal proteins, which in the case of actin lysine 68 trimethylation (ActK68me3) regulates actin polymerization dynamics. Here we show that cells lacking SETD2 exhibit autophagy defects, as well as decreased interaction of the actin nucleation promoting factor WHAMM with its target actin, which is required for initiation of autophagy. Interestingly, the WHAMM actin binding deficit could be rescued with pharmacologic induction of actin polymerization in SETD2-null cells using Jasplakinolide. These data indicate that the decreased interaction between WHAMM and its target actin in SETD2-null cells was secondary to altered actin dynamics rather than loss of the SETD2 ActK68me3 mark itself, and underscores the importance of the functional defect in actin polymerization in SETD2-null cells exhibiting autophagy defects.

Pseudomyxoma peritonei(PMP) is a rare disorder with unique pathological and genetic changes. Although several studies have reported the clinical features and mutational changes of PMP that originates from the appendix, few studies on PMP originating from the ovary have been reported due to its extreme rarity. In order to characterize the somatic mutational landscape and to investigate the prognosis predicting factors of ovary-originating PMP, we examined 830 cases of PMP and identified 16 patients with PMP that originated from the ovary. Whole-exome sequencing(WES) was performed on 12 cases using formalin-fixed, paraffin-embedded(FFPE) tissue samples. We found that 25%(3/12) of the patients carried mutations in cancer driver genes, including TP53, ATM and SETD2,and 16.7%(2/12) of the patients carried mutations in cancer driver genes, including ATRX, EP300, FGFR2, KRAS, NOCR1, and RB1. The MUC16(58.33%), BSN(41.67%), PCNT(41.67%), PPP2R5A(41.67%), PRSS36(41.67%), PTPRK(41.67%),and SBF1(41.67%) genes presented the highest mutational frequencies. The PI3K-Akt signaling pathway, human papillomavirus infection pathway, cell skeleton, cell adhesion, and extracellular matrix and membrane proteins were the major pathways or functions that were affected. Patients were followed up to 174 months(median: 48.26 months). The 5-year OS rate for all patients was 71.2% and the median OS was not reached. PTPRK mutations, pre-surgical CA199 level, completeness of cytoreduction(CCR) and peritoneal cancer index(PCI) were identified as potential predictive factors for patient survival. In conclusion, the mutational landscape for ovary-originating PMP was revealed and exhibited unique features distinct from appendix-originating PMP. PTPRK, CA199, CCR and PCI may predict patient survival. This article is protected by copyright. All rights reserved.

Chondroblastoma is currently classified as a benign neoplasm; however, chondroblastoma and chondroblastoma-like osteosarcoma have morphologic overlap, raising the possibility that some tumors diagnosed as chondroblastoma-like osteosarcoma might actually represent malignant chondroblastoma. The H3F3B K36M point mutation, which has not been reported in osteosarcoma, is identified in 95% of chondroblastomas and is reliably detectable by immunohistochemistry (IHC). We reviewed 11 tumors diagnosed as atypical chondroblastoma, malignant chondroblastoma, or chondroblastoma-like osteosarcoma (median follow-up: 8.8 years; range: 4 months-26.4 years). Seven chondroblastomas with cytologic atypia and permeative growth were designated "malignant chondroblastoma"; six were H3K36M-positive by IHC. Relative to conventional chondroblastoma, malignant chondroblastoma occurred in older individuals (median: 52 years; range: 29-57 years) and arose at unusual sites. Three of four tumors with long-term follow-up recurred, and one patient died of widespread metastases. One was found to have chromosomal copy number alter4ations and a SETD2 mutation in addition to H3F3B K36M. The four remaining tumors were classified as chondroblastoma-like osteosarcoma. Chondroblastoma-like osteosarcoma occurred in younger patients (median: 21 years; range: 19-40 years) than malignant chondroblastoma. In contrast to malignant chondroblastoma, all had regions of malignant cells forming bone. Two of three patients with long-term follow-up developed recurrences, and two died of disease, one with widespread metastases. No mutations in H3F3A/H3F3B were detected by Sanger sequencing. While malignant chondroblastoma and chondroblastoma-like osteosarcoma show significant morphologic overlap, they have distinct clinical presentations and genetic findings. When considering this challenging differential diagnosis, IHC using histone H3 mutation-specific antibodies is a critical diagnostic adjunct.

Background The histone H3K36me3 mark regulates transcription elongation, pre-mRNA splicing, DNA methylation, and DNA damage repair. However, knowledge of the regulation of the enzyme SETD2, which deposits this functionally important mark, is very limited. Results Here, we show that the poorly characterized N-terminal region of SETD2 plays a determining role in regulating the stability of SETD2. This stretch of 1–1403 amino acids contributes to the robust degradation of SETD2 by the proteasome. Besides, the SETD2 protein is aggregate prone and forms insoluble bodies in nuclei especially upon proteasome inhibition. Removal of the N-terminal segment results in the stabilization of SETD2 and leads to a marked increase in global H3K36me3 which, uncharacteristically, happens in a Pol II-independent manner. Conclusion The functionally uncharacterized N-terminal segment of SETD2 regulates its half-life to maintain the requisite cellular amount of the protein. The absence of SETD2 proteolysis results in a Pol II-independent H3K36me3 deposition and protein aggregation.

Summary Conventional chordoma is a rare slow-growing malignant tumor of notochordal origin primarily arising at the base of the skull and sacrococcygeal bones. Chordoma may arise from its benign counterpart, benign notochordal cell tumors, and can also undergo dedifferentiation progressing into dedifferentiated chordoma. No study has directly compared the genomic alterations among these tumors comprising a morphologic continuum. Our prior study identified frequent chromosome 3p loss of heterozygosity and minimal deleted regions on chromosome 3 encompassing SETD2, encoding a histone methyltransferase involved in histone H3 lysine 36 trimethylation (H3K36me3). In the present study, we expanded our study to include 65 sacral conventional chordoma cases, 3 benign notochordal cell tumor cases, and 2 dedifferentiated chordoma cases using single nucleotide polymorphism (SNP) array, targeted next-generation sequencing analysis, and immunohistochemistry. We performed immunohistochemical analysis of histone, H3K36me3, and investigated whether there is any association between the clinical behavior and recurrent chromosome or aneuploidy or H3K36me3 protein expression. We found that there is increased genomic instability from benign notochordal cell tumor to conventional chordoma to dedifferentiated chordoma. The highly recurrent genomic aberration, chromosome 3p loss of heterozygosity (occurred in 70% of conventional chordomas), is correlated with longer relapse-free survival, but not with overall survival or metastasis-free survival in sacral chordoma. Chordomas demonstrate variable patterns and levels of H3K36me3 expression, and reduced expression of H3K36me3 showed marginally significant correlation with longer relapse-free survival. Copy number alterations in the genes encoding the H3K36me3 methylation transferase complex and demethylase may account for the altered H3K36me3 expression levels.

SETD2 encodes an important protein for epigenetic modification of histones which plays an essential role in early development. Variants in SETD2 have been reported in neurodevelopmental disorders including autism spectrum disorder (ASD). However, most de novo SETD2 variants were reported in different large-cohort sequencing studies, mutation pattern and comprehensive genotype-phenotype correlations for SETD2 are still lacking. We have applied target sequencing to identify rare, clinical-relevant SETD2 variants and detected two novel de novo SETD2 variants, including a de novo splicing variant (NM_014159: c.4715+1G>A) and a de novo missense variant (c.3185C>T: p.P1062L) in two individuals with a diagnosis of ASD. To analyze the correlations between SETD2 mutations and corresponding phenotypes, we systematically review the reported individuals with de novo SETD2 variants, classify the pathogenicity, and analyze the detailed phenotypes. We subsequently manually curate 17 SETD2 de novo variants in 17 individuals from published literature. Individuals with de novo SETD2 variants present common phenotypes including speech and motor delay, intellectual disability, macrocephaly, ASD, overgrowth and recurrent otitis media. Our study reveals new SETD2 mutations and provided a relatively homozygous phenotype spectrum of SETD2-related neurodevelopmental disorders which will be beneficial for disease classification and diagnosis in clinical practice.

In recent years, molecular characterization and management of patients with systemic mastocytosis (SM) have greatly benefited from the application of advanced technologies. Highly sensitive and accurate assays for KIT D816V mutation detection and quantification have allowed the switch to non-invasive peripheral blood testing for patient screening; allele burden has prognostic implications and may be used to monitor therapeutic efficacy. Progress in genetic profiling of KIT, together with the use of next-generation sequencing panels for the characterization of associated gene mutations, have allowed the stratification of patients into three subgroups differing in terms of pathogenesis and prognosis: i) patients with mast cell-restricted KIT D816V; ii) patients with multilineage KIT D816V-involvement; iii) patients with “multi-mutated disease”. Thanks to these findings, new prognostic scoring systems combining clinical and molecular data have been developed. Finally, non-genetic SETD2 histone methyltransferase loss of function has recently been identified in advanced SM. Assessment of SETD2 protein levels and activity might provide prognostic information and has opened new research avenues exploring alternative targeted therapeutic strategies. This review discusses how progress in recent years has rapidly complemented previous knowledge improving the molecular characterization of SM, and how this has the potential to impact on patient diagnosis and management.

Inactivating mutations in the SETD2 gene, encoding for a nonredundant histone H3 methyltransferase and regulator of transcription, is a frequent molecular feature in clear cell renal cell carcinomas (ccRCC). SETD2 deficiency is associated with recurrence of ccRCC and bears low prognostic values. Targeting autophagy, a conserved catabolic process with critical functions in maintenance of cellular homeostasis and cell conservation under stress condition, is emerging as a potential therapeutic strategy to combat ccRCC. Epigenetics-based pathways are now appreciated as key components in the regulation of autophagy. However, whether loss of function in the SETD2 histone modifying enzyme occurring in ccRCC cells may impact on their ability to undergo autophagy remained to be explored. Here, we report that SETD2 deficiency in RCC cells is associated with the aberrant accumulation of both free ATG12 and of an additional ATG12-containing complex, distinct from the ATG5–ATG12 complex. Rescue of SETD2 functions in the SETD2 deficiency in RCC cells, or reduction of SETD2 expression level in RCC cells wild type for this enzyme, demonstrates that SETD2 deficiency in RCC is directly involved in the acquisition of these alterations in the autophagic process. Furthermore, we revealed that deficiency in SETD2, known regulator of alternative splicing, is associated with increased expression of a short ATG12 spliced isoform at the depend of the canonical long ATG12 isoform in RCC cells. The defect in the ATG12-dependent conjugation system was found to be associated with a decrease autophagic flux, in accord with the role for this ubiquitin-like protein conjugation system in autophagosome formation and expansion. Finally, we report that SETD2 and ATG12 gene expression levels are associated with favorable respective unfavorable prognosis in ccRCC patients. Collectively, our findings bring further argument for considering the SETD2 gene status of ccRCC tumors, when therapeutic interventions, such as targeting the autophagic process, are considered to combat these kidney cancers.

Resistance to chemotherapy remains a critical barrier to effective cancer treatment. Although cisplatin is one of the most commonly used chemotherapeutic agents in the treatment of non-small cell lung cancer (NSCLC), mechanisms of resistance to this drug are not fully understood. Here, we report a novel cisplatin-resistance mechanism involving SET Domain Containing 2 (SETD2), a histone H3 lysine 36 (H3K36) trimethyltransferase, and cAMP-responsive element-binding protein 1 (CREB1). A549 cells selected in vivo to give brain metastases exhibited cisplatin resistance and decreased expression of phosphorylated CREB1. Next-generation sequencing (NGS) analysis identified a missense mutation in SETD2 (p.T1171K), and we demonstrated that SETD2-mediated trimethylation of H3K36 (H3K36me3) and CREB1 phosphorylation are critical for cellular sensitivity to cisplatin. Moreover, we showed that suppression of SETD2 or CREB1 and ectopic expression of mutant SETD2 conferred cisplatin resistance through inhibition of H3K36me3 and ERK activation in NSCLC cells. Our results provide evidence that SETD2 and CREB1 contribute to cisplatin cytotoxicity via regulation of the ERK signaling pathway, and their inactivation may lead to cisplatin resistance.

SET domain containing protein 2 (SETD2, also known as HYPB) is a 230-kD protein which is located at cytogenetic band p21.31 of chromosome 3. SETD2 is usually transformed or eradicated in multiple forms of tumours in humans. However, its primary function in gastric cancer (GC) remains unclear. In the current study, we investigated the mRNA and protein expression levels of SETD2 using immunohistochemistry, qPCR, RT-PCR, and immunoblotting. The function of SETD2 in GC cells was investigated by MTT and transwell assays. Our results revealed remarkably lower levels of SETD2 mRNA and protein in the tumour samples than in tumour-adjacent tissues. Decreased expression of SETD2 mRNA was observed in 122 (79.7%) of 153 primary tumour tissue samples. On the basis of the overall survival analysis, we could interpret that a low expression of SETD2 was correlated with a poor prognosis (P 0.001, log-rank test). Multivariate survival analysis indicated that SETD2 was an obvious prognostic factor in patients with GC. SETD2 overexpression in GC cell lines significantly inhibited cell proliferation, migration, and invasion. Altogether, the investigation demonstrated the clinical significance of SETD2 expression, supporting the fundamental principle that a decrease in its level is an unfavourable event in the progression and prognosis of GC. Therefore, down-regulated SETD2 can presumably be a potential negative prognostic and progression marker for GC.

Histone methyltransferases (HMTs) have attracted considerable attention as potential targets for pharmaceutical intervention in various malignant diseases. These enzymes are known for introducing methyl marks at specific locations of histone proteins, creating a complex system that regulates epigenetic control of gene expression and cell differentiation. Here, we describe the identification of first-generation cell-permeable non-nucleoside type inhibitors of SETD2, the only mammalian HMT that is able to tri-methylate the K36 residue of histone H3. By generating the epigenetic mark H3K36me3, SETD2 is involved in the progression of acute myeloid leukemia. We developed a structure-based virtual screening protocol that was first validated in retrospective studies. Next, prospective screening was performed on a large library of commercially available compounds. Experimental validation of 22 virtual hits led to the discovery of three compounds that showed dose-dependent inhibition of the enzymatic activity of SETD2. Compound C13 effectively blocked the proliferation of two acute myeloid leukemia (AML) cell lines with MLL rearrangements and led to decreased H3K36me3 levels, prioritizing this chemotype as a viable chemical starting point for drug discovery projects.

Previously, we identified SETD2 loss-of-function mutations in 22% of MLL-rearranged (MLLr) acute leukemia patients, implicating a mechanism for cooperativity between SETD2 mutations and MLL fusions. However, the detailed mechanism of how SETD2-H3K36me3 downregulation accelerates MLLr leukemia remains unclear. Here, we show that in MLLr leukemia, both H3K79me2 and H3K36me3 are aberrantly elevated and co-enriched in a group of genes. SETD2 inactivation leads to a global reduction of H3K36me3 and a further elevation of H3K79me2, but does not change the expression of known MLL fusion target genes. Instead, this pattern of histone changes is associated with transcriptional deregulation of a novel set of genes; downregulating tumor suppressors (for example, ASXL1) and upregulating oncogenes (for example, ERG). Taken together, our findings reveal a global crosstalk between the oncogenic DOT1L-H3K79me2 axis and the tumor suppressive SETD2-H3K36me3 axis in gene regulation, provide molecular insights into how SETD2 mutations accelerate MLLr leukemogenesis through differential regulation of additional tumor suppressors and oncogenes.

Spindle cell tumors are clinically heterogeneous but morphologically similar neoplasms. The term refers to the tumor cells' long and slender microscopic appearance. Distinct subgroups of spindle cell tumors are characterized by chromosomal translocations and also fusion genes. Other spindle cell tumors exist that have not yet been found to have characteristic, let alone pathognomonic, genetic or pathogenetic features. Continuous examination of spindle cell tumors is likely to reveal other subgroups that may, in the future, be seen to correspond to meaningful clinical differences and may even be therapeutically decisive. We analyzed genetically a pediatric spindle cell tumor. Karyotyping showed the tumor cells to carry a t(3;17)(p21;q12) chromosomal translocation whereas RNA sequencing identified a SETD2-NF1 fusion gene caused by the translocation. RT-PCR together with Sanger sequencing verified the presence of the above-mentioned fusion transcript. Interphase FISH analysis confirmed the existence of the chimeric gene and showed that there was no reciprocal fusion. The fusion transcript codes for a protein in which the last 114 amino acids of SETD2, i.e., the entire Set2 Rpb1 interacting (SRI) domain of SETD2, are replaced by 30 amino acids encoded by the NF1 sequence. The result would be similar to that seen with truncating SETD2 mutations in leukemias. Absence of the SRI domain would result in inability to recruit SETD2 to its target gene locus through binding to the phosphor-C-terminal repeat domain of elongating RNA polymerase II and may affect H3K36 methylation. Alternatively, loss of one of two functional SETD2 alleles might be the crucial tumorigenic factor.

Recent studies facilitated by DNA sequencing identified the histone modifying gene SETD2 as the second most frequent mutant gene in sporadic clear cell renal cell carcinoma (ccRCC) patients. SETD2 functions as a tumor suppressor in ccRCC. However, its clinical association and biological functions are not fully delineated. The aim of this study is to evaluate the clinical significance of SETD2 in ccRCC patients. SETD2 and its canonical histone modification product H3K36me3 were analyzed by immunohistochemistry (IHC) in 155 ccRCC patients from two independent cohorts retrospectively. Both SETD2 and H3K36me3 were heterogeneously stained and down-regulated in ccRCC tissues, compared with normal controls. The SETD2 protein deficiency rate was 34.07%, which is much higher than the reported SETD2 gene inactive mutation rate. Furthermore, low SETD2 protein expression, but not H3K36me3 expression, was associated with the aggressive phenotype of ccRCC patients. In addition, cox multivariate analysis identified low SETD2 protein expression as an independent prognostic factor for overall survival of ccRCC patients. Consistently, using RNA-Seq data of ccRCC patients from The Cancer Genome Atlas, we validated our findings that low SETD2 mRNA expression is significantly associated with the aggressive phenotypes, and predicted a worse outcome for ccRCC patients. In conclusion, our study demonstrated a massive down-regulation of SETD2 protein in ccRCC, and identified SETD2 protein, but not H3K36me3, as an independent good prognostic marker, which warrants further study focusing on the non-methyltransferase role of SETD2 in kidney tumor biology.

Significant progress has been made in our understanding of the role of epigenetic modifiers in many types of human cancer. Here, we review currently available studies on the unique histone methyltransferase, SETD2, which is responsible for H3 lysine 36 tri-methylation (H3K36me3). SETD2 plays pivotal roles in RNA alternative splicing regulation, DNA damage repair, and cytoskeleton protein methylation; inactivation of SETD2 and resultant dysregulation of these functions may lead to tumorigenesis. Despite being a newly discovered tumor suppressor, SETD2 has been found to be mutated in multiple types of cancer, including gastrointestinal tumor. Some tumors can acquire a selective growth advantage after SETD2 inactivation, which could happen in different stages in tumor progression. Decreased level of H3K36me3 caused by SETD2 inactivation has been shown to associate with higher tumor grade, tumor stage, metastasis risk, and shorter survival. Some studies also suggest that SETD2 mutation is associated with therapy resistance, therefore these SETD2-deficient tumors may need different therapeutic strategies.

Osteosarcoma (OS) is a rare, metastatic, human adolescent cancer that also occurs in pet dogs. To define the genomic underpinnings of canine OS, we performed multi-platform analysis of OS tumors from 59 dogs, including whole genome sequencing (n = 24) and whole exome sequencing (WES; n = 13) of primary tumors and matched normal tissue, WES (n = 10) of matched primary/metastatic/normal samples and RNA sequencing (n = 54) of primary tumors. We found that canine OS recapitulates features of human OS including low point mutation burden (median 1.98 per Mb) with a trend towards higher burden in metastases, high structural complexity, frequent TP53 (71%), PI3K pathway (37%), and MAPK pathway mutations (17%), and low expression of immune-associated genes. We also identified novel features of canine OS including putatively inactivating somatic SETD2 (42%) and DMD (50%) aberrations. These findings set the stage for understanding OS development in dogs and humans, and establish genomic contexts for future comparative analyses., Heather Gardner et al. report the genomic landscape of canine osteosarcoma, finding recurrent mutations in the histone methyltransferase gene SETD2 and in DMD, the gene encoding dystrophin. The results support the study of naturally-occurring osteosarcoma in dogs for understanding both human disease mechanisms and canine-specific alterations to identify new treatments.

Objectives Epigenetic modifiers were important players in the development of haematological malignancies and sensitivity to therapy. Mutations of SET domain‐containing 2 (SETD2), a methyltransferase that catalyses the trimethylation of histone 3 on lysine 36 (H3K36me3), were found in various myeloid malignancies. However, the detailed mechanisms through which SETD2 confers chronic myeloid leukaemia progression and resistance to therapy targeting on BCR‐ABL remain unclear. Materials and methods The level of SETD2 in imatinib‐sensitive and imatinib‐resistant chronic myeloid leukaemia (CML) cells was examined by immunoblotting and quantitative real‐time PCR. We analysed CD34+CD38− leukaemic stem cells by flow cytometry and colony formation assays upon SETD2 knockdown or overexpression. The impact of SETD2 expression alterations or small‐molecule inhibitor JIB‐04 targeting H3K36me3 loss on imatinib sensitivity was assessed by IC50, cell apoptosis and proliferation assays. Finally, RNA sequencing and ChIP‐quantitative PCR were performed to verify putative downstream targets. Results SETD2 was found to act as a tumour suppressor in CML. The novel oncogenic targets MYCN and ERG were shown to be the direct downstream targets of SETD2, where their overexpression induced by SETD2 knockdown caused imatinib insensitivity and leukaemic stem cell enrichment in CML cell lines. Treatment with JIB‐04, an inhibitor that restores H3K36me3 levels through blockade of its demethylation, successfully improved the cell imatinib sensitivity and enhanced the chemotherapeutic effect. Conclusions Our study not only emphasizes the regulatory mechanism of SETD2 in CML, but also provides promising therapeutic strategies for overcoming the imatinib resistance in patients with CML.

Background SETD2, the single mediator of trimethylation of histone 3 at position lysine 36, has been reported associated with initiation progression and chemotherapy resistance in acute myeloid leukemia (AML). Whether polymorphisms of SETD2 affect prognosis and chemotherapy response of AML remains elusive. Methods Three tag single-nucleotide polymorphisms (tagSNPs) of SETD2 were genotyped in 579 AML patients by using Sequenom Massarray system. Association of the SNPs with complete remission (CR) rate after Ara-C based induction therapy, overall survival (OS) and relapse-free survival (RFS) were analyzed. Result Survival analysis indicated that SETD2 rs76208147 TT genotype was significantly associated with poor prognosis of AML (TT vs. CC + CT hazard ratio: HR = 1.838, 95% confidence interval (CI) 1.005–3.360, p = 0.048). After adjusting for the known prognostic factors including risk stratification, age, allo-SCT, WBC count and LDH count, rs76208147 TT genotype was still associated with OS in the multivariate analysis (TT vs. CC + CT HR = 1.923, 95% CI 1.007–3.675, p = 0.048). In addition, after adjusting by other clinical features, patients with rs4082155 allele G carries showed higher rate of complete remission which indicated by CR rate (AG + GG vs. AA odd ratio (OR) = 0.544, 95% CI 0.338–0.876, p = 0.012). Conclusions SETD2 genetic polymorphism is associated with AML prognosis and chemotherapy outcome, suggesting the possibility for development in AML diagnostics and therapeutics towards SETD2. Electronic supplementary material The online version of this article (10.1186/s12967-019-1848-9) contains supplementary material, which is available to authorized users.

The non-redundant histone methyltransferase SETD2 (SET domain containing 2; KMT3A) is responsible for tri-methylation of lysine 36 on histone H3 (H3K36me3). Presence of the H3K36me3 histone mark across the genome has been correlated with transcriptional activation and elongation, but also with the regulation of DNA mismatch repair, homologous recombination and alternative splicing. The role of SETD2 and the H3K36me3 histone mark in cancer is controversial. SETD2 is lost or mutated in various cancers, supporting a tumor suppressive role of the protein. Alterations in the SETD2 gene are also present in leukemia patients, where they are associated with aggressive disease and relapse. In line, heterozygous SETD2 loss caused chemotherapy resistance in leukemia cell lines and mouse models. In contrast, other studies indicate that SETD2 is critically required for the proliferation of leukemia cells. Thus, although studies of SETD2-dependent processes in cancer have contributed to a better understanding of the SETD2–H3K36me3 axis, many open questions remain regarding its specific role in leukemia. Here, we review the current literature about critical functions of SETD2 in the context of hematopoietic malignancies.

Bi-allelic inactivation of the VHL gene on chromosome 3p is the characteristic feature in most clear cell renal cell carcinomas (ccRCC). Frequent gene alterations were also identified in SETD2, BAP1 and PBRM1, all of which are situated on chromosome 3p and encode histone/chromatin regulators. The relationship between gene mutation, loss of protein expression and the correlations with clinicopathological parameters is important for the understanding of renal cancer progression. We analyzed PBRM1 and BAP1 protein expression as well as the tri-methylation state of H3K36 as a surrogate marker for SETD2 activity in more than 700 RCC samples. In ccRCC loss of nuclear PBRM1 (68%), BAP1 (40%) and H3K36me3 (47%) expression was significantly correlated with each other, advanced tumor stage, poor tumor differentiation (P, Neoplasia, 21 (2), ISSN:1522-8002, ISSN:1476-5586

BACKGROUND The histone methyltransferase (HMT) family includes histone lysine methyltransferases (HKMTs) and histone/protein arginine methyltransferases (PRMTs). The role of HMT gene variants in prostate cancer remains unknown. Therefore, this study aimed to evaluate HMT gene variants in the pathogenesis and prognosis of human prostate cancer, using in vitro cell studies and bioinformatics analysis. MATERIAL AND METHODS Integrative bioinformatics analysis of the expression of 51 HMT genes in human prostate cancer was based on datasets from the Cancer Genome Atlas (TCGA). Correlation and regression analysis were used to identify critical HMTs in prostate cancer. Kaplan-Meier and the area under the receiver operating characteristics curve (AUROC) were performed to evaluate the function of the HMTs on prognosis. Gene expression and function of 22Rv1 human prostate carcinoma cells were studied. RESULTS The HMT genes identified to have a role in the pathogenesis of prostate cancer included the EZH2, SETD5, PRDM12, NSD1, SETD6, SMYD1, and the WHSC1L1 gene. The EZH2, SETD5, and SMYD1 genes were selected as a prognostic panel, with the SUV420H2 HMT gene. SETD2, NSD1, and ASH1L were identified as critical genes in the development of castration-resistant prostate cancer (CRPC), similar to mixed-lineage leukemia (MLL) complex family members. Knockdown of the SETD5 gene in 22Rv1 prostate carcinoma cells in vitro inhibited cancer cell growth and migration. CONCLUSIONS HMT gene variants may have a role in the pathogenesis of prostate cancer. Future studies may determine the role of HMT genes as prognostic biomarkers in patients with prostate cancer.

Renal cell carcinomas with unclassified histology (uRCC) constitute a significant portion of aggressive non-clear cell renal cell carcinomas that have no standard therapy. The oncogenic drivers in these tumours are unknown. Here we perform a molecular analysis of 62 high-grade primary uRCC, incorporating targeted cancer gene sequencing, RNA sequencing, single-nucleotide polymorphism array, fluorescence in situ hybridization, immunohistochemistry and cell-based assays. We identify recurrent somatic mutations in 29 genes, including NF2 (18%), SETD2 (18%), BAP1 (13%), KMT2C (10%) and MTOR (8%). Integrated analysis reveals a subset of 26% uRCC characterized by NF2 loss, dysregulated Hippo–YAP pathway and worse survival, whereas 21% uRCC with mutations of MTOR, TSC1, TSC2 or PTEN and hyperactive mTORC1 signalling are associated with better clinical outcome. FH deficiency (6%), chromatin/DNA damage regulator mutations (21%) and ALK translocation (2%) distinguish additional cases. Altogether, this study reveals distinct molecular subsets for 76% of our uRCC cohort, which could have diagnostic and therapeutic implications., A subset of renal cell carcinomas have uncertain histology and are aggressive in nature. Here, the authors examine this group of unclassified renal cancers using genomics techniques and identify further subclasses of the tumours that have differing prognoses.

The members of the NSD subfamily of lysine methyl transferases are compelling oncology targets due to the recent characterization of gain-of-function mutations and translocations in several hematological cancers. To date, these proteins have proven intractable to small molecule inhibition. Here, we present initial efforts to identify inhibitors of MMSET (aka NSD2 or WHSC1) using solution phase and crystal structural methods. On the basis of 2D NMR experiments comparing NSD1 and MMSET structural mobility, we designed an MMSET construct with five point mutations in the N-terminal helix of its SET domain for crystallization experiments and elucidated the structure of the mutant MMSET SET domain at 2.1 Å resolution. Both NSD1 and MMSET crystal systems proved resistant to soaking or cocrystallography with inhibitors. However, use of the close homologue SETD2 as a structural surrogate supported the design and characterization of N-alkyl sinefungin derivatives, which showed low micromolar inhibition against both SETD2 and MMSET.

The yeast Set2 histone methyltransferase is a critical enzyme that plays a number of key roles in gene transcription and DNA repair. Recently, the human homologue, SETD2, was found to be recurrently mutated in a significant percentage of renal cell carcinomas, raising the possibility that the activity of SETD2 is tumor-suppressive. Using budding yeast and human cell line model systems, we examined the functional significance of two evolutionarily conserved residues in SETD2 that are recurrently mutated in human cancers. Whereas one of these mutations (R2510H), located in the Set2 Rpb1 interaction domain, did not result in an observable defect in SETD2 enzymatic function, a second mutation in the catalytic domain of this enzyme (R1625C) resulted in a complete loss of histone H3 Lys-36 trimethylation (H3K36me3). This mutant showed unchanged thermal stability as compared with the wild type protein but diminished binding to the histone H3 tail. Surprisingly, mutation of the conserved residue in Set2 (R195C) similarly resulted in a complete loss of H3K36me3 but did not affect dimethylated histone H3 Lys-36 (H3K36me2) or functions associated with H3K36me2 in yeast. Collectively, these data imply a critical role for Arg-1625 in maintaining the protein interaction with H3 and specific H3K36me3 function of this enzyme, which is conserved from yeast to humans. They also may provide a refined biochemical explanation for how H3K36me3 loss leads to genomic instability and cancer.

Leukaemia is the leading cause of childhood malignancies. Recent research indicates that the SETD2 gene is associated with acute lymphoblastic leukaemia. This study aims to identify potential lead compounds from traditional Chinese medicine (TCM) using virtual screening for SET domain containing 2 (SETD2) protein against acute lymphoblastic leukaemia. Docking simulation was performed to determine potential candidates which obtain suitable docking poses in the binding domain of the SETD2 protein. We also performed molecular dynamics (MD) simulation to investigate the stability of docking poses of SETD2 protein complexes with the top three TCM candidates and a control. According to the results of docking and MD simulation, coniselin and coniferyl ferulate have high binding affinity and stable interactions with the SETD2 protein. Coniselin is isolated from the alcoholic extract of Comiselinum vaginatum Thell. Coniferyl ferulate can be isolated from Angelica sinensis, Poria cocos (Schw.) Wolf, and Notopterygium forbesii. Although S-adenosyl-L-homocysteine has more stable interactions with key residues in the binding domain than coniselin and coniferyl ferulate during MD simulation, the TCM compounds coniselin and coniferyl ferulate are still potential candidates as lead compounds for further study in the drug development process with the SETD2 protein against acute lymphoblastic leukaemia.

In the past decade important progress has been made in our understanding of the epigenetic regulatory machinery. It has become clear that genetic aberrations in multiple epigenetic modifier proteins are associated with various types of cancer. Moreover, targeting the epigenome has emerged as a novel tool to treat cancer patients. Recently, the first drugs have been reported that specifically target SETD2-negative tumors. In this review we discuss the studies on the associated protein, Set domain containing 2 (SETD2), a histone modifier for which mutations have only recently been associated with cancer development. Our review starts with the structural characteristics of SETD2 and extends to its corresponding function by combining studies on SETD2 function in yeast, Drosophila, Caenorhabditis elegans, mice, and humans. SETD2 is now generally known as the single human gene responsible for trimethylation of lysine 36 of Histone H3 (H3K36). H3K36me3 readers that recruit protein complexes to carry out specific processes, including transcription elongation, RNA processing, and DNA repair, determine the impact of this histone modification. Finally, we describe the prevalence of SETD2-inactivating mutations in cancer, with the highest frequency in clear cell Renal Cell Cancer, and explore how SETD2-inactivation might contribute to tumor development.

SummarySingle nucleotide variants (SNVs), particularly loss-of-function mutations, are significant contributors to autism spectrum disorder (ASD) risk. Here we report the first systematic deep sequencing study of 55 postmortem ASD brains for SNVs in 78 known ASD candidate genes. Remarkably, even without parental samples, we find more ASD brains with mutations that are protein-altering (26/55 cases versus 12/50 controls, p = 0.015), deleterious (16/55 versus 5/50, p = 0.016), or loss-of-function (6/55 versus 0/50, p = 0.028) compared to controls, with recurrent deleterious mutations in ARID1B, SCN1A, SCN2A, and SETD2, suggesting these mutations contribute to ASD risk. In several cases, the identified mutations and medical records suggest syndromic ASD diagnoses. Two ASD and one Fragile X premutation case showed deleterious somatic mutations, providing evidence that somatic mutations occur in ASD cases, and supporting a model in which a combination of germline and/or somatic mutations may contribute to ASD risk on a case-by-case basis.

The level of SETD2-mediated H3K36me3 is inversely correlated with that of EZH2-catalyzed H3K27me3. Nevertheless, it remains unclear whether these two enzymatic activities are molecularly intertwined. Here, we report that SETD2 delays prostate cancer (PCa) metastasis via its substrate EZH2. We show that SETD2 methylates EZH2 which promotes EZH2 degradation. SETD2 deficiency induces a Polycomb-repressive chromatin state that enables cells to acquire metastatic traits. Conversely, mice harboring nonmethylated EZH2 mutant or SETD2 mutant defective in binding to EZH2 develop metastatic PCa. Furthermore, we identify that metformin-stimulated AMPK signaling converges at FOXO3 to stimulate SETD2 expression. Together, our results demonstrate that the SETD2-EZH2 axis integrates metabolic and epigenetic signaling to restrict PCa metastasis.

SUMMARY Ependymomas exist within distinct genetic subgroups, but the molecular diversity within individual ependymomas is unknown. We perform multiplatform molecular profiling of 6 spatially distinct samples from an ependymoma with C11orf95-RELA fusion. DNA methylation and RNA sequencing distinguish clusters of samples according to neuronal development gene expression programs that could also be delineated by differences in magnetic resonance blood perfusion. Exome sequencing and phylogenetic analysis reveal epigenomic intratumor heterogeneity and suggest that chromosomal structural alterations may precede accumulation of single-nucleotide variants during ependymoma tumorigenesis. In sum, these findings shed light on the oncogenesis and intratumor heterogeneity of ependymoma., Graphical Abstract, In Brief Tumor heterogeneity poses a barrier to cancer treatment. Liu etal. investigate radiographically distinct regions of an ependymoma tumor using transcriptomic, genetic, and epigenomic profiling and discover axes of gene expression programs that recapitulate normal brain development in addition to phylogenies that shed light on the tumorigenesis of ependymoma.

Clear cell renal cell carcinoma (ccRCC) is the most common type of RCC in humans. SET domain-containing 2 (SETD2), a lysine methyltransferase for histone and other proteins, has been found to be frequently lost in ccRCC. However, the mechanisms through which deficiency in SETD2 contributes to ccRCC development remain largely unknown. Here, we used a human embryonic kidney epithelial cell line with the SETD2 gene being knocked out using CRISPR/Cas9 technology. Using ChIP-seq analysis, we showed that SETD2 loss leads to diminished occupancy of histone H3K36me3 and H4K16ac on actively transcribed genes. Transcriptome sequencing of the knockout cells revealed diminished expression of genes involved in metabolic pathways and elevated expression of genes involved in regulation of RNA polymerase II-mediated transcription. Quantitative proteomic analysis of chromatin-associated proteins showed that genetic ablation of SETD2 leads to elevated chromatin occupancy of proteins involved in chromatin remodeling and RNA polymerase II transcription regulation, and diminished chromatin binding of proteins involved in translation elongation and RNA splicing. Interestingly, we found that SETD2 depletion attenuates cell proliferation, and this can be rescued by knockdown of CDK1. Taken together, we illustrate multiple SETD2-regulated cellular pathways that suppress cancer development and uncover mechanisms underlying aberrant cell cycle regulation in SETD2-depleted cells.

Mutations in SETD2 are found in many tumors, including central nervous system (CNS) tumors. Previous work has shown these mutations occur specifically in high grade gliomas of the cerebral hemispheres in pediatric and young adult patients. We investigated SETD2 mutations in a cohort of approximately 640 CNS tumors via next generation sequencing; 23 mutations were detected across 19 primary CNS tumors. Mutations were found in a wide variety of tumors and locations at a broad range of allele frequencies. SETD2 mutations were seen in both low and high grade gliomas as well as non-glial tumors, and occurred in patients greater than 55 years of age, in addition to pediatric and young adult patients. High grade gliomas at first occurrence demonstrated either frameshift/truncating mutations or point mutations at high allele frequencies, whereas recurrent high grade gliomas frequently harbored subclones with point mutations in SETD2 at lower allele frequencies in the setting of higher mutational burdens. Comparison with the TCGA dataset demonstrated consistent findings. Finally, immunohistochemistry showed decreased staining for H3K36me3 in our cohort of SETD2 mutant tumors compared to wildtype controls. Our data further describe the spectrum of tumors in which SETD2 mutations are found and provide a context for interpretation of these mutations in the clinical setting.

SETD2 is a histone methyltransferase that catalyzes the trimethylation of lysine 36 on histone 3. SETD2 is frequently found to be mutated or deleted in a variety of human tumors, whereas the role of SETD2 in oncogenesis of osteosarcoma has never been defined. Here in our study, we uncovered that SETD2 regulates tumor growth and chemosensitivity of osteosarcoma. Overexpression of SETD2 significantly inhibited osteosarcoma cell growth in vitro and in vivo. Moreover, SETD2 significantly enhanced cisplatin-induced apoptosis in osteosarcoma cells and inhibited cancer stem cell properties in OS cells. SETD2 regulates Wnt/β-catenin signaling and its downstream gene c-myc, CD133 and cyclin D1. We further revealed that SETD2 upregulates H3K36me3 modification in GSK3B loci and promotes its transcription, which lead to β-catenin degradation. Together, our study delineates SETD2 function in osteosarcoma as an important regulator of Wnt/β-catenin signaling, and suggests SETD2 as a novel target in diagnosis and combined chemotherapy of osteosarcoma.