SETD2 safeguards the genome against isochromosome formation.

Isochromosomes are mirror-imaged chromosomes with simultaneous duplication and deletion of genetic material which may contain two centromeres to create isodicentric chromosomes. Although isochromosomes commonly occur in cancer and developmental disorders and promote genome instability, mechanisms that prevent isochromosomes are not well understood. We show here that the tumor suppressor and methyltransferase SETD2 is essential to prevent these errors. Using cellular and cytogenetic approaches, we demonstrate that loss of SETD2 or its epigenetic mark, histone H3 lysine 36 trimethylation (H3K36me3), results in the formation of isochromosomes as well as isodicentric and acentric chromosomes. These defects arise during DNA replication and are likely due to faulty homologous recombination by RAD52. These data provide a mechanism for isochromosome generation and demonstrate that SETD2 and H3K36me3 are essential to prevent the formation of this common mutable chromatin structure known to initiate a cascade of genomic instability in cancer.

A precision environmental health approach to prevention of human disease.

Human health is determined by the interaction of our environment with the genome, epigenome, and microbiome, which shape the transcriptomic, proteomic, and metabolomic landscape of cells and tissues. Precision environmental health is an emerging field leveraging environmental and system-level ('omic) data to understand underlying environmental causes of disease, identify biomarkers of exposure and response, and develop new prevention and intervention strategies. In this article we provide real-life illustrations of the utility of precision environmental health approaches, identify current challenges in the field, and outline new opportunities to promote health through a precision environmental health framework.

Responding to Natural and Industrial Disasters: Partnerships and Lessons Learned.

OBJECTIVES: The aim of this study was to provide insights learned from disaster research response (DR2) efforts following Hurricane Harvey in 2017 to launch DR2 activities following the Intercontinental Terminals Company (ITC) fire in Deer Park, Texas, in 2019. METHODS: A multidisciplinary group of academic, community, and government partners launched a myriad of DR2 activities. RESULTS: The DR2 response to Hurricane Harvey focused on enhancing environmental health literacy around clean-up efforts, measuring environmental contaminants in soil and water in impacted neighborhoods, and launching studies to evaluate the health impact of the disaster. The lessons learned after Harvey enabled rapid DR2 activities following the ITC fire, including air monitoring and administering surveys and in-depth interviews with affected residents. CONCLUSIONS: Embedding DR2 activities at academic institutions can enable rapid deployment of lessons learned from one disaster to enhance the response to subsequent disasters, even when those disasters are different. Our experience demonstrates the importance of academic institutions working with governmental and community partners to support timely disaster response efforts. Efforts enabled by such experience include providing health and safety training and consistent and reliable messaging, collecting time-sensitive and critical data in the wake of the event, and launching research to understand health impacts and improve resiliency.

Neuronal SETD2 activity links microtubule methylation to an anxiety-like phenotype in mice.

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.

Houston hurricane Harvey health (Houston-3H) study: assessment of allergic symptoms and stress after hurricane Harvey flooding.

BACKGROUND: In August 2017, Hurricane Harvey caused unprecedented flooding across the greater Houston area. Given the potential for widespread flood-related exposures, including mold and sewage, and the emotional and mental toll caused by the flooding, we sought to evaluate the short- and long-term impact of flood-related exposures on the health of Houstonians. Our objectives were to assess the association of flood-related exposures with allergic symptoms and stress among Houston-area residents at two time points: within approximately 30 days (T1) and 12 months (T2) after Hurricane Harvey's landfall. METHODS: The Houston Hurricane Harvey Health (Houston-3H) Study enrolled a total of 347 unique participants from four sites across Harris County at two times: within approximately 1-month of Harvey (T1, n = 206) and approximately 12-months after Harvey (T2, n = 266), including 125 individuals who participated at both time points. Using a self-administered questionnaire, participants reported details on demographics, flood-related exposures, and health outcomes, including allergic symptoms and stress. RESULTS: The majority of participants reported hurricane-related flooding in their homes at T1 (79.1%) and T2 (87.2%) and experienced at least one allergic symptom after the hurricane (79.4% at T1 and 68.4% at T2). In general, flood-exposed individuals were at increased risk of upper respiratory tract allergic symptoms, reported at both the T1 and T2 time points, with exposures to dirty water and mold associated with increased risk of multiple allergic symptoms. The mean stress score of study participants at T1 was 8.0 ± 2.1 and at T2, 5.1 ± 3.2, on a 0-10 scale. Participants who experienced specific flood-related exposures reported higher stress scores when compared with their counterparts, especially 1 year after Harvey. Also, a supplementary paired-samples analysis showed that reports of wheezing, shortness of breath, and skin rash did not change between T1 and T2, though other conditions were less commonly reported at T2. CONCLUSION: These initial Houston-3H findings demonstrate that flooding experiences that occurred as a consequence of Hurricane Harvey had lasting impacts on the health of Houstonians up to 1 year after the hurricane.

An actin-WHAMM interaction linking SETD2 and autophagy.

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.

Clear cell renal cell carcinoma ontogeny and mechanisms of lethality.

The molecular features that define clear cell renal cell carcinoma (ccRCC) initiation and progression are being increasingly defined. The TRACERx Renal studies and others that have described the interaction between tumour genomics and remodelling of the tumour microenvironment provide important new insights into the molecular drivers underlying ccRCC ontogeny and progression. Our understanding of common genomic and chromosomal copy number abnormalities in ccRCC, including chromosome 3p loss, provides a mechanistic framework with which to organize these abnormalities into those that drive tumour initiation events, those that drive tumour progression and those that confer lethality. Truncal mutations in ccRCC, including those in VHL, SET2, PBRM1 and BAP1, may engender genomic instability and promote defects in DNA repair pathways. The molecular features that arise from these defects enable categorization of ccRCC into clinically and therapeutically relevant subtypes. Consideration of the interaction of these subtypes with the tumour microenvironment reveals that specific mutations seem to modulate immune cell populations in ccRCC tumours. These findings present opportunities for disease prevention, early detection, prognostication and treatment.

The Huntingtin-interacting protein SETD2/HYPB is an actin lysine methyltransferase.

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.

Epigenome environment interactions accelerate epigenomic aging and unlock metabolically restricted epigenetic reprogramming in adulthood.

Our early-life environment has a profound influence on developing organs that impacts metabolic function and determines disease susceptibility across the life-course. Using a rat model for exposure to an endocrine disrupting chemical (EDC), we show that early-life chemical exposure causes metabolic dysfunction in adulthood and reprograms histone marks in the developing liver to accelerate acquisition of an adult epigenomic signature. This epigenomic reprogramming persists long after the initial exposure, but many reprogrammed genes remain transcriptionally silent with their impact on metabolism not revealed until a later life exposure to a Western-style diet. Diet-dependent metabolic disruption was largely driven by reprogramming of the Early Growth Response 1 (EGR1) transcriptome and production of metabolites in pathways linked to cholesterol, lipid and one-carbon metabolism. These findings demonstrate the importance of epigenome:environment interactions, which early in life accelerate epigenomic aging, and later in adulthood unlock metabolically restricted epigenetic reprogramming to drive metabolic dysfunction.

Hepatic Tumor Formation in Adult Mice Developmentally Exposed to Organotin.

BACKGROUND: Tributyltin (TBT) is a persistent and bioaccumulative environmental toxicant. Developmental exposure to TBT has been shown to cause fatty liver disease (steatosis), as well as increased adiposity in many species, leading to its characterization as an obesogen. OBJECTIVE: We aimed to determine the long-term effects of developmental TBT exposure on the liver. METHODS: C57BL/6J mice were exposed to a dose of TBT (0.5mg/kg body weight per day; 3.07µM) below the current developmental no observed adverse effect level (NOAEL) via drinking water, or drinking water alone, provided to the dam from preconception through lactation. Sires were exposed during breeding and lactation. Pups from two parity cycles were included in this study. Animals were followed longitudinally, and livers of offspring were analyzed by pathological evaluation, immunohistochemistry, immunoblotting, and RNA sequencing. RESULTS: Developmental exposure to TBT led to increased adiposity and hepatic steatosis at 14 and 20 weeks of age and increased liver adenomas at 45 weeks of age in male offspring. Female offspring displayed increased adiposity as compared with males, but TBT did not lead to an increase in fatty liver or tumor development in female offspring. Liver tumors in male mice were enriched in pathways and gene signatures associated with human and rodent nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC). This includes down-regulation of growth hormone receptor (GHR) and of STAT5 signaling, which occurred in response to TBT exposure and preceded liver tumor development. CONCLUSIONS: These data reveal a previously unappreciated ability of TBT to increase risk for liver tumorigenesis in mice in a sex-specific manner. Taken together, these findings provide new insights into how early life environmental exposures contribute to liver disease in adulthood. https://doi.org/10.1289/EHP5414.

Correction: SETD2 loss sensitizes cells to PI3Kß and AKT inhibition.

[This corrects the article DOI: 10.18632/oncotarget.26567.].

SETD2 regulates the maternal epigenome, genomic imprinting and embryonic development.

The oocyte epigenome plays critical roles in mammalian gametogenesis and embryogenesis. Yet, how it is established remains elusive. Here, we report that histone-lysine N-methyltransferase SETD2, an H3K36me3 methyltransferase, is a crucial regulator of the mouse oocyte epigenome. Deficiency in Setd2 leads to extensive alterations of the oocyte epigenome, including the loss of H3K36me3, failure in establishing the correct DNA methylome, invasion of H3K4me3 and H3K27me3 into former H3K36me3 territories and aberrant acquisition of H3K4me3 at imprinting control regions instead of DNA methylation. Importantly, maternal depletion of SETD2 results in oocyte maturation defects and subsequent one-cell arrest after fertilization. The preimplantation arrest is mainly due to a maternal cytosolic defect, since it can be largely rescued by normal oocyte cytosol. However, chromatin defects, including aberrant imprinting, persist in these embryos, leading to embryonic lethality after implantation. Thus, these data identify SETD2 as a crucial player in establishing the maternal epigenome that in turn controls embryonic development.

SETD2 loss sensitizes cells to PI3Kß and AKT inhibition.

Upregulation of the PI3K pathway has been implicated in the initiation and progression of several types of cancer, including renal cell carcinoma (RCC). Although several targeted therapies have been developed for RCC, durable and complete responses are exceptional. Thus, advanced RCC remains a lethal disease, underscoring the need of robust biomarker-based strategies to treat RCC. We report a synthetic lethal interaction between inhibition of phosphatidylinositol 3-kinase beta (PI3Kß) and loss of SETD2 methyltransferase. Clear cell RCC (ccRCC)-derived SETD2 knockout 786-0 and SETD2 mutant A498 cells treated with TGX221 (PI3Kß-specific) and AZD8186 (PI3Kß- and δ-specific) inhibitors displayed decreased cell viability, cell growth, and migration compared to SETD2 proficient 786-0 cells. Inhibition of the p110 δ and α isoforms alone had modest (δ) and no (α) effect on ccRCC cell viability, growth, and migration. In vivo, treatment of SETD2 mutant A498 cells, but not SETD2 proficient 786-0 cells, with AZD8186 significantly decreased tumor growth. Interestingly, inhibition of the downstream effector AKT (MK2206) recapitulated the effects observed in AZD8186-treated SETD2 deficient cells. Our data show that specific inhibition of PI3Kß causes synthetic lethality with SETD2 loss and suggest targeting of the AKT downstream effector pathway offers a rationale for further translational and clinical investigation of PI3Kß-specific inhibitors in ccRCC.

Updated Recommendations on the Diagnosis, Management, and Clinical Trial Eligibility Criteria for Patients With Renal Medullary Carcinoma.

Renal medullary carcinoma (RMC) is one of the most aggressive renal cell carcinomas. It predominantly afflicts young adults and adolescents with sickle cell trait and other sickle hemoglobinopathies, and is refractory to targeted and antiangiogenic therapies used in patients with clear-cell renal cell carcinoma. Platinum-based cytotoxic chemotherapy is the mainstay for RMC treatment. On the basis of recent advances in the diagnosis, management, and clinical trial development for RMC, a panel of experts met in October 2017 and developed updated consensus recommendations to inform clinicians, researchers, and patients. Because RMC often aggressively recurs while patients are still recovering from nephrectomy, upfront chemotherapy should be considered for most patients, including those with localized disease. After safety and dosing information has been established in adults, phase II and III trials enrolling patients with RMC should allow patients aged 12 years and older to be accrued. Patients with the very rare unclassified renal cell carcinoma with medullary phenotype variant should be included in RMC trials. Medical providers should be aware that RMC can afflict subjects of all races, and not only those of African descent, and that the presence of sickle cell trait, or of other sickle hemoglobinopathies, can affect drug responses and toxicity.

A Model Linking Sickle Cell Hemoglobinopathies and SMARCB1 Loss in Renal Medullary Carcinoma.

Renal medullary carcinoma (RMC) is a highly aggressive malignancy that predominantly afflicts young adults and adolescents with sickle hemoglobinopathies. It is characterized by complete loss of expression of the chromatin remodeler and tumor suppressor SMARCB1 Despite therapy, the outcomes of patients with RMC remain very poor, highlighting the need to understand the etiology of this cancer, and develop new diagnostic, preventive, and therapeutic strategies. A key knowledge gap in RMC biology is why sickle hemoglobinopathies predispose to the development of this cancer. We propose a model wherein the extreme conditions of hypoxia and hypertonicity of the renal medulla, combined with regional ischemia induced by red blood cell sickling, activate DNA repair mechanisms to drive deletions and translocations in SMARCB1, which is localized in a fragile region of chromosome 22. This mechanism would explain the linkage between RMC and sickle hemoglobinopathies, as well as the age dependence and predilection of RMC toward the right kidney.Significance: This perspective proposes an integrated and testable model of renal medullary carcinoma pathogenesis. Insights provided by this model can additionally inform other malignancies arising from the renal medulla and/or associated with loss of the SMARCB1 tumor suppressor gene. Clin Cancer Res; 24(9); 2044-9. ©2018 AACR.

Roadmap for investigating epigenome deregulation and environmental origins of cancer.

The interaction between the (epi)genetic makeup of an individual and his/her environmental exposure record (exposome) is accepted as a determinant factor for a significant proportion of human malignancies. Recent evidence has highlighted the key role of epigenetic mechanisms in mediating gene-environment interactions and translating exposures into tumorigenesis. There is also growing evidence that epigenetic changes may be risk factor-specific ("fingerprints") that should prove instrumental in the discovery of new biomarkers in cancer. Here, we review the state of the science of epigenetics associated with environmental stimuli and cancer risk, highlighting key developments in the field. Critical knowledge gaps and research needs are discussed and advances in epigenomics that may help in understanding the functional relevance of epigenetic alterations. Key elements required for causality inferences linking epigenetic changes to exposure and cancer are discussed and how these alterations can be incorporated in carcinogen evaluation and in understanding mechanisms underlying epigenome deregulation by the environment.

Methylated α-tubulin antibodies recognize a new microtubule modification on mitotic microtubules.

Posttranslational modifications (PTMs) on microtubules differentiate these cytoskeletal elements for a variety of cellular functions. We recently identified SETD2 as a dual-function histone and microtubule methyltransferase, and methylation as a new microtubule PTM that occurs on lysine 40 of α-tubulin, which is trimethylated (α-TubK40me3) by SETD2. In the course of these studies, we generated polyclonal (α-TubK40me3 pAb) and monoclonal (α-TubK40me3 mAb) antibodies to a methylated α-tubulin peptide (GQMPSD-Kme3-TIGGGDC). Here, we characterize these antibodies, and the specific mono-, di- or tri-methylated lysine residues they recognize. While both the pAb and mAb antibodies recognized lysines methylated by SETD2 on microtubules and histones, the clone 18 mAb was more specific for methylated microtubules, with little cross-reactivity for methylated histones. The clone 18 mAb recognized specific subsets of microtubules during mitosis and cytokinesis, and lacked the chromatin staining seen by immunocytochemistry with the pAb. Western blot analysis using these antibodies revealed that methylated α-tubulin migrated faster than unmethylated α-tubulin, suggesting methylation may be a signal for additional processing of α-tubulin and/or microtubules. As the first reagents that specifically recognize methylated α-tubulin, these antibodies are a valuable tool for studying this new modification of the cytoskeleton, and the function of methylated microtubules.

Endocrine-disrupting chemicals and uterine fibroids.

Uterine fibroids are the most frequent gynecologic tumor, affecting 70% to 80% of women over their lifetime. Although these tumors are benign, they can cause significant morbidity and may require invasive treatments such as myomectomy and hysterectomy. Many risk factors for these tumors have been identified, including environmental exposures to endocrine-disrupting chemicals (EDCs) such as genistein and diethylstilbestrol. Uterine development may be a particularly sensitive window to environmental exposures, as some perinatal EDC exposures have been shown to increase tumorigenesis in both rodent models and human epidemiologic studies. The mechanisms by which EDC exposures may increase tumorigenesis are still being elucidated, but epigenetic reprogramming of the developing uterus is an emerging hypothesis. Given the remarkably high incidence of uterine fibroids and their significant impact on women's health, understanding more about how prenatal exposures to EDCs (and other environmental agents) may increase fibroid risk could be key to developing prevention and treatment strategies in the future.

Dual Chromatin and Cytoskeletal Remodeling by SETD2.

Posttranslational modifications (PTMs) of tubulin specify microtubules for specialized cellular functions and comprise what is termed a "tubulin code." PTMs of histones comprise an analogous "histone code," although the "readers, writers, and erasers" of the cytoskeleton and epigenome have heretofore been distinct. We show that methylation is a PTM of dynamic microtubules and that the histone methyltransferase SET-domain-containing 2 (SETD2), which is responsible for H3 lysine 36 trimethylation (H3K36me3) of histones, also methylates α-tubulin at lysine 40, the same lysine that is marked by acetylation on microtubules. Methylation of microtubules occurs during mitosis and cytokinesis and can be ablated by SETD2 deletion, which causes mitotic spindle and cytokinesis defects, micronuclei, and polyploidy. These data now identify SETD2 as a dual-function methyltransferase for both chromatin and the cytoskeleton and show a requirement for methylation in maintenance of genomic stability and the integrity of both the tubulin and histone codes.

Minireview: Epigenomic Plasticity and Vulnerability to EDC Exposures.

The epigenome undergoes significant remodeling during tissue and organ development, which coincides with a period of exquisite sensitivity to environmental exposures. In the case of endocrine-disrupting compounds (EDCs), exposures can reprogram the epigenome of developing tissues to increase susceptibility to diseases later in life, a process termed "developmental reprogramming." Both DNA methylation and histone modifications have been shown to be vulnerable to disruption by EDC exposures, and several mechanisms have been identified by which EDCs can reprogram the epigenome. These include altered methyl donor availability, loss of imprinting control, changes in dioxygenase activity, altered expression of noncoding RNAs, and activation of cell signaling pathways that can phosphorylate, and alter the activity of, histone methyltransferases. This altered epigenomic programming can persist across the life course, and in some instances generations, to alter gene expression in ways that correlate with increased disease susceptibility. Together, these studies on developmental reprogramming of the epigenome by EDCs are providing new insights into epigenomic plasticity that is vulnerable to disruption by environmental exposures.