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Hepatocellular carcinoma (HCC) is the most common primary liver cancer, with high incidence and mortality worldwide. Despite diagnostic and therapeutic advancements, HCC remains poorly responsive to treatment, with a poor prognosis. Understanding the molecular mechanisms driving HCC is crucial for developing effective therapies. Emerging evidence indicates that dysregulated fatty acid metabolism contributes to HCC. Acyl-CoA medium-chain synthetase 5 (ACSM5), involved in fatty acid metabolism, is down-regulated in HCC; however, its role is not well understood. This study was used to analyze ACSM5 expression in HCC patient samples and cell lines. The newly established ACSM5-overexpressing HCC cell lines, Huh7-ACSM5 and Hepa1-6-ACSM5, were used to investigate the effects and regulatory mechanisms of ACSM5. The results showed that ACSM5 was significantly down-regulated in HCC tumor tissues compared with non-tumor tissues. ACSM5 expression was regulated by DNA methylation, with a DNA methyltransferase 1 (DNMT1) inhibitor effectively increasing ACSM5 expression and reducing promoter region methylation. Overexpression of ACSM5 in Huh7 cells reduced fatty acid accumulation, decreased cell proliferation, migration, and invasion in vitro, and inhibited tumor growth in mouse xenografts. Furthermore, ACSM5 overexpression also decreased STAT3 phosphorylation, subsequently affecting downstream cytokine TGFB and FGF12 mRNA levels. These findings suggest that ACSM5 down-regulation contributes to HCC progression, providing insights into its oncogenic role and highlighting its potential as a biomarker and therapeutic target for HCC., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Germline variants in the NSD1 gene are responsible for Sotos syndrome, while somatic variants promote neoplastic cell transformation. Our previous studies revealed three alternative RNA isoforms of NSD1 present in fibroblast cell lines (FBs): the canonical full transcript and 2 alternative transcripts, termed AT2 (NSD1 Δ5Δ7) and AT3 ( NSD1 Δ19-23 at the 5' end). The precise molecular pathways affected by each specific isoform of NSD1 are uncharacterized to date. To elucidate the role of these isoforms, their expression was suppressed by siRNA knockdown in FBs and protein expression and transcriptome data was explored. We demonstrate that one gene target of NSD1 isoform AT2 is ARP3 actin-related protein 3 homolog B ( ACTR3B ). We show that loss of both canonical NSD1 and AT2 isoforms impaired the ability of fibroblasts to regulate the actin cytoskeleton, and we observed that this caused selective loss of stress fibers. Our findings provide novel insights into NSD1 function by distinguishing isoform function and demonstrating an essential role of NSD1 in regulating the actin cytoskeleton and stress fiber formation in fibroblasts.

Epigenetic changes are a common feature of human disease, including liver disease and its progression to liver cancer. The most frequent form of liver cancer, HCC, is unusual because most of its causes, or etiologic drivers, are known and are dominated by environmental exposures, including viral infection, alcohol abuse, and overnutrition/metabolic syndrome. The epigenome is a regulatory system overlayed on the genetic material that regulates when, where, and to what extent genes are expressed in developmental, cell type, and disease-associated contexts. Deregulation of the epigenome has emerged as a major player in the pathologic effects of liver disease driving exposures, particularly during their early phases when genetic changes are uncommon. Although it is inherent in the definition of an epigenetic process to be reversible, emerging evidence indicates that epigenetic changes persist after the removal of the exposure and contribute to long-term risk of disease progression. In other systems, environmental exposures lead to beneficial adaptive changes in expression that facilitate processes such as wound healing, and these too are driven by epigenetic changes. What remains unclear, however, is what drives the transition from a beneficial epigenetic memory to a maladaptive scar, the epigenetic processes involved in forming these memories, and whether this process can be modulated for therapeutic purposes. In this review, we discuss these concepts in relation to liver disease and more broadly using examples from other tissue types and diseases, and finally consider how epigenetic therapies could be used to reprogram maladaptive epigenetic memories to delay and/or prevent hepatocarcinogenesis., (Copyright © 2023 American Association for the Study of Liver Diseases.)

Background and Aims: Disruption of the epigenome is a hallmark of human disease, including liver cirrhosis and HCC. While genetic heterogeneity is an established effector of pathologic phenotypes, epigenetic heterogeneity is less well understood. Environmental exposures alter the liver-specific DNA methylation landscape and influence the onset of liver cancer. Given that currently available treatments are unable to target frequently mutated genes in HCC, there is an unmet need for novel therapeutics to prevent or reverse liver damage leading to hepatic tumorigenesis, which the epigenome may provide., Approach and Results: We performed genome-wide profiling of DNA methylation, copy number, and gene expression from multiple liver regions from 31 patients with liver disease to examine their crosstalk and define the individual and combinatorial contributions of these processes to liver disease progression. We identified epigenetic heterogeneity hotspots that are conserved across patients. Elevated epigenetic heterogeneity is associated with increased gene expression heterogeneity. Cirrhotic regions comprise 2 distinct cohorts-one exclusively epigenetic, and the other where epigenetic and copy number variations collaborate. Epigenetic heterogeneity hotspots are enriched for genes central to liver function (eg, HNF1A ) and known tumor suppressors (eg, RASSF1A ). These hotspots encompass genes including ACSL1 , ACSL5 , MAT1A , and ELFN1 , which have phenotypic effects in functional screens, supporting their relevance to hepatocarcinogenesis. Moreover, epigenetic heterogeneity hotspots are linked to clinical measures of outcome., Conclusions: Substantial epigenetic heterogeneity arises early in liver disease development, targeting key pathways in the progression and initiation of both cirrhosis and HCC. Integration of epigenetic and transcriptional heterogeneity unveils putative epigenetic regulators of hepatocarcinogenesis., (Copyright © 2024 American Association for the Study of Liver Diseases.)

Background: Alcohol use disorders are prevalent mental disorders with significant health implications. Epigenetic alterations may play a role in their pathogenesis, as DNA methylation at several genes has been associated with these disorders. We have previously shown that methylation in the DLGAP2 gene, coding for a synaptic density protein, is associated with alcohol dependence. In this study, we aimed to examine the association between DLGAP2 methylation and treatment response among patients undergoing acamprosate treatment., Methods: 102 patients under acamprosate treatment were included. DNA methylation analysis at DLGAP2 was performed by bisulfite pyrosequencing at the start and after 3-month treatment. Treatment outcomes were having a relapse during the treatment and severity of craving at the end of three months. Cox proportional hazard and linear regression models were performed., Results: Patients whose methylation levels were decreased during the treatment showed an increased risk for relapse within three months in comparison to the ones without methylation change (hazard ratio [HR]=2.44; 95% confidence interval [CI]=1.04, 5.73; p=0.04). For the same group, a positive association for the severity of craving was observed, yet statistical significance was not reached (β=2.97; 95% CI=-0.41, 6.34; p=0.08)., Conclusion: We demonstrate that patients whose DLGAP2 methylation levels decrease during acamprosate treatment are more likely to relapse compared to the ones without changes. This is in line with our previous findings showing that DLGAP2 methylation is lower in alcohol dependent subjects compared to controls, and might suggest a role for changes in DLGAP2 methylation in treatment response., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Histone-lysine N-methyltransferase SETD2 (SETD2), the sole histone methyltransferase that catalyzes trimethylation of lysine 36 on histone H3 (H3K36me3), is often mutated in clear cell renal cell carcinoma (ccRCC). SETD2 mutation and/or loss of H3K36me3 is linked to metastasis and poor outcome in ccRCC patients. Epithelial-to-mesenchymal transition (EMT) is a major pathway that drives invasion and metastasis in various cancer types. Here, using novel kidney epithelial cell lines isogenic for SETD2, we discovered that SETD2 inactivation drives EMT and promotes migration, invasion, and stemness in a transforming growth factor-beta-independent manner. This newly identified EMT program is triggered in part through secreted factors, including cytokines and growth factors, and through transcriptional reprogramming. RNA-seq and assay for transposase-accessible chromatin sequencing uncovered key transcription factors upregulated upon SETD2 loss, including SOX2, POU2F2 (OCT2), and PRRX1, that could individually drive EMT and stemness phenotypes in SETD2 wild-type (WT) cells. Public expression data from SETD2 WT/mutant ccRCC support the EMT transcriptional signatures derived from cell line models. In summary, our studies reveal that SETD2 is a key regulator of EMT phenotypes through cell-intrinsic and cell-extrinsic mechanisms that help explain the association between SETD2 loss and ccRCC metastasis., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: Clear cell renal cell cancer (ccRCC), the 8th leading cause of cancer-related death in the US, is challenging to treat due to high level intratumoral heterogeneity (ITH) and the paucity of druggable driver mutations. CcRCC is unusual for its high frequency of epigenetic regulator mutations, such as the SETD2 histone H3 lysine 36 trimethylase (H3K36me3), and low frequency of traditional cancer driver mutations. In this work, we examined epigenetic level ITH and defined its relationships with pathologic features, aspects of tumor biology, and SETD2 mutations., Results: A multi-region sampling approach coupled with EPIC DNA methylation arrays was conducted on a cohort of normal kidney and ccRCC. ITH was assessed using DNA methylation (5mC) and CNV-based entropy and Euclidian distances. We found elevated 5mC heterogeneity and entropy in ccRCC relative to normal kidney. Variable CpGs are highly enriched in enhancer regions. Using intra-class correlation coefficient analysis, we identified CpGs that segregate tumor regions according to clinical phenotypes related to tumor aggressiveness. SETD2 wild-type tumors overall possess greater 5mC and copy number ITH than SETD2 mutant tumor regions, suggesting SETD2 loss contributes to a distinct epigenome. Finally, coupling our regional data with TCGA, we identified a 5mC signature that links regions within a primary tumor with metastatic potential., Conclusion: Taken together, our results reveal marked levels of epigenetic ITH in ccRCC that are linked to clinically relevant tumor phenotypes and could translate into novel epigenetic biomarkers., (© 2023. The Author(s).)

Chromatin immunoprecipitation (ChIP) is an antibody-based approach that is frequently utilized in chromatin biology and epigenetics. The challenge in experimental variability by unpredictable nature of usable input amounts from samples and undefined antibody titer in ChIP reaction still remains to be addressed. Here, we introduce a simple and quick method to quantify chromatin inputs and demonstrate its utility for normalizing antibody amounts to the optimal titer in individual ChIP reactions. For a proof of concept, we utilized ChIP-seq validated antibodies against the key enhancer mark, acetylation of histone H3 on lysine 27 (H3K27ac), in the experiments. The results indicate that the titration-based normalization of antibody amounts improves assay outcomes including the consistency among samples both within and across experiments for a broad range of input amounts., (© 2023. The Author(s).)

Disrupted liver regeneration following hepatectomy represents an "undruggable" clinical challenge associated with poor patient outcomes. Yes-associated protein (YAP), a transcriptional coactivator that is repressed by the Hippo pathway, is instrumental in liver regeneration. We have previously described an alternative, Hippo-independent mechanism of YAP activation mediated by downregulation of protein tyrosine phosphatase nonreceptor type 11 (PTPN11, also known as SHP2) inhibition. Herein, we examined the effects of YAP activation with a selective SHP1/SHP2 inhibitor, NSC-87877, on liver regeneration in murine partial hepatectomy models. In our studies, NSC-87877 led to accelerated hepatocyte proliferation, improved liver regeneration, and decreased markers of injury following partial hepatectomy. The effects of NSC-87877 were lost in mice with hepatocyte-specific Yap/Taz deletion, and this demonstrated dependence on these molecules for the enhanced regenerative response. Furthermore, administration of NSC-87877 to murine models of nonalcoholic steatohepatitis was associated with improved survival and decreased markers of injury after hepatectomy. Evaluation of transcriptomic changes in the context of NSC-87877 administration revealed reduction in fibrotic signaling and augmentation of cell cycle signaling. Cytoprotective changes included downregulation of Nr4a1, an apoptosis inducer. Collectively, the data suggest that SHP2 inhibition induces a pro-proliferative and cytoprotective enhancement of liver regeneration dependent on YAP.

Background and Aims: Chronic HCV infection is a leading etiologic driver of cirrhosis and ultimately HCC. Of the approximately 71 million individuals chronically infected with HCV, 10%-20% are expected to develop severe liver complications in their lifetime. Epigenetic mechanisms including DNA methylation and histone modifications become profoundly disrupted in disease processes including liver disease., Approach and Results: To understand how HCV infection influences the epigenome and whether these events remain as "scars" following cure of chronic HCV infection, we mapped genome-wide DNA methylation, four key regulatory histone modifications (H3K4me3, H3K4me1, H3K27ac, and H3K27me3), and open chromatin in parental and HCV-infected immortalized hepatocytes and the Huh7.5 HCC cell line, along with DNA methylation and gene-expression analyses following elimination of HCV in these models through treatment with interferon-α (IFN-α) or a direct-acting antiviral (DAA). Our data demonstrate that HCV infection profoundly affects the epigenome (particularly enhancers); HCV shares epigenetic targets with interferon-α targets; and an overwhelming majority of epigenetic changes induced by HCV remain as "scars" on the epigenome following viral cure. Similar findings are observed in primary human patient samples cured of chronic HCV infection. Supplementation of IFN-α/DAA antiviral regimens with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine synergizes in reverting aberrant DNA methylation induced by HCV. Finally, both HCV-infected and cured cells displayed a blunted immune response, demonstrating a functional effect of epigenetic scarring., Conclusions: Integration of epigenetic and transcriptional data elucidate key gene deregulation events driven by HCV infection and how this may underpin the long-term elevated risk for HCC in patients cured of HCV due to epigenome scarring., (© 2021 American Association for the Study of Liver Diseases.)

Myeloid neoplasms are clonal hematopoietic stem cell disorders driven by the sequential acquisition of recurrent genetic lesions. Truncating mutations in the chromatin remodeler ASXL1 (ASXL1 MT ) are associated with a high-risk disease phenotype with increased proliferation, epigenetic therapeutic resistance, and poor survival outcomes. We performed a multi-omics interrogation to define gene expression and chromatin remodeling associated with ASXL1 MT in chronic myelomonocytic leukemia (CMML). ASXL1 MT are associated with a loss of repressive histone methylation and increase in permissive histone methylation and acetylation in promoter regions. ASXL1 MT are further associated with de novo accessibility of distal enhancers binding ETS transcription factors, targeting important leukemogenic driver genes. Chromatin remodeling of promoters and enhancers is strongly associated with gene expression and heterogenous among overexpressed genes. These results provide a comprehensive map of the transcriptome and chromatin landscape of ASXL1 MT CMML, forming an important framework for the development of novel therapeutic strategies targeting oncogenic cis interactions., (© 2022. The Author(s).)

Rapid cycling (RC) burdens bipolar disorder (BD) patients further by causing more severe disability and increased suicidality. Because diagnosing RC can be challenging, RC patients are at risk of rapid decline due to delayed suitable treatment. Here, we aimed to identify the differences in the circulating cell-free DNA (cfDNA) methylome between BD patients with and without RC. The cfDNA methylome could potentially be developed as a diagnostic test for BD RC. We extracted cfDNA from plasma samples of BD1 patients (46 RC and 47 non-RC). cfDNA methylation levels were measured by 850K Infinium MethylationEPIC array. Principal component analysis (PCA) was conducted to assess global differences in methylome. cfDNA methylation levels were compared between RC groups using a linear model adjusted for age and sex. PCA suggested differences in methylation profiles between RC groups ( p = 0.039) although no significant differentially methylated probes (DMPs; q > 0.15) were found. The top four CpG sites which differed between groups at p < 1E-05 were located in CGGPB1 , PEX10 , NR0B2 , and TP53I11 . Gene set enrichment analysis (GSEA) on top DMPs ( p < 0.05) showed significant enrichment of gene sets related to nervous system tissues, such as neurons, synapse, and glutamate neurotransmission. Other top notable gene sets were related to parathyroid regulation and calcium signaling. To conclude, our study demonstrated the feasibility of utilizing a microarray method to identify circulating cfDNA methylation sites associated with BD RC and found the top differentially methylated CpG sites were mostly related to the nervous system and the parathyroid., Competing Interests: MF was a consultant (for Mayo Clinic) to Janssen, Mitsubishi Tanabe Pharma Corporation, Myriad, Sunovion, and Teva Pharmaceuticals; none of this funding contributed to any work carried out in this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ho, Winham, McCauley, Kundakovic, Robertson, Sun, Ordog, Webb, Frye and Veldic.)

Motivation: DNA methylation can be measured at the single CpG level using sodium bisulfite conversion of genomic DNA followed by sequencing or array hybridization. Many analytic tools have been developed, yet there is still a high demand for a comprehensive and multifaceted tool suite to analyze, annotate, QC and visualize the DNA methylation data., Results: We developed the CpGtools package to analyze DNA methylation data generated from bisulfite sequencing or Illumina methylation arrays. The CpGtools package consists of three types of modules: (i) 'CpG position modules' focus on analyzing the genomic positions of CpGs, including associating other genomic and epigenomic features to a given list of CpGs and generating the DNA motif logo enriched in the genomic contexts of a given list of CpGs; (ii) 'CpG signal modules' are designed to analyze DNA methylation values, such as performing the PCA or t-SNE analyses, using Bayesian Gaussian mixture modeling to classify CpG sites into fully methylated, partially methylated and unmethylated groups, profiling the average DNA methylation level over user-specified genomics regions and generating the bean/violin plots and (iii) 'differential CpG analysis modules' focus on identifying differentially methylated CpGs between groups using different statistical methods including Fisher's Exact Test, Student's t-test, ANOVA, non-parametric tests, linear regression, logistic regression, beta-binomial regression and Bayesian estimation., Availability and Implementation: CpGtools is written in Python under the open-source GPL license. The source code and documentation are freely available at https://github.com/liguowang/cpgtools., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Proliferative chronic myelomonocytic leukemia (pCMML), an aggressive CMML subtype, is associated with dismal outcomes. RAS pathway mutations, mainly NRAS G12D , define the pCMML phenotype as demonstrated by our exome sequencing, progenitor colony assays and a Vav-Cre-Nras G12D mouse model. Further, these mutations promote CMML transformation to acute myeloid leukemia. Using a multiomics platform and biochemical and molecular studies we show that in pCMML RAS pathway mutations are associated with a unique gene expression profile enriched in mitotic kinases such as polo-like kinase 1 (PLK1). PLK1 transcript levels are shown to be regulated by an unmutated lysine methyl-transferase (KMT2A) resulting in increased promoter monomethylation of lysine 4 of histone 3. Pharmacologic inhibition of PLK1 in RAS mutant patient-derived xenografts, demonstrates the utility of personalized biomarker-driven therapeutics in pCMML.

Monocyte homing to the liver and adhesion to the liver sinusoidal endothelial cells (LSECs) are key elements in nonalcoholic steatohepatitis (NASH) pathogenesis. We reported previously that VCAM-1 mediates monocyte adhesion to LSECs. However, the pathogenic role of VCAM-1 in NASH is unclear. Herein, we report that VCAM-1 was a top upregulated adhesion molecule in the NASH mouse liver transcriptome. Open chromatin landscape profiling combined with genome-wide transcriptome analysis showed robust transcriptional upregulation of LSEC VCAM-1 in murine NASH. Moreover, LSEC VCAM-1 expression was significantly increased in human NASH. LSEC VCAM-1 expression was upregulated by palmitate treatment in vitro and reduced with inhibition of the mitogen-activated protein 3 kinase (MAP3K) mixed lineage kinase 3 (MLK3). Likewise, LSEC VCAM-1 expression was reduced in the Mlk3-/- mice with diet-induced NASH. Furthermore, VCAM-1 neutralizing Ab or pharmacological inhibition attenuated diet-induced NASH in mice, mainly via reducing the proinflammatory monocyte hepatic population as examined by mass cytometry by time of flight (CyTOF). Moreover, endothelium-specific Vcam1 knockout mice were also protected against NASH. In summary, lipotoxic stress enhances the expression of LSEC VCAM-1, in part, through MLK3 signaling. Inhibition of VCAM-1 was salutary in murine NASH and might serve as a potential therapeutic strategy for human NASH.

Background: Despite using prognostic algorithms and standard surveillance guidelines, 17% of patients initially diagnosed with low risk clear cell renal cell carcinoma (ccRCC) ultimately relapse and die of recurrent disease, indicating additional molecular parameters are needed for improved prognosis., Results: To address the gap in ccRCC prognostication in the lower risk population, we performed a genome-wide analysis for methylation signatures capable of distinguishing recurrent and non-recurrent ccRCCs within the subgroup classified as 'low risk' by the Mayo Clinic Stage, Size, Grade, and Necrosis score (SSIGN 0-3). This approach revealed that recurrent patients have globally hypermethylated tumors and differ in methylation significantly at 5929 CpGs. Differentially methylated CpGs (DMCpGs) were enriched in regulatory regions and genes modulating cell growth and invasion. A subset of DMCpGs stratified low SSIGN groups into high and low risk of recurrence in independent data sets, indicating that DNA methylation enhances the prognostic power of the SSIGN score., Conclusions: This study reports a global DNA hypermethylation in tumors of recurrent ccRCC patients. Furthermore, DMCpGs were capable of discriminating between aggressive and less aggressive tumors, in addition to SSIGN score. Therefore, DNA methylation presents itself as a potentially strong biomarker to further improve prognostic power in patients with low risk SSIGN score (0-3).

The 5-carbon positions on cytosine nucleotides preceding guanines in genomic DNA (CpG) are common targets for DNA methylation (5mC). DNA methylation removal can occur through both active and passive mechanisms. Ten-eleven translocation enzymes (TETs) oxidize 5mC in a stepwise manner to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5mC can also be removed passively through sequential cell divisions in the absence of DNA methylation maintenance. In this chapter, we describe approaches that couple TET-assisted bisulfite (TAB) and oxidative bisulfite (OxBS) conversion to the Illumina MethylationEPIC BeadChIP (EPIC array) and show how these technologies can be used to distinguish active versus passive DNA demethylation. We also describe integrative bioinformatics pipelines to facilitate this analysis.

Loss-of-function TET2 mutations (TET2 MT ) are frequent early clonal events in myeloid neoplasms and are thought to confer a fitness advantage to hematopoietic precursors. This large, multi-institutional study (n = 1084), investigated the TET2 mutational landscape and prognostic implications of the number, type, and location of TET2 MT and the epistatic relationship with other somatic events in chronic myelomonocytic leukemia (CMML). Nine hundred and forty-two TET2 MT were identified in 604 (56%) patients, of which 710 (75%) were predicted to be truncating (involving the catalytic domain). Three hundred and sixteen (29%) patients had ≥1 TET2 MT , with 28%, 1%, and 0.2% harboring 2, 3, and 5 mutations, respectively. In comparison to TET2 WT , TET2 MT patients were older in age, more likely to have dysplastic CMML, a higher number of co-occurring mutations, and lower-risk stratification. Importantly, TET2 MT were associated with a survival advantage (49 vs. 30 months, p < 0.0001), especially in the context of multiple TET2 MT (≥2; 57 months, p < 0.001), and truncating TET2 MT (51 months, p < 0.001). In addition, the adverse prognostic impact of ASXL1 MT was partially mitigated by concurrent TET2 MT , with the ASXL1 WT /TET2 MT genotype having better outcomes and resulting in further risk stratification of ASXL1 inclusive CMML prognostic models, in comparison to ASXL1 MT alone.

Background: The two most common repetitive elements (REs) in humans, long interspersed nuclear element-1 (LINE-1) and Alu element (Alu), have been linked to various cancers. Hepatitis C virus (HCV) may cause hepatocellular carcinoma (HCC) by suppressing host defenses, through DNA methylation that controls the mobilization of REs. We aimed to investigate the role of RE methylation in HCV-induced HCC (HCV-HCC)., Results: We studied methylation of over 30,000 locus-specific REs across the genome in HCC, cirrhotic, and healthy liver tissues obtained by surgical resection. Relative to normal liver tissue, we observed the largest number of differentially methylated REs in HCV-HCC followed by alcohol-induced HCC (EtOH-HCC). After excluding EtOH-HCC-associated RE methylation (FDR < 0.001) and those unable to be validated in The Cancer Genome Atlas (TCGA), we identified 13 hypomethylated REs (11 LINE-1 and 2 Alu) and 2 hypermethylated REs (1 LINE-1 and 1 Alu) in HCV-HCC (FDR < 0.001). A majority of these REs were located in non-coding regions, preferentially enriched with chromatin repressive marks H3K27me3, and positively associated with gene expression (median correlation r = 0.32 across REs). We further constructed an HCV-HCC RE methylation score that distinguished HCV-HCC (lowest score), HCV-cirrhosis, and normal liver (highest score) in a dose-responsive manner (p for trend < 0.001). HCV-cirrhosis had a lower score than EtOH-cirrhosis (p = 0.038) and HCV-HCC had a lower score than EtOH-HCC in TCGA (p = 0.024)., Conclusions: Our findings indicate that HCV infection is associated with loss of DNA methylation in specific REs, which could implicate molecular mechanisms in liver cancer development. If our findings are validated in larger sample sizes, methylation of these REs may be useful as an early detection biomarker for HCV-HCC and/or a target for prevention of HCC in HCV-positive individuals.

Dorsolateral prefrontal cortex (DLPFC) and temporal pole (TP) are brain regions that display abnormalities in bipolar disorder (BD) patients. DNA methylation - an epigenetic mechanism both heritable and sensitive to the environment - may be involved in the pathophysiology of BD. To study BD-associated DNA methylomic differences in these brain regions, we extracted genomic DNA from the postmortem tissues of Brodmann Area (BA) 9 (DLPFC) and BA38 (TP) gray matter from 20 BD, ten major depression (MDD), and ten control age-and-sex-matched subjects. Genome-wide methylation levels were measured using the 850 K Illumina MethylationEPIC BeadChip. We detected striking differences between cortical regions, with greater numbers of between-brain-region differentially methylated positions (DMPs; i.e., CpG sites) in all groups, most pronounced in the BD group, and with substantial overlap across groups. The genes of DMPs common to both BD and MDD (hypothetically associated with their common features such as depression) and those distinct to BD (hypothetically associated with BD-specific features such as mania) were enriched in pathways involved in neurodevelopment including axon guidance. Pathways enriched only in the BD-MDD shared list pointed to GABAergic dysregulation, while those enriched in the BD-only list suggested glutamatergic dysregulation and greater impact on synaptogenesis and synaptic plasticity. We further detected group-specific between-brain-region gene expression differences in ODC1, CALY, GALNT2, and GABRD, which contained significant between-brain-region DMPs. In each brain region, no significant DMPs or differentially methylated regions (DMRs) were found between diagnostic groups. In summary, the methylation differences between DLPFC and TP may provide molecular targets for further investigations of genetic and environmental vulnerabilities associated with both unique and common features of various mood disorders and suggest directions of future development of individualized treatment strategies., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hepatocellular carcinoma (HCC), the most prevalent form of liver cancer, is growing in incidence but treatment options remain limited, particularly for late stage disease. As liver cirrhosis is the principal risk state for HCC development, markers to detect early HCC within this patient population are urgently needed. Perturbation of epigenetic marks, such as DNA methylation (5mC), is a hallmark of human cancers, including HCC. Identification of regions with consistently altered 5mC levels in circulating cell free DNA (cfDNA) during progression from cirrhosis to HCC could therefore serve as markers for development of minimally-invasive screens of early HCC diagnosis and surveillance. Methods : To discover DNA methylation derived biomarkers of HCC in the background of liver cirrhosis, we profiled genome-wide 5mC landscapes in patient cfDNA using the Infinium HumanMethylation450k BeadChip Array. We further linked these findings to primary tissue data available from TCGA and other public sources. Using biological and statistical frameworks, we selected CpGs that robustly differentiated cirrhosis from HCC in primary tissue and cfDNA followed by validation in an additional independent cohort. Results : We identified CpGs that segregate patients with cirrhosis, from patients with HCC within a cirrhotic liver background, through genome-wide analysis of cfDNA 5mC landscapes. Lasso regression analysis pinpointed a panel of probes in our discovery cohort that were validated in two independent datasets. A panel of five CpGs (cg04645914, cg06215569, cg23663760, cg13781744, and cg07610777) yielded area under the receiver operating characteristic (AUROC) curves of 0.9525, 0.9714, and 0.9528 in cfDNA discovery and tissue validation cohorts 1 and 2, respectively. Validation of a 5-marker panel created from combining hypermethylated and hypomethylated CpGs in an independent cfDNA set by bisulfite pyrosequencing yielded an AUROC of 0.956, compared to the discovery AUROC of 0.996. Conclusion : Our finding that 5mC markers derived from primary tissue did not perform well in cfDNA, compared to those identified directly from cfDNA, reveals potential advantages of starting with cfDNA to discover high performing markers for liquid biopsy development., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

There are currently no effective treatments for advanced-stage papillary renal cell carcinoma (PRCC). The goal of this study is to define potential DNA methylation-based markers and treatment targets for advanced-stage type 2 PRCC. Progressive DNA methylation changes and copy number variation (CNV) from localized to advanced-stage type 2 PRCC are analyzed by using methylation data generated by TCGA's kidney renal papillary cell carcinoma (TCGA-KIRP, 450k array) project. Survival analyses are performed for the identified biomarkers and genes with CNV. In addition, expression of the corresponding genes is investigated by RNA-seq analysis. Progressive methylation changes in several CpGs from localized to advanced-stage type 2 PRCC are observed. Four CpGs (cg00489401, cg27649239, cg20555674, and cg07196505) in particular are identified as markers for differentiating between localized and advanced-stage type 2 PRCC. Copy number analysis reveals that copy gain of PTK7 mostly occurs in advanced-stage type 2 PRCC. Both the four CpG methylation changes and PTK7 copy number gain are associated with patient survival. RNA-seq analysis demonstrates that PTK7 copy gain leads to higher PTK7 expression relative to tumors without copy number gain. Moreover, PTK7 is significantly upregulated from localized to advanced-stage type 2 PRCC and is linked to cancer cell invasion. In conclusion, DNA methylation markers that differentiate between localized and advanced-stage type 2 PRCC may serve as useful markers for disease staging or outcome, while PTK7 copy gain represents a potential treatment target for advanced-stage type 2 PRCC. Stepwise methylation changes and copy number gain also associate with disease stage in PRCC patients., (© 2019 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Alpha-1 antitrypsin deficiency (AATD) liver disease is characterized by marked heterogeneity in presentation and progression, despite a common underlying gene mutation, strongly suggesting the involvement of other genetic and/or epigenetic modifiers. Variation in clinical phenotype has added to the challenge of detection, diagnosis, and testing of new therapies in patients with AATD. We examined the contribution of DNA methylation (5-methylcytosine [5mC]) to AATD liver disease heterogeneity because 5mC responds to environmental and genetic cues and its deregulation is a major driver of liver disease. Using liver biopsies from adults with early-stage AATD and the ZZ genotype, genome-wide 5mC patterns were interrogated. We compared DNA methylation among patients with early AATD, and among patients with normal liver, cirrhosis, and hepatocellular carcinoma derived from multiple etiologic exposures, and linked patient clinical/demographic features. Global analysis revealed significant genomic hypomethylation in AATD liver-impacting genes related to liver cancer, cell cycle, and fibrosis, as well as key regulatory molecules influencing growth, migration, and immune function. Further analysis indicated that 5mC changes are localized, with hypermethylation occurring within a background of genome-wide 5mC loss and with patients with AATD manifesting distinct epigenetic landscapes despite their mutational homogeneity. By integrating clinical data with 5mC landscapes, we observed that CpGs differentially methylated among patients with AATD disease are linked to hallmark clinical features of AATD (e.g., hepatocyte degeneration and polymer accumulation) and further reveal links to well-known sex-specific effects of liver disease progression. Conclusion: Our data reveal molecular epigenetic signatures within this mutationally homogeneous group that point to ways to stratify patients for liver disease risk., (© 2019 by the American Association for the Study of Liver Diseases.)

Background: Epigenetic dysregulation is involved in the etiology and progression of various human diseases. Formalin-fixed paraffin-embedded (FFPE) samples represent the gold standard for archiving pathology samples, and thus FFPE samples are a major resource of samples in clinical research. However, chromatin-based epigenetic assays in the clinical settings are limited to fresh or frozen samples, and are hampered by low chromatin yield in FFPE samples due to the lack of a reliable and efficient chromatin preparation method. Here, we introduce a new chromatin extraction method from FFPE tissues (Chrom-EX PE) for chromatin-based epigenetic assays., Results: During rehydration of FFPE tissues, applying a tissue-level cross-link reversal into the deparaffinized tissue at 65 °C dramatically increased chromatin yield in the soluble fraction. The resulting chromatin is compatible with targeted ChIP-qPCR and genome-wide ChIP-seq approaches. The chromatin prepared by Chrom-EX PE showed a gradual fragmentation pattern with varying incubation temperature. At temperatures below 37 °C, the majority of soluble chromatin is over 1 kb. The soluble chromatin prepared in the range of 45-60 °C showed a typical nucleosomal pattern. And the majority of chromatin prepared at 65 °C is close to mononucleosomal size. These observations indicate that chromatin preparation from FFPE samples can be controlled for downstream chromatin-based epigenetic assays., Conclusions: This study provided a new method that achieves efficient extraction of high-quality chromatin suitable for chromatin-based epigenetic assays with less damage on chromatin. This approach may provide a way to circumvent the over-fixed nature of FFPE tissues for future technology development.

Disruption of epigenetic mechanisms has been intimately linked to the etiology of human cancer. Understanding how these epigenetic mechanisms (including DNA methylation [5mC], hydroxymethylation [5hmC], and histone post-translational modifications) work in concert to drive cancer initiation and progression remains unknown. Hepatocellular carcinoma (HCC) is increasing in frequency in Western countries but lacks efficacious treatments. The epigenome of HCC remains understudied. To better understand the epigenetic underpinnings of HCC, we performed a genome-wide assessment of 5mC, 5hmC, four histone modifications linked to promoter/enhancer function (H3K4me1, H3K27ac, H3K4me3, and H3K27me3), and transcription across normal, cirrhotic, and HCC liver tissue. Implementation of bioinformatic strategies integrated these epigenetic marks with each other and with transcription to provide a comprehensive epigenetic profile of how and when the liver epigenome is perturbed during progression to HCC. Our data demonstrate significant deregulation of epigenetic regulators combined with disruptions in the epigenome hallmarked by profound loss of 5hmC, locus-specific gains in 5mC and 5hmC, and markedly altered histone modification profiles, particularly remodeling of enhancers. Data integration demonstrates that these marks collaborate to influence transcription (e.g., hyper-5hmC in HCC-gained active enhancers is linked to elevated expression) of genes regulating HCC proliferation. Two such putative epigenetic driver loci identified through our integrative approach, COMT and FMO3, increase apoptosis and decrease cell viability in liver-derived cancer cell lines when ectopically re-expressed. Conclusion: Altogether, integration of multiple epigenetic parameters is a powerful tool for identifying epigenetically regulated drivers of HCC and elucidating how epigenome deregulation contributes to liver disease and HCC., (© 2018 by the American Association for the Study of Liver Diseases.)

SETD2, a histone H3 lysine trimethyltransferase, is frequently inactivated and associated with recurrence of clear cell renal cell carcinoma (ccRCC). However, the impact of SETD2 loss on metabolic alterations in ccRCC is still unclear. In this study, SETD2 null isogenic 38E/38F clones derived from 786-O cells were generated by zinc finger nucleases, and subsequent metabolic, genomic, and cellular phenotypic changes were analyzed by targeted metabolomics, RNA sequencing, and biological methods, respectively. Our results showed that compared with parental 786-O cells, 38E/38F cells had elevated levels of MTT/Alamar blue levels, ATP, glycolytic/mitochondrial respiratory capacity, citrate synthase (CS) activity, and TCA metabolites such as aspartate, malate, succinate, fumarate, and α-ketoglutarate. The 38E/38F cells also utilized alternative sources beyond pyruvate to generate acetyl-CoA for the TCA cycle. Moreover, 38E/38F cells showed disturbed gene networks mainly related to mitochondrial metabolism and the oxidation of fatty acids and glucose, which was associated with increased PGC1α, mitochondrial mass, and cellular size/complexity. Our results indicate that SETD2 deficiency induces a metabolic switch toward enhanced oxidative phosphorylation in ccRCC, which can be related to PGC1α-mediated metabolic networks. Therefore, this current study lays the foundation for the further development of a global metabolic analysis of cancer cells in individual patients, which ultimately will have significant potential for the discovery of novel therapeutics and precision medicine in SETD2-inactivated ccRCC.

The interplay between transcription factors and epigenetic writers like the DNA methyltransferases (DNMTs), and the role of this interplay in gene expression, is being increasingly appreciated. ZBTB24, a poorly characterized zinc-finger protein, or the de novo methyltransferase DNMT3B, when mutated, cause Immunodeficiency, Centromere Instability, and Facial anomalies (ICF) syndrome, suggesting an underlying mechanistic link. Chromatin immunoprecipitation coupled with loss-of-function approaches in model systems revealed common loci bound by ZBTB24 and DNMT3B, where they function to regulate gene body methylation. Genes coordinately regulated by ZBTB24 and DNMT3B are enriched for molecular mechanisms essential for cellular homeostasis, highlighting the importance of the ZBTB24-DNMT3B interplay in maintaining epigenetic patterns required for normal cellular function. We identify a ZBTB24 DNA binding motif, which is contained within the promoters of most of its transcriptional targets, including CDCA7, AXIN2, and OSTC. Direct binding of ZBTB24 at the promoters of these genes targets them for transcriptional activation. ZBTB24 binding at the promoters of RNF169 and CAMKMT, however, targets them for transcriptional repression. The involvement of ZBTB24 targets in diverse cellular programs, including the VDR/RXR and interferon regulatory pathways, suggest that ZBTB24's role as a transcriptional regulator is not restricted to immune cells.

With the epidemic of obesity, nonalcoholic fatty liver disease (NAFLD) has become the most common pediatric liver disease. The influence of a perinatal obesity-inducing diet (OID) on the development and progression of NAFLD in offspring is important but incompletely studied. Hence, we fed breeding pairs of C57BL/6J mice during gestation and lactation (perinatally) either chow or an OID rich in fat, fructose, and cholesterol (FFC). The offspring were weaned to either chow or an FFC diet, generating four groups: perinatal (p)Chow-Chow, pChow-FFC, pFFC-Chow, and pFFC-FFC. Mice were sacrificed at 10 weeks of age. We examined the whole-liver transcriptome by RNA sequencing (RNA-seq) and whole-liver genome methylation by reduced representation bisulfite sequencing (RRBS). Our results indicated that the pFFC-FFC mice had a significant increase in hepatic steatosis, injury, inflammation, and fibrosis, as assessed histologically and biochemically. We identified 189 genes that were differentially expressed and methylated in the pFFC-FFC mice versus the pChow-FFC mice. Gene set enrichment analysis identified hepatic fibrosis/hepatic stellate cell activation as the top canonical pathway, suggesting that the differential DNA methylation events in the mice exposed to the FFC diet perinatally were associated with a profibrogenic transcriptome. To verify that this finding was consistent with perinatal nutritional reprogramming of the methylome, we exposed pFFC-Chow mice to an FFC diet in adulthood. These mice developed significant hepatic steatosis, injury, inflammation, and more importantly fibrosis when compared to the appropriate controls. Conclusion : Perinatal exposure to an OID primes the immature liver for an accentuated fibrosing nonalcoholic steatohepatitis (NASH) phenotype, likely through nutritional reprogramming of the offspring methylome. These data have potential clinical implications for monitoring children of obese mothers and risk stratification of children with NAFLD.

Alcohol dependence (ALC) is a chronic, relapsing disorder that increases the burden of chronic disease and significantly contributes to numerous premature deaths each year. Previous research suggests that chronic, heavy alcohol consumption is associated with differential DNA methylation patterns. In addition, DNA methylation levels at certain CpG sites have been correlated with age. We used an epigenetic clock to investigate the potential role of excessive alcohol consumption in epigenetic aging. We explored this question in five independent cohorts, including DNA methylation data derived from datasets from blood (n = 129, n = 329), liver (n = 92, n = 49), and postmortem prefrontal cortex (n = 46). One blood dataset and one liver tissue dataset of individuals with ALC exhibited positive age acceleration (p < 0.0001 and p = 0.0069, respectively), whereas the other blood and liver tissue datasets both exhibited trends of positive age acceleration that were not significant (p = 0.83 and p = 0.57, respectively). Prefrontal cortex tissue exhibited a trend of negative age acceleration (p = 0.19). These results suggest that excessive alcohol consumption may be associated with epigenetic aging in a tissue-specific manner and warrants further investigation using multiple tissue samples from the same individuals.

Background: Glioma stem cells (GSCs) are a subpopulation of stem-like cells that contribute to glioblastoma (GBM) aggressiveness, recurrence, and resistance to radiation and chemotherapy. Therapeutically targeting the GSC population may improve patient survival, but unique vulnerabilities need to be identified., Results: We isolate GSCs from well-characterized GBM patient-derived xenografts (PDX), characterize their stemness properties using immunofluorescence staining, profile their epigenome including 5mC, 5hmC, 5fC/5caC, and two enhancer marks, and define their transcriptome. Fetal brain-derived neural stem/progenitor cells are used as a comparison to define potential unique and common molecular features between these different brain-derived cells with stem properties. Our integrative study reveals that abnormal expression of ten-eleven-translocation (TET) family members correlates with global levels of 5mC and 5fC/5caC and may be responsible for the distinct levels of these marks between glioma and neural stem cells. Heterogenous transcriptome and epigenome signatures among GSCs converge on several genes and pathways, including DNA damage response and cell proliferation, which are highly correlated with TET expression. Distinct enhancer landscapes are also strongly associated with differential gene regulation between glioma and neural stem cells; they exhibit unique co-localization patterns with DNA epigenetic mark switching events. Upon differentiation, glioma and neural stem cells exhibit distinct responses with regard to TET expression and DNA mark changes in the genome and GSCs fail to properly remodel their epigenome., Conclusions: Our integrative epigenomic and transcriptomic characterization reveals fundamentally distinct yet potentially targetable biologic features of GSCs that result from their distinct epigenomic landscapes.

Hepatocellular carcinoma (HCC) is the most prevalent primary tumor of the liver, and is steadily becoming one of the most lethal cancers worldwide. Liver resection, which is the recommended procedure for early localized HCC, results in frequent recurrence (50-70%), while the standard of care for late-stage HCC, multikinase inhibitors, only improves survival by a few months. The lack of success for these treatment modalities is attributable, at least in part, to marked phenotypic heterogeneity within the tumor. Intratumoral heterogeneity (ITH) has emerged as a defining characteristic of human tumors, with individual cancer cells displaying distinct differences in properties including growth rate, metastatic capacity, and response to treatment. This heterogeneity, which is unlikely to be captured from a biopsy, impacts outcome because a single treatment targeting one cancer-specific pathway would spare tumor cells having distinct characteristics. Development of effective biomarkers remains a major challenge for similar reasons. Understanding, interpreting, and circumventing the impact of ITH is therefore paramount for developing reliable biomarkers and designing effective individualized treatment strategies for HCC., Competing Interests: Disclosure The authors report no conflicts of interest in this work., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Background: Chromatin immunoprecipitation-sequencing (ChIP-seq) is a widely used epigenetic approach for investigating genome-wide protein-DNA interactions in cells and tissues. The approach has been relatively well established but several key steps still require further improvement. As a part of the procedure, immnoprecipitated DNA must undergo purification and library preparation for subsequent high-throughput sequencing. Current ChIP protocols typically yield nanogram quantities of immunoprecipitated DNA mainly depending on the target of interest and starting chromatin input amount. However, little information exists on the performance of reagents used for the purification of such minute amounts of immunoprecipitated DNA in ChIP elution buffer and their effects on ChIP-seq data. Here, we compared DNA recovery, library preparation efficiency, and ChIP-seq results obtained with several commercial DNA purification reagents applied to 1 ng ChIP DNA and also investigated the impact of conditions under which ChIP DNA is stored., Results: We compared DNA recovery of ten commercial DNA purification reagents and phenol/chloroform extraction from 1 to 50 ng of immunopreciptated DNA in ChIP elution buffer. The recovery yield was significantly different with 1 ng of DNA while similar in higher DNA amounts. We also observed that the low nanogram range of purified DNA is prone to loss during storage depending on the type of polypropylene tube used. The immunoprecipitated DNA equivalent to 1 ng of purified DNA was subject to DNA purification and library preparation to evaluate the performance of four better performing purification reagents in ChIP-seq applications. Quantification of library DNAs indicated the selected purification kits have a negligible impact on the efficiency of library preparation. The resulting ChIP-seq data were comparable with the dataset generated by ENCODE consortium and were highly correlated between the data from different purification reagents., Conclusions: This study provides comparative data on commercial DNA purification reagents applied to nanogram-range immunopreciptated ChIP DNA and evidence for the importance of storage conditions of low nanogram-range purified DNA. We verified consistent high performance of a subset of the tested reagents. These results will facilitate the improvement of ChIP-seq methodology for low-input applications.

Motivation: Chromatin accessibility plays a key role in epigenetic regulation of gene activation and silencing. Open chromatin regions allow regulatory elements such as transcription factors and polymerases to bind for gene expression while closed chromatin regions prevent the activity of transcriptional machinery. Recently, Methyltransferase Accessibility Protocol for individual templates-Bisulfite Genome Sequencing (MAPit-BGS) and nucleosome occupancy and methylome sequencing (NOMe-seq) have been developed for simultaneously profiling chromatin accessibility and DNA methylation on single molecules. Therefore, there is a great demand in developing computational methods to identify chromatin accessibility from MAPit-BGS and NOMe-seq., Results: In this article, we present CAME (Chromatin Accessibility and Methylation), a seed-extension based approach that identifies chromatin accessibility from NOMe-seq. The efficiency and effectiveness of CAME were demonstrated through comparisons with other existing techniques on both simulated and real data, and the results show that our method not only can precisely identify chromatin accessibility but also outperforms other methods., Availability and Implementation: CAME is implemented in java and the program is freely available online at http://sourceforge.net/projects/came/., Contacts: jechoi@gru.edu or khryu@dblab.chungbuk.ac.kr., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com)

While intratumor heterogeneity contributes to disease progression, metastasis, and resistance to chemotherapy, it also provides a route to understanding the evolution and drivers of disease. Defects in epigenetic landscapes are intimately linked to pathogenesis of a variety of human diseases, with epigenetic deregulation promoting tumorigenesis. Understanding epigenetic heterogeneity is crucial in hepatocellular carcinoma (HCC), where epigenetic alterations are frequent, early, and pathogenic events. We determined genome-wide DNA methylation and copy number variation leveraging the Infinium 450K in a series of regenerative nodules from within single patient livers. Bioinformatics strategies were used to ascertain within-patient heterogeneity, link epigenetic changes to clinical features, and determine their relevance to disease pathogenesis. Our data demonstrate that DNA methylation and copy number alterations evolve during the pre-neoplastic phase of HCC and independently segregate regenerative nodules into distinct clusters. Regenerative nodules with a high frequency of epigenetic changes have significantly lower copy number variation, suggesting that individual nodules have differential enrichment of epigenetic and genetic components, with both contributing to disease progression. Regenerative nodules were scored based on 'epigenetic progression' with higher scores associated with increased proliferation measured by Ki67 staining. Early events observed in epigenetically 'aggressive' nodules are enriched for genes involved in liver cancer. Our study demonstrates that marked epigenetic and genetic heterogeneity exists in early pre-neoplastic liver tissue within individual patients, emphasizing the potential contributions of each mechanism to driving liver disease progression, and it unveils strategies for identifying epigenetic drivers of hepatocellular carcinoma.

High-fat diet consumption and sedentary lifestyle elevates risk for obesity, non-alcoholic fatty liver disease, and cancer. Exercise training conveys health benefits in populations with or without these chronic conditions. Diet and exercise regulate gene expression by mediating epigenetic mechanisms in many tissues; however, such effects are poorly documented in the liver, a central metabolic organ. To dissect the consequences of diet and exercise on the liver epigenome, we measured DNA methylation, using reduced representation bisulfite sequencing, and transcription, using RNA-seq, in mice maintained on a fast food diet with sedentary lifestyle or exercise, compared with control diet with and without exercise. Our analyses reveal that genome-wide differential DNA methylation and expression of gene clusters are induced by diet and/or exercise. A combination of fast food and exercise triggers extensive gene alterations, with enrichment of carbohydrate/lipid metabolic pathways and muscle developmental processes. Through evaluation of putative protective effects of exercise on diet-induced DNA methylation, we show that hypermethylation is effectively prevented, especially at promoters and enhancers, whereas hypomethylation is only partially attenuated. We assessed diet-induced DNA methylation changes associated with liver cancer-related epigenetic modifications and identified significant increases at liver-specific enhancers in fast food groups, suggesting partial loss of liver cell identity. Hypermethylation at a subset of gene promoters was associated with inhibition of tissue development and promotion of carcinogenic processes. Our study demonstrates extensive reprogramming of the epigenome by diet and exercise, emphasizing the functional relevance of epigenetic mechanisms as an interface between lifestyle modifications and phenotypic alterations.

Background: Aberrant DNA methylation has been identified as a key molecular event regulating the pathogenesis of myelodysplastic syndromes (MDS); myeloid neoplasms with an inherent risk of transformation to acute myeloid leukemia (AML). Based on the above findings, DNA hypomethylating agents (HMA) have been widely used to treat AML and MDS, especially in elderly patients and in those who are not eligible for allogeneic stem cell transplantation (SCT). Our goal was to determine if there is any therapeutic advantage of HMA vs. conventional care regimens (CCR) and indirectly compare the efficacy of azacitidine and decitabine in this patient population., Methods: Eligible studies were limited to randomized controlled trials comparing HMA to CCR in adult patients with AML or MDS., Results: Overall survival (OS) rate was 33.2 vs. 21.4 % (RR 0.83, 95 % CI 0.71-0.98) and overall response rate (ORR) 23.7 vs. 13.4 % (RR 0.87, 95 % CI 0.81-0.93) for HMA and CCR, respectively. In subgroup analyses, only azacitidine treatment showed OS improvement (RR 0.75, 95 % CI 0.64-0.98) and not decitabine. Cytogenetic risk or bone marrow blast count did not have independent prognostic impact., Conclusion: Collectively, these results demonstrate that HMA have superior outcomes compared to CCR and suggest that azacitidine in comparison to decitabine, may be more effective.

Nucleosomes are the basic unit of chromatin. Nucleosome positioning (NP) plays a key role in transcriptional regulation and other biological processes. To better understand NP we used MNase-seq to investigate changes that occur as human embryonic stem cells (hESCs) transition to nascent mesoderm and then to smooth muscle cells (SMCs). Compared to differentiated cell derivatives, nucleosome occupancy at promoters and other notable genic sites, such as exon/intron junctions and adjacent regions, in hESCs shows a stronger correlation with transcript abundance and is less influenced by sequence content. Upon hESC differentiation, genes being silenced, but not genes being activated, display a substantial change in nucleosome occupancy at their promoters. Genome-wide, we detected a shift of NP to regions of higher G+C content as hESCs differentiate to SMCs. Notably, genomic regions with higher nucleosome occupancy harbor twice as many G↔C changes but fewer than half A↔T changes, compared to regions with lower nucleosome occupancy. Finally, our analysis indicates that the hESC genome is not rearranged and has a sequence mutation rate resembling normal human genomes. Our study reveals another unique feature of hESC chromatin, and sheds light on the relationship between nucleosome occupancy and sequence G+C content.

Clear cell renal cell carcinomas (ccRCCs) harbor frequent mutations in epigenetic modifiers including SETD2, the H3K36me3 writer. We profiled DNA methylation (5mC) across the genome in cell line-based models of SETD2 inactivation and SETD2 mutant primary tumors because 5mC has been linked to H3K36me3 and is therapeutically targetable. SETD2 depleted cell line models (long-term and acute) exhibited a DNA hypermethylation phenotype coinciding with ectopic gains in H3K36me3 centered across intergenic regions adjacent to low expressing genes, which became upregulated upon dysregulation of the epigenome. Poised enhancers of developmental genes were prominent hypermethylation targets. SETD2 mutant primary ccRCCs, papillary renal cell carcinomas, and lung adenocarcinomas all demonstrated a DNA hypermethylation phenotype that segregated tumors by SETD2 genotype and advanced grade. These findings collectively demonstrate that SETD2 mutations drive tumorigenesis by coordinated disruption of the epigenome and transcriptome,and they have important implications for future therapeutic strategies targeting chromatin regulator mutant tumors.

Motivation: Tag density plots are very important to intuitively reveal biological phenomena from capture-based sequencing data by visualizing the normalized read depth in a region., Results: We have developed iTagPlot to compute tag density across functional features in parallel using multicores and a grid engine and to interactively explore it in a graphical user interface. It allows us to stratify features by defining groups based on biological function and measurement, summary statistics and unsupervised clustering., Availability and Implementation: http://sourceforge.net/projects/itagplot/., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Background: Heart development is tightly regulated by signaling events acting on a defined number of progenitor and differentiated cardiac cells. Although loss of function of these signaling pathways leads to congenital malformation, the consequences of cardiac progenitor cell or embryonic cardiomyocyte loss are less clear. In this study, we tested the hypothesis that embryonic mouse hearts exhibit a robust mechanism for regeneration after extensive cell loss., Methods and Results: By combining a conditional cell ablation approach with a novel blastocyst complementation strategy, we generated murine embryos that exhibit a full spectrum of cardiac progenitor cell or cardiomyocyte ablation. Remarkably, ablation of up to 60% of cardiac progenitor cells at embryonic day 7.5 was well tolerated and permitted embryo survival. Ablation of embryonic cardiomyocytes to a similar degree (50% to 60%) at embryonic day 9.0 could be fully rescued by residual myocytes with no obvious adult cardiac functional deficit. In both ablation models, an increase in cardiomyocyte proliferation rate was detected and accounted for at least some of the rapid recovery of myocardial cellularity and heart size., Conclusion: Our study defines the threshold for cell loss in the embryonic mammalian heart and reveals a robust cardiomyocyte compensatory response that sustains normal fetal development., (© 2015 American Heart Association, Inc.)

Despite much effort, pancreatic cancer survival rates are still dismally low. Novel therapeutics may hold the key to improving survival. YM155 is a small molecule inhibitor that has shown antitumor activity in a number of cancers by reducing the expression of survivin. The aim of our study is to understand the mechanisms by which YM155 functions in pancreatic cancer cells. We established the antitumor effect of YM155 with in vitro studies in cultured cells, and in vivo studies using a mouse xenograft model. Our data demonstrated that YM155 reduced the expression of survivin; however, downregulation of survivin itself is insufficient to induce apoptosis in pancreatic cancer cells. We showed for the first time that treatment with YM155 increased death receptor 5 (DR5) expression in pancreatic cancer cells. We found that YM155 induced apoptosis by broad-spectrum inhibition of IAP family member proteins (e.g., CIAP1/2 and FLIP) and induced proapoptotic Bak protein upregulation and activation; the antitumor effect of YM155 treatment with either the DR5 agonist lexatumumab or gemcitabine on pancreatic cancer cells was synergistic. Our data also revealed that YM155 inhibits tumor growth in vivo, without apparent toxicity to the noncancerous human pancreatic ductal epithelial cell line. Together, these findings suggest that YM155 could be a novel therapeutic agent for pancreatic cancer., (©2014 American Association for Cancer Research.)