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Obesity is accompanied by low-grade systemic inflammation that etiologically contributes to obesity-induced cardiovascular disease (CVD). Growing evidence supports that neutrophil, the most abundant type of leukocytes in human, is most likely to be the target peripheral leukocyte subtype initiating the inflammatory cascade in obesity. However, few studies have systematically assessed the genome wide changes in neutrophils associated with obesity. In this study, a hypothesis-free OMIC approach (i.e. the discovery phase) and a target approach (i.e. the validation phase) were used to identify obesity related neutrophil activation markers and their roles on CVD risks. In the discovery phase, genome wide DNA methylation, RNA-sequencing and quantitative proteomics were obtained from purified neutrophils (12 obese vs. 12 lean). In the validation phase, gene expression levels of the promising genes from the OMIC platforms were measured in 81 obese cases vs. 83 lean controls, and the association between the expression levels and CVD risks were evaluated. Significant difference was found for one gene, alkaline phosphatase, liver/bone/kidney (ALPL), across 3 OMIC platforms. In the validation phase, the gene expression levels of ALPL in leukocytes were significantly higher in obese compared with lean subjects (p ALPL expression level showed significantly positive association with CVD risk factors (p ALPL of neutrophil activation in response to obesity and provided evidence that obesity induced change in ALPL expression was associated with CVD risk factors.

Background While aberrant DNA methylation is a characteristic feature of tumor cells, our knowledge of how these DNA methylation patterns are established and maintained is limited. DNA methyltransferases and ten-eleven translocation methylcytosine dioxygenases (TETs) function has been found altered in a variety of cancer types. Results Here, we report that in T cell acute lymphoblastic leukemia (T-ALL) the MYC oncogene controls the expression of TET1 and TET2 to maintain 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) patterns, which is associated with tumor cell-specific gene expression. We found that cellular senescence and tumor regression upon MYC inactivation in T-ALL was associated with genome-wide changes in 5mC and 5hmC patterns. Correlating with the changes in DNA (hydroxy)methylation, we found that T-ALL overexpress TET1, while suppressing TET2 in a MYC-dependent fashion. Consequently, MYC inactivation led to an inverse expression pattern, decreasing TET1, while increasing TET2 levels. Knockdown of TET1 or ectopic expression of TET2 in T-ALL was associated with genome-wide changes in 5mC and 5hmC enrichment and decreased cell proliferation, suggesting a tumor promoting function of TET1, and a tumor suppressing role for TET2. Among the genes and pathways controlled by TET1, we found ribosomal biogenesis and translational control of protein synthesis highly enriched. Conclusions Our finding that MYC directly deregulates the expression of TET1 and TET2 in T-ALL provides novel evidence that MYC controls DNA (hydroxy)methylation in a genome-wide fashion. It reveals a coordinated interplay between the components of the DNA (de)methylating machinery that contribute to MYC-driven tumor maintenance, highlighting the potential of specific TET enzymes for therapeutic strategies. Electronic supplementary material The online version of this article (10.1186/s13072-019-0278-5) contains supplementary material, which is available to authorized users.

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/ Supplementary information Supplementary data are available at Bioinformatics online.

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.

Triple-negative breast cancer (TNBC) cells are modulated in reaction to tumor-infiltrating lymphocytes. However, their specific responses to this immune pressure are unknown. In order to address this question, we first used mRNA sequencing to compare the immunophenotype of the TNBC cell line MDA-MB-231 and the luminal breast cancer cell line MCF7 after both were cocultured with activated human T cells. Despite similarities in the cytokine-induced immune signatures of the two cell lines, MDA-MD-231 cells were able to transcribe more IDO1 than MCF7 cells. The two cell lines had similar upstream JAK/STAT1 signaling and IDO1 mRNA stability. However, using a series of breast cancer cell lines, IFNγ stimulated IDO1 protein expression and enzymatic activity only in ER−, not ER+, cell lines. Treatment with 5-aza-deoxycytidine reversed the suppression of IDO1 expression in MCF7 cells, suggesting that DNA methylation was potentially involved in IDO1 induction. By analyzing several breast cancer datasets, we discovered subtype-specific mRNA and promoter methylation differences in IDO1, with TNBC/basal subtypes exhibiting lower methylation/higher expression and ER+/luminal subtypes exhibiting higher methylation/lower expression. We confirmed this trend of IDO1 methylation by bisulfite pyrosequencing breast cancer cell lines and an independent cohort of primary breast tumors. Taken together, these findings suggest that IDO1 promoter methylation regulates anti-immune responses in breast cancer subtypes and could be used as a predictive biomarker for IDO1 inhibitor–based immunotherapy. Cancer Immunol Res; 5(4); 330–44. ©2017 AACR.

// Ryan A. Hlady 1 , Rochelle L. Tiedemann 1,2 , William Puszyk 3 , Ivan Zendejas 4 , Lewis R. Roberts 5 , Jeong-Hyeon Choi 2 , Chen Liu 3 and Keith D. Robertson 1 1 Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN, USA 2 Cancer Center, Georgia Regents University, Augusta, GA, USA 3 Department of Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL, USA 4 Department of Surgery, University of Florida, Gainesville, FL, USA 5 Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA Correspondence: Keith D. Robertson, email: // Chen Liu, email: // Keywords : hepatocellular carcinoma, cirrhosis, etiology, epigenetics, DNA methylation Received : August 28, 2014 Accepted : September 07, 2014 Published : September 08, 2014 Abstract Hepatocellular carcinoma (HCC) is the second most common cause of cancer deaths worldwide. Deregulated DNA methylation landscapes are ubiquitous in human cancers. Interpretation of epigenetic aberrations in HCC is confounded by multiple etiologic drivers and underlying cirrhosis. We globally profiled the DNA methylome of 34 normal and 122 liver disease tissues arising in settings of hepatitis B (HBV) or C (HCV) viral infection, alcoholism (EtOH), and other causes to examine how these environmental agents impact DNA methylation in a manner that contributes to liver disease. Our results demonstrate that each ‘exposure’ leaves unique and overlapping signatures on the methylome. CpGs aberrantly methylated in cirrhosis-HCV and conserved in HCC were enriched for cancer driver genes, suggesting a pathogenic role for HCV-induced methylation changes. Additionally, large genomic regions displaying stepwise hypermethylation or hypomethylation during disease progression were identified. HCC-HCV/EtOH methylomes overlap highly with cryptogenic HCC, suggesting shared epigenetically deregulated pathways for hepatocarcinogenesis. Finally, overlapping methylation abnormalities between primary and cultured tumors unveil conserved epigenetic signatures in HCC. Taken together, this study reveals profound epigenome deregulation in HCC beginning during cirrhosis and influenced by common environmental agents. These results lay the foundation for defining epigenetic drivers and clinically useful methylation markers for HCC.

// Emily L. Putiri 1 , Rochelle L. Tiedemann 1,2 , Chunsheng Liu 1 , Jeong-Hyeon Choi 2 and Keith D. Robertson 1 1 Department of Molecular Pharmacology and Experimental Therapeutics and Center for Individualized Medicine, Mayo Clinic, Rochester, MN 2 Cancer Center, Georgia Regents University, Augusta, GA Correspondence: Keith D. Robertson, email: // Keywords : DNA methylation, epigenetics, cancer, histone modification, histone methylation Received : May 05, 2014 Accepted : July 13, 2014 Published : July 14, 2014 Abstract The correlation between DNA methylation and a subset of histone post-translational modifications (positive and negative) has hinted at an underlying regulatory crosstalk between histone marks and DNA methylation in patterning the human DNA methylome, an idea further supported by corresponding alterations to both histone marks and DNA methylation during malignant transformation. This study investigated the framework by which histone marks influence DNA methylation at a genome-wide level. Using RNAi in a pluripotent human embryonic carcinoma cell line we depleted essential components of the MLL/COMPASS, polycomb repressive complex 2 (PRC2), and PRC1 histone modifying complexes that establish, respectively, the post-translational modifications H3K4me3, H3K27me3, and H2AK119ub, and assayed the impact of the subsequent depletion of these marks on the DNA methylome. Absence of H2AK119ub resulted predominantly in hypomethylation across the genome. Depletion of H3K4me3 and, surprisingly, H3K27me3 caused CpG island hypermethylation at a subset of loci. Intriguingly, many promoters were co-regulated by all three histone marks, becoming hypermethylated with loss of H3K4me3 or H3K27me3 and hypomethylated with depletion of H2AK119ub, and many of these co-regulated loci were among those commonly targeted for aberrant hypermethylation in cancer. Taken together, our results elucidate novel roles for polycomb and MLL/COMPASS in regulating DNA methylation and define targets of this regulation.

Immunosuppression is a prevalent clinical feature in chronic lymphocytic leukemia (CLL) patients, with many patients demonstrating increased susceptibility to infections as well as increased failure of an antitumor immune response. However, much is currently not understood regarding the precise mechanisms that attribute to this immunosuppressive phenotype in CLL. To provide further clarity to this particular phenomenon, we analyzed the T-cell profile of CLL patient samples within a large cohort and observed that patients with an inverted CD4/CD8 ratio had a shorter time to first treatment as well as overall survival. These observations coincided with higher expression of the immune checkpoint receptor PD-1 in CLL patient CD8+ T cells when compared to age-matched healthy donors. Interestingly, we discovered that increased PD-1 expression in CD8+ T cells corresponds with decreased DNA methylation levels in a distal upstream locus of the PD-1 gene PDCD1. Further analysis using luciferase reporter assays suggests that the identified PDCD1 distal upstream region acts as an enhancer for PDCD1 transcription and this region becomes demethylated during activation of naive CD8+ T cells by anti-CD3/anti-CD28 antibodies and IL2. Finally, we conducted a genome-wide DNA methylation analysis comparing CD8+ T cells from CLL patients against healthy donors and identified additional differentially methylated genes with known immune regulatory functions including CCR6 and KLRG1. Taken together, our findings reveal the occurrence of epigenetic reprogramming taking place within CLL patient CD8+ T cells and highlight the potential mechanism of how immunosuppression is accomplished in CLL.

It is well known that cancer cells are diverse and heterogeneous among patients, and that this heterogeneity makes cancer diagnosis and cure difficult. Intra-tumoral heterogeneity has very recently become important because a small proportion of drug-resistant or tumor-initiating cells can ultimately determine a patient's outcome. In this study, we propose new approaches to use variance tests, including the Lepage test, to identify inter-patient cancer heterogeneity, as well as multinomial logistic regression to identify intra-patient cancer heterogeneity, i.e., different epiallele composition. We conduct experiments to show the performance of the proposed approaches.

The Fas–FasL effector mechanism plays a key role in cancer immune surveillance by host T cells, but metastatic human colon carcinoma often uses silencing Fas expression as a mechanism of immune evasion. The molecular mechanism under FAS transcriptional silencing in human colon carcinoma is unknown. We performed genome-wide chromatin immunoprecipitation sequencing analysis and identified that the FAS promoter is enriched with H3K9me3 in metastatic human colon carcinoma cells. The H3K9me3 level in the FAS promoter region is significantly higher in metastatic than in primary cancer cells, and it is inversely correlated with Fas expression level. We discovered that verticillin A is a selective inhibitor of histone methyltransferases SUV39H1, SUV39H2, and G9a/GLP that exhibit redundant functions in H3K9 trimethylation and FAS transcriptional silencing. Genome-wide gene expression analysis identified FAS as one of the verticillin A target genes. Verticillin A treatment decreased H3K9me3 levels in the FAS promoter and restored Fas expression. Furthermore, verticillin A exhibited greater efficacy than decitabine and vorinostat in overcoming colon carcinoma resistance to FasL-induced apoptosis. Verticillin A also increased DR5 expression and overcame colon carcinoma resistance to DR5 agonist drozitumab-induced apoptosis. Interestingly, verticillin A overcame metastatic colon carcinoma resistance to 5-fluorouracil in vitro and in vivo. Using an orthotopic colon cancer mouse model, we demonstrated that tumor-infiltrating cytotoxic T lymphocytes are FasL+ and that FasL-mediated cancer immune surveillance is essential for colon carcinoma growth control in vivo. Our findings determine that H3K9me3 of the FAS promoter is a dominant mechanism underlying FAS silencing and resultant colon carcinoma immune evasion and progression.

Glioblastoma (GBM) is the most common and most aggressive primary brain tumor in adults. The existence of a small population of stem-like tumor cells that efficiently propagate tumors and resist cytotoxic therapy is one proposed mechanism leading to the resilient behavior of tumor cells and poor prognosis. In this study, we performed an in-depth analysis of the DNA methylation landscape in GBM-derived cancer stem cells (GSCs). Parallel comparisons of primary tumors and GSC lines derived from these tumors with normal controls (a neural stem cell (NSC) line and normal brain tissue) identified groups of hyper- and hypomethylated genes that display a trend of either increasing or decreasing methylation levels in the order of controls, primary GBMs, and their counterpart GSC lines, respectively. Interestingly, concurrent promoter hypermethylation and gene body hypomethylation were observed in a subset of genes including MGMT, AJAP1 and PTPRN2. These unique DNA methylation signatures were also found in primary GBM-derived xenograft tumors indicating that they are not tissue culture-related epigenetic changes. Integration of GSC-specific epigenetic signatures with gene expression analysis further identified candidate tumor suppressor genes that are frequently down-regulated in GBMs such as SPINT2, NEFM and PENK. Forced re-expression of SPINT2 reduced glioma cell proliferative capacity, anchorage independent growth, cell motility, and tumor sphere formation in vitro. The results from this study demonstrate that GSCs possess unique epigenetic signatures that may play important roles in the pathogenesis of GBM.

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/. Contact: jechoi@gru.edu and jeochoi@gmail.com Supplementary information: Supplementary data are available at Bioinformatics online.

DNA methylation is the most studied epigenetic event in cancer, with focus being placed on studying the entire DNA methylation landscape in specific cancers. Due to the recent advances of next-generation sequencing technology, several effective methods have been developed for high-throughput analysis of DNA methylation, enabling DNA methylation markers to be innovative diagnostic and therapeutic strategies in cancer. In this review, we discuss various current and emerging technologies in DNA methylation analysis that integrate next-generation sequencing with the basic principles of methylation detections including methylation sensitive restriction enzyme digestion, affinity purification with antibody or binding proteins, and bisulfite treatment. Variations to these described methods have also allowed for detection of 5-hydroxymethylcytosine marks on a genome-wide scale. We also describe several of the bioinformatic tools used to properly analyze methylome-sequencing data. Finally, recently developed artificial transcription-factor (ATF) targeting tools may provide flexible manipulation of DNA methylation events in specific gene regions, revealing the functional consequences of particular DNA methylation events.

DNA methylation is the most studied epigenetic event in cancer, with focus being placed on studying the entire DNA methylation landscape in specific cancers. Due to the recent advances of next-generation sequencing technology, several effective methods have been developed for high-throughput analysis of DNA methylation, enabling DNA methylation markers to be innovative diagnostic and therapeutic strategies in cancer. In this review, we discuss various current and emerging technologies in DNA methylation analysis that integrate next-generation sequencing with the basic principles of methylation detections including methylation sensitive restriction enzyme digestion, affinity purification with antibody or binding proteins, and bisulfite treatment. Variations to these described methods have also allowed for detection of 5-hydroxymethylcytosine marks on a genome-wide scale. We also describe several of the bioinformatic tools used to properly analyze methylome-sequencing data. Finally, recently developed artificial transcription-factor (ATF) targeting tools may provide flexible manipulation of DNA methylation events in specific gene regions, revealing the functional consequences of particular DNA methylation events.

The parasitoid wasp Nasonia vitripennis is an emerging genetic model for functional analysis of DNA methylation. Here, we characterize genome-wide methylation at a base-pair resolution, and compare these results to gene expression across five developmental stages and to methylation patterns reported in other insects. An accurate assessment of DNA methylation across the genome is accomplished using bisulfite sequencing of adult females from a highly inbred line. One-third of genes show extensive methylation over the gene body, yet methylated DNA is not found in non-coding regions and rarely in transposons. Methylated genes occur in small clusters across the genome. Methylation demarcates exon-intron boundaries, with elevated levels over exons, primarily in the 5′ regions of genes. It is also elevated near the sites of translational initiation and termination, with reduced levels in 5′ and 3′ UTRs. Methylated genes have higher median expression levels and lower expression variation across development stages than non-methylated genes. There is no difference in frequency of differential splicing between methylated and non-methylated genes, and as yet no established role for methylation in regulating alternative splicing in Nasonia. Phylogenetic comparisons indicate that many genes maintain methylation status across long evolutionary time scales. Nasonia methylated genes are more likely to be conserved in insects, but even those that are not conserved show broader expression across development than comparable non-methylated genes. Finally, examination of duplicated genes shows that those paralogs that have lost methylation in the Nasonia lineage following gene duplication evolve more rapidly, show decreased median expression levels, and increased specialization in expression across development. Methylation of Nasonia genes signals constitutive transcription across developmental stages, whereas non-methylated genes show more dynamic developmental expression patterns. We speculate that loss of methylation may result in increased developmental specialization in evolution and acquisition of methylation may lead to broader constitutive expression., Author Summary Insects use methylation to modulate genome function in a different manner from vertebrates. Here, we quantified the global methylation profile in a parasitic wasp species, Nasonia vitripennis, a model with some advantages over ant and honeybee for functional and genetic analyses of methylation, such as short generation time, inbred lines, and inter-fertile species. Using a highly inbred line permitted us to precisely characterize DNA methylation, which is compared to gene expression variation across developmental stages, and contrasted to other insect species. DNA methylation is almost exclusively on the 5′-most 1 kbp coding exons, and ∼1/3 of protein coding genes are methylated. Methylated genes tend to occur in small clusters in the genome. Unlike many organisms, Nasonia leaves nearly all transposable element genes non-methylated. Methylated genes exhibit more uniform expression across developmental stages for both moderately and highly expressed genes, suggesting that DNA methylation is marking the genes for constitutive expression. Among pairs of differentially methylated duplicated genes, the paralogs that lose DNA methylation after duplication in the Nasonia lineage show lower expression and greater specialization of expression. Finally, by comparative analysis, we show that methylated genes are more conserved at three different time scales during evolution.

DNA methylation, mediated by the combined action of three DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B), is essential for mammalian development and is a major contributor to cellular transformation. To elucidate how DNA methylation is targeted, we mapped the genome-wide localization of all DNMTs and methylation, and examined the relationships among these markers, histone modifications, and nucleosome structure in a pluripotent human tumor cell line in its undifferentiated and differentiated states. Our findings reveal a strong link between DNMTs and transcribed loci, and that DNA methylation is not a simple sum of DNMT localization patterns. A comparison of the epigenomes of normal and cancerous stem cells, and pluripotent and differentiated states shows that the presence of at least two DNMTs is strongly associated with loci targeted for DNA hypermethylation. Taken together, these results shed important light on the determinants of DNA methylation and how it may become disrupted in cancer cells., National Institutes of Health (U.S.) (Grant RC1HG005334), National Science Foundation (U.S.) (Postdoctoral Fellowship 0905968)

We conducted a genome-wide DNA methylation analysis in CD19 (+) B-cells from chronic lymphocytic leukemia (CLL) patients and normal control samples using reduced representation bisulfite sequencing (RRBS). The methylation status of 1.8-2.3 million CpGs in the CLL genome was determined; about 45% of these CpGs were located in more than 23,000 CpG islands (CGIs). While global CpG methylation was similar between CLL and normal B-cells, 1764 gene promoters were identified as being differentially methylated in at least one CLL sample when compared with normal B-cell samples. Nineteen percent of the differentially methylated genes were involved in transcriptional regulation. Aberrant hypermethylation was found in all HOX gene clusters and a significant number of WNT signaling pathway genes. Hypomethylation occurred more frequently in the gene body including introns, exons, and 3'-UTRs in CLL. The NFATc1 P2 promoter and first intron was found to be hypomethylated and correlated with upregulation of both NFATc1 RNA and protein expression levels in CLL suggesting that an epigenetic mechanism is involved in the constitutive activation of NFAT activity in CLL cells. This comprehensive DNA methylation analysis will further our understanding of the epigenetic contribution to cellular dysfunction in CLL.

Bisulfite sequencing (BS-seq) is one of the most important sequencing protocols for genome-wide DNA methylation studies. However, it causes significant challenges in computation because it converts unmethylated cytosines to thymines. This paper presents a software package to address those challenges and to provide various kinds of analysis modules, from mapping to comparing multiple samples.

Background Follicular lymphoma (FL) is a form of non-Hodgkin's lymphoma (NHL) that arises from germinal center (GC) B-cells. Despite the significant advances in immunotherapy, FL is still not curable. Beyond transcriptional profiling and genomics datasets, there currently is no epigenome-scale dataset or integrative biology approach that can adequately model this disease and therefore identify novel mechanisms and targets for successful prevention and treatment of FL. Methodology/Principal Findings We performed methylation-enriched genome-wide bisulfite sequencing of FL cells and normal CD19+ B-cells using 454 sequencing technology. The methylated DNA fragments were enriched with methyl-binding proteins, treated with bisulfite, and sequenced using the Roche-454 GS FLX sequencer. The total number of bases covered in the human genome was 18.2 and 49.3 million including 726,003 and 1.3 million CpGs in FL and CD19+ B-cells, respectively. 11,971 and 7,882 methylated regions of interest (MRIs) were identified respectively. The genome-wide distribution of these MRIs displayed significant differences between FL and normal B-cells. A reverse trend in the distribution of MRIs between the promoter and the gene body was observed in FL and CD19+ B-cells. The MRIs identified in FL cells also correlated well with transcriptomic data and ChIP-on-Chip analyses of genome-wide histone modifications such as tri-methyl-H3K27, and tri-methyl-H3K4, indicating a concerted epigenetic alteration in FL cells. Conclusions/Significance This study is the first to provide a large scale and comprehensive analysis of the DNA methylation sequence composition and distribution in the FL epigenome. These integrated approaches have led to the discovery of novel and frequent targets of aberrant epigenetic alterations. The genome-wide bisulfite sequencing approach developed here can be a useful tool for profiling DNA methylation in clinical samples.

Indoleamine 2, 3-dioxygenase 1, encoded by IDO1, is a key immunosuppressive enzyme that catabolizes essential amino acid tryptophan to kynurenine in the tumor microenvironment. Severe depletion of tryptophan by IDO1 affects proliferation of T cells and tryptophan metabolites cause T-cell anergy and induce apoptosis. In this study, we investigated the epigenetic regulation of IDO1 expression by DNA methylation in breast cancer cells. Bisulfite pyrosequencing analysis of IDO1 promoter methylation performed on a panel of 10 breast cancer cell lines revealed that triple negative breast cancer (TNBC) cell lines (i.e. MDA-MB-231, Hs578t, SUM159) are hypomethylated compared to estrogen receptor positive (ER+) cell lines (i.e. MCF7, BT474, T47D). The same analysis was extended to 30 primary breast tumor and normal control samples and the results demonstrated that TNBC tumors had lower IDO1 promoter methylation compared to ER+ tumors. RT-PCR analysis showed that IDO1 mRNA expression is higher in TNBC cell lines than ER+ cell lines. This inverse correlation between IDO1 promoter methylation and mRNA expression can also be observed in TCGA 450K methylation and RNA-seq data sets in which promoter is hypomethylated and mRNA is up-regulated in basal-like molecular subtypes as compared to other breast cancer subtypes. IDO1 promoter is relatively CpG poor with most CpG sites concentrated near interferon γ (IFNg) responsive GAS and ISRE transcription factor binding sites. To investigate the role of CpG methylation in regulating IDO1 induction by IFNg, we cloned either methylated or unmethylated IDO1 promoter DNA into a luciferase reporter plasmid. Methylated promoter reporter exhibited significantly lower luciferase activity at basal or with IFNg stimulation compared to unmethylated reporter plasmid when transfected into MCF-7 or MDA-MB-231 cell lines. Treatment with a demethylating agent, 5-aza-deoxycytidine, synergistically up-regulated IDO1 mRNA expression with IFNg in MCF7 cells which have hypermethylated IDO1 promoter further supporting the influence of CpG methylation on IDO1 expression. IFNg stimulation or co-culture with activated T-cells significantly induced IDO1 protein expression in MDA-MB-231 cells, but not in MCF7 cells. We found no difference in IDO1 mRNA stability and IFNg induced JAK/STAT signaling pathway between MDA-MB-231 and MCF7 cell lines except promoter methylation. Furthermore, RNA-seq analysis of MDA-MB-231 and MCF7 cell lines co-cultured with activated T-cells revealed an active immune signaling mediated through JAK/STAT pathway shared by both cell lines, but also differential induction of IDO1 transcription between these two cell lines. These findings indicate IDO1 promoter methylation status as an important factor that regulates anti-immune responses by tumor cells towards tumor infiltrating lymphocytes and it could be used as a useful biomarker for IDO inhibitor based immunotherapy. Citation Format: Satish Kumar Reddy Noonepalle, Eun Joon Lee, Maria Ouzounova, Jaejik Kim, Jeong-Hyeon Choi, Austin Shull, Lirong Pei, Ravindra Kolhe, Pei-Yin Hsu, Nagireddy Putluri, Tim Hui-Ming Huang, Arun Sreekumar, Hasan Korkaya, David Munn, Huidong Shi. Promoter methylation regulates interferon-γ induced indoleamine 2,3-dioxygenase expression in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4060. doi:10.1158/1538-7445.AM2015-4060

Mammary stem/progenitor cells (MaSCs) maintain self-renewal of the mammary epithelium during puberty and pregnancy. DNA methylation provides a potential epigenetic mechanism for maintaining cellular memory during self-renewal. Although DNA methyltransferases (DNMTs) are dispensable for embryonic stem cell maintenance, their role in maintaining MaSCs and cancer stem cells (CSCs) in constantly replenishing mammary epithelium is unclear. Here we show that DNMT1 is indispensable for MaSC maintenance. Furthermore, we find that DNMT1 expression is elevated in mammary tumours, and mammary gland-specific DNMT1 deletion protects mice from mammary tumorigenesis by limiting the CSC pool. Through genome-scale methylation studies, we identify ISL1 as a direct DNMT1 target, hypermethylated and downregulated in mammary tumours and CSCs. DNMT inhibition or ISL1 expression in breast cancer cells limits CSC population. Altogether, our studies uncover an essential role for DNMT1 in MaSC and CSC maintenance and identify DNMT1-ISL1 axis as a potential therapeutic target for breast cancer treatment.

DNA methyltransferases (DNMTs) are responsible for establishing (DNMT3A, 3B, 3L) and maintaining (DNMT1) DNA methylation genome-wide. Aberrant DNA methylation is frequently observed in cancer; however, little is known about how regulation of this modification goes awry. In this study, we aim to understand how DNA methylation is regulated by the DNMTs throughout the genome by identifying specific and broad changes in methylation patterning upon depletion of the DNMTs. We utilize siRNA technology to acutely deplete NCCIT embryonal carcinoma cells of DNMT mRNA (individually and in combination), and then assay the impact on genome-wide DNA methylation patterns using the HumanMethylation450 Bead Chip (450K array). Depletion of DNMT1 (individual/combination) resulted in widespread hypomethylation, most notably in gene bodies, 3′UTRs, and intergenic sequences. DNMT3 knockdown resulted in more specific changes in DNA methylation, but surprisingly, more hypermethylation (predominately in gene bodies) than hypomethylation events occurred. These specific hypermethylation events, particularly in samples with DNMT3B KD, significantly overlapped with sites hypomethylated in DNMT1 KD conditions, indicating a potential cross-regulatory role for DNMT1 and DNMT3B in regulating DNA methylation across gene bodies. To gain a more comprehensive genome-wide view of DNA methylation in the absence of DNMT3B, we performed Methyl-CpG-Binding-Domain(MBD)-seq on DNMT3B KD cells. MBD-seq revealed dynamic changes in methylation with minor hypermethylation in promoters and subtle hypomethylation across gene bodies; however, analysis of only the most significant methylation changes (> 4-fold) revealed that more hypermethylation events occur in intronic sequences, consistent with results obtained using the 450K array. To further investigate the overlap between DNMT1 hypomethylated and DNMT3B hypermethylated sites, we examined DNA methylation in HCT116 colorectal carcinoma cells lacking (KO) or over-expressing (KI) DNMT1/DNMT3B. Interestingly, a marked number of CpG sites that gained methylation in the DNMT3B KO overlapped significantly with sites that became hypermethylated in DNMT1 and DNMT3B KI, and hypomethylated in DNMT1 KO. Additionally, these HCT116 hypermethylated CpG sites gained methylation in NCCIT DNMT3B KD (individual/combination) and lost methylation in DNMT1 KD. Taken together, these results suggest that DNMT1 and DNMT3B co-regulate DNA methylation at conserved loci across cell types in an opposing fashion, providing novel insight into a potential regulatory mechanism for DNA methylation patterning. Further elucidation of this DNMT1 and DNMT3B co-regulation holds the potential to yield novel therapeutic strategies for correcting aberrant methylation events in cancer. Citation Format: Rochelle Tiedemann, Jeong-Hyeon Choi, Keith Robertson. Acute depletion reveals novel co-regulation of DNA methylation at conserved loci by DNMT1 and DNMT3B. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2305. doi:10.1158/1538-7445.AM2014-2305

The Ten-eleven translocation (TET) family of dioxygenases hydroxylate 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. 5mC provides important epigenetic instructions during development, and its aberrant control is a major contributor to cellular transformation; however TET functions in regulating the epigenome, particularly in cancer, remain largely unknown. We targeted TET1, TET2, and TET3 for siRNA-mediated depletion in pluripotent human embryonic carcinoma cells and examined the impact on 5mC and 5hmC genome-wide localization. TET1, TET2, and TET3 co-regulate 5hmC at many sites, and depletion of only one of the TETs is sufficient to reduce 5hmC at these co-regulated sites, suggesting a functional co-dependence for TETs. Depletion of TET1 and TET2 had the greatest impact on 5hmC levels at high and low CpG density promoters, respectively, indicating that TETs exhibit DNA sequence-based functional specificity. All TETs prevent hypermethylation throughout the genome, especially in CpG island shores, where TET depletion resulted in prolific hypermethylation. Promoter hypermethylation resulting from TET depletion was associated with histone H2AK119 monoubiquitination, DNMT1, and DNMT3B occupancy. Surprisingly, TETs also promote cytosine methylation, as many loci became hypomethylated following TET depletion. Induction of differentiation generally caused 5hmC reduction, except at transcriptionally activated genes, which become enriched for 5hmC. Importantly, genes prone to promoter hypermethylation in cancer become depleted of intragenic 5hmC and 5mC with TET deficiency. This study highlights the multi-dimensional functions of TETs in mediating DNA methylation, hydroxymethylation, and gene expression patterns, and the results reveal that chromatin landscape and DNA sequence composition are regulators of TET function. Citation Format: Emily L. Putiri, Rochelle L. Tiedemann, Jeong-Hyeon Choi, Keith D. Robertson. Dynamics of TET methylcytosine dioxygenases in 5-methylcytosine and 5-hydroxymethylcytosine patterning in human cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2319. doi:10.1158/1538-7445.AM2014-2319

Glioblastoma (GBM) is the most common and most aggressive brain tumor in adults. Its frequent recurrence after resection and dismal prognosis are thought to be due to a small population of stem-like tumor cells that efficiently propagate tumors and resist cytotoxic therapy. In this study, we performed an in depth analysis of the DNA methylation landscape in GBM-derived cancer stem cells (GSCs). Parallel comparisons of primary GBM tumors and GSC lines derived from these tumors with normal controls (a neural stem cell (NSC) line and normal brain tissue) identified two groups hyper- and hypomethylated of genes that display a trend of either increasing or decreasing methylation levels in the order of controls, primary GBMs, and their counterpart GSC lines, respectively. Interestingly, concurrent promoter hypermethylation and gene body hypomethylation were observed in a subset of genes including MGMT, AJAP1 and PTPRN2. These unique DNA methylation signatures were also be found in primary GBM-derived xenograft tumors suggesting that they were not tissue culture-related epigenetic changes. Integration of GSC-specific epigenetic signatures with gene expression analysis further identified candidate tumor suppressor genes that are frequently down regulated in GBMs such as SPINT2, NEFM, and PENK. The comparison between the NSC line and the normal brain tissue sample leads to the identification of a group of NSC-specific differentially methylated regions (nsDMRs). Cluster analysis using nsDMRs revealed the presence of stem cell-specific DNA methylation signatures in both primary GBM and GSC lines. Higher expression of genes associated with stem cell-specific DNA methylation signatures such as DNMT3A, STAT5A, CSTB, PMEPA1 and G6PD in GBM patients was found to be associated with poor patient survival. The results from this study demonstrate the utility of using cancer stem cell models for advancing understanding of the pathobiology of gliomas. Citation Format: Eun Joon Lee, Prakash Rath, Jimei Liu, Dungsheng Ryu, Alan Free, Lirong Pei, Douglas C Anthony, Suash Sharma, Mark D Kirk, John J. Laterra, Duck Hwan Ryu, Jeong-Hyeon Choi, Huidong Shi, Douglas C. Miller, N. Scott Litofsky, Qi Feng. Identification of global DNA methylation signatures in glioblastoma-derived cancer stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1379. doi:10.1158/1538-7445.AM2014-1379

Dysregulation of the intrinsic DNA methylation landscape is a ubiquitous feature of human carcinogenesis, manifested by global hypomethylation and promoter-specific hypermethylation, ultimately resulting in genome instability and tumor suppressor gene silencing, respectively. Alterations in DNA methylation are particularly apparent in hepatocellular carcinoma (HCC) which afflicts roughly 750,000 new patients each year [1]. Indeed, it has been demonstrated that a variety of tumor suppressor genes (e.g. p53, E-cadherin) are hypermethylated and silenced in HCC [2-4]. Importantly, HCC is accompanied by the premalignant stage of cirrhosis in 80% of cases. One major roadblock to understanding the methylome in HCC is the presence of multiple etiologies such as Hepatitis C virus (HCV), Hepatitis B virus (HBV), and chronic alcoholism. Therefore, we performed genome-wide methylation profiling to dissect the methylation patterns of more than 170 primary liver samples to stratify etiologic and stage-specific changes in the DNA methylation landscape. Our results profile the DNA methylation landscape across normal, cirrhotic, and HCC livers in the largest study of its kind to date. We unveil distinct locus-specific and large-scale effects of HCV, HBV, and chronic alcoholism in hepatocarcinogenesis. Furthermore, analysis indicates a specific methylation profile for individual etiologies as well as conserved patterns throughout cirrhosis and hepatocellular carcinoma. Our study demonstrates that each etiology contains potential biomarkers and targets for downstream clinical therapeutics. This study is our first step toward defining the epigenome in cirrhosis and hepatocellular carcinoma and will be combined with future genomic and epimutational data (e.g. transcription, histone modifications, miRNA) to determine the true extent and interplay between epigenetic marks across different stages of liver cancer. Overall, this research has the potential to improve our understanding of epigenetics and result in diagnostic, prognostic, and therapeutic epigenetic signatures in cirrhosis and hepatocellular carcinoma, which are expected to allow for more timely and efficient detection of disease. 1. Jemal, A., et al., Global cancer statistics. CA: a cancer journal for clinicians, 2011. 61(2): p. 69-90. 2. Tischoff, I. and A. Tannapfe, DNA methylation in hepatocellular carcinoma. World journal of gastroenterology : WJG, 2008. 14(11): p. 1741-8. 3. Calvisi, D.F., et al., Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma. The Journal of clinical investigation, 2007. 117(9): p. 2713-22. 4. Lambert, M.P., et al., Aberrant DNA methylation distinguishes hepatocellular carcinoma associated with HBV and HCV infection and alcohol intake. Journal of hepatology, 2011. 54(4): p. 705-15. Citation Format: Ryan A. Hlady, Rochelle Tiedemann, William Puszyk, Chen Liu, Jeong-Hyeon Choi, Keith D. Robertson. Identification of common and unique epigenetic signatures of chronic hepatitis infection and alcohol abuse in human liver disease. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2309. doi:10.1158/1538-7445.AM2014-2309

Background: The TET family of dioxygenases catalyze conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), but their involvement in establishing normal 5mC patterns during mammalian development and their contributions to aberrant control of 5mC during cellular transformation remain largely unknown. We depleted TET1, TET2, and TET3 in a pluripotent embryonic carcinoma cell model and examined the impact on genome-wide 5mC, 5hmC, and transcriptional patterns. Results: TET1 depletion yields widespread reduction of 5hmC, while depletion of TET2 and TET3 reduces 5hmC at a subset of TET1 targets suggesting functional co-dependence. TET2 or TET3 depletion also causes increased 5hmC, suggesting these proteins play a major role in 5hmC removal. All TETs prevent hypermethylation throughout the genome, a finding dramatically illustrated in CpG island shores, where TET depletion results in prolific hypermethylation. Surprisingly, TETs also promote methylation, as hypomethylation was associated with 5hmC reduction. TET function is highly specific to chromatin environment: 5hmC maintenance by all TETs occurs at polycomb-marked chromatin and genes expressed at moderate levels; 5hmC removal by TET2 is associated with highly transcribed genes enriched for H3K4me3 and H3K36me3. Importantly, genes prone to hypermethylation in cancer become depleted of 5hmC with TET deficiency, suggesting that TETs normally promote 5hmC at these loci. Finally, all three TETs, but especially TET2, are required for 5hmC enrichment at enhancers, a condition necessary for expression of adjacent genes. Conclusions: These results provide novel insight into the division of labor among TET proteins and reveal important connections between TET activity, the chromatin landscape, and gene expression. Background Vertebrate cellular identity arises through intricate differentiation events orchestrated by epigenetic regulation of gene expression. One key epigenetic mechanism is methylation of DNA. DNA is covalently modified by methylation of the carbon-5 position within cytosine nucleotides (5mC), an epigenetic mark that, when occurring in gene promoters, is associated with transcriptional repression. DNA methylation primarily occurs in the context of cytosine followed by guanine (CpG), and normal CpG methylation patterns have been extensively characterized

DNA methyltransferases (DNMTs) are responsible for establishing (DNMT3A, DNMT3B, DNMT3L) and maintaining (DNMT1) DNA methylation genome-wide. Hypomethylation of repetitive sequences and transposable elements coupled with gene-specific promoter hypermethylation events contribute to the genomic instability and loss of tumor suppressor gene transcription observed in cancer. Regulation of aberrant methylation in cancer remains poorly understood. The aim of our study was to identify unique and overlapping target sites for each of the DNMTs to better understand regulation of normal and aberrant DNA methylation. We hypothesized that acute depletion of the DNMTs (individual and combination) mediated by siRNA technology in NCCIT human embryonic carcinoma cells would result in both distinct and broad changes in DNA methylation patterning. Genome-wide methylation was assayed using the HumanMethylation450 Bead Chip (450K array) allowing for specific CpG site methylation status determination. Select target sites were verified by bisulfite genomic sequencing. DNMT1 knockdown samples (individual/combination) revealed genome-wide hypomethylation, with the strongest demethylation occurring in gene bodies, 3′UTR, and intergenic sequences. Interestingly, only the DNMT1 individual knockdown showed significant hypermethylation in gene promoters. DNMT3 knockdowns showed more specific changes in DNA methylation, but surprisingly, more hypermethylation events occurred than hypomethylation at CpG dinucleotides. Hypermethylation observed in DNMT3 knockdown occurred primarily in gene bodies and 3′UTR, and overlapped significantly with those genes that become hypomethylated in DNMT1 knockdown, indicating a potential cross-regulatory role for the DNMTs to maintain proper regulation of DNA methylation at specific gene termini. Conversely, gene promoters were targeted for hypomethylation in DNMT3 knockdown, and did not significantly overlap with genes that become hypermethylated in DNMT1 knockdown. Of particular interest was that DNMT3B knockdown resulted in widespread non-CpG hypomethylation. In contrast, DNMT3L knockdown showed non-CpG hypermethylation, indicating a potential mechanism for regulation of non-CpG methylation where DNMT3B is responsible for non-CpG methylation, and DNMT3L acts to restrict DNMT3B's activity at non-CpG dinucleotides. Our results reveal a complex view of DNA methylation regulation, in which DNMTs not only target specific sites for methylation, but also cooperate to establish and maintain proper levels of DNA methylation at CpG and non-CpG dinucleotides. Moving forward, we believe our results will provide the framework needed to define the regulatory mechanisms by which DNA methylation is conferred and ultimately develop therapeutic strategies to correct aberrant methylation events that occur in cancer. Citation Format: Rochelle L. Tiedemann, Jeong-Hyeon Choi, Keith D. Robertson. Acute depletion reveals novel divisions of labor among human DNA methyltransferases in cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B34.

DNA methyltransferases (DNMTs) are responsible for establishing (DNMT3A, DNMT3B, DNMT3L) and maintaining (DNMT1) DNA methylation to regulate gene transcription and promote overall genome stability. Global changes in DNA methylation, such as hypomethylation of repetitive sequences and hypermethylation of tumor supressor gene promoters, are commonly observed in various types of cancer; however, the DNMTs’ contribution to this aberrant methylation remains largely unknown. In this study, we aim to identify unique and overlapping target sites for each of the DNMTs in order to better understand aberrant DNA methylation in cancer that will ultimately permit development of new therapeutic strategies. We utilize siRNA-mediated knockdown technology to acutely deplete the mRNA for each of the DNMTs in both individual and combinatorial fashion in NCCIT embryonal carcinoma cells. Subsequently, DNA methylation is observed genome-wide in each DNA sample using two different methodologies: (1) Methyl-CpG Binding Domain (MBD)-seq, which identifies regions of the genome that are enriched for DNA methylation, and (2) Illumina Infinium HumanMethylation450 BeadChip (450K array) allowing for specific CpG site methylation status determination. Aligned MBD-seq sequences for individual DNMT knockdowns are analyzed by read coverage nomalization within 100Kbp windows and using various peak-calling algorithms (e.g. MACS, BALM). For the 450K array, DNA samples for both individual and combination knockdowns undergo bisulfite conversion and array processing; β-values for each CpG site are derived from the array signal intensities using GenomeStudio and R/Bioconductor (minfi). Genome-wide DNA methylation analysis reveals that DNMT1 depletion, both individual and in combination with knockdown of other DNMTs, results in global demethylation among all genomic features. Interestingly, a small population of genes exhibit hypermethylation in gene-promoter CpG islands for DNMT1 (individual only) depletion. In contrast, de novo methyltransferase depletion (individual and combination) shows more specific demethylation effects. In particular, hypomethylation events resulting from DNMT3B depletion (individual and combination (not 3B+3L)) occur within gene bodies and largely outside of CpG islands and flanking regions (shores, shelves). Additionally, a number of hypermethylation events occurring within the 3’UTR region of genes are observed in de novo methyltransferase depleted samples. We anticipate further analysis will reveal unique and overlapping target sites for each of the DNMTs that will lay the ground-work necessary to characterize and understand DNMT recruitment both in normal and cancerous tissues. Citation Format: Rochelle L. Tiedemann, Jeong-Hyeon Choi, Keith D. Robertson. Acute depletion of DNA methyltransferases reveals unique and overlapping target sites in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2970. doi:10.1158/1538-7445.AM2013-2970

We applied a solution hybrid selection approach to the enrichment of CpG islands (CGIs) and promoter sequences from the human genome for targeted high-throughput bisulfite sequencing. A single lane of Illumina sequences allowed accurate and quantitative analysis of ~1 million CpGs in more than 21,408 CGIs and more than 15,946 transcriptional regulatory regions. Of the CpGs analyzed, 77-84% fell on or near capture probe sequences; 69-75% fell within CGIs. More than 85% of capture probes successfully yielded quantitative DNA methylation information of targeted regions. Differentially methylated regions (DMRs) were identified in the 5'-end regulatory regions, as well as the intra- and intergenic regions, particularly in the X-chromosome among the three breast cancer cell lines analyzed. We chose 46 candidate loci (762 CpGs) for confirmation with PCR-based bisulfite sequencing and demonstrated excellent correlation between two data sets. Targeted bisulfite sequencing of three DNA methyltransferase (DNMT) knockout cell lines and the wild-type HCT116 colon cancer cell line revealed a significant decrease in CpG methylation for the DNMT1 knockout and DNMT1, 3B double knockout cell lines, but not in DNMT3B knockout cell line. We demonstrated the targeted bisulfite sequencing approach to be a powerful method to uncover novel aberrant methylation in the cancer epigenome. Since all targets were captured and sequenced as a pool through a series of single-tube reactions, this method can be easily scaled up to deal with a large number of samples.

Chronic lymphocytic leukemia (CLL) is the most common leukemia of adults in the western world. The clinical course of CLL is highly variable. Some patients are able to live years with no need for treatment; whereas others progress rapidly requiring therapy within a short time after diagnosis. The identification and validation of prognostic molecular markers (including surface markers, cytogenetic abnormalities, and IGHV mutational status) have resulted in refinements in the approach to the management of these patients. Further discovery of biologically relevant factors that influence the heterogeneity and progression of CLL will not only promote our understanding of the disease process, but also allow us to identify rational therapeutic approaches. In this study, we conducted genome-wide DNA methylation analyses in purified CD19+ B-cells from 11 CLL patients with a range of CD38 expression using reduced representation bisulfite sequencing (RRBS). Using one lane of the Illumina sequencing data, we were able to consistently determine the methylation status of approximately 1.8 million CpGs; 45% of these CpGs are located in more than 23,000 CpG islands (CGIs), accounting for more than 80% of all annotated CGIs across the genome. We have identified a large number of CGIs that were differentially methylated between CLL cells and normal CD19+ B-cells. The promoter hypermethylation patterns of many genes previously reported in CLL, such as FOXD3, GRM7, DLEU7 etc, were determined in the 11 CLL samples. In addition, we have observed aberrant DNA methylation changes in all 4 HOX gene clusters in CLL. For instance, HOXD8, HOXD9, and HOXD11 were hypermethylated in 11 out of 11 CLL samples. In addition, we also identified a significant number of DNA methylation alterations in the WNT signaling pathway genes. One-way ANOVA analysis of DNA methylation profiles of 764,984 CpGs shared by all CLL samples identified 570 CpGs that were differentially methylated (p20% CLL cells expressing CD38 as CD38high group). The cluster analysis of the 570 CpGs reveals that it is possible to separate these CLL samples into two distinct groups based on the DNA methylation profiles. The study further confirms our early findings that CLL is affected by CpG island methylation in some genes that segregate with CD38 expression levels, while most others show similar methylation patterns across all levels. The aberrant promoter hypermethylation in certain functional gene groups and pathway-associated genes that are known to be deregulated in CLL provides additional insights into the CLL methylome and epigenetic contribution to cellular dysfunction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4791. doi:10.1158/1538-7445.AM2011-4791

Summary At sites of inflammation, certain regulatory T cells (Treg cells) can undergo rapid reprogramming into helper-like cells without loss of the transcription factor Foxp3. We show that reprogramming is controlled by downregulation of the transcription factor Eos ( Ikzf4 ), an obligate corepressor for Foxp3. Reprogramming was restricted to a specific subset of "Eos-labile" Treg cells that was present in the thymus and identifiable by characteristic surface markers and DNA methylation. Mice made deficient in this subset became impaired in their ability to provide help for presentation of new antigens to naive T cells. Downregulation of Eos required the proinflammatory cytokine interleukin-6 (IL-6), and mice lacking IL-6 had impaired development and function of the Eos-labile subset. Conversely, the immunoregulatory enzyme IDO blocked loss of Eos and prevented the Eos-labile Treg cells from reprogramming. Thus, the Foxp3 + lineage contains a committed subset of Treg cells capable of rapid conversion into biologically important helper cells.

Global patterns of DNA methylation, mediated by the DNA methyltransferases (DNMTs), are disrupted in all cancers by mechanisms that remain largely unknown, hampering their development as therapeutic targets. Combinatorial acute depletion of all DNMTs in a pluripotent human tumor cell line, followed by epigenome and transcriptome analysis, revealed DNMT functions in unprecedented detail. DNMT3B occupancy regulates methylation during differentiation, while an unexpected interplay was discovered in which DNMT1 and DNMT3B antithetically regulate methylation and hydroxymethylation in gene bodies, a finding confirmed in other cell types. DNMT3B mediated nonCpG methylation, while DNMT3L influenced the activity of DNMT3B toward nonCpG versus CpG site methylation. Taken together, these data reveal new functional targets of each DNMT suggesting that isoform selective inhibition would be therapeutically advantageous.