CpG Methylome Research Articles

Abstract 3170: Epigenetic identification and functional characterization of a novel RING finger protein tumor suppressor in nasopharyngeal carcinoma and esophageal carcinoma

Abstract RING finger proteins play crucial roles in normal physiology and pathophysiology as important regulators of ubiquitin-dependent proteasomal protein degradation. Many RING finger proteins have also been implicated in tumorigenesis either as oncogenes or tumor suppressors. Through cancer CpG methylome studies, we identified RING finger protein 150 (RNF150) as a methylated target gene in nasopharyngeal carcinoma (NPC) and esophageal carcinoma (ESCC). RNF150 was found to be ubiquitously expressed in human normal tissues but frequently downregulated in NPC and ESCC cells due to its promoter methylation. Its downregulation is associated with poor survival in patients with head and neck cancer and esophageal cancer. We also found that RNF150 is a functional tumor suppressor by inhibiting tumor cell growth and inducing cell cycle arrest in NPC and ESCC cells. Furthermore, ectopic expression of RNF150 inhibited NPC and ESCC cell migration/invasion by suppressing epithelial-mesenchymal transition (EMT) through upregulation of epithelial marker E-cadherin and downregulation of mesenchymal markers (vimentin, N-cadherin, Snail, and Slug). RNF150 is located in the cytoplasm and represses several oncogenic signaling pathways including the Wnt/β-catenin and NF-kB signaling pathways. Liquid chromatography-mass spectrometry (LC-MS) was performed to elucidate RNF150 binding proteins. KEGG enrichment analysis showed that glycolysis/gluconeogenesis, pyruvate metabolism, central carbon metabolism in cancer, and AMPK signaling were the top 4 pathways with significant enrichment, suggesting potential targeting of metabolic molecules by RNF150. Thus, our results provide insights into a new ring finger protein member, RNF150, as a functional tumor suppressor for NPC and ESCC. Our study also suggests a potential epigenetic biomarker for the early dection of NPC and ESCC. Citation Format: Xiaoxue Chai, Feifei Luo, Shujie Bao, Liuzhen Meng, Anthony T. Chan, Brigette B. MA, Qian Tao, li LiLi. Epigenetic identification and functional characterization of a novel RING finger protein tumor suppressor in nasopharyngeal carcinoma and esophageal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3170.

Abstract 6023: Plasma methylome landscape across the spectrum of prostate cancer progression, and differentially methylated region (DMR) based biomarkers of hormonal therapy failure in metastatic hormone-sensitive prostate cancer

Abstract Background: DNA silencing of gene expression can affect tumorigenesis, treatment outcomes and prognosis. Epigenetic silencing of tumor suppressor genes in prostate cancer detected in plasma at different stages of progression and its effect on clinical outcomes has not been determined. In a cohort study across different stages of prostate cancer (PC) progression, we performed plasma cell-free DNA (cfDNA)-based epigenomic profiling for determining the landscape of methylation and Differentially Methylated Region (DMR) classifiers and the potential impact on clinical outcomes in metastatic hormone-sensitive prostate cancer (mHSPC) state. Methods: Plasma was collected prospectively in a longitudinal cohort study of 108 PC patients (pts) of which 24 had non-metastatic Local PC; 28 had mHSPC; 56 had mCRPC. cfDNA was used for enzymatic methylation sequencing (EM-seq) of a targeted panel of genes (N=441) implicated in prostate cancer biology. Methylation status at CpG islands of these genes were examined using Twist targeted cfDNA methylation assay. The Bismark program was used for methylation calls. Temporal trends of differentially methylated regions (DMRs) were identified in different states of progression by comparing the methylation status of target genes in localized PC, mHSPC and mCRPC states using a False Discovery Rate (FDR-q-value) of 0.2 or less. DMRs uniquely associated with mHSPC state were identified and determination of genes with DMRs in pts experiencing early failure of androgen deprivation therapy (ADT)-based therapies was performed. Early failure was defined as progression on ADT within 12 months. Results: We identified 304/441 genes that were differentially methylated between localized PC and advanced PC (mHSPC and mCRPC) (FDR<0.2), of which 237 genes differed between mHSPC and mCRPC (p<0.05) states. mHSPC pts were serially profiles before and after 3 months of ADT-based therapies. All 28 pts experienced a PSA response at 3-months and 112/237 genes showed significant change in methylation status in these serial samples taken before/after 3 months of ADT-based therapies. The median follow up of the cohort was 18 months (range 10-24). 6/28 mHSPC pts experienced early ADT failure (<12 months; median 10 months). 8/112 genes were significantly associated with early failures and included DMRs in RB1, FOXA1, SOX11. GSC, ALDH1L1, SCGB3A1, FLT4, and PTPRN2. Conclusions. A targeted cfDNA plasma liquid-biopsy based epigenomic profiling of PC cancer related genes was able to capture significant differences in DMRs across different states of cancer progression. Candidate differentially expressed CpG methylome gene biomarkers detected in plasma also identified biomarkers associated with early ADT-based treatment failure in mHSPC. Citation Format: Manish Kohli, Jodie Wong, Yijun Tian, Amir Bitaraf, Claire Hanson, Matt Larsen, Jennifer Lloyd, Julia Batten, Neeraj Agarwal, Umang Swami, Benjamin Maughan, Sumati Gupta, Jonathan Tward. Plasma methylome landscape across the spectrum of prostate cancer progression, and differentially methylated region (DMR) based biomarkers of hormonal therapy failure in metastatic hormone-sensitive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6023.

A novel tumor suppressor encoded by a 1p36.3 lncRNA functions as a phosphoinositide-binding protein repressing AKT phosphorylation/activation and promoting autophagy.

Peptides/small proteins, encoded by noncanonical open reading frames (ORF) of previously claimed non-coding RNAs, have recently been recognized possessing important biological functions, but largely uncharacterized. 1p36 is an important tumor suppressor gene (TSG) locus frequently deleted in multiple cancers, with critical TSGs like TP73, PRDM16, and CHD5 already validated. Our CpG methylome analysis identified a silenced 1p36.3 gene KIAA0495, previously thought coding long non-coding RNA. We found that the open reading frame 2 of KIAA0495 is actually protein-coding and translating, encoding a small protein SP0495. KIAA0495 transcript is broadly expressed in multiple normal tissues, but frequently silenced by promoter CpG methylation in multiple tumor cell lines and primary tumors including colorectal, esophageal and breast cancers. Its downregulation/methylation is associated with poor survival of cancer patients. SP0495 induces tumor cell apoptosis, cell cycle arrest, senescence and autophagy, and inhibits tumor cell growth in vitro and in vivo. Mechanistically, SP0495 binds to phosphoinositides (PtdIns(3)P, PtdIns(3,5)P2) as a lipid-binding protein, inhibits AKT phosphorylation and its downstream signaling, and further represses oncogenic AKT/mTOR, NF-κB, and Wnt/β-catenin signaling. SP0495 also regulates the stability of autophagy regulators BECN1 and SQSTM1/p62 through modulating phosphoinositides turnover and autophagic/proteasomal degradation. Thus, we discovered and validated a 1p36.3 small protein SP0495, functioning as a novel tumor suppressor regulating AKT signaling activation and autophagy as a phosphoinositide-binding protein, being frequently inactivated by promoter methylation in multiple tumors as a potential biomarker.

The developmental regulator HAND1 inhibits gastric carcinogenesis through enhancing ER stress apoptosis via targeting CHOP and BAK which is augmented by cisplatin.

Epigenetic disruption of tumor suppressor genes, particularly aberrant CpG methylation, plays a crucial role in gastric cancer (GC) pathogenesis. Through CpG methylome and expression profiling, a developmental transcription factor - Hand-And-Neural-crest-Derivative-expressed 1 (HAND1), was identified methylated and downregulated in GC. However, its role and underlying mechanisms in GC progression are poorly understood. Here, we show that HAND1 was frequently downregulated in GC by promoter methylation, and significantly correlated with tumor progression and poor prognosis of GC patients. High expression of HAND1 in GC patients was associated with significantly higher 5-year overall survival rates. Ectopic expression of HAND1 inhibited GC cell growth and migration in vitro and in vivo. HAND1 expression increased ROS levels and cytosolic Ca2+ concentration, enhanced cisplatin-induced apoptosis through endoplasmic reticulum (ER) stress/mitochondria-mediated apoptosis. Knockdown of CHOP and BAK attenuated HAND1-induced cell apoptosis. Overexpression of CHOP increased BAK expression. HAND1 interacts with CHOP, also directly binds to CHOP and BAK promoters and positively regulates BAK transcription. Thus, the present study demonstrates that HAND1 is a tumor suppressor gene methylated in GC, induces ER stress and apoptosis via CHOP and BAK, which is augmented by cisplatin. Low HAND1 expression is an independent poor prognostic factor for GC. The tumor-specific methylation of HAND1 promoter could be a candidate biomarker for GC.

Capture Methylation-Sensitive Restriction Enzyme Sequencing (Capture MRE-Seq) for Methylation Analysis of Highly Degraded DNA Samples.

Understanding the impact of DNA methylation within different disease contexts often requires accurate assessment of these modifications in a genome-wide fashion. Frequently, patient-derived tissues stored in long-term hospital tissue banks have been preserved using formalin-fixation paraffin-embedding (FFPE). While these samples can comprise valuable resources for studying disease, the fixation process ultimately compromises the DNA's integrity and leads to degradation. Degraded DNA can complicate CpG methylome profiling using traditional techniques, particularly when performing methylation-sensitive restriction enzyme sequencing (MRE-seq), yielding high backgrounds and resulting in lowered library complexity. Here, we describe Capture MRE-seq, a new MRE-seq protocol tailored to preserving unmethylated CpG information when using samples with highly degraded DNA. The results using Capture MRE-seq correlate well (0.92) with traditional MRE-seq calls when profiling non-degraded samples, and can recover unmethylated regions in highly degraded samples when traditional MRE-seq fails, which we validate using bisulfite sequencing-based data (WGBS) as well as methylated DNA immunoprecipitation followed by sequencing (MeDIP-seq).

DNA Methylome and Transcriptome Study of Triterpenoid CDDO in TPA-Mediated Skin Carcinogenesis Model.

Overexposure to ultraviolet radiation and environmental carcinogens drive skin cancer development through redox imbalance and gene mutation. Antioxidants such as triterpenoids have exhibited anti-oxidative and anti-inflammatory potentials to alleviate skin carcinogenesis. This study investigated the methylome and transcriptome altered by tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or TPA with 2-cyano 2,3-dioxoolean-1,9-dien-28-oic acid (CDDO). The results show that CDDO blocks TPA-induced transformation dose dependently. Several differential expressed genes (DEGs) involved in skin cell transformation, while counteracted by CDDO, were revealed by differential expression analysis including Lyl1, Lad1, and Dennd2d. In CpG methylomic profiles, the differentially methylated regions (DMRs) in the promoter region altered by TPA while showing the opposite methylation status in the CDDO treatment group were identified. The correlation between DNA methylation and RNA expression has been established and DMRs showing inverse correlation were further studied as potential therapeutic targets. From the CpG methylome and transcriptome results, CDDO significantly restored gene expression of NAD(P)H:quinone oxidoreductase 1 (Nqo1) inhibited by TPA by decreasing their promoter CpG methylation. Ingenuity Pathways Analysis (IPA) shows that CDDO neutralized the effect of TPA through modulating cell cycles, cell migration, and inflammatory and immune response regulatory pathways. Notably, Tumor Necrosis Factor Receptor 2 (TNFR2) signaling was significantly downregulated by CDDO potentially contributing to prevention of TPA-induced cell transformation. Overall, incorporating the transcriptome, CpG methylome, and signaling pathway network, we reveal potential therapeutic targets and pathways by which CDDO could reverse TPA-induced carcinogenesis. The results could be useful for future human study and targets development for skin cancer.

UVB Drives Metabolic Rewiring and Epigenetic Reprograming and Protection by Sulforaphane in Human Skin Keratinocytes.

Sulforaphane (SFN) is a potent anticancer agent which could protect the skin from ultraviolet (UV) radiation-induced insults. Currently, the metabolic rewiring and epigenetic reprograming induced by UVB and the role of SFN in UVB-mediated skin cell transformation remain largely unknown. Herein, we study the metabolome, epigenome, and transcriptome of human keratinocytes (HaCaT cells) exposed to UVB with or without SFN using liquid chromatography-mass spectroscopy, DNA methylation sequencing, and RNA sequencing. UVB increases intracellular reactive oxygen species (ROS) and SFN enhances ROS acutely in post-UVB-exposed HaCaT cells. UVB and SFN alter multiple metabolites and metabolism-related signaling pathways. Pathway analysis shows that UVB impacts numerous signaling pathways including STAT3, inhibition of matrix metalloproteases, and TGF-β, among others. DNA/CpG methylation analysis shows that SFN could partially reverse some of the alterations of UVB-induced CpG methylome. Integrating RNA-seq and Methyl-seq data, starburst plots show the correlation of mRNA expression and CpG methylation status. The potential linkages between the metabolome, CpG methylome, and transcriptome suggest that metabolites produced during metabolism act as cofactors or substrates for catalytic epigenetic modification and transcriptional regulation. These results indicate that UVB drives metabolic rewiring, epigenetic reprograming, and phenotypic transcriptomic alterations and SFN would block or attenuate many of these aberrations, potentially contributing to the overall protective effect of SFN against UVB-induced skin damage.

Tools and applications for integrative analysis of DNA methylation in social insects.

DNA methylation is a common epigenetic signalling tool and an important biological process which is widely studied in a large array of species. The presence, level and function of DNA methylation vary greatly across species. In some insects, DNA methylation systems are minimal, and overall methylation rates tend to be low in all studied insect species. Low methylation levels probed by whole-genome bisulphite sequencing require great care with respect to data quality control and interpretation. Here, we introduce BWASP/R, a complete workflow that allows efficient, scalable and entirely reproducible analyses of raw DNA methylation sequencing data. Consistent application of quality control filters and analysis parameters provides fair comparisons among different studies and an integrated view of all experiments on one species. We describe the capabilities of the BWASP/R workflow by re-analysing several publicly available social insect WGBS data sets, comprising 70samples and cumulatively 147 replicates from four different species. We show that the CpG methylome comprises only about 1.5% of CpG sites in the honeybee genome and that the cumulative data are consistent with genetic signatures of site accessibility and physiological control of methylation levels.

B1a and B2 cells are characterized by distinct CpG modification states at DNMT3A-maintained enhancers

The B1 and B2 lineages of B cells contribute to protection from pathogens in distinct ways. The role of the DNA CpG methylome in specifying these two B-cell fates is still unclear. Here we profile the CpG modifications and transcriptomes of peritoneal B1a and follicular B2 cells, as well as their respective proB cell precursors in the fetal liver and adult bone marrow from wild-type and CD19-Cre Dnmt3a floxed mice lacking DNMT3A in the B lineage. We show that an underlying foundational CpG methylome is stably established during B lineage commitment and is overlaid with a DNMT3A-maintained dynamic methylome that is sculpted in distinct ways in B1a and B2 cells. This dynamic DNMT3A-maintained methylome is composed of novel enhancers that are closely linked to lineage-specific genes. While DNMT3A maintains the methylation state of these enhancers in both B1a and B2 cells, the dynamic methylome undergoes a prominent programmed demethylation event during B1a but not B2 cell development. We propose that the methylation pattern of DNMT3A-maintained enhancers is determined by the coincident recruitment of DNMT3A and TET enzymes, which regulate the developmental expression of B1a and B2 lineage-specific genes.

Epigenomic, Transcriptomic, and Protective Effect of Carotenoid Fucoxanthin in High Glucose-Induced Oxidative Stress in Mes13 Kidney Mesangial Cells

Diabetic nephropathy (DN) is the major cause of kidney related diseases in patients induced by high glucose (HG) affecting around 40% of type 1 and 2 diabetic patients. It is characterized by excessive inflammation inducing factors, reactive oxygen species (ROS) overproduction, and potential epigenomic related changes. Fucoxanthin (FX), a carotenoid found in brown seaweed, has a structure which includes an allenic bond and a 5,6-monoepoxide in the molecule, with strong antioxidant and anti-inflammatory activity. However, understanding of the impact of FX on DN was lacking. In this study we tested the early effects of high glucose (HG) on mouse mesangial kidney Mes13 cells, a potential in vitro cell culture model of DN. Our results show that HG induced oxidative stress on kidney mesangial Mes13 cells, while FX treatment attenuates the oxidative stress by decreasing the ROS, demonstrated by flow cytometry. Next, we utilized next-generation sequencing (NGS) to profile the HG-induced early epigenomic and transcriptomic changes in this in vitro DN model and the protective effects of FX. Differentially expressed genes (DEGs) and differentially methylated regions (DMRs) were analyzed using R software in HG and FX treated groups. Differential regulation of signaling pathways was studied using Reactome Pathway Analysis in the comparison. DEG analysis shows that novel biomarkers with specific pathways, including interleukin regulation, Toll-like receptor pathway, and PKA phosphorylation pathways, were found to be modulated by the FX treatment. TGF β 1i1 (TGFB 1i1), MAP-3-kinase-13(MAP3K13) involved in crucial cellular processes including glucose metabolism, phosphodiesterase regulation was methylated in HG, which was demethylated with FX treatment. Integrated transcriptomic and CpG methylome analysis of DEGs and DMRs revealed that genes like adenylate cyclase (Adcy7), calponin 1 (CNN1), potassium voltage-gated channel interacting protein 2 (KCNIP2), phosphatidylinositol-4-phosphate 5-kinase type 1 β (PIP5K1B), and transmembrane protein with EGF-like and two follistatin-like domains 1 (TMEFF1), which were modulated by FX in HG-exposed Mes13 cells, potentially modulate ion channel transport and glucose metabolism. In summary, our current study shows that novel early epigenomic and transcriptomic biomarkers were altered during the disease progression of HG-induced DN and that FX modified these alterations potentially contributing to the protective effects of mesangial cells from the HG-induced oxidative stress and damage.

Transcriptomic and epigenomics atlas of myotubes reveals insight into the circadian control of metabolism and development

Aim: Innate circadian rhythms are critical for optimal tissue-specific functions, including skeletal muscle, a major insulin-sensitive tissue responsible for glucose homeostasis. We determined whether transcriptional oscillations are associated with CpG methylation changes in skeletal muscle. Materials & methods: We performed rhythmicity analysis on the transcriptome and CpG methylome of circadian synchronized myotubes. Results: We identified several transcripts and CpG-sites displaying oscillatory behavior, which were enriched with Gene Ontology terms related to metabolism and development. Oscillating CpG methylation was associated with rhythmic expression of 31 transcripts. Conclusion: Although circadian oscillations may be regulated by rhythmic DNA methylation, strong rhythmic associations between transcriptome and CpG methylation were not identified. This resource constitutes a transcriptomic/epigenomic atlas of skeletal muscle and regulation of circadian rhythms.

DNA methylome and transcriptome alterations and cancer prevention by triterpenoid ursolic acid in UVB-induced skin tumor in mice.

Nonmelanoma skin cancers (NMSCs) are the most common type of skin cancers. Major risk factors for NMSCs include exposure to ultraviolet (UV) irradiation. Ursolic acid (UA) is a natural triterpenoid enriched in blueberries and herbal medicinal products, and possess anticancer activities. This study focuses on the impact of UA on epigenomic, genomic mechanisms and prevention of UVB-mediated NMSC. CpG methylome and RNA transcriptome alterations of early, promotion and late stages of UA treated on UVB-induced NMSC in SKH-1 hairless mice were conducted using CpG methyl-seq and RNA-seq. Samples were collected at weeks 2, 15, and 25, and integrated bioinformatic analyses were performed to identify key pathways and genes modified by UA against UVB-induced NMSC. Morphologically, UA significantly reduced NMSC tumor volume and tumor number. DNA methylome showed inflammatory pathways IL-8, NF-κB, and Nrf2 pathways were highly involved. Antioxidative stress master regulator Nrf2, cyclin D1, DNA damage, and anti-inflammatory pathways were induced by UA. Nrf2, cyclin D1, TNFrsf1b, and Mybl1 at early (2 weeks) and late (25 weeks) stages were identified and validated byquantitative polymerase chain reaction. In summary, integration of CpG methylome and RNA transcriptome studies show UA alters antioxidative, anti-inflammatory, and anticancer pathways in UVB-induced NMSC carcinogenesis. Particularly, UA appears to drive Nrf2 and its upstream/downstream genes, anti-inflammatory (at early stages) and cell cycle regulatory (both early and late stages) genes, of which might contribute to the overall chemopreventive effects of UVB-induced MNSC. This study may provide potential biomarkers/targets for chemoprevention of early stage of UVB-induced NMSC in human.

Epigenetic regulation of diabetogenic adipose morphology

ObjectiveHypertrophic white adipose tissue (WAT) morphology is associated with insulin resistance and type 2 diabetes. The mechanisms governing hyperplastic versus hypertrophic WAT expansion are poorly understood. We assessed if epigenetic modifications in adipocytes are associated with hypertrophic adipose morphology. A subset of genes with differentially methylated CpG-sites (DMS) in the promoters was taken forward for functional evaluation. MethodsThe study included 126 women who underwent abdominal subcutaneous biopsy to determine adipose morphology. Global transcriptome profiling was performed on WAT from 113 of the women, and CpG methylome profiling on isolated adipocytes from 78 women. Small interfering RNAs (siRNA) knockdown in human mesenchymal stem cells (hMSCs) was used to assess influence of specific genes on lipid storage. ResultsA higher proportion of CpG-sites were methylated in hypertrophic compared to hyperplastic WAT. Methylation at 35,138 CpG-sites was found to correlate to adipose morphology. 2,102 of these CpG-sites were also differentially methylated in T2D; 98% showed directionally consistent change in methylation in WAT hypertrophy and T2D. We identified 2,508 DMS in 638 adipose morphology-associated genes where methylation correlated with gene expression. These genes were over-represented in gene sets relevant to WAT hypertrophy, such as insulin resistance, lipolysis, extracellular matrix organization, and innate immunity. siRNA knockdown of ADH1B, AZGP1, C14orf180, GYG2, HADH, PRKAR2B, PFKFB3, and AQP7 influenced lipid storage and metabolism. ConclusionCpG methylation could be influential in determining adipose morphology and thereby constitute a novel antidiabetic target. We identified C14orf180 as a novel regulator of adipocyte lipid storage and possibly differentiation.

CpG methyl-seq and RNA-seq epigenomic and transcriptomic studies on the preventive effects of Moringa isothiocyanate in mouse epidermal JB6 cells induced by the tumor promoter TPA

Epigenetic mechanisms play an important role in the early stages of carcinogenesis. Moringa isothiocyanate (MIC-1) is a major bioactive component derived from Moringa oleifera that has considerable antioxidant and anti-inflammatory effects. However, how MIC-1 influences epigenomic alterations in TPA-mediated JB6 cell carcinogenic transformation has not been evaluated. In this study, DNA and RNA isolated from TPA-induced JB6 cells in the presence or absence of MIC-1 were subjected to DNA Methyl-seq and RNA-seq to identify differentially methylated regions (DMRs) and differentially expressed genes (DEGs), respectively. When JB6 cells were challenged with TPA alone, there was a significant alteration of DEGs and DMRs; importantly, MIC-1 treatment reversed the patterns of some of the DEGs and DMRs. Transcriptome and CpG methylome profiling was performed in Ingenuity® Pathway Analysis (IPA) software to analyze the altered signaling pathways. Several anti-inflammatory responses, antioxidative stress-related pathways, and anticancer-related pathways were identified to be affected by MIC-1. These pathways included NF-kB, IL-1, LPS/IL-1-mediated inhibition of RXR function, Nrf2-mediated oxidative stress response, p53, and PTEN signaling pathways. Examination of correlations between transcriptomic and CpG methylome profiles yielded a small subset of genes, including the cancer-related genes Tmpt, Tubb3, and Muc2; the GTPases Gchfr and Igtp; and the cell cycle-related gene Cdc7. Taken together, our results show the potential contributions of epigenomic changes in DNA CpG methylation to gene expression to molecular pathways active in TPA-induced JB6 cells and demonstrate that MIC-1 can reverse these changes, supporting the potential preventive/treatment effects of MIC-1 against skin carcinogenesis.

47 - METHYLOME-WIDE ASSOCIATION STUDIES FOR MAJOR DEPRESSIVE DISORDER IN BLOOD OVERLAP WITH METHYLATION RESULTS FROM BRAIN AND LARGE-SCALE GWAS

Background Epigenetic modifications such as DNA methylation provide stability and diversity to the cellular phenotype and aberrant methylation has been implicated in processes underlying psychiatric disorders. Therefore, studies combining DNA methylation and genotype information provide a promising approach to study disorders where genotype information alone has failed to reveal the full etiology. Methods We applied an optimized MBD-seq protocol to assay the complete CpG methylome in cases with Major Depressive Disorder (MDD) and controls using blood samples (N=1,132) from Netherlands Study of Depression and Anxiety and brain samples (N=64) from the Victorian Brain Bank Network. Data were analyzed with RaMWAS, a novel Bioconductor package specifically designed for Methylome-Wide Association Studies (MWAS). To study the overlap between top MWAS findings in blood and brain, we used a permutation based enrichment test (shiftR) that accounted for the dependency between adjacent CpG sites. Furthermore, we utilized the methylation data in combination with existing genotype information from the same individuals in a MWAS of CpGs created or destroyed by SNPs. Next, we tested whether top results from this CpG-SNP MWAS overlapped with recent large-scale GWAS to identify robust associations with genomic loci of importance for MDD etiology. Results The MWAS in blood identified five methylome-wide significant sites (P Discussion This work involves the largest MWAS for MDD performed to date. Our integrated analysis with brain tissue, genotype information and GWAS results highlighted biological functions of potential value for MDD etiology. Part of the associated methylation marks in blood overlapped with MWAS finding in brain. As blood can easily be collected in a clinical setting, these loci may be of direct value as potential diagnostic biomarkers for MDD.

A Statistical Method for Observing Personal Diploid Methylomes and Transcriptomes with Single-Molecule Real-Time Sequencing

We address the problem of observing personal diploid methylomes, CpG methylome pairs of homologous chromosomes that are distinguishable with respect to phased heterozygous variants (PHVs), which is challenging due to scarcity of PHVs in personal genomes. Single molecule real-time (SMRT) sequencing is promising as it outputs long reads with CpG methylation information, but a serious concern is whether reliable PHVs are available in erroneous SMRT reads with an error rate of ∼15%. To overcome the issue, we propose a statistical model that reduces the error rate of phasing CpG site to 1%, thereby calling CpG hypomethylation in each haplotype with >90% precision and sensitivity. Using our statistical model, we examined GNAS complex locus known for a combination of maternally, paternally, or biallelically expressed isoforms, and observed allele-specific methylation pattern almost perfectly reflecting their respective allele-specific expression status, demonstrating the merit of elucidating comprehensive personal diploid methylomes and transcriptomes.

Epstein-Barr Virus-Induced Epigenetic Pathogenesis of Viral-Associated Lymphoepithelioma-Like Carcinomas and Natural Killer/T-Cell Lymphomas.

Cancer genome studies of Epstein-Barr virus (EBV)-associated tumors, including lymphoepithelioma-like carcinomas (LELC) of nasopharyngeal (NPC), gastric (EBVaGC) and lung tissues, and natural killer (NK)/T-cell lymphoma (NKTCL), reveal a unique feature of genomic alterations with fewer gene mutations detected than other common cancers. It is known now that epigenetic alterations play a critical role in the pathogenesis of EBV-associated tumors. As an oncogenic virus, EBV establishes its latent and lytic infections in B-lymphoid and epithelial cells, utilizing hijacked cellular epigenetic machinery. EBV-encoded oncoproteins modulate cellular epigenetic machinery to reprogram viral and host epigenomes, especially in the early stage of infection, using host epigenetic regulators. The genome-wide epigenetic alterations further inactivate a series of tumor suppressor genes (TSG) and disrupt key cellular signaling pathways, contributing to EBV-associated cancer initiation and progression. Profiling of genome-wide CpG methylation changes (CpG methylome) have revealed a unique epigenotype of global high-grade methylation of TSGs in EBV-associated tumors. Here, we have summarized recent advances of epigenetic alterations in EBV-associated tumors (LELCs and NKTCL), highlighting the importance of epigenetic etiology in EBV-associated tumorigenesis. Epigenetic study of these EBV-associated tumors will discover valuable biomarkers for their early detection and prognosis prediction, and also develop effective epigenetic therapeutics for these cancers.

The new 6q27 tumor suppressor DACT2, frequently silenced by CpG methylation, sensitizes nasopharyngeal cancer cells to paclitaxel and 5-FU toxicity via \u03b2-catenin/Cdc25c signaling and G2/M arrest

BackgroundNasopharyngeal carcinoma (NPC) is prevalent in South China, including Hong Kong and Southeast Asia, constantly associated with Epstein-Barr virus (EBV) infection. Epigenetic etiology attributed to EBV plays a critical role in NPC pathogenesis. Through previous CpG methylome study, we identified Disheveled-associated binding antagonist of beta-catenin 2 (DACT2) as a methylated target in NPC. Although DACT2 was shown to regulate Wnt signaling in some carcinomas, its functions in NPC pathogenesis remain unclear.MethodsRT-PCR, qPCR, MSP, and BGS were applied to measure expression levels and promoter methylation of DACT2 in NPC. Transwell, flow cytometric analysis, colony formation, and BrdU-ELISA assay were used to assess different biological functions affected by DACT2. Immunofluorescence, Western blot, and dual-luciferase reporter assay were used to explore the mechanisms of DACT2 functions. Chemosensitivity assay was used to measure the impact of DACT2 on chemotherapy drugs.ResultsWe found that DACT2 is readily expressed in multiple normal adult tissues including upper respiratory tissues. However, it is frequently downregulated in NPC and correlated with promoter methylation. DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine restored its expression in NPC cells. DACT2 methylation was further detected in 29/32 (91%) NPC tumors but not in any (0/8) normal nasopharyngeal tissue samples. Ectopic expression of DACT2 in NPC cells suppressed their proliferation, migration, and invasion through downregulating matrix metalloproteinases. DACT2 expression also induced G2/M arrest in NPC cells through directly suppressing β-catenin/Cdc25c signaling, which sensitized NPC cells to paclitaxel and 5-FU, but not cisplatin.ConclusionOur results demonstrate that DACT2 is frequently inactivated epigenetically by CpG methylation in NPC, while it inhibits NPC cell proliferation and metastasis via suppressing β-catenin/Cdc25c signaling. Our study suggests that DACT2 promoter methylation is a potential epigenetic biomarker for the detection and chemotherapy guidance of NPC.

Epigenomic characterization of a p53-regulated 3p22.2 tumor suppressor that inhibits STAT3 phosphorylation via protein docking and is frequently methylated in esophageal and other carcinomas.

Rationale: Oncogenic STAT3 signaling activation and 3p22-21.3 locus alteration are common in multiple tumors, especially carcinomas of the nasopharynx, esophagus and lung. Whether these two events are linked remains unclear. Our CpG methylome analysis identified a 3p22.2 gene, DLEC1, as a methylated target in esophageal squamous cell (ESCC), nasopharyngeal (NPC) and lung carcinomas. Thus, we further characterized its epigenetic abnormalities and functions.Methods: CpG methylomes were established by methylated DNA immunoprecipitation. Promoter methylation was analyzed by methylation-specific PCR and bisulfite genomic sequencing. DLEC1 expression and clinical significance were analyzed using TCGA database. DLEC1 functions were analyzed by transfections followed by various cell biology assays. Protein-protein interaction was assessed by docking, Western blot and immunoprecipitation analyses.Results: We defined the DLEC1 promoter within a CpG island and p53-regulated. DLEC1 was frequently downregulated in ESCC, lung and NPC cell lines and primary tumors, but was readily expressed in normal tissues and immortalized normal epithelial cells, with mutations rarely detected. DLEC1 methylation was frequently detected in ESCC tumors and correlated with lymph node metastasis, tumor recurrence and progression, with DLEC1 as the most frequently methylated among the established 3p22.2 tumor suppressors (RASSF1A, PLCD1 and ZMYND10/BLU). DLEC1 inhibits carcinoma cell growth through inducing cell cycle arrest and apoptosis, and also suppresses cell metastasis by reversing epithelial-mesenchymal transition (EMT) and cell stemness. Moreover, DLEC1 represses oncogenic signaling including JAK/STAT3, MAPK/ERK, Wnt/β-catenin and AKT pathways in multiple carcinoma types. Particularly, DLEC1 inhibits IL-6-induced STAT3 phosphorylation in a dose-dependent manner. DLEC1 contains three YXXQ motifs and forms a protein complex with STAT3 via protein docking, which blocks STAT3-JAK2 interaction and STAT3 phosphorylation. IL-6 stimulation enhances the binding of DLEC1 with STAT3, which diminishes their interaction with JAK2 and further leads to decreased STAT3 phosphorylation. The YXXQ motifs of DLEC1 are crucial for its inhibition of STAT3 phosphorylation, and disruption of these motifs restores STAT3 phosphorylation through abolishing DLEC1 binding to STAT3.Conclusions: Our study demonstrates, for the first time, predominant epigenetic silencing of DLEC1 in ESCC, and a novel mechanistic link of epigenetic DLEC1 disruption with oncogenic STAT3 signaling in multiple carcinomas.

Methyl-CpG-Binding Domain Sequencing: MBD-seq.

Detailed biological knowledge about the potential importance of the methylome is typically lacking for common diseases. Therefore, methylome-wide association studies (MWAS) are critical to detect disease relevant methylation sites. Methyl-CpG-binding domain sequencing (MBD-seq) offers potential advantages compared to antibody-based enrichment, but performance depends critically on using an optimal protocol. Using an optimized protocol, MBD-seq can approximate the sensitivity/specificity obtained with whole-genome bisulfite sequencing, but at a fraction of the costs and time to complete the project. Thus, MBD-seq offers a comprehensive first pass at the CpG methylome and is economically feasible with the samples sizes required for MWAS.