Estimating and accounting for tumor purity in the analysis of DNA methylation data from cancer studies. Zheng Xiaoqi,Zhang Naiqian,Wu Hua-Jun,Wu Hao Genome biology We present a set of statistical methods for the analysis of DNA methylation microarray data, which account for tumor purity. These methods are an extension of our previously developed method for purity estimation; our updated method is flexible, efficient, and does not require data from reference samples or matched normal controls. We also present a method for incorporating purity information for differential methylation analysis. In addition, we propose a control-free differential methylation calling method when normal controls are not available. Extensive analyses of TCGA data demonstrate that our methods provide accurate results. All methods are implemented in InfiniumPurify. 10.1186/s13059-016-1143-5
    Epigenomic analysis detects aberrant super-enhancer DNA methylation in human cancer. Heyn Holger,Vidal Enrique,Ferreira Humberto J,Vizoso Miguel,Sayols Sergi,Gomez Antonio,Moran Sebastian,Boque-Sastre Raquel,Guil Sonia,Martinez-Cardus Anna,Lin Charles Y,Royo Romina,Sanchez-Mut Jose V,Martinez Ramon,Gut Marta,Torrents David,Orozco Modesto,Gut Ivo,Young Richard A,Esteller Manel Genome biology BACKGROUND:One of the hallmarks of cancer is the disruption of gene expression patterns. Many molecular lesions contribute to this phenotype, and the importance of aberrant DNA methylation profiles is increasingly recognized. Much of the research effort in this area has examined proximal promoter regions and epigenetic alterations at other loci are not well characterized. RESULTS:Using whole genome bisulfite sequencing to examine uncharted regions of the epigenome, we identify a type of far-reaching DNA methylation alteration in cancer cells of the distal regulatory sequences described as super-enhancers. Human tumors undergo a shift in super-enhancer DNA methylation profiles that is associated with the transcriptional silencing or the overactivation of the corresponding target genes. Intriguingly, we observe locally active fractions of super-enhancers detectable through hypomethylated regions that suggest spatial variability within the large enhancer clusters. Functionally, the DNA methylomes obtained suggest that transcription factors contribute to this local activity of super-enhancers and that trans-acting factors modulate DNA methylation profiles with impact on transforming processes during carcinogenesis. CONCLUSIONS:We develop an extensive catalogue of human DNA methylomes at base resolution to better understand the regulatory functions of DNA methylation beyond those of proximal promoter gene regions. CpG methylation status in normal cells points to locally active regulatory sites at super-enhancers, which are targeted by specific aberrant DNA methylation events in cancer, with putative effects on the expression of downstream genes. 10.1186/s13059-016-0879-2
    DNA methylation at enhancers identifies distinct breast cancer lineages. Fleischer Thomas,Tekpli Xavier,Mathelier Anthony,Wang Shixiong,Nebdal Daniel,Dhakal Hari P,Sahlberg Kristine Kleivi,Schlichting Ellen, ,Børresen-Dale Anne-Lise,Borgen Elin,Naume Bjørn,Eskeland Ragnhild,Frigessi Arnoldo,Tost Jörg,Hurtado Antoni,Kristensen Vessela N Nature communications Breast cancers exhibit genome-wide aberrant DNA methylation patterns. To investigate how these affect the transcriptome and which changes are linked to transformation or progression, we apply genome-wide expression-methylation quantitative trait loci (emQTL) analysis between DNA methylation and gene expression. On a whole genome scale, in cis and in trans, DNA methylation and gene expression have remarkably and reproducibly conserved patterns of association in three breast cancer cohorts (n = 104, n = 253 and n = 277). The expression-methylation quantitative trait loci associations form two main clusters; one relates to tumor infiltrating immune cell signatures and the other to estrogen receptor signaling. In the estrogen related cluster, using ChromHMM segmentation and transcription factor chromatin immunoprecipitation sequencing data, we identify transcriptional networks regulated in a cell lineage-specific manner by DNA methylation at enhancers. These networks are strongly dominated by ERα, FOXA1 or GATA3 and their targets were functionally validated using knockdown by small interfering RNA or GRO-seq analysis after transcriptional stimulation with estrogen. 10.1038/s41467-017-00510-x
    Genome-wide DNA methylation analysis reveals a prognostic classifier for non-metastatic colorectal cancer (ProMCol classifier). Gündert Melanie,Edelmann Dominic,Benner Axel,Jansen Lina,Jia Min,Walter Viola,Knebel Phillip,Herpel Esther,Chang-Claude Jenny,Hoffmeister Michael,Brenner Hermann,Burwinkel Barbara Gut OBJECTIVE:Pathological staging used for the prediction of patient survival in colorectal cancer (CRC) provides only limited information. DESIGN:Here, a genome-wide study of DNA methylation was conducted for two cohorts of patients with non-metastatic CRC (screening cohort (n=572) and validation cohort (n=274)). A variable screening for prognostic CpG sites was performed in the screening cohort using marginal testing based on a Cox model and subsequent adjustment of the p-values via independent hypothesis weighting using the methylation difference between 34 pairs of tumour and normal mucosa tissue as auxiliary covariate. From the 1000 CpG sites with the smallest adjusted p-value, 20 CpG sites with the smallest Brier score for overall survival (OS) were selected. Applying principal component analysis, we derived a prognostic methylation-based classifier for patients with non-metastatic CRC (ProMCol classifier). RESULTS:This classifier was associated with OS in the screening (HR 0.51, 95% CI 0.41 to 0.63, p=6.2E-10) and the validation cohort (HR 0.61, 95% CI 0.45 to 0.82, p=0.001). The independent validation of the ProMCol classifier revealed a reduction of the prediction error for 3-year OS from 0.127, calculated only with standard clinical variables, to 0.120 combining the clinical variables with the classifier and for 4-year OS from 0.153 to 0.140. All results were confirmed for disease-specific survival. CONCLUSION:The ProMCol classifier could improve the prognostic accuracy for patients with non-metastatic CRC. 10.1136/gutjnl-2017-314711
    MICMIC: identification of DNA methylation of distal regulatory regions with causal effects on tumorigenesis. Tong Yin,Sun Jianlong,Wong Chi Fat,Kang Qingzheng,Ru Beibei,Wong Ching Ngar,Chan April Sheila,Leung Suet Yi,Zhang Jiangwen Genome biology Aberrant promoter methylation is a common mechanism for tumor suppressor inactivation in cancer. We develop a set of tools to identify genome-wide DNA methylation in distal regions with causal effect on tumorigenesis called MICMIC. Many predictions are directly validated by dCas9-based epigenetic editing to support the accuracy and efficiency of our tool. Oncogenic and lineage-specific transcription factors are shown to aberrantly shape the methylation landscape by modifying tumor-subtype core regulatory circuitry. Notably, the gene regulatory networks orchestrated by enhancer methylation across different cancer types are seen to converge on a common architecture. MICMIC is available on https://github.com/ZhangJlab/MICMIC . 10.1186/s13059-018-1442-0
    DNA Methylation Clocks in Aging: Categories, Causes, and Consequences. Field Adam E,Robertson Neil A,Wang Tina,Havas Aaron,Ideker Trey,Adams Peter D Molecular cell Age-associated changes to the mammalian DNA methylome are well documented and thought to promote diseases of aging, such as cancer. Recent studies have identified collections of individual methylation sites whose aggregate methylation status measures chronological age, referred to as the DNA methylation clock. DNA methylation may also have value as a biomarker of healthy versus unhealthy aging and disease risk; in other words, a biological clock. Here we consider the relationship between the chronological and biological clocks, their underlying mechanisms, potential consequences, and their utility as biomarkers and as targets for intervention to promote healthy aging and longevity. 10.1016/j.molcel.2018.08.008
    Hypermethylation of gene body CpG islands predicts high dosage of functional oncogenes in liver cancer. Arechederra Maria,Daian Fabrice,Yim Annie,Bazai Sehrish K,Richelme Sylvie,Dono Rosanna,Saurin Andrew J,Habermann Bianca H,Maina Flavio Nature communications Epigenetic modifications such as aberrant DNA methylation reshape the gene expression repertoire in cancer. Here, we used a clinically relevant hepatocellular carcinoma (HCC) mouse model (Alb-R26) to explore the impact of DNA methylation on transcriptional switches associated with tumorigenesis. We identified a striking enrichment in genes simultaneously hypermethylated in CpG islands (CGIs) and overexpressed. These hypermethylated CGIs are located either in the 5'-UTR or in the gene body region. Remarkably, such CGI hypermethylation accompanied by gene upregulation also occurs in 56% of HCC patients, which belong to the "HCC proliferative-progenitor" subclass. Most of the genes upregulated and with hypermethylated CGIs in the Alb-R26 HCC model undergo the same change. Among reprogrammed genes, several are well-known oncogenes. For others not previously linked to cancer, we demonstrate here their action together as an "oncogene module". Thus, hypermethylation of gene body CGIs is predictive of elevated oncogene levels in cancer, offering a novel stratification strategy and perspectives to normalise cancer gene dosages. 10.1038/s41467-018-05550-5
    DNA methylation loss promotes immune evasion of tumours with high mutation and copy number load. Jung Hyunchul,Kim Hong Sook,Kim Jeong Yeon,Sun Jong-Mu,Ahn Jin Seok,Ahn Myung-Ju,Park Keunchil,Esteller Manel,Lee Se-Hoon,Choi Jung Kyoon Nature communications Mitotic cell division increases tumour mutation burden and copy number load, predictive markers of the clinical benefit of immunotherapy. Cell division correlates also with genomic demethylation involving methylation loss in late-replicating partial methylation domains. Here we find that immunomodulatory pathway genes are concentrated in these domains and transcriptionally repressed in demethylated tumours with CpG island promoter hypermethylation. Global methylation loss correlated with immune evasion signatures independently of mutation burden and aneuploidy. Methylome data of our cohort (n = 60) and a published cohort (n = 81) in lung cancer and a melanoma cohort (n = 40) consistently demonstrated that genomic methylation alterations counteract the contribution of high mutation burden and increase immunotherapeutic resistance. Higher predictive power was observed for methylation loss than mutation burden. We also found that genomic hypomethylation correlates with the immune escape signatures of aneuploid tumours. Hence, DNA methylation alterations implicate epigenetic modulation in precision immunotherapy. 10.1038/s41467-019-12159-9
    DNA methylation in childhood asthma: an epigenome-wide meta-analysis. Xu Cheng-Jian,Söderhäll Cilla,Bustamante Mariona,Baïz Nour,Gruzieva Olena,Gehring Ulrike,Mason Dan,Chatzi Leda,Basterrechea Mikel,Llop Sabrina,Torrent Maties,Forastiere Francesco,Fantini Maria Pia,Carlsen Karin C Lødrup,Haahtela Tari,Morin Andréanne,Kerkhof Marjan,Merid Simon Kebede,van Rijkom Bianca,Jankipersadsing Soesma A,Bonder Marc Jan,Ballereau Stephane,Vermeulen Cornelis J,Aguirre-Gamboa Raul,de Jongste Johan C,Smit Henriette A,Kumar Ashish,Pershagen Göran,Guerra Stefano,Garcia-Aymerich Judith,Greco Dario,Reinius Lovisa,McEachan Rosemary R C,Azad Raf,Hovland Vegard,Mowinckel Petter,Alenius Harri,Fyhrquist Nanna,Lemonnier Nathanaël,Pellet Johann,Auffray Charles, ,van der Vlies Pieter,van Diemen Cleo C,Li Yang,Wijmenga Cisca,Netea Mihai G,Moffatt Miriam F,Cookson William O C M,Anto Josep M,Bousquet Jean,Laatikainen Tiina,Laprise Catherine,Carlsen Kai-Håkon,Gori Davide,Porta Daniela,Iñiguez Carmen,Bilbao Jose Ramon,Kogevinas Manolis,Wright John,Brunekreef Bert,Kere Juha,Nawijn Martijn C,Annesi-Maesano Isabella,Sunyer Jordi,Melén Erik,Koppelman Gerard H The Lancet. Respiratory medicine BACKGROUND:DNA methylation profiles associated with childhood asthma might provide novel insights into disease pathogenesis. We did an epigenome-wide association study to assess methylation profiles associated with childhood asthma. METHODS:We did a large-scale epigenome-wide association study (EWAS) within the Mechanisms of the Development of ALLergy (MeDALL) project. We examined epigenome-wide methylation using Illumina Infinium Human Methylation450 BeadChips (450K) in whole blood in 207 children with asthma and 610 controls at age 4-5 years, and 185 children with asthma and 546 controls at age 8 years using a cross-sectional case-control design. After identification of differentially methylated CpG sites in the discovery analysis, we did a validation study in children (4-16 years; 247 cases and 2949 controls) from six additional European cohorts and meta-analysed the results. We next investigated whether replicated CpG sites in cord blood predict later asthma in 1316 children. We subsequently investigated cell-type-specific methylation of the identified CpG sites in eosinophils and respiratory epithelial cells and their related gene-expression signatures. We studied cell-type specificity of the asthma association of the replicated CpG sites in 455 respiratory epithelial cell samples, collected by nasal brushing of 16-year-old children as well as in DNA isolated from blood eosinophils (16 with asthma, eight controls [age 2-56 years]) and compared this with whole-blood DNA samples of 74 individuals with asthma and 93 controls (age 1-79 years). Whole-blood transcriptional profiles associated with replicated CpG sites were annotated using RNA-seq data of subsets of peripheral blood mononuclear cells sorted by fluorescence-activated cell sorting. FINDINGS:27 methylated CpG sites were identified in the discovery analysis. 14 of these CpG sites were replicated and passed genome-wide significance (p<1·14 × 10) after meta-analysis. Consistently lower methylation levels were observed at all associated loci across childhood from age 4 to 16 years in participants with asthma, but not in cord blood at birth. All 14 CpG sites were significantly associated with asthma in the second replication study using whole-blood DNA, and were strongly associated with asthma in purified eosinophils. Whole-blood transcriptional signatures associated with these CpG sites indicated increased activation of eosinophils, effector and memory CD8 T cells and natural killer cells, and reduced number of naive T cells. Five of the 14 CpG sites were associated with asthma in respiratory epithelial cells, indicating cross-tissue epigenetic effects. INTERPRETATION:Reduced whole-blood DNA methylation at 14 CpG sites acquired after birth was strongly associated with childhood asthma. These CpG sites and their associated transcriptional profiles indicate activation of eosinophils and cytotoxic T cells in childhood asthma. Our findings merit further investigations of the role of epigenetics in a clinical context. FUNDING:EU and the Seventh Framework Programme (the MeDALL project). 10.1016/S2213-2600(18)30052-3
    DNA Methylation Patterns Separate Senescence from Transformation Potential and Indicate Cancer Risk. Xie Wenbing,Kagiampakis Ioannis,Pan Lixia,Zhang Yang W,Murphy Lauren,Tao Yong,Kong Xiangqian,Kang Byunghak,Xia Limin,Carvalho Filipe L F,Sen Subhojit,Chiu Yen Ray-Whay,Zahnow Cynthia A,Ahuja Nita,Baylin Stephen B,Easwaran Hariharan Cancer cell Overall shared DNA methylation patterns between senescence (Sen) and cancers have led to the model that tumor-promoting epigenetic patterns arise through senescence. We show that transformation-associated methylation changes arise stochastically and independently of programmatic changes during senescence. Promoter hypermethylation events in transformation involve primarily pro-survival and developmental genes, similarly modified in primary tumors. Senescence-associated hypermethylation mainly involves metabolic regulators and appears early in proliferating "near-senescent" cells, which can be immortalized but are refractory to transformation. Importantly, a subset of transformation-associated hypermethylated developmental genes exhibits highest methylation gains at all age-associated cancer risk states across tissue types. These epigenetic changes favoring cell self-renewal and survival, arising during tissue aging, are fundamentally important for stratifying cancer risk and concepts for cancer prevention. 10.1016/j.ccell.2018.01.008
    Colorectal cancer: CpG island methylation indicates inferior survival outcomes. Sidaway Peter Nature reviews. Clinical oncology 10.1038/nrclinonc.2016.107
    Integration of CpG-free DNA induces de novo methylation of CpG islands in pluripotent stem cells. Takahashi Yuta,Wu Jun,Suzuki Keiichiro,Martinez-Redondo Paloma,Li Mo,Liao Hsin-Kai,Wu Min-Zu,Hernández-Benítez Reyna,Hishida Tomoaki,Shokhirev Maxim Nikolaievich,Esteban Concepcion Rodriguez,Sancho-Martinez Ignacio,Belmonte Juan Carlos Izpisua Science (New York, N.Y.) CpG islands (CGIs) are primarily promoter-associated genomic regions and are mostly unmethylated within highly methylated mammalian genomes. The mechanisms by which CGIs are protected from de novo methylation remain elusive. Here we show that insertion of CpG-free DNA into targeted CGIs induces de novo methylation of the entire CGI in human pluripotent stem cells (PSCs). The methylation status is stably maintained even after CpG-free DNA removal, extensive passaging, and differentiation. By targeting the DNA mismatch repair gene CGI, we could generate a PSC model of a cancer-related epimutation. Furthermore, we successfully corrected aberrant imprinting in induced PSCs derived from an Angelman syndrome patient. Our results provide insights into how CpG-free DNA induces de novo CGI methylation and broaden the application of targeted epigenome editing for a better understanding of human development and disease. 10.1126/science.aag3260
    DNA Hypermethylation Encroachment at CpG Island Borders in Cancer Is Predisposed by H3K4 Monomethylation Patterns. Skvortsova Ksenia,Masle-Farquhar Etienne,Luu Phuc-Loi,Song Jenny Z,Qu Wenjia,Zotenko Elena,Gould Cathryn M,Du Qian,Peters Timothy J,Colino-Sanguino Yolanda,Pidsley Ruth,Nair Shalima S,Khoury Amanda,Smith Grady C,Miosge Lisa A,Reed Joanne H,Kench James G,Rubin Mark A,Horvath Lisa,Bogdanovic Ozren,Lim Sue Mei,Polo Jose M,Goodnow Christopher C,Stirzaker Clare,Clark Susan J Cancer cell Promoter CpG islands are typically unmethylated in normal cells, but in cancer a proportion are subject to hypermethylation. Using methylome sequencing we identified CpG islands that display partial methylation encroachment across the 5' or 3' CpG island borders. CpG island methylation encroachment is widespread in prostate and breast cancer and commonly associates with gene suppression. We show that the pattern of H3K4me1 at CpG island borders in normal cells predicts the different modes of cancer CpG island hypermethylation. Notably, genetic manipulation of Kmt2d results in concordant alterations in H3K4me1 levels and CpG island border DNA methylation encroachment. Our findings suggest a role for H3K4me1 in the demarcation of CpG island methylation borders in normal cells, which become eroded in cancer. 10.1016/j.ccell.2019.01.004
    CpG Island Methylation in Sessile Serrated Adenomas Increases With Age, Indicating Lower Risk of Malignancy in Young Patients. Liu Cheng,Bettington Mark L,Walker Neal I,Dwine Joel,Hartel Gunter F,Leggett Barbara A,Whitehall Vicki L J Gastroenterology Among sessile serrated adenomas (SSAs) with identical histologic features, some never progress, whereas others become dysplastic and develop into invasive cancers. Development of the CpG island methylator phenotype is a feature of SSA progression; we examined the CIMP status of 448 SSAs and examined the association with patient clinical data. Overall, 190 SSAs were CpG island methylator phenotype-positive. CpG island methylator phenotype positivity was associated with older patient age (P < .001) and proximal polyp site (P < .001), but not with patient sex (P = .94) or polyp size (P = .34). These results might be used to improve SSA surveillance guidelines. 10.1053/j.gastro.2018.07.012
    CpG Island Hypermethylation Mediated by DNMT3A Is a Consequence of AML Progression. Spencer David H,Russler-Germain David A,Ketkar Shamika,Helton Nichole M,Lamprecht Tamara L,Fulton Robert S,Fronick Catrina C,O'Laughlin Michelle,Heath Sharon E,Shinawi Marwan,Westervelt Peter,Payton Jacqueline E,Wartman Lukas D,Welch John S,Wilson Richard K,Walter Matthew J,Link Daniel C,DiPersio John F,Ley Timothy J Cell DNMT3A mutations occur in ∼25% of acute myeloid leukemia (AML) patients. The most common mutation, DNMT3A, has dominant negative activity that reduces DNA methylation activity by ∼80% in vitro. To understand the contribution of DNMT3A-dependent methylation to leukemogenesis, we performed whole-genome bisulfite sequencing of primary leukemic and non-leukemic cells in patients with or without DNMT3A mutations. Non-leukemic hematopoietic cells with DNMT3A displayed focal methylation loss, suggesting that hypomethylation antedates AML. Although virtually all AMLs with wild-type DNMT3A displayed CpG island hypermethylation, this change was not associated with gene silencing and was essentially absent in AMLs with DNMT3A mutations. Primary hematopoietic stem cells expanded with cytokines were hypermethylated in a DNMT3A-dependent manner, suggesting that hypermethylation may be a response to, rather than a cause of, cellular proliferation. Our findings suggest that hypomethylation is an initiating phenotype in AMLs with DNMT3A, while DNMT3A-dependent CpG island hypermethylation is a consequence of AML progression. 10.1016/j.cell.2017.01.021