Methylome Data Research Articles

Functional complementation between transcriptional methylation regulation and post-transcriptional microRNA regulation in the human genome

BackgroundDNA methylation in the 5' promoter regions of genes and microRNA (miRNA) regulation at the 3' untranslated regions (UTRs) are two major epigenetic regulation mechanisms in most eukaryotes. Both DNA methylation and miRNA regulation can suppress gene expression and their corresponding protein product; thus, they play critical roles in cellular processes. Although there have been numerous investigations of gene regulation by methylation changes and miRNAs, there is no systematic genome-wide examination of their coordinated effects in any organism.ResultsIn this study, we investigated the relationship between promoter methylation at the transcription level and miRNA regulation at the post-transcription level by taking advantage of recently released human methylome data and high quality miRNA and other gene annotation data. We found methylation level in the promoter regions and expression level was negatively correlated. Then, we showed that miRNAs tended to target the genes with a low DNA methylation level in their promoter regions. We further demonstrated that this observed pattern was not attributed to the gene expression level, expression broadness, or the number of transcription factor binding sites. Interestingly, we found miRNA target sites were significantly enriched in the genes located in differentially methylated regions or partially methylated domains. Finally, we explored the features of DNA methylation and miRNA regulation in cancer genes and found cancer genes tended to have low methylation level and more miRNA target sites.ConclusionThis is the first genome-wide investigation of the combined regulation of gene expression. Our results supported a complementary regulation between DNA methylation (transcriptional level) and miRNA function (post-transcriptional level) in the human genome. The results were helpful for our understanding of the evolutionary forces towards organisms' complexity beyond traditional sequence level investigation.

Abstract C22: Next-generation sequencing reveals CpG island shore methylation regulates caveolin-1 expression in breast cancer

Abstract Caveolin-1 (Cav1), a multifunctional membrane protein associated with cell surface caveolae, regulates caveolae-dependent lipid trafficking, vesicular transport and signal transduction. While reported to function as a tumor suppressor at early stages of cancer progression, a growing body of evidence indicates that Cav1 also performs a tumor-promoting role in various cancer types. Cav1 expression level is decreased in breast carcinomas compared to normal tissue, and loss of Cav1 expression promotes mammary tumorigenesis and is associated with the development of tamoxifen resistance. However, gene expression profiling of breast cancer cell lines revealed an inverse relationship between expression of Cav1 and estrogen receptor-alpha (ERα), and Cav1 overexpression was observed in a subset of basal-like breast carcinomas. To investigate the mechanism regulating Cav1 expression, we performed genome-wide profiling of DNA methylation using affinity purification and subsequent next-generation sequencing of eluted DNA (MethylCap-seq) on 30 breast cancer cell lines. In breast cancer cell lines displaying low Cav1 expression (“Cav1-low;” n=15 of primarily luminal subtype), Cav1 promoter CpG island (CGI) hypermethylation was observed in only four cell lines. In the remaining Cav1-low lines, dense methylation at “CGI shores” (methylation at regions outside of CGI) was seen, and Cav1 promoter CGI methylation was strikingly low or absent. In “Cav1-high” cell lines (n=15 of basal-like subtype), hypomethylation of both promoter CGI and CGI shores was observed. By integrating the methylome data with Cav1 mRNA expression profiles, we found a significant negative correlation between Cav1 CGI shore methylation and Cav1 gene expression. Treatment of Cav1-low cells with the DNA methyltransferase inhibitor 5-aza-deoxycytidine resulted in demethylation of CGI shores and Cav1 re-expression, further confirming the regulation of Cav1 expression by CGI shore methylation. MethylCap-seq results were validated by pyrosequencing, and we observed a similar phenomenon in a panel of antiestrogen-sensitive and -resistant cell lines previously generated by our lab, with hypermethylation at CGI shores for a Cav1-low, tamoxifen-resistant line, and CGI shore hypomethylation in Cav1-high, fulvestrant-resistant lines (basal-like). SiRNA-mediated Cav1 knockdown resulted in a significant cell growth inhibition, indicating an important role of Cav1 in supporting breast cancer cell proliferation. Furthermore, by using an on-line survival analysis tool (Kaplan Meier Plotter), we determined a significant positive correlation between Cav1 levels and poor overall survival for patients with ERα-negative breast tumors. The results of this study demonstrate that DNA methylation at Cav1 CGI shores is associated with tumor progression, displaying hypomethylation in normal breast epithelial cells, hypermethylation in luminal breast cancer cells, and hypomethylation in basal-like breast cancer cells. In addition, Cav1 CGI shore methylation may represent a novel prognostic factor for ERα-negative, fulvestrant-resistant breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C22.

Structure and Function of Mammalian DNA Methyltransferases

DNA methylation plays an important role in epigenetic signalling, having an impact on gene regulation, chromatin structure, development and disease. Here, we review the structures and functions of the mammalian DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b, including their domain structures, catalytic mechanisms, localisation, regulation, post-translational modifications and interaction with chromatin and other proteins, summarising data obtained in genetic, cell biology and enzymatic studies. We focus on the question of how the molecular and enzymatic properties of these enzymes are connected to the dynamics of DNA methylation patterns and to the roles the enzymes play in the processes of de novo and maintenance DNA methylation. Recent enzymatic and genome-wide methylome data have led to a new model of genomic DNA methylation patterns based on the preservation of average levels of DNA methylation in certain regions, rather than the methylation states of individual CG sites.

Abstract 158: The landscape of DNA hypomethylation in liver cancer, its correlation with other cancers, and its potential role in cancer

Abstract DNA demethylation, although a common hallmark of cancer, has been relatively ignored. We used hepatic cellular carcinoma, which is one of the most common human cancers, as a model to delineate the landscape of hypomethylation in cancer, unravel the driving forces of hypomethylation and determine its potential role in cancer progression, metastasis and invasion. Using liver cancer cell lines as a model system, we tested whether the demethylation observed in liver cancer is driven by MBD2, a protein previously implicated in demethylation, which we found to be overexpressed in liver cancer. We used genome-wide promoter arrays and methylated DNA immunoprecipitation to map the sites in the genome that lost methyl marks in liver cancer patients. Affymetrix arrays were used for gene expression studies. The array data were validated by pyrosequencing and QPCR. For functional analyses, we used siRNA transfection, soft agar and invasion assays. We discovered that about 3700 transcript-encoding genes were demethylated in liver cancer samples compared to adjacent normal tissue. At least 350 of these 3700 genes were significantly induced in tumors. In the group of demethylated and induced genes, we identified a distinct subgroup of over 100 genes with CpG dense promoters that were highly methylated in normal liver, but demethylated in the tumors. Gene ontology analyses revealed that these genes are involved in cell growth, cell adhesion, signal transduction and invasion. Examination of the Ensemble methylome data suggests that these promoters are differentially methylated in a tissue specific manner but are always methylated in liver. All of the demethylated genes in liver cancer were found to be demethylated in other cancers suggesting that the demethylation of these genes is a basic requirement in cancer. The experiments in HepG2 and SkHep1 cells showed that knock-down of MBD2 with siRNA results in suppression of the majority of the hypomethylated genes and in a relevant decrease in cancer cell growth and invasion. Furthermore, the down-regulation of these genes was accompanied by an increase in promoter methylation and a decrease in MBD2 binding. It suggests that MBD2 is required for demethylation and up-regulation of these genes, which appear to be putative targets for MBD2. Our results demonstrated that DNA hypomethylation drives the coordinated expression of several gene circuitries involved in cancer growth, survival and metastasis. We showed that MBD2 is involved in driving this processes by affecting methylation and expression of important players in liver cancer growth and metastasis. Our studies established for the first time the rules governing hypomethylation in liver cancer and defined the potential functional role of hypomethylation. This study was supported by a grant from the MDEEI program of the government of Quebec and the National Cancer Institute of Canada to MS and MOST to ZGH Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 158.

Left Ventricular Hypertrophy in New Hemodialysis Patients without Symptomatic Cardiac Disease

Although left ventricular hypertrophy (LVH) is a characteristic finding in hemodialysis (HD) populations, few risk factors for progressive LVH have been identified. As part of a multinational, blinded, randomized, controlled trial that demonstrated no effect of hemoglobin targets on LV size, 596 incident HD patients, without symptomatic cardiac disease or cardiac dilation, had baseline echocardiograms within 18 months of starting dialysis and subsequently at 24, 48, and 96 weeks later. A wide array of baseline risk factors were assessed, as were BP and hemoglobin levels during the trial. The median age and duration of dialysis were 51.5 years and 9 months, respectively. LV mass index (LVMI) rose substantially during follow-up (114.2 g/m(2) at baseline, 121 at week 48, 123.4 at week 48, and 128.3 at week 96), as did fractional shortening, whereas LV volume (68.7, 70.1, 68.7, and 68.1 ml/m(2)) and E/A ratio remained unchanged. At baseline, the only multivariate associations of LVMI were gender and N terminal pro-B type natriuretic peptide. Comparing first and last echocardiograms in those without LVH at baseline, independent predictors of increase in LVMI were higher time-integrated systolic BP and cause of ESRD. An unadjusted association between baseline LVMI and subsequent cardiovascular events or death was eliminated by adjusting for age, diabetes, systolic BP, and N terminal pro-B type natriuretic peptide. Progressive concentric LVH and hyperkinesis occur in HD patients, which is partly explained by hypertension but not by a wide array of potential risk factors, including anemia.

Oral presentations

Considerable progress has been made in the past few years, and methylome characterization is likely to become a routine and biologically valid application of many laboratories worldwide. Molecular biomarkers should help to improve personalized medicine, and in the near future could be able to characterize patients for specific therapeutic options. We have discussed the main findings regarding DNA methylation in cancer and non-neoplastic diseases. A growing body of evidence shows that changes in the population’s exposure to diet- and lifestyle-related factors induce epigenetic changes that in turn contribute to diseases. However, the causal relationship is, to date, often uncertain. There is also increasing evidence implicating the epigenome in the development of inflammatory and rheumatic diseases. The hypothesis that methyl donors restore DNA methylation is currently under investigation. Studies gathering genomic, methylomic, transcriptomic (including microRNA and non-coding long RNAs), and proteonomic data on the same samples will emerge in the next few years.