Distinct functional patterns of gene promoter hypomethylation and hypermethylation in cancer genomes

Background

Aberrant DNA methylation plays important roles in carcinogenesis. However, the functional significance of genome-wide hypermethylation and hypomethylation of gene promoters in carcinogenesis currently remain unclear.

Principal Findings

Based on genome-wide methylation data for five cancer types, we showed that genes with promoter hypermethylation were highly consistent in function across different cancer types, and so were genes with promoter hypomethylation. Functions related to “developmental processes” and “regulation of biology processes” were significantly enriched with hypermethylated genes but were depleted of hypomethylated genes. In contrast, functions related to “cell killing” and “response to stimulus”, including immune and inflammatory response, were associated with an enrichment of hypomethylated genes and depletion of hypermethylated genes. We also observed that some families of cytokines secreted by immune cells, such as IL10 family cytokines and chemokines, tended to be hypomethylated in various cancer types. These results provide new hints for understanding the distinct functional roles of genome-wide hypermethylation and hypomethylation of gene promoters in carcinogenesis.

Conclusions

Genes with promoter hypermethylation and hypomethylation are highly consistent in function across different cancer types, respectively, but these two groups of genes tend to be enriched in different functions associated with cancer. Especially, we speculate that hypomethylation of gene promoters may play roles in inducing immunity and inflammation disorders in precancerous conditions, which may provide hints for improving epigenetic therapy and immunotherapy of cancer.     

Specific methylation events contribute to the transcriptional repression of the mouse tissue inhibitor of metalloproteinases-3 gene in neoplastic cells.

The tissue inhibitor of metalloproteinases-3 (TIMP-3) gene is specifically down-regulated in neoplastic cells of the mouse JB6 progression model, suggesting a role for TIMP-3 inactivation in neoplastic progression. On the basis of 5-azacytidine reversal, the mechanism for this down-regulation appears to involve changes in the methylation state of the TIMP-3 promoter. Although total genomic methylation levels are comparable, specific differences in the methylation of the TIMP-3 promoter were observed between preneoplastic and neoplastic JB6 cells at three Hpall sites, with preneoplastic cells being less methylated. Expression of antisense methyltransferase in a neoplastic JB6 variant known to be hypermethylated in TIMP-3 resulted in reactivation of the endogenous TIMP-3 gene and restoration of hypomethylated status to the three implicated Hpall sites. Thus, hypermethylation at specific sequences in the TIMP-3 promoter appears to contribute to the silencing of the gene in neoplastic cells.     

Tissue of origin determines cancer-associated CpG island promoter hypermethylation patterns

Background

Aberrant CpG island promoter DNA hypermethylation is frequently observed in cancer and is believed to contribute to tumor progression by silencing the expression of tumor suppressor genes. Previously, we observed that promoter hypermethylation in breast cancer reflects cell lineage rather than tumor progression and occurs at genes that are already repressed in a lineage-specific manner. To investigate the generality of our observation we analyzed the methylation profiles of 1,154 cancers from 7 different tissue types.

Results

We find that 1,009 genes are prone to hypermethylation in these 7 types of cancer. Nearly half of these genes varied in their susceptibility to hypermethylation between different cancer types. We show that the expression status of hypermethylation prone genes in the originator tissue determines their propensity to become hypermethylated in cancer; specifically, genes that are normally repressed in a tissue are prone to hypermethylation in cancers derived from that tissue. We also show that the promoter regions of hypermethylation-prone genes are depleted of repetitive elements and that DNA sequence around the same promoters is evolutionarily conserved. We propose that these two characteristics reflect tissue-specific gene promoter architecture regulating the expression of these hypermethylation prone genes in normal tissues.

Conclusions

As aberrantly hypermethylated genes are already repressed in pre-cancerous tissue, we suggest that their hypermethylation does not directly contribute to cancer development via silencing. Instead aberrant hypermethylation reflects developmental history and the perturbation of epigenetic mechanisms maintaining these repressed promoters in a hypomethylated state in normal cells.     

Maternal cocaine administration in mice alters DNA methylation and gene expression in hippocampal neurons of neonatal and prepubertal offspring

Previous studies documented significant behavioral changes in the offspring of cocaine-exposed mothers. We now explore the hypothesis that maternal cocaine exposure could alter the fetal epigenetic machinery sufficiently to cause lasting neurochemical and functional changes in the offspring. Pregnant CD1 mice were administered either saline or 20 mg/kg cocaine twice daily on gestational days 8-19. Male pups from each of ten litters of the cocaine and control groups were analyzed at 3 (P3) or 30 (P30) days postnatum. Global DNA methylation, methylated DNA immunoprecipitation followed by CGI(2) microarray profiling and bisulfite sequencing, as well as quantitative real-time RT-PCR gene expression analysis, were evaluated in hippocampal pyramidal neurons excised by laser capture microdissection. Following maternal cocaine exposure, global DNA methylation was significantly decreased at P3 and increased at P30. Among the 492 CGIs whose methylation was significantly altered by cocaine at P3, 34% were hypermethylated while 66% were hypomethylated. Several of these CGIs contained promoter regions for genes implicated in crucial cellular functions. Endogenous expression of selected genes linked to the abnormally methylated CGIs was correspondingly decreased or increased by as much as 4-19-fold. By P30, some of the cocaine-associated effects at P3 endured, reversed to opposite directions, or disappeared. Further, additional sets of abnormally methylated targets emerged at P30 that were not observed at P3. Taken together, these observations indicate that maternal cocaine exposure during the second and third trimesters of gestation could produce potentially profound structural and functional modifications in the epigenomic programs of neonatal and prepubertal mice.     

Genome-Wide Analysis of DNA Methylation before-and after Exercise in the Thoroughbred Horse with MeDIP-Seq

Athletic performance is an important criteria used for the selection of superior horses. However, little is known about exercise-related epigenetic processes in the horse. DNA methylation is a key mechanism for regulating gene expression in response to environmental changes. We carried out comparative genomic analysis of genome-wide DNA methylation profiles in the blood samples of two different thoroughbred horses before and after exercise by methylated-DNA immunoprecipitation sequencing (MeDIP-Seq). Differentially methylated regions (DMRs) in the pre-and post-exercise blood samples of superior and inferior horses were identified. Exercise altered the methylation patterns. After 30 min of exercise, 596 genes were hypomethylated and 715 genes were hypermethylated in the superior horse, whereas in the inferior horse, 868 genes were hypomethylated and 794 genes were hypermethylated. These genes were analyzed based on gene ontology (GO) annotations and the exercise-related pathway patterns in the two horses were compared. After exercise, gene regions related to cell division and adhesion were hypermethylated in the superior horse, whereas regions related to cell signaling and transport were hypermethylated in the inferior horse. Analysis of the distribution of methylated CpG islands confirmed the hypomethylation in the gene-body methylation regions after exercise. The methylation patterns of transposable elements also changed after exercise. Long interspersed nuclear elements (LINEs) showed abundance of DMRs. Collectively, our results serve as a basis to study exercise-based reprogramming of epigenetic traits.     

Demethylation of CpG islands in the 5' upstream regions mediates the expression of the human testis-specific gene MAGEB16 and its mouse homolog Mageb16

Tissue-specific gene expression is regulated by epigenetic modification involving trans-acting factors. Here, we identified that the human MAGEB16 gene and its mouse homolog, Mageb16, are only expressed in the testis. To investigate the mechanism governing their expression, the promoter methylation status of these genes was examined in different samples. Two CpG islands (CGIs) in the 5'' upstream region of MAGEB16 were highly demethylated in human testes, whereas they were methylated in cells without MAGEB16 expression. Similarly, the CGI in Mageb16 was hypomethylated in mouse testes but hypermethylated in other tissues and cells without Mageb16 expression. Additionally, the expression of these genes could be activated by treatment with the demethylation agent 5''-aza-2''-deoxycytidine (5''-aza-CdR). Luciferase assays revealed that both gene promoter activities were inhibited by methylation of the CGI regions. Therefore, we propose that the testis-specific expression of MAGEB16 and Mageb16 is regulated by the methylation status of their promoter regions. [BMB Reports 2014; 47(2): 86-91]     

A Comprehensive Microarray-Based DNA Methylation Study of 367 Hematological Neoplasms

Background

Alterations in the DNA methylation pattern are a hallmark of leukemias and lymphomas. However, most epigenetic studies in hematologic neoplasms (HNs) have focused either on the analysis of few candidate genes or many genes and few HN entities, and comprehensive studies are required.

Methodology/Principal Findings

Here, we report for the first time a microarray-based DNA methylation study of 767 genes in 367 HNs diagnosed with 16 of the most representative B-cell (n = 203), T-cell (n = 30), and myeloid (n = 134) neoplasias, as well as 37 samples from different cell types of the hematopoietic system. Using appropriate controls of B-, T-, or myeloid cellular origin, we identified a total of 220 genes hypermethylated in at least one HN entity. In general, promoter hypermethylation was more frequent in lymphoid malignancies than in myeloid malignancies, being germinal center mature B-cell lymphomas as well as B and T precursor lymphoid neoplasias those entities with highest frequency of gene-associated DNA hypermethylation. We also observed a significant correlation between the number of hypermethylated and hypomethylated genes in several mature B-cell neoplasias, but not in precursor B- and T-cell leukemias. Most of the genes becoming hypermethylated contained promoters with high CpG content, and a significant fraction of them are targets of the polycomb repressor complex. Interestingly, T-cell prolymphocytic leukemias show low levels of DNA hypermethylation and a comparatively large number of hypomethylated genes, many of them showing an increased gene expression.

Conclusions/Significance

We have characterized the DNA methylation profile of a wide range of different HNs entities. As well as identifying genes showing aberrant DNA methylation in certain HN subtypes, we also detected six genes—DBC1, DIO3, FZD9, HS3ST2, MOS, and MYOD1—that were significantly hypermethylated in B-cell, T-cell, and myeloid malignancies. These might therefore play an important role in the development of different HNs.     

Hierarchical Clustering of Breast Cancer Methylomes Revealed Differentially Methylated and Expressed Breast Cancer Genes

Oncogenic transformation of normal cells often involves epigenetic alterations, including histone modification and DNA methylation. We conducted whole-genome bisulfite sequencing to determine the DNA methylomes of normal breast, fibroadenoma, invasive ductal carcinomas and MCF7. The emergence, disappearance, expansion and contraction of kilobase-sized hypomethylated regions (HMRs) and the hypomethylation of the megabase-sized partially methylated domains (PMDs) are the major forms of methylation changes observed in breast tumor samples. Hierarchical clustering of HMR revealed tumor-specific hypermethylated clusters and differential methylated enhancers specific to normal or breast cancer cell lines. Joint analysis of gene expression and DNA methylation data of normal breast and breast cancer cells identified differentially methylated and expressed genes associated with breast and/or ovarian cancers in cancer-specific HMR clusters. Furthermore, aberrant patterns of X-chromosome inactivation (XCI) was found in breast cancer cell lines as well as breast tumor samples in the TCGA BRCA (breast invasive carcinoma) dataset. They were characterized with differentially hypermethylated XIST promoter, reduced expression of XIST, and over-expression of hypomethylated X-linked genes. High expressions of these genes were significantly associated with lower survival rates in breast cancer patients. Comprehensive analysis of the normal and breast tumor methylomes suggests selective targeting of DNA methylation changes during breast cancer progression. The weak causal relationship between DNA methylation and gene expression observed in this study is evident of more complex role of DNA methylation in the regulation of gene expression in human epigenetics that deserves further investigation.     

Short‐term calorie restriction ameliorates genomewide,age‐related alterations in DNA methylation

DNA methylation plays major roles in many biological processes, including aging, carcinogenesis, and development. Analyses of DNA methylation using next‐generation sequencing offer a new way to profile and compare methylomes across the genome in the context of aging. We explored genomewide DNA methylation and the effects of short‐term calorie restriction (CR) on the methylome of aged rat kidney. Whole‐genome methylation of kidney in young (6 months old), old (25 months old), and OCR (old with 4‐week, short‐term CR) rats was analyzed by methylated DNA immunoprecipitation and next‐generation sequencing (MeDIP‐Seq). CpG islands and repetitive regions were hypomethylated, but 5′‐UTR, exon, and 3′‐UTR hypermethylated in old and OCR rats. The methylation in the promoter and intron regions was decreased in old rats, but increased in OCR rats. Pathway enrichment analysis showed that the hypermethylated promoters in old rats were associated with degenerative phenotypes such as cancer and diabetes. The hypomethylated promoters in old rats related significantly to the chemokine signaling pathway. However, the pathways significantly enriched in old rats were not observed from the differentially methylated promoters in OCR rats. Thus, these findings suggest that short‐term CR could partially ameliorate age‐related methylation changes in promoters in old rats. From the epigenomic data, we propose that the hypermethylation found in the promoter regions of disease‐related genes during aging may indicate increases in susceptibility to age‐related diseases. Therefore, the CR‐induced epigenetic changes that ameliorate age‐dependent aberrant methylation may be important to CR's health‐ and life‐prolonging effects.