Genome-wide profiling of DNA methylation reveals a class of normally methylated CpG island promoters

The role of CpG island methylation in normal development and cell differentiation is of keen interest, but remains poorly understood. We performed comprehensive DNA methylation profiling of promoter regions in normal peripheral blood by methylated CpG island amplification in combination with microarrays. This technique allowed us to simultaneously determine the methylation status of 6,177 genes, 92% of which include dense CpG islands. Among these 5,549 autosomal genes with dense CpG island promoters, we have identified 4.0% genes that are nearly completely methylated in normal blood, providing another exception to the general rule that CpG island methylation in normal tissue is limited to X inactivation and imprinted genes. We examined seven genes in detail, including ANKRD30A, FLJ40201, INSL6, SOHLH2, FTMT, C12orf12, and DPPA5. Dense promoter CpG island methylation and gene silencing were found in normal tissues studied except testis and sperm. In both tissues, bisulfite cloning and sequencing identified cells carrying unmethylated alleles. Interestingly, hypomethylation of several genes was associated with gene activation in cancer. Furthermore, reactivation of silenced genes could be induced after treatment with a DNA demethylating agent or in a cell line lacking DNMT1 and/or DNMT3b. Sequence analysis identified five motifs significantly enriched in this class of genes, suggesting that cis-regulatory elements may facilitate preferential methylation at these promoter CpG islands. We have identified a group of non-X-linked bona fide promoter CpG islands that are densely methylated in normal somatic tissues, escape methylation in germline cells, and for which DNA methylation is a primary mechanism of tissue-specific gene silencing.     

DNA methylation in states of cell physiology and pathology

DNA methylation is one of epigenetic mechanisms regulating gene expression. The methylation pattern is determined during embryogenesis and passed over to differentiating cells and tissues. In a normal cell, a significant degree of methylation is characteristic for extragenic DNA (cytosine within the CG dinucleotide) while CpG islands located in gene promoters are unmethylated, except for inactive genes of the X chromosome and the genes subjected to genomic imprinting. The changes in the methylation pattern, which may appear as the organism age and in early stages of cancerogenesis, may lead to the silencing of over ninety endogenic genes. It has been found, that these disorders consist not only of the methylation of CpG islands, which are normally unmethylated, but also of the methylation of other dinucleotides, e.g. CpA. Such methylation has been observed in non-small cell lung cancer, in three regions of the exon 5 of the p53 gene (so-called "non-CpG" methylation). The knowledge of a normal methylation process and its aberrations appeared to be useful while searching for new markers enabling an early detection of cancer. With the application of the Real-Time PCR technique (using primers for methylated and unmethylated sequences) five new genes which are potential biomarkers of lung cancer have been presented.     

Genome-wide analysis of DNA methylation status of CpG islands in embryoid bodies, teratomas, and fetuses

Differentiation of embryonic stem (ES) cells into embryoid bodies (EBs) provides an in vitro system for the study of early lineage determination during mammalian development. We have previously reported that there are 247 CpG islands that potentially have tissue-dependent and differentially methylated regions (T-DMRs). This provided evidence that the formation of DNA methylation patterns at CpG islands is a crucial epigenetic event underlying mammalian development. Here we present an analysis by the restriction landmark genomic scanning (RLGS) using NotI as a landmark enzyme of the genome-wide methylation status of CpG islands of ES cells and EBs and of teratomas produced from ES cells. These results are considered in relation to the methylation status of CpG islands of genomic DNA from normal fetus (10.5 dpc) and adult tissues. We have prepared a DNA methylation panel that consists of 259 T-DMRs and includes novel T-DMRs that are distinctly methylated or unmethylated in the teratomas. The DNA methylation pattern was complex and differed for the ES cells, EBs, and teratomas, providing evidence that differentiation of cells involves both de novo DNA methylation as well as demethylation. Comparison of the numbers of T-DMRs, that were differentially methylated or unmethylated among the cells and tissue types studied, revealed that the teratomas were the most epigenetically different from ES cells. Thus, analysis of the DNA methylation profiles prepared in this study provides new insights into the differentiation of ES cells and development of fetus, EB, teratoma, and somatic tissues.     

DNA methylation establishment of CpG islands near maternally imprinted genes on chromosome 7 during mouse oocyte growth

The genome methylation is globally erased in early fetal germ cells, and it is gradually re‐established during gametogenesis. The expression of some imprinted genes is regulated by the methylation status of CpG islands, while the exact time of DNA methylation establishment near maternal imprinted genes during oocyte growth is not well known. Here, growing oocytes were divided into three groups based on follicle diameters including the S‐group (60–100 μm), M‐group (100–140 μm), and L‐group (140–180 μm). The fully grown germinal vesicle (GV)‐stage and metaphase II (M2)‐stage mature oocytes were also collected. These oocytes were used for single‐cell bisulfite sequencing to detect the methylation status of CpG islands near imprinted genes on chromosome 7. The results showed that the CpG islands near Ndn, Magel2, Mkrn3, Peg12, and Igf2 were completely unmethylated, but those of Peg3, Snrpn, and Kcnq1ot1 were hypermethylated in MII‐stage oocytes. The methylation of CpG islands near different maternal imprinted genes occurred asynchronously, being completed in later‐stage growing oocytes, fully grown GV oocytes, and mature MII‐stage oocytes, respectively. These results show that CpG islands near some maternally imprinted genes are not necessarily methylated, and that the establishment of methylation of other maternally imprinted genes is completed at different stages of oocyte growth, providing a novel understanding of the establishment of maternally imprinted genes in oocytes.     

The CpG island promoter of the human proopiomelanocortin gene is methylated in nonexpressing normal tissue and tumors and represses expression

Ectopic secretion of ACTH, from sites such as small cell lung cancer (SCLC), results in severe Cushing's syndrome. ACTH is cleaved from POMC. The syndrome may occur when the highly tissue-specific promoter of the human POMC gene (POMC) is activated. The mechanism of activation is not fully understood. This promoter is embedded within a defined CpG island, and CpG islands are usually considered to be unmethylated in all tissues. We demonstrate that much of this CpG island is methylated in normal nonexpressing tissues, in contrast to somatically expressed CpG island promoters reported to date, and is specifically unmethylated in expressing tissues, tumors, and the POMC-expressing DMS-79 SCLC cell line. A narrow 100-bp region is free of methylation in all tissues. E2F factors binding to the upstream domain IV region of the promoter have been shown to be involved in the expression of POMC in SCLC. We show that these sites are methylated in normal nonexpressing tissues, which will prevent binding of E2F, but are unmethylated in expressing tissue. Methylation in vitro is sufficient for silencing of expression, which is not reversed by treatment with Trichostatin A, suggesting that inhibition of expression may be mediated by means other than recruitment of histone deacetylase activity. The DMS-79 cells lack POMC demethylating activity, implying that the methylation and expression patterns are likely to be set early or before neoplastic transformation, and that targeted de novo methylation might be a potential therapeutic strategy.     

小鼠骨髓细胞中p16和Ralb基因启动区CpG岛的甲基化位点分析

抑癌基因p16和白血病致癌因子Ralb与白血病的发生密切相关,其启动子区CpG岛的甲基化对基因表达具有重要作用.本文旨在分析p16、Ralb基因启动子区CpG岛甲基化位点信息,并比较这两个基因在小鼠骨髓细胞和原代培养的骨髓细胞中甲基化状态的差异.运用"MethPrimer"软件预测p16、Ralb基因启动子区的CpG岛,设计甲基化特异性引物.利用重亚硫酸盐测序法(BSP)检测甲基化位点信息.结果显示,p16有1个CpG岛,岛上21个CpG位点全部未发生甲基化;Ralb有2个CpG岛,CpG岛1上的5个CpG位点全部呈甲基化状态,而CpG岛2上的17个CpG位点全部呈非甲基化状态,且小鼠骨髓细胞和体外原代培养的骨髓细胞中两基因的甲基化状态一致.表明p16、Ralb基因甲基化状态未受外界培养条件的影响而改变,提示在与两基因甲基化相关的研究中体外试验可替代体内试验.     

DNA methylome profiling identifies novel methylated genes in African American patients with colorectal neoplasia

The identification of genes that are differentially methylated in colorectal cancer (CRC) has potential value for both diagnostic and therapeutic interventions specifically in high-risk populations such as African Americans (AAs). However, DNA methylation patterns in CRC, especially in AAs, have not been systematically explored and remain poorly understood. Here, we performed DNA methylome profiling to identify the methylation status of CpG islands within candidate genes involved in critical pathways important in the initiation and development of CRC. We used reduced representation bisulfite sequencing (RRBS) in colorectal cancer and adenoma tissues that were compared with DNA methylome from a healthy AA subject’s colon tissue and peripheral blood DNA. The identified methylation markers were validated in fresh frozen CRC tissues and corresponding normal tissues from AA patients diagnosed with CRC at Howard University Hospital. We identified and validated the methylation status of 355 CpG sites located within 16 gene promoter regions associated with CpG islands. Fifty CpG sites located within CpG islands—in genes ATXN7L1 (2), BMP3 (7), EID3 (15), GAS7 (1), GPR75 (24), and TNFAIP2 (1)—were significantly hypermethylated in tumor vs. normal tissues (P < 0.05). The methylation status of BMP3, EID3, GAS7, and GPR75 was confirmed in an independent, validation cohort. Ingenuity pathway analysis mapped three of these markers (GAS7, BMP3 and GPR) in the insulin and TGF-β1 network—the two key pathways in CRC. In addition to hypermethylated genes, our analysis also revealed that LINE-1 repeat elements were progressively hypomethylated in the normal-adenoma-cancer sequence. We conclude that DNA methylome profiling based on RRBS is an effective method for screening aberrantly methylated genes in CRC. While previous studies focused on the limited identification of hypermethylated genes, ours is the first study to systematically and comprehensively identify novel hypermethylated genes, as well as hypomethylated LINE-1 sequences, which may serve as potential biomarkers for CRC in African Americans. Our discovered biomarkers were intimately linked to the insulin/TGF-B1 pathway, further strengthening the association of diabetic disorders with colon oncogenic transformation.     

CpG island methylation in human lymphocytes is highly correlated with DNA sequence, repeats, and predicted DNA structure

CpG island methylation plays an important role in epigenetic gene control during mammalian development and is frequently altered in disease situations such as cancer. The majority of CpG islands is normally unmethylated, but a sizeable fraction is prone to become methylated in various cell types and pathological situations. The goal of this study is to show that a computational epigenetics approach can discriminate between CpG islands that are prone to methylation from those that remain unmethylated. We develop a bioinformatics scoring and prediction method on the basis of a set of 1,184 DNA attributes, which refer to sequence, repeats, predicted structure, CpG islands, genes, predicted binding sites, conservation, and single nucleotide polymorphisms. These attributes are scored on 132 CpG islands across the entire human Chromosome 21, whose methylation status was previously established for normal human lymphocytes. Our results show that three groups of DNA attributes, namely certain sequence patterns, specific DNA repeats, and a particular DNA structure, are each highly correlated with CpG island methylation (correlation coefficients of 0.64, 0.66, and 0.49, respectively). We predicted, and subsequently experimentally examined 12 CpG islands from human Chromosome 21 with unknown methylation patterns and found more than 90% of our predictions to be correct. In addition, we applied our prediction method to analyzing Human Epigenome Project methylation data on human Chromosome 6 and again observed high prediction accuracy. In summary, our results suggest that DNA composition of CpG islands (sequence, repeats, and structure) plays a significant role in predisposing CpG islands for DNA methylation. This finding may have a strong impact on our understanding of changes in CpG island methylation in development and disease.     

Demethylation of somatic and testis-specific histone H2A and H2B genes in F9 embryonal carcinoma cells.

In contrast to many other genes containing a CpG island, the testis-specific H2B (TH2B) histone gene exhibits tissue-specific methylation patterns in correlation with gene activity. Characterization of the methylation patterns within a 20-kb segment containing the TH2A and TH2B genes in comparison with that in a somatic histone cluster revealed that: (i) the germ cell-specific unmethylated domain of the TH2A and TH2B genes is defined as a small region surrounding the CpG islands of the TH2A and TH2B genes and (ii) somatic histone genes are unmethylated in both liver and germ cells, like other genes containing CpG islands, whereas flanking sequences are methylated. Transfection of in vitro-methylated TH2B, somatic H2B, and mouse metallothionein I constructs into F9 embryonal carcinoma cells revealed that the CpG islands of the TH2A and TH2B genes were demethylated like those of the somatic H2A and H2B genes and the metallothionein I gene. The demethylation of those CpG islands became significantly inefficient at a high number of integrated copies and a high density of methylated CpG dinucleotides. In contrast, three sites in the somatic histone cluster, of which two sites are located in the long terminal repeat of an endogenous retrovirus-like sequence, were efficiently demethylated even at a high copy number and a high density of methylated CpG dinucleotides. These results suggest two possible mechanisms for demethylation in F9 cells and methylation of CpG islands of the TH2A and TH2B genes at the postblastula stage during embryogenesis.     

A human CpG island randomly inserted into a plant genome is protected from methylation

In vertebrate genomes the dinucleotide CpG is heavily methylated, except in CpG islands, which are normally unmethylated. It is not clear why the CpG islands are such poor substrates for DNA methyltransferase. Plant genomes display methylation, but otherwise the genomes of plants and animals represent two very divergent evolutionary lines. To gain a further understanding of the resistance of CpG islands to methylation, we introduced a human CpG island from the proteasome-like subunit I gene into the genome of the plant Arabidopsis thaliana. Our results show that prevention of methylation is an intrinsic property of CpG islands, recognized even if a human CpG island is transferred to a plant genome. Two different parts of the human CpG island – the promoter region/ first exon and exon2–4 – both displayed resistance against methylation, but the promoter/ exon1 construct seemed to be most resistant. In contrast, certain sites in a plant CpG-rich region used as a control transgene were always methylated. The frequency of silencing of the adjacent nptII (Km^R) gene in the human CpG constructs was lower than observed for the plant CpG-rich region. These results have implications for understanding DNA methylation, and for construction of vectors that will reduce transgene silencing.