A DNA signal from the Thy-1 gene defines de novo methylation patterns in embryonic stem cells.

Although DNA can be extensively methylated de novo when introduced into pluripotent cells, the CpG island in the Thy-1 gene does not become methylated either in the mouse embryo or in embryonic stem cells. A 214-base-pair region near the promoter of the Thy-1 gene protects itself as well as heterologous DNA sequences from de novo methylation. We propose that this nucleotide sequence is representative of a class of important signals that limits de novo methylation in the embryo and establishes the pattern of hypomethylated CpG dinucleotides found in somatic tissues.     

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.     

Specific protection of methylated CpGs in mammalian nuclei

We have compared nuclear accessibility of methylated and nonmethylated sequences using restriction enzymes. MspI, which cuts CpG sites in naked DNA regardless of methylation, cut DNA in intact mouse liver or brain nuclei almost exclusively at CpG islands. Bulk chromatin was not significantly cleaved by MspI but was cleaved extensively by enzymes that do not recognize CpG. Quantitative analysis of limit digests showed that MspI and another methyl-CpG insensitive enzyme, Tth, have a strong bias against cutting methylated sites in these nuclei. Southern analysis confirmed this at three genomic loci. Our results suggest that resistance to nucleases is mediated by factors that are bound specifically to methylated CpGs. MeCP, a protein that binds to methylated DNA in vitro, may be one such factor, since nuclease resistance was significantly reduced in an MeCP-deficient cell line.     

Methylation-mediated silencing of TMS1/ASC is accompanied by histone hypoacetylation and CpG island-localized changes in chromatin architecture.

Aberrant methylation of CpG-dense islands in the promoter regions of genes is an acquired epigenetic alteration associated with the silencing of tumor suppressor genes in human cancers. In a screen for endogenous targets of methylation-mediated gene silencing, we identified a novel CpG island-associated gene, TMS1, which is aberrantly methylated and silenced in response to the ectopic expression of DNA methyltransferase-1. TMS1 functions in the regulation of apoptosis and is frequently methylated and silenced in human breast cancers. In this study, we characterized the methylation pattern and chromatin architecture of the TMS1 locus in normal fibroblasts and determined the changes associated with its progressive methylation. In normal fibroblasts expressing TMS1, the CpG island is defined by an unmethylated domain that is separated from densely methylated flanking DNA by distinct 5' and 3' boundaries. Analysis of the nucleoprotein architecture of the locus in intact nuclei revealed three DNase I-hypersensitive sites that map within the CpG island. Strikingly, two of these sites coincided with the 5'- and 3'-methylation boundaries. Methylation of the TMS1 CpG island was accompanied by loss of hypersensitive site formation, hypoacetylation of histones H3 and H4, and gene silencing. This altered chromatin structure was confined to the CpG island and occurred without significant changes in methylation, histone acetylation, or hypersensitive site formation at a fourth DNase I-hypersensitive site 2 kb downstream of the TMS1 CpG island. The data indicate that there are sites of protein binding and/or structural transitions that define the boundaries of the unmethylated CpG island in normal cells and that aberrant methylation overcomes these boundaries to direct a local change in chromatin structure, resulting in gene silencing.     

De novo DNA methylation at the CpG island of the zebrafish no tail gene

The zebrafish no tail gene (ntl) is indispensable for the formation of the notochord and the tail structure. Here we showed that de novo DNA methylation occurred at the CpG island of ntl. The methylation started at the segmentation stage and continued after the larval stage. However, it occurred predominantly between 14 and 48 h postfertilization, which overlaps the period in which ntl expression disappears in the notochord and the tailbud. This inverse correlation, together with the methylation-associated formation of an inaccessible chromatin structure at the ntl CpG island region, suggested the involvement of the de novo methylation in ntl repression. Since no changes in methylation patterns were observed at the CpG islands of four other zebrafish genes, there must be a mechanism in zebrafish for specific methylation of the ntl CpG island.     

A Distinct DNA-Methylation Boundary in the 5′- Upstream Sequence of the FMR1 Promoter Binds Nuclear Proteins and Is Lost in Fragile X Syndrome

We have discovered a distinct DNA-methylation boundary at a site between 650 and 800 nucleotides upstream of the CGG repeat in the first exon of the human FMR1 gene. This boundary, identified by bisulfite sequencing, is present in all human cell lines and cell types, irrespective of age, gender, and developmental stage. The same boundary is found also in different mouse tissues, although sequence homology between human and mouse in this region is only 46.7%. This boundary sequence, in both the unmethylated and the CpG-methylated modes, binds specifically to nuclear proteins from human cells. We interpret this boundary as carrying a specific chromatin structure that delineates a hypermethylated area in the genome from the unmethylated FMR1 promoter and protecting it from the spreading of DNA methylation. In individuals with the fragile X syndrome (FRAXA), the methylation boundary is lost; methylation has penetrated into the FMR1 promoter and inactivated the FMR1 gene. In one FRAXA genome, the upstream terminus of the methylation boundary region exhibits decreased methylation as compared to that of healthy individuals. This finding suggests changes in nucleotide sequence and chromatin structure in the boundary region of this FRAXA individual. In the completely de novo methylated FMR1 promoter, there are isolated unmethylated CpG dinucleotides that are, however, not found when the FMR1 promoter and upstream sequences are methylated in vitro with the bacterial M-SssI DNA methyltransferase. They may arise during de novo methylation only in DNA that is organized in chromatin and be due to the binding of specific proteins.     

DNA methylation down-regulates CDX1 gene expression in colorectal cancer cell lines

CDX1 is a homeobox protein that inhibits proliferation of intestinal epithelial cells and regulates intestine-specific genes involved in differentiation. CDX1 expression is developmentally and spatially regulated, and its expression is aberrantly down-regulated in colorectal cancers and colon cancer-derived cell lines. However, very little is known about the molecular mechanism underlying the regulation of CDX1 gene expression. In this study, we characterized the CDX1 gene structure and identified that its gene promoter contained a typical CpG island with a CpG observed/expected ratio of 0.80, suggesting that the CDX1 gene is a target of aberrant methylation. Alterations of DNA methylation in the CDX1 gene promoter were investigated in a series of colorectal cancer cell lines. Combined Bisulfite Restriction Analysis (COBRA) and bisulfite sequencing analysis revealed that the CDX1 promoter is methylated in CDX1 non-expressing colorectal cancer cell lines but not in human normal colon tissue and T84 cells, which express CDX1. Treatment with 5'-aza-2'-deoxycytidine (5-azaC), a DNA methyltransferase inhibitor, induced CDX1 expression in the colorectal cancer cell lines. Furthermore, de novo methylation was determined by establishing stably transfected clones of the CDX1 promoter in SW480 cells and demethylation by 5-azaC-activated reporter gene expression. These results indicate that aberrant methylation of the CpG island in the CDX1 promoter is one of the mechanisms that mediate CDX1 down-regulation in colorectal cancer cell lines.     

脑胶质瘤MGMT、hMLH1和hMSH2基因启动子甲基化状态研究

目的：探讨脑胶质瘤患者O6-甲基鸟嘌呤-DNA甲基转移酶基因MGMT和错配修复基因hMLH1、hMSH2启动子CpG岛甲基化状态,及其在烷化剂化疗中的意义。方法：采用甲基化特异性PCR（MSP）方法检测39例脑胶质瘤和6例正常脑组织MGMT、hMLH1和hMSH2基因启动子区的甲基化状态,免疫组化方法测定蛋白表达。结果：脑胶质瘤患者组织MGMT、hMLH1和hMSH2基因启动子区甲基化发生率分别为46.2%、10.3%和20.5%,3种基因启动子未甲基化模式与其对应蛋白表达模式相似,并与患者性别、年龄、病理类型和病理分级无明显相关性。回顾性分析患者资料,显示39例脑胶质瘤患者中,MGMT基因甲基化的患者生存期显著高于MGMT基因未甲基化患者（P〈0.05,Log-rank检验）。结论：MGMT及错配修复基因甲基化是脑胶质瘤发生过程中常见的分子事件,可能与肿瘤的发生有关;检测MGMT、hMLH1和hMSH2基因启动子甲基化状态,在判断脑胶质瘤患者预后和预测烷化剂化疗耐药性中可能具有重要意义。