CpG island methylation pattern in different human tissues and its correlation with gene expression

The study on DNA methylation pattern in different human tissues attracts increasing interest nowadays, but a systematic analysis of CpG island methylation pattern between both somatic tissues and gametocyte is still lacking. In this work, we analyzed the CpG island methylation data of sperm and other 11 somatic tissues from Human Epigenome Project, and found that the CpG island methylation profiles are highly correlated between somatic tissues, while the methylation profile in sperm is quite distinct. Furthermore, we observed that in the six tissues investigated, there is no obvious correlation between the methylation level of promoter CpG islands and corresponding gene expression across different tissues.     

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.     

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.     

Tissue specific DNA methylation of CpG islands in normal human adult somatic tissues distinguishes neural from non-neural tissues

Although most CpG islands are generally thought to remain unmethylated in all adult somatic tissues, recent genome-wide approaches have found that some CpG islands have distinct methylation patterns in various tissues, with most differences being seen between germ cells and somatic tissues. Few studies have addressed this among human somatic tissues and fewer still have studied the same sets of tissues from multiple individuals. In the current study, we used Restriction Landmark Genomic Scanning to study tissue specific methylation patterns in a set of 12 human tissues collected from multiple individuals. We identified 34 differentially methylated CpG islands among these tissues, many of which showed consistent patterns in multiple individuals. Of particular interest were striking differences in CpG island methylation, not only among brain regions, but also between white and grey matter of the same region. These findings were confirmed for selected loci by quantitative bisulfite sequencing. Cluster analysis of the RLGS data indicated that several tissues clustered together, but the strongest clustering was in brain. Tissues from different brain regions clustered together, and, as a group, brain tissues were distinct from either mesoderm or endoderm derived tissues which demonstrated limited clustering. These data demonstrate consistent tissue specific methylation for certain CpG islands, with clear differences between white and grey matter of the brain. Furthermore, there was an overall pattern of tissue specifically methylated CpG islands that distinguished neural tissues from non-neural.Key words: Tissue specific methylation, CpG island methylation, neural, brain tissue, grey matter, white matter     

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.     

Tissue specific DNA methylation of CpG islands in normal human adult somatic tissues distinguishes neural from non-neural tissues

Although most CpG islands are generally thought to remain unmethylated in all adult somatic tissues, recent genome-wide approaches have found that some CpG islands have distinct methylation patterns in various tissues, with most differences being seen between germ cells and somatic tissues. Few studies have addressed this among human somatic tissues and fewer still have studied the same sets of tissues from multiple individuals. In the current study, we used Restriction Landmark Genomic Scanning to study tissue specific methylation patterns in a set of twelve human tissues collected from multiple individuals. We identified 34 differentially methylated CpG islands among these tissues, many of which showed consistent patterns in multiple individuals. Of particular interest were striking differences in CpG island methylation, not only among brain regions, but also between white and grey matter of the same region. These findings were confirmed for selected loci by quantitative bisulfite sequencing. Cluster analysis of the RLGS data indicated that several tissues clustered together, but the strongest clustering was in brain. Tissues from different brain regions clustered together, and, as a group, brain tissues were distinct from either mesoderm or endoderm derived tissues which demonstrated limited clustering. These data demonstrate consistent tissue specific methylation for certain CpG islands, with clear differences between white and grey matter of the brain. Furthermore, there was an overall pattern of tissue specifically methylated CpG islands that distinguished neural tissues from non-neural.     

Comparative Analysis of CpG Islands among HBV Genotypes

DNA methylation is being increasingly recognized to play a role in regulation of hepatitis B virus (HBV) gene expression. The aim of this study was to compare the CpG island distribution among different HBV genotypes. We analyzed 176 full-length HBV genomic sequences obtained from the GenBank database, belonging to genotypes A through J, to identify the CpG islands in the HBV genomes. Our results showed that while 79 out of 176 sequences contained three conventional CpG islands (I–III) as previously described, 83 HBV sequences harbored only two of the three known islands. Novel CpG islands were identified in the remaining 14 HBV isolates and named as CpG island IV, V, and VI. Among the eight known HBV genotypes and two putative genotypes, while HBV genomes containing three CpG islands were predominant in genotypes A, B, D, E, and I; genotypes C, F, G, and H tended to contain only two CpG islands (II and III). In conclusion, the CpG islands, which are potential targets for DNA methylation mediated by the host functions, differ among HBV genotypes, and these genotype-specific differences in CpG island distribution could provide new insights into the understanding of epigenetic regulation of HBV gene expression and hepatitis B disease outcome.     

宫颈癌患者血浆和组织中FHIT基因5′端CpG岛甲基化状态的研究

为了检测宫颈癌患者血浆和组织中FHIT基因5′端CpG岛甲基化状态, 以找到无创伤性诊断宫颈癌的新指标, 选取151例宫颈癌患者的血浆和30例患者的宫颈癌组织为研究对象,用MSP的方法检测FHIT基因5′端CpG岛甲基化状态, 并对MSP产物进行克隆和测序。结果在宫颈癌患者血浆和组织中, FHIT基因5′端CpG岛甲基化率为30.46%和53.33%, 血浆和组织的总体符合率为80%。而对照中均未检测到甲基化状态。随着患者临床分期和组织学分级的增加, FHIT基因甲基化的检出率也在逐渐的增加。表明宫颈癌患者的血浆和肿瘤组织中FHIT基因5′端CpG岛甲基化的发生是高频事件, 使用FHIT基因作为标记可以对宫颈癌患者进行无创伤诊断和预后的评估。     

宫颈癌患者血浆和组织中FHIT基因5′端CpG岛甲基化状态的研究

为了检测宫颈癌患者血浆和组织中FHIT基因5′端CpG岛甲基化状态, 以找到无创伤性诊断宫颈癌的新指标, 选取151例宫颈癌患者的血浆和30例患者的宫颈癌组织为研究对象,用MSP的方法检测FHIT基因5′端CpG岛甲基化状态, 并对MSP产物进行克隆和测序。结果在宫颈癌患者血浆和组织中, FHIT基因5′端CpG岛甲基化率为30.46%和53.33%, 血浆和组织的总体符合率为80%。而对照中均未检测到甲基化状态。随着患者临床分期和组织学分级的增加, FHIT基因甲基化的检出率也在逐渐的增加。表明宫颈癌患者的血浆和肿瘤组织中FHIT基因5′端CpG岛甲基化的发生是高频事件, 使用FHIT基因作为标记可以对宫颈癌患者进行无创伤诊断和预后的评估。     

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.     

The neuronatin gene resides in a "micro-imprinted" domain on human chromosome 20q11.2.

A small fraction of the genome contains genes that are imprinted and thus expressed exclusively from one parental allele.We report here that the human neuronatin gene (NNAT) on chromosome 20q11.2 is imprinted and transcribed specifically from the paternal allele. The region containing NNAT has multiple CpG islands, and methylation analysis showed that a 1.8-kb CpG island in its promoter region exhibits differential methylation in all tissues examined. This finding is consistent with the island acting as a component of the NNAT imprint control domain. NNAT lies within the singular 8.5-kb intron of the gene encoding bladder cancer-associated protein (BLCAP), which, as we demonstrate, is not imprinted. This study provides the first example, to our knowledge, in humans of an imprinted gene contained within the genomic structure of a nonimprinted gene. Thus, NNAT is in an imprinted "microdomain," making this locus uniquely suited for the investigation of mechanisms of localized imprint regulation.     

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.     

Two CpG islands in the <Emphasis Type="Italic">SEMA3B</Emphasis> gene: Methylation in clear cell renal cell carcinoma

Earlier, methylation of a CpG island in the SEMA3B gene (3p21.31) was observed in cell lines of small-cell and non-small-cell lung carcinoma. According to NCBI (Build 36), that island belonged to intron 1 of the gene. Our study concerns the methylation of two CpG islands, promoter and intronic, in the SEMA3B gene in patients with clear cell renal cell carcinoma (RCC). Methylation-specific PCR and bisulfite sequencing revealed a high frequency of methylation in the promoter CpG island (34/61, 56%) and somewhat lower, in the intronic (17/48, 35%). A significant inverse correlation was found between the SEMA3B mRNA level and methylation of the promoter CpG island in RCC (P < 0.05 according to Fisher’s exact test). The intronic island showed no such correlation. Thus, we suggest that the methylation of the promoter CpG island contributes to the inactivation of the SEMA3B suppressor gene in RCC tissue.