CpG islands: features and distribution in the genomes of vertebrates

We have investigated the distribution of unmethylated CpG islands in vertebrate genomes fractionated according to their base composition. Genomes from warm-blooded vertebrates (man, mouse and chicken) are characterized by abundant CpG islands, whose frequency increases in DNA fractions of increasing % of guanine + cytosine; % G + C (GC), in parallel with the distribution of genes and CpG doublets. Small, yet significant, differences in the distribution of CpG islands were found in the three genomes. In contrast, genomes from cold-blooded vertebrates (two reptiles, one amphibian, and two fishes) were characterized by an extreme scarcity or absence of CpG islands (detected in these experiments as HpaII tiny fragments or HTF). CpG islands associated with homologous genes from cold- and warm-blooded vertebrates were then compared by analyzing CpG frequencies, GC levels, HpaII sites, rare-cutter sites and G/C boxes (GGGGCGGGGC and closely related motifs) in sequences available in gene banks. Small, yet significant, differences were again detected among the CpG islands associated with homologous genes from warm-blooded vertebrates, in that CpG islands associated with mouse or rat genes often showed low CpG and/or GC levels, as well as low numbers of HpaII sites, rare-cutter sites and G/C boxes, compared to homologous human genes; more rarely, CpG islands were just absent. As far as cold-blooded vertebrates were concerned, a number of genes showed CpG islands, which exhibited a much lower frequency of CpG doublets than that found in CpG islands of warm-blooded vertebrates, but still approached the statistically expected frequency; none of the other features of CpG islands associated with genes from warm-blooded vertebrates were present. Other genes did not show any associated CpG islands, unlike their homologues from warm-blooded vertebrates.     

In vitro DNA cytosine methylation of cis-regulatory elements modulates c-Ha-ras promoter activity in vivo.

The effect of DNA cytosine methylation on promoter activity was assessed using a transient expression system employing pHrasCAT. This 551 bp Ha-ras-1 gene promoter region is enriched with 84 CpG dinucleotides, six functional GC boxes, and is prototypic of many genes possessing CpG islands in their promoter regions. Bacterial modification enzymes HhaI methyl transferase (MTase) and HpaII MTase, alone or in combination with a human placental DNA methyltransferase (HP MTase) that methylates CpG sites in a generalized manner, including asymmetric elements such as GC box CpG's, were used to methylate at different types of sites in the promoter. Methylation of HhaI and HpaII sites reduced CAT expression by approximately 70%-80%, whereas methylation at generalized CpG sites with HP MTase inactivated the promoter by greater than 95%. The inhibition of H-ras promoter activity was not attributable to methylation-induced differences in DNA uptake or stability in the cell, topological form of the plasmid, or methylation effects in non-promoter regions.     

Effect of CpG methylation on gene expression in transfected plant protoplasts

Activity of the cat gene driven by the cauliflower mosaic virus 35S promoter has been assayed by transfecting petunia protoplasts with the pUC8CaMVCAT plasmid. In vitro methylation of this plasmid with M.HpaII (methylates C in CCGG sites) and M.HhaI (methylates GCGC sites) did not affect bacterial chloramphenicol acetyltransferase (CAT) activity. It should be noted, however, that no HpaII or HhaI sites are present in the promoter sequence. In contrast, in vitro methylation of the plasmid with the spiroplasma methylase M.SssI, which methylates all CpG sites, resulted in complete inhibition of CAT activity. The promoter sequence contains 16 CpG sites and 13 CpNpG sites that are known to be methylation sites in plant DNA. In the light of this fact, and considering the results of the experiments presented here, we conclude that methylation at all CpG sites leaving CpNpG sites unmethylated is sufficient to block gene activity in a plant cell. Methylation of CpNpG sites in plant cells may, therefore, play a role other than gene silencing.     

Active X chromosome DNA is unmethylated at eight CCGG sites clustered in a guanine-plus-cytosine-rich island at the 5'' end of the gene for phosphoglycerate kinase.

The 5' control region and first exon for human X-chromosome-linked phosphoglycerate kinase is contained in a G + C-rich island. We measured methylation at all HpaII sites in this 5' region of leukocyte DNA. By use of a blotting procedure that allows analysis of small DNA fragments, we found that the HpaII sites are entirely methylated when from an inactive X chromosome and entirely unmethylated when from an active one. In contrast, methylation of HpaII sites in more downstream regions of the gene is essentially the same in active and inactive X chromosomes.     

The distribution of genes on human chromosomes as studied by in situ nick translation.

We have studied the distribution of potentially active genes on human chromosomes, using two methods: DNAse I hypersensitivity and restriction enzyme--nick translation with enzymes sensitive to methylation of CpG doublets. DNAse hypersensitivity is known to be associated with potentially active genes, and, when the reaction is detected by "in situ" nick translation, produces an R-banding pattern. Digestion of chromosomes with HpaII or CfoI, both of which should preferentially cut unmethylated sequences in the CpG islands associated with the majority of genes, also produces R-banding patterns. Deviations are attributable to overdigestion of the chromosomes, leading to extraction of DNA and loss of the specific sites that were to be detected. Contrary to the results of a number of previous workers, we have failed to demonstrate any differences between the DNAse I hypersensitivity or the degree of methylation of the active and inactive X chromosomes in metaphases from females.     

A sensitive new method for rapid detection of abnormal methylation patterns in global DNA and within CpG islands.

To assess alterations in DNA methylation density in both global DNA and within CpG islands, we have developed a simple method based on the use of methylation-sensitive restriction endonucleases that leave a 5' guanine overhang after DNA cleavage, with subsequent single nucleotide extension with radiolabeled [(3)H]dCTP. The methylation-sensitive restriction enzymes HpaII and AciI have relatively frequent recognition sequences at CpG sites that occur randomly throughout the genome. BssHII is a methylation sensitive enzyme that similarly leaves a guanine overhang, but the recognition sequence is nonrandom and occurs predominantly at unmethylated CpG sites within CpG islands. The selective use of these enzymes can be used to screen for alterations in genome-wide methylation and CpG island methylation status, respectively. The extent of [(3)H]dCTP incorporation opposite the exposed guanine after restriction enzyme treatment is directly proportional to the number of unmethylated (cleaved) CpG sites. The "cytosine-extension assay" has several advantages over existing methods because (a) radiolabel incorporation is independent of the integrity of the DNA, (b) methylation detection does not require PCR amplification or DNA methylase reactions, and (c) it is applicable to ng quantities of DNA. Using DNA extracted from normal human liver and from human hepatocellular carcinoma, the applicability of the assay is demonstrated by the detection of an increase in genome-wide hypomethylation and CpG island hypermethylation in the tumor DNA.     

Isolation and enrichment of human genomic CpG islands by methylation-sensitive mirror orientation selection

CpG islands (CGIs) in human genomic DNA are GC-rich fragments whose aberrant methylation is associated with human disease development. In the current study, methylation-sensitive mirror orientation selection (MS-MOS) was developed to efficiently isolate and enrich unmethylated CGIs from human genomic DNA. The unmethylated CGIs prepared by the MS-MOS procedure subsequently were used to construct a CGI library. Then the sequence characteristics of cloned inserts of the library were analyzed by bioinformatics tools, and the methylation status of CGI clones was analyzed by HpaII PCR. The results showed that the MS-MOS method could be used to isolate up to 0.001% of differentially existed unmethylated DNA fragments in two complex genomic DNA. In the CGI library, 34.1% of clones had insert sequences satisfying the minimal criteria for CGIs. Excluding duplicates, 22.0% of the 80,000 clones were unique CGI clones, representing 60% of all the predicted CGIs (about 29,000) in human genomic DNA, and most or all of the CGI clones were unmethylated in human normal cell DNA based on the HpaII PCR analysis results of randomly selected CGI clones. In conclusion, MS-MOS was an efficient way to isolate and enrich human genomic CGIs. The method has powerful potential application in the comprehensive identification of aberrantly methylated CGIs associated with human tumorigenesis to improve understanding of the epigenetic mechanisms involved.     

Evaluation of single CpG sites as proxies of CpG island methylation states at the genome scale

Methylation of a CpG island is a faithful marker of silencing of its associated gene. Different approaches report the methylation status of a CpG island based on the determination of one or a few CpG sites by assuming the homogeneity of methylation along the element. This strategy is frequently applied in both locus-specific and genome-wide studies, but often without a validation of the representativeness of the interrogated CpG site compared with the whole element. We have evaluated the predictive informativeness of the HpaII sites located in CpG islands using data from high-resolution methylome maps, which offer the possibility to assess the methylation homogeneity of each CpG island and to determine the reporter accuracy of single sites as surrogate markers. An excellent correlation was observed between the HpaII and CpG island methylation levels (r > 0.93). At the qualitative level, the predictive sensitivity of HpaII was >95% with >92% specificity for methylated CpG islands and >90% sensitivity with >95% specificity for unmethylated CpG islands. This analysis provides a global validation framework for strategies based on the use of the methylation-sensitive HpaII restriction enzyme.     

Key Features of Cereal Genome Organization as Revealed by the Use of Cytosine Methylation-Sensitive Restriction Endonucleases

Unlike mammalian genomes, cereal (Gramineae) genomes exhibit little suppression of CpG dinucleotides. In cereal genomes, however, most of the numerous potential recognition sites for CpG methylation-sensitive restriction enzymes are methylated. Analysis of cereal genomic libraries and of regions flanking genes indicates that unmethylated NotI sites are useful landmarks for regions containing genes/single-copy sequences. Studies of a rye chromosome arm indicate that its pericentromeric region has a reduced density of unmethylated NotI (and MluI) sites and therefore of genes. Unmethylated MluI and NruI sites are distributed nonrandomly in the genomes of wheat, barley, and rice. Analysis of the genomic blocks defined by these sites in wheat and barley indicates that they are most likely to have arisen by amplification. These observations form the basis of a proposed model for the organization and evolution of the wheat, barley, and rice genomes.     

Regionally Specific and Genome-Wide Analyses Conclusively Demonstrate the Absence of CpG Methylation in Human Mitochondrial DNA

Although CpG methylation clearly distributes genome-wide in vertebrate nuclear DNA, the state of methylation in the vertebrate mitochondrial genome has been unclear. Several recent reports using immunoprecipitation, mass spectrometry, and enzyme-linked immunosorbent assay methods concluded that human mitochondrial DNA (mtDNA) has much more than the 2 to 5% CpG methylation previously estimated. However, these methods do not provide information as to the sites or frequency of methylation at each CpG site. Here, we have used the more definitive bisulfite genomic sequencing method to examine CpG methylation in HCT116 human cells and primary human cells to independently answer these two questions. We found no evidence of CpG methylation at a biologically significant level in these regions of the human mitochondrial genome. Furthermore, unbiased next-generation sequencing of sodium bisulfite treated total DNA from HCT116 cells and analysis of genome-wide sodium bisulfite sequencing data sets from several other DNA sources confirmed this absence of CpG methylation in mtDNA. Based on our findings using regionally specific and genome-wide approaches with multiple human cell sources, we can definitively conclude that CpG methylation is absent in mtDNA. It is highly unlikely that CpG methylation plays any role in direct control of mitochondrial function.     

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.     

The loss of CpC dinucleotides from DNA. II. Methylated and non-methylated genes of vertebrates

The frequencies of neighboring b.p. in more than 1100 genes of vertebrates in the EMBL bank (1000 kb) have been analysed. It has been found that the majority of such genes exhibit a lack of CpG duplexes and an excess of TpG+CpA. The loss of CpG may indicate that the major part of these sites in the genome is methylated and has been subjected to the pressure of CpG----TpG+CpA mutations. The methylated genes grouped into compartment M+ are represented by a fraction of repeated sequences and by genes of the most rapidly diverging families of proteins (globins, immunoglobulins, structural proteins, etc.). The genes of this compartment are characterized by a correlation between the G+C content and the value of CpG-suppression. A group of genes has been detected in which the CpG mutation process has gone so far that nearly all of these dinucleotides have disappeared from DNA. Judging by the value of CpG-suppression, these genes, grouped in the Mo+ compartment, used to be strongly methylated before. However, in the now extant vertebrates they have fully depleted their CpG reserve and for this reason lost the methylation capacity. Transitions in methylated CpG may be one of the sources of spontaneous mutagenesis resulting in the enhanced genetic instability of the cell. A gene compartment has been detected with an intermediate level of CpG deficiency; this compartment has been designated as M+. In these genes only a few of the available CpGs have been steadily methylated (and subjected to mutation). It has been found that the genome of vertebrates contains a specific CpG-rich fraction which exhibits no CpG-suppression, irrespective of the overall content of G+C. Probably, CpG sites have persisted unmethylated throughout the existence of these genes. We suggest them to constitute a M- compartment. This compartment comprises the genes of tRNA and rRNA (5S, 5.8S, 18S, 28S) and small nuclear RNAs U2-U6, as well as the genes of core histones, some enzymes, viruses and 5'-flanking sequences of certain protein-coding genes. In the genome of vertebrates, the genes of the evolutionary most conserved proteins and RNAs have not undergone methylation. A list of genes, belonging to different compartments of the vertebrate genome, is given. Compartment Mo+ constitutes 19%, M(+)--35%, M(+/-)--28% and M(-)--8% of all the vertebrate genes studied. Possible mechanisms, protecting the functionally most significant genes of vertebrates from methylation, and discussed.     

The molecular basis for the lack of immunostimulatory activity of vertebrate DNA

Macrophages and B cells are activated by unmethylated CpG-containing sequences in bacterial DNA. The lack of activity of self DNA has generally been attributed to CpG suppression and methylation, although the role of methylation is in doubt. The frequency of CpG in the mouse genome is 12.5% of Escherichia coli, with unmethylated CpG occurring at approximately 3% the frequency of E. coli. This suppression of CpG alone is insufficient to explain the inactivity of self DNA; vertebrate DNA was inactive at 100 micro g/ml, 3000 times the concentration at which E. coli DNA activity was observed. We sought to resolve why self DNA does not activate macrophages. Known active CpG motifs occurred in the mouse genome at 18% of random occurrence, similar to general CpG suppression. To examine the contribution of methylation, genomic DNAs were PCR amplified. Removal of methylation from the mouse genome revealed activity that was 23-fold lower than E. coli DNA, although there is only a 7-fold lower frequency of known active CpG motifs in the mouse genome. This discrepancy may be explained by G-rich sequences such as GGAGGGG, which potently inhibited activation and are found in greater frequency in the mouse than the E. coli genome. In summary, general CpG suppression, CpG methylation, inhibitory motifs, and saturable DNA uptake combined to explain the inactivity of self DNA. The immunostimulatory activity of DNA is determined by the frequency of unmethylated stimulatory sequences within an individual DNA strand and the ratio of stimulatory to inhibitory sequences.     

Methylation pattern of mouse mitochondrial DNA.

The pattern of methylation of mouse mitochondrial DNA (mtDNA) was studied using several techniques. By employing a sensitive analytical procedure it was possible to show that this DNA contains the modified base 5-methylcytosine (m5Cyt). This residue occurred exclusively at the dinucleotide sequence CpG at a frequency of 3 to 5%. The pattern of methylation was further investigated by determining the state of methylation of several MspI (HpaII) sites. Different sites were found to be methylated to a different extent, implying that methylation of mtDNA is nonrandom. Based on the known base composition and nucleotide sequence of mouse mtDNA, the dinucleotide sequence CpG was found to be underrepresented in this DNA. The features of mtDNA methylation (CpG methylation, partial methylation of specific sites and CpG underrepresentation) are also characteristic of vertebrate nuclear DNA. This resemblance may reflect functional relationship between the mitochondrial and nuclear genomes.     

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.     

In vitro methylation of CpG-rich islands.

CpG islands are distinguishable from the bulk of vertebrate DNA for being unmethylated and CpG-rich. Since CpG doublets are the specific target of eukaryotic DNA methyltransferases, CpG-rich sequences might be expected to be good methyl-accepting substrates in vitro, despite their unmethylated in vivo condition. This was tested using a partially purified DNA-methyltransferase from human placenta and several cloned CpG-rich or CpG-depleted sequences. The efficiency of methylation was found to be proportional to the CpG content for CpG-depleted regions, which are representative of the bulk genome. However, methylation was much less efficient for CpG frequencies higher than 1 in 12 nucleotides, reaching only 60% of the expected level. That suggests that the close CpG spacing typical of CpG-islands somehow inhibits mammalian DNA methyltransferase. The implications of these findings on the in vivo pattern of DNA methylation are discussed.