Frequency of abnormal human haemoglobins caused by C----T transitions in CpG dinucleotides

A large part of human genetic disease apparently arises from deamination of cytosine residues in methylated CpG dinucleotides. Their mutation rate is known to be high when C is present as 5-methyl-cytosine, but is believed to be normal when it is unmethylated. The beta-globin gene contains five, the gamma-globin gene two, and each of the alpha-globin genes contains 35 CpG dinucleotides. The CpG dinucleotides in the beta and gamma-globin genes are methylated, while those in the alpha-globin genes are under-methylated. One would therefore have expected the CpG dinucleotides to be a frequent source of mutations in the beta and gamma-globin genes, but not in the alpha-globin genes. In fact, the evidence points to CpG dinucleotides being a frequent source of mutations in both the alpha and beta-globin genes. This suggests either that the mutation rates of both methylated and unmethylated CpG dinucleotides are abnormally high, which conflicts with published evidence, or that there is a finite chance of some of these in the alpha-globin genes of certain individuals being methylated and therefore subject to mutation.     

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.     

Immune stimulation in the silkworm, Bombyx mori L., by CpG oligodeoxynucleotides

Synthetic ODNs containing unmethylated CpG dinucleotides are known to stimulate immune responses in vertebrates, but so far the effect has not been studied in insects. In this report, we describe an induction of immune response following injection of oligodeoxynucleotides (ODNs) into the insect hemocoel. The fifth instar silkworm (Bombyx mori L.) larvae were injected with several synthetic ODNs containing variable number of unmethylated CpG motifs, heat-denatured genomic DNA of B. mori itself, or intact genomic DNA to observe a new induction pattern in the insect immune mechanism. When the induction of immune response was examined based on the expression rates of genes for antibacterial peptides such as attacin and cecropin, we could confirm that it was triggered upon injection of ODNs. The expression was, however, neither dependent on numbers of CpG motifs nor methylation of CpGs in ODNs. Furthermore, it was confirmed that the presence of CpG in ODN was not involved in the induction pattern of insect immunity caused by ODNs, although it has been reported that vertebrates respond in a specific manner against invading ODNs containing CpG dinucleotides. In addition, insect immunity was not stimulated by injection of intact DNA from host. In contrast, the injection of denatured genomic DNA provoked the host immune reaction. Taken together, our data suggest that foreignness of ODNs or DNA might be a key factor in the induction of insect immunity.     

De novo DNA methylation at nonrandom founder sites 5' from an unmethylated minimal origin of DNA replication in latent Epstein-Barr virus genomes

Latent episomal genomes of Epstein-Barr virus, a human gammaherpesvirus, represent a suitable model system for studying replication and methylation of chromosomal DNA in mammals. We analyzed the methylation patterns of CpG dinucleotides in the latent origin of DNA replication of Epstein-Barr virus using automated fluorescent genomic sequencing of bisulfite-modified DNA samples. We observed that the minimal origin of DNA replication was unmethylated in 8 well-characterized human cell lines or clones carrying latent Epstein-Barr virus genomes as well as in a prototype virus producer marmoset cell line. This observation suggests that unmethylated DNA domains can function as initiation sites or zones of DNA replication in human cells. Furthermore, 5' from this unmethylated region we observed focal points of de novo DNA methylation in nonrandom positions in the majority of Burkitt's lymphoma cell lines and clones studied while the corresponding CpG dinucleotides in viral genomes carried by lymphoblastoid cell lines and marmoset cells were completely unmethylated. Clustering of highly methylated CpG dinucleotides suggests that de novo methylation of unmethylated double-stranded episomal viral genomes starts at discrete founder sites in vivo. This is the first comparative high-resolution methylation analysis of a latent viral origin of DNA replication in human cells.     

Developmental differences in methylation of human Alu repeats.

Alu repeats are especially rich in CpG dinucleotides, the principal target sites for DNA methylation in eukaryotes. The methylation state of Alus in different human tissues is investigated by simple, direct genomic blot analysis exploiting recent theoretical and practical advances concerning Alu sequence evolution. Whereas Alus are almost completely methylated in somatic tissues such as spleen, they are hypomethylated in the male germ line and tissues which depend on the differential expression of the paternal genome complement for development. In particular, we have identified a subset enriched in young Alus whose CpGs appear to be almost completely unmethylated in sperm DNA. The existence of this subset potentially explains the conservation of CpG dinucleotides in active Alu source genes. These profound, sequence-specific developmental changes in the methylation state of Alu repeats suggest a function for Alu sequences at the DNA level, such as a role in genomic imprinting.     

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.     

Methylation of CpG dinucleotides in the open reading frame of a testicular germ cell-specific intronless gene,Tact1/Actl7b,represses its expression in somatic cells

Methylation of CpG islands spanning promoter regions is associated with control of gene expression. However, it is considered that methylation of exonic CpG islands without promoter is not related to gene expression, because such exonic CpG islands are usually distant from the promoter. Whether methylation of exonic CpG islands near the promoter, as in the case of a CpG-rich intronless gene, causes repression of the promoter remains unknown. To gain insight into this issue, we investigated the distribution and methylation status of CpG dinucleotides in the mouse Tact1/Actl7b gene, which is intronless and expressed exclusively in testicular germ cells. The region upstream to the gene was poor in CpG, with CpG dinucleotides absent from the core promoter. However, a CpG island was found inside the open reading frame (ORF). Analysis of the methylation status of the Tact1/Actl7b gene including the 5′-flanking area demonstrated that all CpG sites were methylated in somatic cells, whereas these sites were unmethylated in the Tact1/Actl7b-positive testis. Trans fection experiments with in vitro-methylated constructs indicated that methylation of the ORF but not 5′ upstream repressed Tact1/Actl7b promoter activity in somatic cells. Similar effects of ORF methylation on the promoter activity were observed in testicular germ cells. These are the first results indicating that methylation of the CpG island in the ORF represses its promoter in somatic cells and demethylation is necessary for gene expression in spermatogenic cells.     

Promoter DNA Methylation Regulates Murine SUR1 (Abcc8) and SUR2 (Abcc9) Expression in HL-1 Cardiomyocytes

Two mammalian genes encode the SURx (SUR1, Abcc8 and SUR2, Abcc9) subunits that combine with Kir6.2 (Kcnj11) subunits to form the ATP-sensitive potassium (KATP) channel in cardiac myocytes. Different isoform combinations endow the channel with distinct physiological and pharmacological properties, and we have recently reported that the molecular composition of sarcolemmal KATP channels is chamber specific in the mouse heart. KATP channel composition is determined by what subunits are expressed in a cell or tissue. In the present study, we explore the role of CpG methylation in regulating SUR1 and SUR2 expression. In HL-1 cardiomyocytes, as in atrial myocytes, SUR1 expression is markedly greater than SUR2. Consistent with CpG methylation-dependent silencing of SUR2 expression, bisulfite sequencing of genomic DNA isolated from HL-1 cells demonstrates that 57.6% of the CpGs in the promoter region of the SUR2 gene are methylated, compared with 0.14% of the the CpG residues in the SUR1 sequence. Moreover, treatment with 10 μM 5-aza-2'-deoxycytidine (Aza-dC) significantly increased both the unmethylated fraction of the SUR2 CpG island and mRNA expression. However, we cannot rule out additional mechanisms of Aza-dC action, as Aza-dC also causes a decrease in SUR1 expression and lower doses of Aza-dC do not alter the unmethylated DNA fraction but do elicit a small increase in SUR2 expression. The conclusion that DNA methylation alone is not the only regulator of SUR subunit expression is also consistent with observations in native myocytes, where the CpG islands of both SUR genes are essentially unmethylated in both atrial and ventricular myocytes. Collectively, these data demonstrate the potential for CpG methylation to regulate SURx subunit expression and raises the possibility that regulated or aberrant CpG methylation might play a role in controlling channel structure and function under different physiological conditions or different species.     

Methylation of the mouse DIx5 and Osx gene promoters regulates cell type-specific gene expression

Dlx5 and Osx are master regulatory proteins essential for initiating the cascade leading to osteoblast differentiation in mammals, but the mechanism of osteoblast-specific expression is not fully understood. DNA methylation at CpG sequences is involved in tissue and cell type-specific gene expression. We investigated the methylation status of Dlx5 and Osx in osteogenic and nonosteogenic cell lines by methylation-specific PCR (MSP). The CpG dinucleotides of the Dlx5 and Osx promoter regions were unmethylated in osteogenic cell lines transcribing these genes but methylated in nonosteogenic cell lines. Treatment of C2C12 cells with 5-AzadC induced dose- and time-dependent expression of Dlx5 and Osx mRNA by demethylating the corresponding promoters. Furthermore the mRNAs for the osteoblast markers ALP and OC, which were undetectable in untreated cells, gradually increased after 5-AzadC treatment. In addition, BMP-2 stimulation induced Dlx5 expression by hypomethylating its promoter. These findings suggest that DNA methylation plays an important role in cell type-specific expression of Dlx5 and Osx.     

CpG methylation of the CENP-B box reduces human CENP-B binding

In eukaryotes, CpG methylation is an epigenetic DNA modification that is important for heterochromatin formation. Centromere protein B (CENP-B) specifically binds to the centromeric 17 base-pair CENP-B box DNA, which contains two CpG dinucleotides. In this study, we tested complex formation by the DNA-binding domain of CENP-B with methylated and unmethylated CENP-B box DNAs, and found that CENP-B preferentially binds to the unmethylated CENP-B box DNA. Competition analyses revealed that the affinity of CENP-B for the CENP-B box DNA is reduced nearly to the level of nonspecific DNA binding by CpG methylation.     

DNA Methylation-Dependent Epigenetic Regulation of Gene Expression in MCF-7 Breast Cancer Cells

To identify epigenetically-regulated genes in breast cancer, MCF-7 cells were exposed to 250nM 5-aza or 5-aza + 50nM TSA for 3 weeks followed by a 5 week recovery period after treatment withdrawal and gene expression patterns were examined by microarray analysis. We identified 20 genes that are associated with a >2-fold increase in expression in response to the demethylating treatment but returned to control levels after treatment withdrawal. RT-PCR verified that the genes identified were expressed at low or undetectable levels in control MCF-7 cells, but increased expression in treated cells. Most of these putative epigentically-regulated genes in MCF-7 cells do not contain CpG islands. In fact, these genes could be classified based upon their promoter CpG features, including genes with: (i) typical CpG features (CpG islands), (ii) intermediate CpG features (weak CpG islands), and (iii) atypical CpG features (no CpG islands). Prototype genes from each class (including CpG-deficient genes) were shown to be methylation-sensitive (subject to CpG methylation and responsive to demethylating agents), suggesting that not all gene targets of DNA methylation in breast cancer will contain a CpG island. Based upon the results of the current study and observations from the literature, we propose expansion of the current model for methylation-dependent regulation of gene expression to include genes lacking typical CpG islands. The expanded model we propose recognizes that all promoter CpG dinucleotides represent legitimate targets for DNA methylation and that the methylation of specific CpG dinucleotides in critical domains of regulatory regions can result in gene silencing.      

The loss of CpG dinucleotides from DNA. III. Methylation and evolution of histone genes

From nucleotide sequences of more than 70 histones genes in 15 species of eucaryotes the probable frequency was determined for CpG----TpG + CpA substitutions, occurring as a result of deamination of 5-methylcytosine residues in DNA. It was found that histone genes differ in the character of CpG methylation with respect to the species studied and may be divided into three groups differing in the value of CpG suppression. In one of them, M-, CpG dinucleotides must have not been methylated throughout the existence of these genes; in another, M+, nearly every other CpG has undergone transition. In the third group, M +/-, no more than 20% of CpG have steadily undergone methylation (and mutation). The CpG deficiency in M+ and M +/- histone genes is in general proportional to the level of methylation of total DNA in different species. It has been noted that the genes of different core histones in the same organism are characterized, as a rule, by the same type of CpG methylation and belong to the same group. Genes H1 and H5 show a higher level of CpG suppression and thus have a higher degree of methylation than the genes of core histones from the same organism. The most conserved among the histone genes, those for H3 and H4 in particular, must have not been methylated in the majority of the species studied. The distribution of methylated and non-methylated spacers and coding sequences of histone genes of man, mouse, hen and yeast reveals a mosaic pattern. It has been found that 5'-flanked regions in most cases are methylated more than respective genes, while the G + C content in them is significantly lower, compared with the coding gene sequences. The absence of methylation in the 5'-regulatory regions does not appear to be mandatory for histone genes. It has been established that the genes of the same histones may differ in the level of methylation even in more or less closely related species. Group M- comprises genes of core histones of man, hen, sea urchin, Drosophila, Neurospora and wheat; group M +/- includes analogous genes of mouse, Xenopus, trout and sea urchins. The results obtained testify against the possible universal involvement of methylation in the regulation of histone gene expression.     

The influence of CpG (5′-d(CpG)-3′ dinucleotides) methylation on ultrasonic DNA fragmentation

Abstract

DNA methylation is an important way of gene regulation. The variety of methods for DNA methylation analysis based on chemical modification or enzyme digestion has been proposed. However, DNA is able to undergo transformations under physical power. Here, we report that the cytosine methylation in CpG dinucleotides determines the difference in fragmentation rate of methylated and unmethylated DNA under sonication. We found that at the beginning of sonication, methylated DNAs are degraded faster than unmethylated one, and the difference in fragmentation degree can be evaluated with high reliability by quantitative polymerase chain reaction (qPCR). The optimal parameters that provide the greatest difference in amount of amplifiable DNA targets corresponding to fragmentation degree are the following: moderate amplicon size (about 150–250?bp), medium CpG sparseness (one CpG dinucleotide per ～12–14 nucleotides of the chain), and short sonication time (less than 5?min). Along with CpG, the CpA and CpT contents of amplified regions should be taken into account for proper DNA fragmentation by ultrasound as well. The obtained data could be used for elaboration of a method for comparative methylation testing, when there is no need to detect methylation of certain CpG dinucleotides. This method will be simple (can be used by any technician familiar with PCR), low cost (no need to use an expensive reagents), and fast (only brief DNA sonication and conventional qPCR are carried out).

Communicated by Ramaswamy H. Sarma