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 loss of dinucleotides CpG from DNA. IV. Methylation and divergence of genes and pseudogenes of small nuclear RNA

The frequency of neighboring base pairs in nucleotide sequences of over 80 genes and pseudogenes of low molecular weight RNAs U1-U8, 4.5S and 7S in different eukaryotes was determined. The probable frequency of CpG----TpG + CpA substitutions, caused as a result of the deamination of 5-methylcytosine residues in DNA, was determined. It was found that the genes of small RNAs do not reveal a single level of CpG methylation for all the species studied. In most cases CpG in the genes of U1, 4.5S and 7S RNA are methylated, whereas in the genes of U2-U6-RNA these sites must have never been subjected to methylation. Nearly all the investigated pseudogenes of different small RNAs are strongly methylated due to a considerable lack of CpG. It was established that CpG----TpG + CpA transitions may amount to as much third of all the mutations accumulated in the genes of the same RNAs in different species. Such transitions in pseudogenes may account for 40% of all the nucleotide substitutions. This disproportionately high level of mutations in CpG dinucleotides (3-5-fold higher than in other DNA dupletes) must be the direct result of the methylation of these sites. Consequently, CpG methylation causes a dramatic acceleration of the divergence rate of DNA sequences. It has been concluded that protection of most vital genes against methylation is one of the essential conditions for sustaining the high level of stability of the macromolecular structure and for the reliability of macromolecular functioning in a cell.     

Increased G + C content of DNA stabilizes methyl CpG dinucleotides.

The vertebrate genome is a mosaic of regions differing dramatically in their G + C content. Those regions with a high G + C content contain the expected number of CpG dinucleotides and we propose that following methylation these have been protected from deamination by the increased stability of the surrounding DNA duplex. This argument applies both to the microenvironment of the CpG dinucleotide and to whole gene regions.     

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.     

Excision of 8-oxoguanine from methylated CpG dinucleotides by human 8-oxoguanine DNA glycosylase

CpG dinucleotides are targets for epigenetic methylation, many of them bearing 5-methylcytosine (mCyt) in the human genome. Guanine in this context can be easily oxidized to 8-oxoguanine (oxoGua), which is repaired by 8-oxoguanine-DNA glycosylase (OGG1). We have studied how methylation affects the efficiency of oxoGua excision from damaged CpG dinucleotides. Methylation of the adjacent cytosine moderately decreased the oxoGua excision rate while methylation opposite oxoGua lowered the rate of product release. Cytosine methylation abolished stimulation of OGG1 by repair endonuclease APEX1. The OGG1 S326C polymorphic variant associated with lung cancer showed poorer base excision and lost sensitivity to the opposite-base methylation. The overall repair in the system reconstituted from purified proteins decreased for CpG with mCyt in the damaged strand.     

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.     

Unmethylated and methylated CpG dinucleotides distinctively regulate the physical properties of DNA

In eukaryotic cells, DNA has to bend significantly to pack inside the nucleus. Physical properties of DNA such as bending flexibility and curvature are expected to affect DNA packaging and partially determine the nucleosome positioning patterns inside a cell. DNA CpG methylation, the most common epigenetic modification found in DNA, is known to affect the physical properties of DNA. However, its detailed role in nucleosome formation is less well‐established. In this study, we evaluated the effect of defined CpG patterns (unmethylated and methylated) on DNA structure and their respective nucleosome‐forming ability. Our results suggest that the addition of CpG dinucleotides, either as a (CG)_n stretch or (CGX₈)_n repeats at 10 bp intervals, lead to reduced hydrodynamic radius and decreased nucleosome‐forming ability of DNA. This effect is more predominant for a DNA stretch ((CG)₅) located in the middle of a DNA fragment. Methylation of CpG sites, surprisingly, seems to reduce the difference in DNA structure and nucleosome‐forming ability among DNA constructs with different CpG patterns. Our results suggest that unmethylated and methylated CpG patterns can play very different roles in regulating the physical properties of DNA. CpG methylation seems to reduce the DNA conformational variations affiliated with defined CpG patterns. Our results can have significant bearings in understanding the nucleosome positioning pattern in living organisms modulated by DNA sequences and epigenetic features. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 517–524, 2014.     

CpG dinucleotides in the hMSH2 and hMLHI genes are hotspots for HNPCC mutations

Hereditary nonpolyposis colon cancer (HN-PCC) is an autosomally inherited predisposition to cancer that has recently been linked to defects in the human mismatch repair genes hMSH2 and hMLHI. The identification of the causative mutations in HNPCC families is desirable, since it confirms the diagnosis and allows the carrier status of unaffected relatives at risk to be determined. We report six different new mutations identified in the hMSH2 and hMLH1 genes of Russian and Moldavian HNPCC families. Three of these mutations occur in CpG dinucleotides and lead to a premature stop codon, a splicing defect or an amino-acid substitution in an evolutionary conserved residue. Analysis of a compilation of published mutations including our new data suggests that CpG dinucleotides within the coding regions of the hMSH2 and hMLH1 genes are hotspots for single base-pair substitutions.     

The loss of CpG dinucleotides from DNA. I. Methylated and non-methylated genome compartments in eukaryotes with different levels of 5-methylcytosine in DNA

The methylation of cytosine residues in CpG significantly increases the frequency of m5CpG----TpG transitions in DNA and CpG dinucleotides are eliminated from the genome (CpG-suppression). In the millions of years of vertebrates evolution about 3 mol% of 5-methylcytosine have disappeared from their genome, i.e., 2-3-fold more than the amount persisting in the DNA of the now extant species. A computer analysis has been carried out of neighboring b.p. frequencies in more than 2500 sequenced genes of different species in the EMBL bank with an overall extension of over 3000 kb. It has been found that CpG methylated sites exhibit a highly irregular distribution pattern in the genome of eucaryotes. The majority of the vertebrate sequences (92%) bears the impress of a significant lack of CpG and an excess of TpG+CpA; therefore they may be referred to the genome methylated compartment. A group of genes has been discovered (about 8%) where CpG must have never been subjected to methylation. In invertebrates, such a nonmethylated compartment makes up 59% of the genome and in eubacteria--85%. A brief list of genes, belonging to the methylated and the non-methylated compartments of the invertebrate and yeast genome, is given. It has been established that the mean value of CpG-suppression in genes is directly proportional to the methylation level of total DNA in different species.     

Methylation of CpG dinucleotides in the lacI gene of the Big Blue® transgenic mouse

Cytosine residues at CpG dinucleotides can be methylated by endogenous methyltransferases in mammalian cells. The resulting 5-methylcytosine base may undergo spontaneous deamination to form thymine causing G/C to A/T transition mutations. Methylated CpGs also can form preferential targets for environmental mutagens and carcinogens. The Big Blue® transgenic mouse has been used to investigate tissue and organ specificity of mutations and to deduce mutational mechanisms in a mammal in vivo. The transgenic mouse contains approximately 40 concatenated lambda-like shuttle vectors, each of which contains one copy of an Escherichia coli lacI gene as a mutational target. lacI mutations in lambda transgenic mice are characterized by a high frequency of spontaneous mutations targeted to CpG dinucleotides suggesting an important contribution from methylation-mediated events. To study the methylation status of CpGs in the lacI gene, we have mapped the distribution of 5-methylcytosines along the DNA-binding domain and flanking sequences of the lacI gene of transgenic mice. We analyzed genomic DNA from various tissues including thymus, liver, testis, and DNA derived from two thymic lymphomas. The mouse genomic DNAs and methylated and unmethylated control DNAs were chemically cleaved, then the positions of 5-methylcytosines were mapped by ligation-mediated PCR which can be used to distinguish methylated from unmethylated cytosines. Our data show that most CpG dinucleotides in the DNA binding domain of the lacI gene are methylated to a high extent (>98%) in all tissues tested; only a few sites are partially (70–90%) methylated. We conclude that tissue-specific methylation is unlikely to contribute significantly to tissue-specific mutational patterns, and that the occurrence of common mutation sites at specific CpGs in the lacI gene is not related to selective methylation of only these sequences. The data confirm previous suggestions that the high frequency of CpG mutations in lacI transgenes is related to the presence of 5-methylcytosine bases.     

Cellular content of chloroplast DNA and chloroplast ribosomal RNA genes in Euglena gracilis during chloroplast development.

The cellular content of chloroplast DNA in Euglena gracilis has been quantitatively determined. DNA was extracted from Euglena cells at various stages of chloroplast development and renatured in the presence of trace amounts of 3H-labeled chloroplast DNA. From the kinetics of renaturation of the 3H-labeled chloroplast DNA, compared with the kinetics of renaturation of excess nonradioactive chloroplast DNA, the fraction of cellular DNA represented by chloroplast DNA was calculated. The content of chloroplast DNA was found to increase from 4.9 to 14.6% of cellular DNA during light-induced chloroplast development. Correcting for the change in DNA mass per cell, the number of copies of chloroplast DNA is found to vary from 1400 to 2900 per cell. During this developmental transition, the cellular content of the chloroplast ribosomal RNA genes varies from 1900 to 5200 copies per cell. The ratio of the number of copies of rRNA genes to chloroplast genomes per cell remains in the range of 1-2 throughout chloroplast development, ruling out selective amplification of chloroplast rRNA genes as a means of regulation of rRNA gene expression. Direct measurement of the number of rRNA cistrons per 9.2 X 10(7) dalton genome yields a value of 1 or 2.     

Lupin chloroplast and mitochondrial tRNA populations and their genes

Transfer RNAs isolated from lupin chloroplasts and mitochondria were compared by two-dimensional gel electrophoresis. Twenty chloroplast and 24 mitochondrial tRNA species were identified. The saturation hybridization between lupin chloroplast DNA and 125I-labelled lupin chloroplast tRNAs pointed to the presence of about 34 tRNA genes in lupin chloroplast DNA. The number of mitochondrial tRNA genes estimated by the same method was about 30 genes. EcoRI restriction digest of lupin mitochondrial DNA probed with 32P-labelled lupin mitochondrial tRNAs revealed only a small number of positive restriction fragments. Some of these mitochondrial restriction fragments hybridized with 32P-labelled chloroplast tRNA.     

CpG甲基化与基因调控

CpG双核苷酸中的胞嘧啶甲基化和去甲基化在哺乳动物的基因表达中有重要的调控作用.哺乳动物基因组中有两类启动子:CpG岛启动子和CpG缺乏启动子.两种蛋白质因子通过与甲基化CpG的相互作用影响基因表达,CpG岛在基因组分析中也有广泛的用途.     

Study of Tissue-Specific CpG Methylation of DNA in Extended Genomic Loci

Modern approaches for studies on genome functioning include investigation of its epigenetic regulation. Methylation of cytosines in CpG dinucleotides is an inherited epigenetic modification that is responsible for both functional activity of certain genomic loci and total chromosomal stability. This review describes the main approaches for studies on DNA methylation. Under consideration are site-specific approaches based on bisulfite sequencing and methyl-sensitive PCR, whole-genome approaches aimed at searching for new methylation hot spots, and also mapping of unmethylated CpG sites in extended genomic loci.