Asymmetrical distribution of CpG in an 'average' mammalian gene.

The frequency and distribution of the rare dinucleotide CpG was examined in 15 mammalian genes. CpG is highly methylated at cytosine in mammalian DNA (1,2) and 5-methylcytosine (5mC) is thought to undergo a transition mutation via deamination to produce thymine (3). This would result in the accumulation of TpG and CpA and depletion of CpG during evolution (4). Consistent with this hypothesis, the gene sample of 26,541 dinucleotides contained CpG at 40% the frequency expected by base composition and the CpG transition products, TpG+CpA, were significantly elevated at 124% of expected random frequency. However, because CpG occurs at only 25% of expected random frequency in the genome, the sampled genes were considerably enriched in this dinucleotide. CpGs were asymmetrically distributed in sequences flanking the genes. 5'-flanking sequences were enriched in CpG at 135% of the frequency expected assuming a symmetrical distribution of all the CpGs in the sampled genes (p less than 0.01), while 3'-flanking regions were depleted in CpG at 40% of expected values (p less than 0.0001). This asymmetry may reflect the role of 5-methylcytosine in gene expression. In contrast the frequencies of GpC and GpT+ ApC did not differ significantly from that predicted by base composition and these dinucleotides were not asymmetrically distributed.     

An overview of the analysis of DNA methylation in mammalian genomes

DNA methylation at position C5 of the pyrimidine ring of cytosine in mammalian genomes has received a great deal of research interest due to its importance in many biological phenomena. It is associated with events such as epigenetic gene silencing and the maintenance of genome integrity. Aberrant DNA methylation, particularly that of chromosomal regions called CpG islands, is an important step in carcinogenesis. In order to elucidate methylation profiling of complex genomes, various methods have been developed. Many of these methods are based on the differential reactivity of cytosine and 5-methylcytosine to various chemicals. The combined use of these chemical reactions and other preexisting methods has enabled the discrimination of cytosine and 5-methylcytosine in complex genomes. The use of proteins that preferentially bind to methylated DNA has also successfully been used to discriminate between methylated and unmethylated sites. The chemical and structural dissection of the in vivo processes of enzymatic methylation and the binding of methyl-CpG binding proteins provides evidence for the complex mechanisms that nature has acquired. In this review we summarize the methods available for the discrimination between cytosine and 5-methylcytosine in complex genomes.     

Methylation sites in angiosperm genes

Summary The extent to which CpG dinucleotides were depleted in a large set of angiosperm genes was, on average, very similar to the extent of CpG depletion in total angiosperm genomic DNA and far less than the extent of CpG depletion in vertebrate genes. Gene sequences from Arabidopsis thaliana, a dicotyledonous species with relatively low levels of total 5-methylcytosine, were just as CpG depleted as the angiosperm genes in general. Furthermore, levels of TpG and CpA, the potential deamination mutation products of methylated CpG, were elevated in A. thaliana genes, supporting a high rate of deamination mutation as the cause of the CpG deficiency. Using a method that takes into account the dinucleotide frequencies within each sequence of interest, we calculated the expected frequencies of CpNpG trinucleotides, which are also highly methylated in angiosperm genomes. CpNpG trinucleotides were not extensively enriched or depleted in the angiosperm genes. Two hypotheses could account for our results. Differential depletion of CpG and CpNpG within angiosperm genes and differential depletion of CpG in angiosperm and vertebrate genes could arise from different efficiencies of mismatch repair or from different levels of cytosine methylation in the cell lineages that contribute to germ cells.Offprint requests to: M. Gardiner-Garden     

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.     

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.     

Active mammalian replication origins are associated with a high-density cluster of mCpG dinucleotides.

ori-beta is a well-characterized origin of bidirectional replication (OBR) located approximately 17 kb downstream of the dihydrofolate reductase gene in hamster cell chromosomes. The approximately 2-kb region of ori-beta that exhibits greatest replication initiation activity also contains 12 potential methylation sites in the form of CpG dinucleotides. To ascertain whether DNA methylation might play a role at mammalian replication origins, the methylation status of these sites was examined with bisulfite to chemically distinguish cytosine (C) from 5-methylcytosine (mC). All of the CpGs were methylated, and nine of them were located within 356 bp flanking the minimal OBR, creating a high-density cluster of mCpGs that was approximately 10 times greater than average for human DNA. However, the previously reported densely methylated island in which all cytosines were methylated regardless of their dinucleotide composition was not detected and appeared to be an experimental artifact. A second OBR, located at the 5' end of the RPS14 gene, exhibited a strikingly similar methylation pattern, and the organization of CpG dinucleotides at other mammalian origins revealed the potential for high-density CpG methylation. Moreover, analysis of bromodeoxyuridine-labeled nascent DNA confirmed that active replication origins were methylated. These results suggest that a high-density cluster of mCpG dinucleotides may play a role in either the establishment or the regulation of mammalian replication origins.     

The DeltauvrB mutations in the Ames strains of Salmonella span 15 to 119 genes

The lacI transgene used in the Big Blue (BB) mouse and rat mutation assays typically displays spontaneous mutation frequencies in the 5x10(-5) range. Recently, the bone marrow and bladder of the Big Blue rat were reported to have, by an order of magnitude, the lowest spontaneous mutation frequencies ever observed for lacI in a transgenic animal, approaching the value for endogenous targets such as hprt ( approximately 10(-6)). Since spontaneous mutations in transgenes have been attributed in part to deamination of 5-methylcytosine in CpG sequences, we have investigated the methylation status of the lacI transgene in bone marrow of BB rats and compared it to that present in other tissues including liver, spleen, and breast. The first 400 bases of the lacI gene were investigated using bisulfite genomic sequencing since this region contains the majority of both spontaneous and induced mutations. Surprisingly, all the CpG cytosines in the lacI sequence were fully methylated in all the tissues examined from both 2- and 14-week-old rats. Thus, there is no correlation between 5-methylcytosine content at CpG sites in lacI and the frequency of spontaneous mutation at this marker. We also investigated the methylation status of another widely used transgenic mutation target, the cII gene. The CpG sites in cII in BB rats were fully methylated while those in BB mice were partially methylated (each site approximately 50% methylated). Since spontaneous mutation frequency at cII is comparable in rat and mouse, the methylation status of CpG sequences in this gene also does not correlate with spontaneous frequency. We conclude that other mechanisms besides spontaneous deamination of 5-methylcytosine at CpG sites are driving spontaneous mutation at BB transgenic loci.     

The covariation between TpA deficiency, CpG deficiency, and G+C content of human isochores is due to a mathematical artifact

CpG and TpA dinucleotides are underrepresented in the human genome. The CpG deficiency is due to the high mutation rate from C to T in methylated CpG's. The TpA suppression was thought to reflect a counterselection against TpA's destabilizing effect in RNA. Unexpectedly, the TpA and CpG deficiencies vary according to the G+C contents of sequences. It has been proposed that the variation in CpG suppression was correlated with a particular chromatin organization in G+C-rich isochores. Here, we present an improved model of dinucleotide evolution accounting for the overlap between successive dinucleotides. We show that an increased mutation rate from CpG to TpG or CpA induces both an apparent TpA deficiency and a correlation between CpG and TpA deficiencies and G+C content. Moreover, this model shows that the ratio of observed over expected CpG frequency underestimates the real CpG deficiency in G+C-rich sequences. The predictions of our model fit well with observed frequencies in human genomic data. This study suggests that previously published selectionist interpretations of patterns of dinucleotide frequencies should be taken with caution. Moreover, we propose new criteria to identify unmethylated CpG islands taking into account this bias in the measure of CpG depletion.     

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.     

Factors to preserve CpG-rich sequences in methylated CpG islands

Background

Mammalian CpG islands (CGIs) normally escape DNA methylation in all adult tissues and developmental stages. However, in our previous study we unexpectedly identified many methylated CGIs in human peripheral blood leukocytes. Methylated CpG dinucleotides convert to TpG dinucleotides through deaminization of their cytosine bases more frequently than hypomethylated CpG dinucleotides. Therefore, we wondered how methylated CGIs in germline or non-germline cells maintain their CpG-rich sequences. It is known that events such as germline hypomethylation, CpG selection, biased gene conversion (BGC), and frequent CpG fixation can contribute to the maintenance of CpG-rich sequences in methylated CGIs in germline or non-germline cells. However, it has not been investigated which of the processes maintain CpG-rich sequences of methylated CGIs in each genomic position.

Results

In this study, we comprehensively examined the contribution of the processes described above to the maintenance of CpG-rich sequences in methylated CGIs in germline and non-germline cells which were classified by genomic positions. Approximately 60–80% of CGIs with high methylation in H1 cell line (H1-HM) in all the genomic positions showed a low average CpG → TpG/CpA substitution rate. In contrast, fewer than half the numbers of CGIs with H1-HM in all the genomic positions showed a low average CpG → TpG/CpA substitution rate and low levels of methylation in sperm cells (SPM-LM). Furthermore, a small fraction of CGIs with a low average CpG → TpG/CpA substitution rate and high levels of methylation in sperm cells (SPM-HM) showed CpG selection.On the other hand, independent of the positions in genes, most CGIs with SPM-HM showed a slightly higher average TpG/CpA → CpG substitution rate compared with those with SPM-LM.

Conclusions

Relatively high numbers (approximately 60–80%) of CGIs with H1-HM in all the genomic positions preserve their CpG-rich sequences by a low CpG → TpG/CpA substitution rate caused mainly by their SPM-LM, and for those with SPM-HM partly by CpG selection and TpG/CpA → CpG fixation. BGC has little contribution to the maintenance of CpG-rich sequences of CGIs with SPM-HM which were classified by genomic positions.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1286-x) contains supplementary material, which is available to authorized users.     

Activation-induced cytidine deaminase deaminates 5-methylcytosine in DNA and is expressed in pluripotent tissues: implications for epigenetic reprogramming

DNA deaminases of the Aid/Apobec family convert cytosine into uracil and play key roles in acquired and innate immunity. The epigenetic modification by methylation of cytosine in CpG dinucleotides is also mutagenic, but this is thought to occur by spontaneous deamination. Here we show that Aid and Apobec1 are 5-methylcytosine deaminases resulting in a thymine base opposite a guanine. Their action can thus lead to C --> T transition mutations in methylated DNA, or in conjunction with repair of the T:G mismatch, to demethylation. The Aid and Apobec1 genes are located in a cluster of pluripotency genes including Nanog and Stella and are co-expressed with these genes in oocytes, embryonic germ cells, and embryonic stem cells. These results suggest that Aid and perhaps some of its family members may have roles in epigenetic reprogramming and cell plasticity. Transition in CpG dinucleotides is the most frequent mutation in human genetic diseases, and sequence context analysis of CpG transitions in the APC tumor suppressor gene suggests that DNA deaminases may play a significant role in tumor etiology.     

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.     

Comparative Genomic Study Reveals a Transition from TA Richness in Invertebrates to GC Richness in Vertebrates at CpG Flanking Sites： An Indication for Context-Dependent Mutagenicity of Methylated CpG Sites

Vertebrate genomes are characterized with CpG deficiency, particularly for GCpoor regions. The GC content-related CpG deficiency is probably caused by context-dependent deamination of methylated CpG sites. This hypothesis was examined in this study by comparing nucleotide frequencies at CpG flanking positions among invertebrate and vertebrate genomes. The finding is a transition of nucleotide preference of 5＇ T to 5＇ A at the invertebrate-vertebrate boundary, indicating that a large number of CpG sites with 5＇ Ts were depleted because of global DNA methylation developed in vertebrates. At genome level, we investigated CpG observed/expected （obs/exp） values in 500 bp fragments, and found that higher CpG obs/exp value is shown in GC-poor regions of invertebrate genomes （except sea urchin） but in GC-rich sequences of vertebrate genomes. We next compared GC content at CpG flanking positions with genomic average, showing that the GC content is lower than the average in invertebrate genomes, but higher than that in vertebrate genomes. These results indicate that although 5＇ T and 5＇ A are different in inducing deamination of methylated CpG sites, GC content is even more important in affecting the deamination rate. In all the tests, the results of sea urchin are similar to vertebrates perhaps due to its fractional DNA methylation. CpG deficiency is therefore suggested to be mainly a result of high mutation rates of methylated CpG sites in GC-poor regions.     

The effect of C(5) cytosine methylation at CpG sequences on mitomycin-DNA bonding profiles

Recent studies have documented that cytosine C(5) methylation of CpG sequences enhances mitomycin C (1) adduction. The reports differ on the extent and uniformity of 1 modification at the nucleotide level. We have determined the bonding profiles for mitomycin monoalkylation in two DNA restriction fragments where the CpG sequences were methylated. Three mitomycin substrates were used and two different enzymatic assays employed to monitor the extent of drug modification at the individual base sites. Drug DNA modification was accomplished with I and 10-decarbamoylmitomycin C (2) under reductive (Na2S2O4) condilions and with N-methyl-7-methoxyaziridinomitosene (3) under nonreductive conditions. The UvrABC incision assay permitted us to quantitate the sites of drug adduction, and the lambda-exonuclease stop assay provided a qualitative estimation of drug-DNA modification consistent with the UvrABC data. We learned that C(5) cytosine methylation (m5C) enhanced the extent of overall DNA modification. Using the UvrABC endonuclease assay, we found that modification by 1 increased 2.0 and 7.4 times for the two DNA restriction fragments. Analysis of the modification sites at the nucleotide sequence level revealed that guanine (G) was the only base modified and that the overall increased level of DNA adduction was due to enhanced modification of select m5CpG* (G* = mitomycin (mitosene) adduction sites) loci compared with CpG* sites: the largest differences reached two orders of magnitude. Significantly, not all CpG* sites underwent increased drug adduction upon C(5) cytosine methylation. The effect of C(5) cytosine methylation on the drug adduction profiles was less pronounced for G* sites located within dinucleotide sequences other than CpG*. We observed that DNA methylation often led to slightly diminished adduction levels at these sites. The different m5CpG* adduction patterns provided distinctive sequence-selective bonding profiles for 1-3. We have attributed the large differences in guanine reactivity to DNA structural factors created, in part, by C(5) cytosine methylation. The significance of these findings in cancer chemotherapy is briefly discussed.     

5-Methylcytosine content and methylation status in six millet DNAs

High performance liquid chromatographic analysis of the total nuclear DNAs of 6 millets plant species indicates that the 5-methylcytosine content ranges from 3% in barn yard millet to 9.6% in great millet while the fraction of cytosines methylated varies between 14% in little millet to 31 % in pearl millet. Digestion of millet DNAs with MspI/HpaII suggests that CpG methylation is more in great millet DNA while CpC methylation is more in the other 5 millet DNAs. Digestion of millet DNAs with MboI, Sau3AI andDpnI indicates that some of the^5’ GATC^3’ sequences are methylated at adenine and/or cytosine residues except in little millet where adenine methylation of the^5’GATC^3’ sequences is insignificant and there is a predominance of cytosine methylation in these sequences.