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.     

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.     

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.     

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 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.     

Primate CpG islands are maintained by heterogeneous evolutionary regimes involving minimal selection

Mammalian CpG islands are key epigenomic elements that were first characterized experimentally as genomic fractions with low levels of DNA methylation. Currently, CpG islands are defined based on their genomic sequences alone. Here, we develop evolutionary models to show that several distinct evolutionary processes generate and maintain CpG islands. One central evolutionary regime resulting in enriched CpG content is driven by low levels of DNA methylation and consequentially low rates of CpG deamination. Another major force forming CpG islands is biased gene conversion that stabilizes constitutively methylated CpG islands by balancing rapid deamination with CpG fixation. Importantly, evolutionary analysis and population genetics data suggest that selection for high CpG content is not?a significant factor contributing to conservation of CpGs in differentially methylated regions. The heterogeneous, but not selective, origins of CpG islands have direct implications for the understanding of DNA methylation patterns in healthy and diseased cells.     

CpG methylation of DNA restricts prereplication complex assembly in Xenopus egg extracts

In a Xenopus egg replication system, the origin recognition complex (ORC) does not bind to CpG methylated DNA and DNA replication is inhibited. Insertion of low density CpG DNA of at least 1.2 kb into methylated plasmids rescues both replication and ORC binding. Using this pseudo-origin, we find that ORC binding is restricted to low-CpG-density DNA; however, MCM is loaded onto both weakly and highly methylated DNA and occupies at least approximately 2 kb of DNA. Replication initiates coincident with MCM, and even the most distally bound MCM is associated with sites of replication initiation. These results suggest that in metazoans MCM is loaded onto and initiates replication over a large region distant from ORC.     

Genome-wide profiling of sperm DNA methylation in relation to buffalo (Bubalus bubalis) bull fertility

The DNA methylation pattern in spermatozoa of buffalo bulls of different fertility status was investigated. Spermatozoa isolated DNA from two groups of buffalo bulls (n = 5), selected based on their artificial insemination–generated conception rate data followed by IVF efficiency, were studied for global methylation changes using a custom-designed 180 K buffalo (Bubalus bubalis) CpG island/promoter microarray. A total of 96 individual genes with another 55 genes covered under CpG islands were found differentially methylated in sperm of high-fertile and subfertile buffalo bulls. Important genes associated with biological processes, cellular components, and functions were identified to be differentially methylated in buffalo bulls with differential fertility status. The identified differentially methylated genes were found to be involved in germ cell development, spermatogenesis, capacitation, and embryonic development. The observations hint that methylation defects of sperm DNA may play a crucial role in determining the fertility of breeding bulls. This growing field of sperm epigenetics will be of great benefit in understanding the graded fertility conditions of breeding bulls in commercial livestock production system.     

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.     

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.     

Segmental distribution of genes harboring a CpG island-like region on rice chromosomes

In plant genomes, there exist discrete regions rich in CpG dinucleotides, namely CpG clusters. In rice, most of these CpG clusters are associated with genes. Rice genes are grouped into one of the five classes according to the position of an associated CpG cluster. Among them, class 1 genes, which harbor a CpG cluster at the 5′-terminus, share similarities with human genes having CpG islands. In the present study, by analyzing plant genome sequence data, primarily from rice, we investigated the chromosomal distribution of genes of each class, mainly class 1 genes. Class 1 genes were not uniformly distributed across the rice genome, but were clustered into discrete chromosomal segments. EST-based analysis of the distribution of expressed genes indicates that this segmental distribution of class 1 genes caused a preferential distribution of expressed genes within class 1 gene-rich segments. We then compared the methylation status of genes of each class to examine the possibility that differential DNA methylation, if any, is relevant to the observed differential expression level of genes inside and outside the class 1 segments. The difference in the methylation level between these genes was revealed to be fairly small, which does not support the above-mentioned possibility. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

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.     

Hydration and recognition of methylated CpG steps in DNA.

The analysis of the hydration pattern around methylated CpG steps in three high resolution (1.7, 2.15 and 2.2 A) crystal structures of A-DNA decamers reveals that the methyl groups of cytosine residues are well hydrated. In comparing the native structure with two structurally distinct forms of the decamer d(CCGCCGGCGG) fully methylated at its CpG steps, this study shows also that in certain structural and sequence contexts, the methylated cytosine base can be more hydrated that the unmodified one. These water molecules seem to be stabilized in front of the methyl group through the formation C-H...O interactions. In addition, these structures provide the first observation of magnesium cations bound to the major groove of A-DNA and reveal two distinct modes of metal binding in methylated and native duplexes. These findings suggest that methylated cytosine bases could be recognized by protein or DNA polar residues through their tightly bound water molecules.