Метилированные CpG сайты и регуляция генов

Region of often methylated DNA with a cytosine followed by a guanine

The CpG sites or CG sites are regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5' → 3' direction. CpG sites occur with high frequency in genomic regions called CpG islands. Cytosines in CpG dinucleotides can be methylated to form 5 methylcytosines. Enzymes that add a methyl group are called DNA methyltransferases. In mammals, 70% to 80% of CpG cytosines are methylated. Methylating the cytosine within a gene can change its expression, a mechanism that is part of a larger field of science studying gene regulation that is called epigenetics. Methylated cytosines often mutate to thymines. In humans, about 70% of promoters located near the transcription start site of a gene (proximal promoters) contain a CpG island.

CpG is shorthand for 5'—C—phosphate—G—3' , that is, cytosine and guanine separated by only one phosphate group; phosphate links any two nucleosides together in DNA. The CpG notation is used to distinguish this single stranded linear sequence from the CG base pairing of cytosine and guanine for double stranded sequences. The CpG notation is therefore to be interpreted as the cytosine being 5 prime to the guanine base. CpG should not be confused with GpC, the latter meaning that a guanine is followed by a cytosine in the 5' → 3' direction of a single stranded sequence.

CpG dinucleotides have long been observed to occur with a much lower frequency in the sequence of vertebrate genomes than would be expected due to random chance. For example, in the human genome, which has a 42% GC content, a pair of nucleotides consisting of cytosine followed by guanine would be expected to occur of the time. The frequency of CpG dinucleotides in human genomes is less than one fifth of the expected frequency. This underrepresentation is a consequence of the high mutation rate of methylated CpG sites: the spontaneously occurring deamination of a methylated cytosine results in a thymine, and the resulting G:T mismatched bases are often improperly resolved to A:T; whereas the deamination of unmethylated cytosine results in a uracil, which as a foreign base is quickly replaced by a cytosine by the base excision repair mechanism. The C to T transition rate at methylated CpG sites is ~10 fold higher than at unmethylated sites.

+ CpG sites GpC sites Distribution of CpG sites (left: in red) and GpC sites (right: in green) in the human APRT gene. CpG are more abundant in the upstream region of the gene, where they form a CpG island, whereas GpC are more evenly distributed. The 5 exons of the APRT gene are indicated (blue), and the start (ATG) and stop (TGA) codons are emphasized (bold blue). CpG dinucleotides frequently occur in CpG islands (see definition of CpG islands, below). There are 28,890 CpG islands in the human genome, (50,267 if one includes CpG islands in repeat sequences). This is in agreement with the 28,519 CpG islands found by Venter et al. since the Venter et al. genome sequence did not include the interiors of highly similar repetitive elements and the extremely dense repeat regions near the centromeres. Since CpG islands contain multiple CpG dinucleotide sequences, there appear to be more than 20 million CpG dinucleotides in the human genome.

CpG islands (or CG islands) are regions with a high frequency of CpG sites. Though objective definitions for CpG islands are limited, the usual formal definition is a region with at least 200 bp, a GC percentage greater than 50%, and an observed to expected CpG ratio greater than 60%. The "observed to expected CpG ratio" can be derived where the observed is calculated as: and the expected as or (promoter regions). Because of this, the presence of a CpG island is used to help in the prediction and annotation of genes. In mammalian genomes, CpG islands are typically 300–3,000 base pairs in length, and have been found in or near approximately 40% of promoters of mammalian genes. Over 60% of human genes and almost all house keeping genes have their promoters embedded in CpG islands. Given the frequency of GC two nucleotide sequences, the number of CpG dinucleotides is much lower than would be expected. A 2002 study revised the rules of CpG island prediction to exclude other GC rich genomic sequences such as Alu repeats. Based on an extensive search on the complete sequences of human chromosomes 21 and 22, DNA regions greater than 500 bp were found more likely to be the "true" CpG islands associated with the 5' regions of genes if they had a GC content greater than 55%, and an observed to expected CpG ratio of 65%. CpG islands are characterized by CpG dinucleotide content of at least 60% of that which would be statistically expected (~4–6%), whereas the rest of the genome has much lower CpG frequency (~1%), a phenomenon called CG suppression. Unlike CpG sites in the coding region of a gene, in most instances the CpG sites in the CpG islands of promoters are unmethylated if the genes are expressed. This observation led to the speculation that methylation of CpG sites in the promoter of a gene may inhibit gene expression. Methylation, along with histone modification, is central to imprinting. Most of the methylation differences between tissues, or between normal and cancer samples, occur a short distance from the CpG islands (at "CpG island shores") rather than in the islands themselves. CpG islands typically occur at or near the transcription start site of genes, particularly housekeeping genes, in vertebrates.

In humans, about 70% of promoters located near the transcription start site of a gene (proximal promoters) contain a CpG island. CpG islands also occur frequently in promoters for functional noncoding RNAs such as microRNAs.

In humans, DNA methylation occurs at the 5 position of the pyrimidine ring of the cytosine residues within CpG sites to form 5 methylcytosines. The presence of multiple methylated CpG sites in CpG islands of promoters causes stable silencing of genes. Silencing of a gene may be initiated by other mechanisms, but this is often followed by methylation of CpG sites in the promoter CpG island to cause the stable silencing of the gene. In contrast, in one study of colon tumors compared to adjacent normal appearing colonic mucosa, 1,734 CpG islands were heavily methylated in tumors whereas these CpG islands were not methylated in the adjacent mucosa. Half of the CpG islands were in promoters of annotated protein coding genes, A third study found more than 2,000 genes differentially methylated between colon cancers and adjacent mucosa. Using gene set enrichment analysis, 569 out of 938 gene sets were hypermethylated and 369 were hypomethylated in cancers. Hypomethylation of CpG islands in promoters results in overexpression of the genes or gene sets affected. One 2012 study listed 147 specific genes with colon cancer associated hypermethylated promoters, along with the frequency with which these hypermethylations were found in colon cancers. At least 10 of those genes had hypermethylated promoters in nearly 100% of colon cancers. They also indicated 11 microRNAs whose promoters were hypermethylated in colon cancers at frequencies between 50% and 100% of cancers. MicroRNAs (miRNAs) are small endogenous RNAs that pair with sequences in messenger RNAs to direct post transcriptional repression. On average, each microRNA represses several hundred target genes. Thus microRNAs with hypermethylated promoters may be allowing over expression of hundreds to thousands of genes in a cancer. The information above shows that, in cancers, promoter CpG hyper/hypo methylation of genes and of microRNAs causes loss of expression (or sometimes increased expression) of far more genes than does mutation.

DNA repair genes are frequently repressed in cancers due to hypermethylation of CpG islands within their promoters. In head and neck squamous cell carcinomas at least 15 DNA repair genes have frequently hypermethylated promoters; these genes are XRCC1, MLH3, PMS1, RAD51B, XRCC3, RAD54B, BRCA1, SHFM1, GEN1, FANCE, FAAP20, SPRTN, SETMAR, HUS1, and PER1. About seventeen types of cancer are frequently deficient in one or more DNA repair genes due to hypermethylation of their promoters. As an example, promoter hypermethylation of the DNA repair gene MGMT occurs in 93% of bladder cancers, 88% of stomach cancers, 74% of thyroid cancers, 40% 90% of colorectal cancers and 50% of brain cancers. Promoter hypermethylation of LIG4 occurs in 82% of colorectal cancers. Promoter hypermethylation of NEIL1 occurs in 62% of head and neck cancers and in 42% of non small cell lung cancers. Promoter hypermethylation of ATM occurs in 47% of non small cell lung cancers. Promoter hypermethylation of MLH1 occurs in 48% of non small cell lung cancer squamous cell carcinomas. Promoter hypermethylation of FANCB occurs in 46% of head and neck cancers. On the other hand, the promoters of two genes, PARP1 and FEN1, were hypomethylated and these genes were over expressed in numerous cancers. PARP1 and FEN1 are essential genes in the error prone and mutagenic DNA repair pathway microhomology mediated end joining. If this pathway is over expressed the excess mutations it causes can lead to cancer. PARP1 is over expressed in tyrosine kinase activated leukemias, in neuroblastoma, in testicular and other germ cell tumors, and in Ewing's sarcoma, FEN1 is over expressed in the majority of cancers of the breast, prostate, stomach, neuroblastomas, pancreatic, and lung. DNA damage appears to be the primary underlying cause of cancer. If accurate DNA repair is deficient, DNA damages tend to accumulate. Such excess DNA damage can increase mutational errors during DNA replication due to error prone translesion synthesis. Excess DNA damage can also increase epigenetic alterations due to errors during DNA repair. Such mutations and epigenetic alterations can give rise to cancer (see malignant neoplasms). Thus, CpG island hyper/hypo methylation in the promoters of DNA repair genes are likely central to progression to cancer.

Since age has a strong effect on DNA methylation levels on tens of thousands of CpG sites, one can define a highly accurate biological clock (referred to as epigenetic clock or DNA methylation age) in humans and chimpanzees.

Unmethylated CpG dinucleotide sites can be detected by Toll like receptor 9 (TLR 9) on plasmacytoid dendritic cells, monocytes, natural killer (NK) cells, and B cells in humans. This is used to detect intracellular viral infection.

In mammals, DNA methyltransferases (which add methyl groups to DNA bases) exhibit a sequence preference for cytosines within CpG sites. In the mouse brain, 4.2% of all cytosines are methylated, primarily in the context of CpG sites, forming 5mCpG. Most hypermethylated 5mCpG sites increase the repression of associated genes. In 2016 Halder et al. using mice, and in 2017 Duke et al. As reviewed in 2018, in brain neurons, 5mC is oxidized by the ten eleven translocation (TET) family of dioxygenases (TET1, TET2, TET3) to generate 5 hydroxymethylcytosine (5hmC). In successive steps TET enzymes further hydroxylate 5hmC to generate 5 formylcytosine (5fC) and 5 carboxylcytosine (5caC). Thymine DNA glycosylase (TDG) recognizes the intermediate bases 5fC and 5caC and excises the glycosidic bond resulting in an apyrimidinic site (AP site). In an alternative oxidative deamination pathway, 5hmC can be oxidatively deaminated by activity induced cytidine deaminase/apolipoprotein B mRNA editing complex (AID/APOBEC) deaminases to form 5 hydroxymethyluracil (5hmU) or 5mC can be converted to thymine (Thy). 5hmU can be cleaved by TDG, single strand selective monofunctional uracil DNA glycosylase 1 (SMUG1), Nei Like DNA Glycosylase 1 (NEIL1), or methyl CpG binding protein 4 (MBD4). AP sites and T:G mismatches are then repaired by base excision repair (BER) enzymes to yield cytosine (Cyt). Two reviews summarize the large body of evidence for the critical and essential role of ROS in memory formation. The DNA demethylation of thousands of CpG sites during memory formation depends on initiation by ROS. In 2016, Zhou et al.,

CpG depletion has been observed in the process of DNA methylation of Transposable Elements (TEs) where TEs are not only responsible in the genome expansion but also CpG loss in a host DNA. TEs can be known as "methylation centers" whereby the methylation process, the TEs spreads into the flanking DNA once in the host DNA. This spreading might subsequently result in CpG loss over evolutionary time. Older evolutionary times show a higher CpG loss in the flanking DNA, compared to the younger evolutionary times. Therefore, the DNA methylation can lead eventually to the noticeably loss of CpG sites in neighboring DNA.

Previous studies have confirmed the variety of genomes sizes among species, where invertebrates and vertebrates have smaller and larger genomes compared to humans. The genome size is strongly connected to the number of transposable elements. However, there is a correlation between the number of TEs methylation versus the CpG amount. This negative correlation consequently causes depletion of CpG due to intergenic DNA methylation which is mostly attributed to the methylation of TEs. Overall, this contributes to a noticeable amount of CpG loss in different species' genomes.

Alu elements are known as the most abundant type of transposable elements. Some studies have used Alu elements as a way to study the factors responsible for genome expansion. Alu elements are CpG rich in a longer amount of sequence, unlike LINEs and ERVs. Alus can work as a methylation center, and the insertion into a host DNA can produce DNA methylation and provoke a spreading into the Flanking DNA area. This spreading is why there are is considerable CpG loss and genome expansion. However, this is a result that is analyzed over time because older Alus elements show more CpG loss in sites of neighboring DNA compared to younger ones.