Dna Methylation

DNA methylation is instrumental for both imprinting and X-chromosome inactivation. In mammals, physiological methylation of DNA is restricted to the 5-position of cytosine residues, and again only to those in CpG dinucleotides. Since CG is a palindromic sequence, a CpG site can be non-methylated, hemi-methylated, i.e. in one strand only, or fully methylated, i.e. symmetrically in both strands (Figure 8.4). Except during replication, the usual state of methylated sites in human DNA is symmetrical methylation. After replication, which creates a hemimethylated site, symmetrical methylation is re-established by a maintenance DNA methyltransferase. If no re-methylation occurs, the site remains hemi-methylated and can become unmethylated in one daughter strand during the next round of replication. So, normally, removal of DNA methylation requires at least two cell cycles.

Methylation levels vary somewhat among normal tissues, with more pronounced changes during germ cell and embryonic development. In typical somatic cells, 3.54% of all cytosines are methylated. This is an average value, since DNA methylation is unequally distributed across the genome. Most methylcytosines are contained in repetitive sequences such as LINE and SINE retrotransposons interspersed in the genome and in CpG-rich satellites concentrated in peri- and juxtacentromeric regions. Genes and intergenic regions, too, are mostly methylated.

Dnmt3a Dnmt3b Novo Methylation

Figure 8.4 Establishment and changes of methylation status at CpG sites Hemimethylated DNA formed during DNA replication from symmetrically methylated DNA is reconverted by a maintenance methylase (normally DNMT1). Insufficient activity of the enzyme can lead to loss of methylation after two rounds of replication. Unmethylated sites can be methylated by the successive action of de-novo-methylases (normally DNMT3A or DNMT3B) and the maintenance enzyme. All enzymes use S-adenosylmethionine (SAM) as the methyl group donor, converting it to S-adenosylhomocysteine (SAH).

Figure 8.4 Establishment and changes of methylation status at CpG sites Hemimethylated DNA formed during DNA replication from symmetrically methylated DNA is reconverted by a maintenance methylase (normally DNMT1). Insufficient activity of the enzyme can lead to loss of methylation after two rounds of replication. Unmethylated sites can be methylated by the successive action of de-novo-methylases (normally DNMT3A or DNMT3B) and the maintenance enzyme. All enzymes use S-adenosylmethionine (SAM) as the methyl group donor, converting it to S-adenosylhomocysteine (SAH).

In contrast, regulatory regions of active genes are generally undermethylated. Specifically, in <50% of all human genes, 0.5 - 2 kb stretches around the transcriptional start site, including the basal promoter, are richer in CpG-dinucleotides than the rest of the genome, with a frequency of >0.6 found/expected in a random sequence and a higher GC content than the rest of the genome (Figure 8.5). These sequences are called 'CpG-islands'. As a rule, they remain unmethylated throughout development and in all tissues. A prominent exception are the CpG-islands on the inactive X-chromosome which are methylated and the respective genes are silenced.

Figure 8.5 CpG islands in the human genome Top: Schematic illustration of variations in GC content and distribution of CpG sites (stick and circle symbols) in the human genome. Note that gene 2 does not possess a CpG island, like »40% of human genes. Bottom: methylation patterns in normal and cancer cells. As customary, methylated sites are indicated by filled and unmethylated sites by open circles.

Figure 8.5 CpG islands in the human genome Top: Schematic illustration of variations in GC content and distribution of CpG sites (stick and circle symbols) in the human genome. Note that gene 2 does not possess a CpG island, like »40% of human genes. Bottom: methylation patterns in normal and cancer cells. As customary, methylated sites are indicated by filled and unmethylated sites by open circles.

Apparently, the lack of methylation helps to mark CpG-islands as regions of potential transcription in the genome. Typically, genes with CpG-island type promoters can be transcribed in several different cell types. DNA methylation also regulates the transcription of some other genes without CpG-islands, including some with cell-type specific expression.

CpG-islands stand out from the rest of the genome, because they contain more CpG-dinucleotides. More precisely, the rest of the genome contains less, as a consequence of cytosine loss during evolution. Hydrolytic deamination of cytosine occurs frequently, spontaneously or induced by chemicals, and yields uracil. This base is very efficiently recognized as incongruous and accordingly repaired (^3.1).

In contrast, methylcytosine yields methyluracil, i.e. thymine, albeit in a G-T mismatch. Such mismatches are accordingly repaired preferentially towards G-C, with the help of the protein MBD4 which recognizes the methylcytosine in the opposite strand of the CpG palindrome (^3.1).

In spite of such precautionary mechanisms, over evolutionary periods, CpGs have become depleted from heavily methylated sequences by mutating to TpG (or CpA). This depletion has not affected sequences exempt from methylation and in this fashion has sculpted CpG-islands out of the genome background. In fact, the mutation rate at methylated cytosines remains higher in the present. Therefore, methylated CpGs are preferential sites of mutations not only in the human germline, but also in cancers.

DNA methylation patterns change substantially during development. During germ cell development, DNA is first widely demethylated and then remethylated to yield distinctive patterns in oocytes and sperm. Differential methylation at imprinted genes is also established during this period. Following fertilization, methylation again decreases across the genome, although some specific sites, e.g. in imprinted genes, are exempt from these changes. Extraembryonal tissues remain strongly demethylated, whereas in the cells of the fetus proper the genome is subjected to a wave of de-novo-methylation during gastrulation. This process largely establishes the overall level of methylation found in the DNA of somatic cells. Demethylation of genes expressed in a cell-type specific fashion then leads to the DNA methylation patterns of the various cell types. Of note, CpG-islands are in general exempt from these changes and remain unmethylated throughout development. Likewise, methylation patterns of imprinted genes follow their own rules.

These wide swings in overall methylation levels likely reflect the necessity to completely reprogram the expression of the genome, once during the development of germ cells and then again during embryonic development. A major reason why cloned embryos are often defective appears to be a failure to achieve this reprogramming properly. Very low levels of DNA methylation in germ cells may moreover signify a state of chromatin that facilitates recombination during meiosis.

Given this background, it is not unexpected that the altered state of cancer cells is often also accompanied by alterations in DNA methylation. Basically, two types of alterations can be distinguished (Figure 8.6). Both can occur in the same cell. In many cancers, overall DNA methylation levels are diminished by up to 70% compared to the corresponding normal cell type. This decrease affects mostly methylcytosine contained in repetitive sequences and is therefore termed 'global' or 'genome-wide' 'hypomethylation'. In contrast, specific sites can become 'hypermethylated'. Hypermethylation occurs, in particular, at CpG-islands which are never methylated otherwise. Like the extent of hypomethylation, that of hypermethylation differs widely between cancers, even of the same histological type. In some cancers, only individual CpG-islands become hypermethylated, whereas several hundreds are afflicted in others. Moreover, hypermethylation affects different genes in different cancers, although some genes are prone to hypermethylation in many cancer types (Table 8.2). Alterations of DNA methylation may also affect the expression of imprinted genes (^8.2).

Figure 8.6 Alterations of DNA methylation in human cancers In normal somatic cells, most of the genome is densely methylated, specifically repetitive sequences. CpG-islands form a prominent class of sequences exempt from methylation. In cancer cells, up to several hundred CpG-islands become hypermethylated (black dots), whereas repetitive sequences, in particular, are hypomethylated.

Figure 8.6 Alterations of DNA methylation in human cancers In normal somatic cells, most of the genome is densely methylated, specifically repetitive sequences. CpG-islands form a prominent class of sequences exempt from methylation. In cancer cells, up to several hundred CpG-islands become hypermethylated (black dots), whereas repetitive sequences, in particular, are hypomethylated.

Hypermethylation of CpG-islands is almost invariably associated with stable silencing of the affected genes. Therefore, hypermethylation is a very efficient means of gene inactivation in cancers. It is now regarded as a mechanism of tumor suppressor gene inactivation comparable to mutation and deletion. For instance, the CDKN2A locus (^5.2) is inactivated in a wide variety of human cancers. In almost every cancer type, mutation, deletion, and promoter hypermethylation are all observed as mechanisms of inactivation, although their relative contributions vary. One important difference towards mutation and deletion, however, is that hypermethylation is in principle reversible by inhibitors of DNA methylation. Of course, in cancers with hundreds of genes inactivated by hypermethylation, not each one of them is a tumor suppressor like CDKN2A. Rather, gene silencing via DNA methylation may reflect a 'slimming' of gene expression.

By comparison, global hypomethylation would be expected to increase gene expression across the genome at large. There is, indeed, some evidence for hypomethylation to increase the level of 'transcriptional noise' and to cause inappropriate expression of certain sequences, e.g. of retrotransposon sequences and of 'cancer testis antigens', i.e. genes that are normally restricted to developing male germ cells. More importantly, perhaps, global hypomethylation is associated with enhanced chromosomal instability. The underlying mechanism are under investigation. Possibly, decreased methylation in pericentromeric repeats and interspersed repetitive sequences facilitates illegitimate recombination and chromosomal loss during mitosis.

Table 8.2. A selection of hypermethylatedgenes in human cancers

Gene

Function

Cancers with hypermethylation

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