Biology Notes on DNA Methylation | Genetics

The below mentioned article provides notes on DNA methylation.

The DNA of many higher eukaryotes is modified after replication by methylation of cytosine bases at the 5-carbon position. The cytosines are incorporated in their normal unmodified form in the course of DNA replication; the methyl group (CH₃) is then added by an enzyme DNA methylase.

In mammals DNA is methylated specifically at the cytosines that precede guanosine residues, that is CpG di-nucleotides (5′-CG-3′). Many mammalian genes have CG-rich regions upstream of the coding region that provide multiple sites for methylation. These are called CpG islands where “p” represents the phosphate group in the polynucleotide backbone.

In higher plants 3—7% of the bases contain methylated cytosine, while in animal DNA the proportion is less. In some protozoa, the adenine groups are methylated, while in the green alga Chlamydomonas both adenine and cytosine are methylated. There is a higher concentration of methylated cytosines in highly repetitious eukaryotic DNA.

Methylation is found to correlate with reduced transcriptional activity of genes. Genes that contain high frequency of CpG di-nucleotides in the vicinity of their promoters show a low rate of transcriptional activity. Methylation inhibits transcription of these genes through the action of a protein, MeCP2, that specifically binds to methylated DNA and brings about repression of transcription.

Heavy methylation associated with reduced transcription is seen in the inactive X chromosome in mammalian cells which is extensively methylated. In fact, in adult mammals, the majority of CpG di-nucleotides in all chromosomes are methylated in adult cells. The un-methylated CpGs are usually associated with the promoters of housekeeping genes that are transcriptionally active.

Treatment of cells with the cytosine analog azacytidine reverses methylation and can restore transcriptional activity. For example, in cell culture, some lineages of rat pituitary tumour cells are able to express the gene for prolactin, whereas other related lineages cannot do so.

The reason being that the gene is methylated in the non-producing cells but is not methylated in cells that produce prolactin. Treatment of non-producing cells with azacytidine reverses methylation, resulting in prolactin expression in these cells.

The regulatory role of DNA methylation has been determined through an unusual type of epigenetic silencing known as genomic imprinting, which controls the expression of some genes involved in development of embryos in mammals. Usually, both paternal and maternal alleles of a gene are expressed in diploid cells.

However, a few imprinted genes have been found in mouse and humans whose expression depends upon whether they have been transmitted (inherited) from the mother or from the father. In some cases, only the paternal allele of an imprinted gene is expressed, whereas the maternal allele is transcriptionally inactive.

In other imprinted genes, the maternal allele is expressed while the paternal allele is inactive. It seems that DNA methylation is able to distinguish between paternal and maternal alleles of imprinted genes. For example, the gene H19 is specifically methylated during development of male germ cells, but not female germ cells. Thus H19 is transcribed only from the maternal copy.

After fertilisation, the zygote develops into an embryo containing a methylated paternal allele and an un-methylated maternal allele of this gene. The paternal H19 allele therefore, remains methylated and transcriptionally inactive in embryonic cells and somatic tissues.

The process of genomic imprinting displays following characteristics. Imprinting occurs in the germ line; it affects at the most a few hundred genes, many of them located in clusters; it is accompanied by heavy methylation; imprinted genes are differentially methylated in the female and male germ lines; once imprinted and methylated, a silenced gene remains transcriptionally inactive during embryogenesis.

Although mammalian gametes are extensively methylated, most of the DNA is de-methylated in pre-implantation development, except for imprinted genes that have a sex-specific gene silencing pattern. After implantation, embryonic DNA is re-methylated to the heavy methylation levels that are present in adult somatic cells. In the germ line of both males and females, all re-methylated genes acquire methylation to an identical level.

Except for a few genes that have sex-specific patterns of imprinting and different levels of methylation in female and male in females, imprinted genes undergo methylation during growth of oocytes prior to ovulation. Because methylation associated with imprinting is retained throughout embryonic development, any gene that is imprinted in either the female or male germ line has, therefore, only one active copy in the embryo.

Epigenetic sex-specific gene silencing is seen in a pair of human syndromes called Prader-Willi syndrome and Angelman syndrome that display neuromuscular defects, mental retardation and some other abnormalities. Both conditions result from rare spontaneous deletions involving the chromosomal region 15qll.

If the deletion takes place in the father, it results in Prader-Willi syndrome, whereas if the deletion occurs in the mother, it produces Angelman syndrome. The reason is that, the DNA in the deleted region 15qll includes at least three genes that are imprinted and differentially methylated in the male and female gametes.

Ribosomal RNA Gene Silencing:

Eukaryotes have hundreds of rRNA genes whose transcription by RNA polymerase I maintains ribosome production and protein synthesis. However, only a subset of the total rRNA gene pool is active at any one time, thus maintaining a dynamic balance between gene silencing and activation.

Since rRNA genes are essentially identical in sequence in a pure species, it is not possible to distinguish between active and inactive genes. The explanation is provided by the epigenetic phenomenon called nucleolar dominance. Epigenetic silencing of one parental set of rRNA genes and transcription of rRNA genes from the other parent is associated and is called nucleolar dominance, commonly observed in interspecific hybrids.

Parental genes in hybrids typically differ in sequence as well as expression, and have allowed understanding of chromatin modifications of rRNA genes in active and inactive states.

Since only active rRNA genes initiate formation of a nucleolus in the nucleolus organizer region of the chromosome, hence the name nucleolar dominance. The genes that encode ribosomal RNA are present in two types of chromatin, one that permits transcription, the other that is transcriptionally inactive.

As in other epigenetic phenomena, chromatin modifications result in selective gene silencing. Although the epigenetic mechanisms that discriminate between parental sets of rRNA genes are not known, it seems that the level of cytosine methylation and histone modifications (acetylation) that alter chromatin structure effectively turn rRNA genes on and off resulting in active and inactive rRNA genes.