DNA Methylation & Gene Expression

In mammals DNA methylation occurs after DNA replication and involves the transfer of a methyl group from S-adenosyl-methionine (SAM) to the 5’ – position of cytosine residues, in a reaction catalysed by the enzyme DNA methyltransferase (Dnmt). Three enzyme families associated with DNA, identified are as follows :

DNA methyltransferase I   : primarily mediates maintenance of methyl    transferase  activity during the  S phase.

DNA methyltransferase II       :  plays a role in some aspects of  centromere function.

DNA methyltransferase III           :    plays an essential role in the de novo

[ Dnmt3a and Dnmt3b ]                    methylation in vivo.

DNA Methylation & Gene Expression :

CpG island is usually the site for DNA methylation. CpG islands are genomic regions that contain a high frequency of CG nucleotides. They are regions with at least 200 bp and with a GC percentage that is greater than 50% and with an observed/expected CpG ratio that is greater than 60%. CpG islands typically occur at or near the transcription start site of genes, particularly housekeeping genes. They act as strong promoters and have also been proposed to function as replication origins. It is estimated that CpG islands are associated with about half of all mammalian genes. Unmethylated CpG islands are associated house keeping genes, while the CpG islands of many tissue-specific genes are methylated, except in the tissues where they are expressed. Interactions between proteins and DNA are changed by methylation, leading to alterations in chromatin structure and a transcription rate change. Thus DNA methylation mechanisms for gene regulation can be as follows :

·         Preventing the binding of transcription factors to their target sequences via proteins that bind preferentially to methylated promoters.

·         Interfering with the binding of transcription factors to the methylated cytosine.

·         Altering the chromatin structure leading to a change in the rate of transcription.

The best-characterized cluster that follows a strict insulator model for imprinted expression is the cluster containing maternally expressed H19 and paternally expressed insulin-like growth factor 2 (IGF2). This cluster resides at 11p15.5 in humans and is found in conserved synteny on distal chromosome 7 in mice.

Human chromosome 11p15.5 contains a large cluster of imprinted genes spanning a region of 1-Mb. This cluster can be divided into two independent imprinting sub-domains: one containing H19, IGF2 and INS2 (H19 domain) and other containing CDKN1C, KCNQ1, ASCL2 and TSSC3 (KCNQ1 domain). These two domains contain several genes that play a role in embryo growth. Regulatory disturbances of a number of genes in this cluster result in imprinting disorder like Beckwith-Wiedemann Syndrome (BWS), Silver Russell Syndrome (SRS) and increased predisposition to development of specific embryonic tumors, like Wilm’s Tumor (Verona et al., 2003).

While most studies on H19 and IGF2 have been performed in the mouse, many attributes of these genes including their expression profile and regulatory mechanisms are similar in humans. In both mouse and human, H19 and IGF2 are widely expressed during embryonic development and postnatally down regulated in most tissues.

H19 encodes a fully processed 2.3 kb non-coding RNA and was initially implicated as a tumor suppressor. However, it has also been shown to have oncogenic properties. IGF2 encodes a protein that plays a major role in promoting embryonic and placental growth and development.

As with all imprinted clusters, imprinted expression of H19 and IGF2 is regulated by Imprinting Control Region [designated imprinting center 1 (IC1) in humans and ICR or differentially methylated domain (DMD) in mouse] located between the two genes. This region is approximately 5 kb and 2 kb long in humans and mice, respectively.

The H19 and IGF2 genes are co-coordinately regulated, most probably through interaction of either promoter with a common ‘enhancer element’ and the action of a further insulator.

Insulator proteins, such as CTCF (a zinc finger CCCTC-binding factor) and BORIS have been demonstrated to bind to specific DNA elements and prevent promoter and enhancer interaction. CTCF is present in both somatic and germ cells, whereas BORIS is expressed specifically in the male germ line and has been suggested to be associated with both demethylases mediating erasure of imprinting marks and methylases mediating de novo methylation.