Modification of DNA sequences and their transcripts in gene expression
Methylation and gene control. During 1980s, a definite role of methylation and demethylation in gene control was also established. It was shown in many genes, that methylation of specific sites in the vicinity of a gene may lead to suppression of gene activity and demethylation may lead to expression of gene. With the help of restriction enzymes like HpaIIand MspII(methylation sensitive enzymes), degree of methylation, particularly in CpG (cytosine- guanine) rich sites located in the vicinity of certain genes could be studied and it was shown that these sites are undermethylated in tissues with gene activity in comparison to the tissues where the gene is inactive. Evidence of suppression of gene activity due to methylation is available from several experiments, which will be described briefly withqut giving details.
Herpes thymidine kinase (tk)gene was first methylated in vitro and then used for transformation of mouse tk cells. It was found that efficiency of transformation was low and the few transformed tk+ cells available had partially demethylated DNA in comparison to the methylated tk gene used for transformation. Similar results for another gene apart+in mouse were also available.
In mouse retroviruses, using transfection
experiments, it was shown that the inactive provirus sequences were heavily methylated, while active provirus sequences were undermethylated. It was also shown that non-methylated proviral sequences were infectious, and methylated sequences were
In cultured mouse embryo cells, differentiation could be artificially induced by inhibiting methylation using a base analogue 5 zacytidine (5 azaC), which is specific in inhibiting DNA methylation in vivo. It was also shown in these differentiated embryo cells that the DNA actually became demethylated. Effect of 5 azaC was also shown in avian retrovirus, where it induced gene expression associated with demethylation at an endogenous level. In human inactivated X-chromosome in mouse-human cell hybrids also, 5 azaC enhanced reactivation, presumably due to demethylation.
The above evidences suggest and support the model that absence of methylation is necessary for gene expression in many cases. It is believed that methylation or demethylation takes place at specific critical sites, which represent a small fraction of sites available in DNA. This results in alteration of a site in such a manner that a particular regulating protein may interact with the site in a very specific manner, thus regulating gene activity.