The significance of DNA methylation patterns: promoter inhibition by sequence-specific methylation is one functional consequence

Abstract

This paper presents a review of previously published results from my laboratory on the inactivating or inhibiting function of sequence-specific methylation on promoter activity. In this study, viral promoters, mostly those from adenovirus type 2 (Ad2) or type 12 (Ad12), have been used. It has also been shown that the transcriptional block of these methylated viral promoters can, at least partly, be reversed by a transactivating protein or by the presence of a strong enhancer. We have also adduced evidence that the methylation of the late E2A promoter of Ad2 DNA at positions +6 and +24 from the cap site of this promoter interferes with the binding of one or several proteins at these particular sites, at least when 50 or 73 base-pair long fragments of this promoter have been used for studies on protein binding. With a 377 base-pair fragment, binding differences between the unmethylated and the 5'-CCGG-3' methylated late E2A promoter are not obvious. By applying the genomic sequencing technique developed by Church & Gilbert (1984), the patterns of methylation in all 5'-CG-3' dinucleotides in the late E2A promoter in the active or inactive state in different Ad2-transformed cell lines have been determined. It has been found that 5-methyldeoxycytidine residues introduced into foreign DNA, which is then integrated into the mammalian cell genome, can lead to the spreading of methylation starting from the point of initial methylation. We have begun to investigate whether certain patterns of methylation in mammalian DNA can also influence biological processes other than promoter activity. We have developed a cell-free system using nuclear extracts from hamster cells to study recombination between Ad12 DNA and hamster pre-insertion sites into which Ad12 DNA had previously integrated. The DNAs used in recombination experiments are in the unmethylated or the methylated form. Some speculative aspects have also been discussed in this review. Could existing patterns of methylation in mammalian (human) DNA represent composites of several interdigitating patterns each one of which might have a different signal value? Can a 5-methyldeoxycytidine group in DNA modulate DNA-protein interactions in a positive or negative way, for proteins which could have positively or negatively regulating functions? Patterns of methylation appear to be relatively stable over many years for cell lines propagated in culture. Are patterns of methylation stable also in different parts of the human chromosome? To what extent are these patterns inheritable?