DNA Methylation Density Influences the Stability of an Epigenetic Imprint and Dnmt3a/b-Independent De Novo Methylation

Abstract

DNA methylation plays an important role in transcriptional repression. To gain insight into the dynamics of demethylation and de novo methylation, we introduced a proviral reporter, premethylated at different densities, into a defined chromosomal site in murine erythroleukemia cells and monitored the stability of the introduced methylation and reporter gene expression. A high density of methylation was faithfully propagated in vivo. In contrast, a low level of methylation was not stable, with complete demethylation and associated transcriptional activation or maintenance-coupled de novo methylation and associated silencing occurring with equal probability. Deletion of the proviral enhancer increased the probability of maintenance-coupled de novo methylation, suggesting that this enhancer functions in part to antagonize such methylation. The DNA methyltransferases (MTases) Dnmt3a and Dnmt3b are thought to be the sole de novo MTases in the mammalian genome. To determine whether these enzymes are responsible for maintenance-coupled de novo methylation, the unmethylated or premethylated proviral reporter was introduced into DNA MTase-deficient embryonic stem cells. These studies revealed the presence of a Dnmt3a/Dnmt3b-independent de novo methyltransferase activity that is stimulated by the presence of preexisting methylation.DNA methylation is essential for mammalian development (27, 37), playing an important role in maintaining transcriptional silencing of genes on the inactive X chromosome, imprinted genes, and parasitic elements (2, 5). In mammals, DNA methylation occurs predominantly on cytosines (m 5 C) in the context of the 5Ј-CpG-3Ј dinucleotide, and this epigenetic mark is propagated on both parent and nascent strands after DNA replication. Recent experiments suggest that DNA methylation may serve as a tag for the recruitment of methyl DNA binding domain proteins and the histone deacetylase complexes with which they interact (24, 35) to generate a chromatin structure that is repressive for transcription (33). However, why some genes or CpG sites are susceptible to methylation while others remain methylation free remains to be determined.Using in vitro-methylated constructs and transient-transfection assays, a number of studies have shown that enhancers and associated transcription factor complexes play an important role in overcoming the repressive effects of methylation (25, 51). Transcription of an ␣-globin reporter construct methylated at low density, for example, only occurs in the presence of an enhancer (6, 7), but even in the presence of this enhancer, dense methylation prevents transcription. Similarly, the degree of repression of an episomal Rous sarcoma virus promoter construct is correlated with increasing methylation density, with a high level of methylation extinguishing expression completely (20). While these experiments reveal the antagonistic relationship between DNA methylation density and transcriptional activity, the methods used preclude analysis of the dynamic relationship between trans...

Methylation of the 3p22 region encompassing MLH1 is representative of the CpG island methylator phenotype in colorectal cancer

“…55 Mechanistically, it has been suggested that the low level and mosaic patterns of methylation can provide a nidus of methylation, around which subsequent methylation can spread through stimulation of de novo methyltransferase activity. 56 An interesting observation was that four individuals in whom concomitant 3p22 methylation occurred in conjunction with the BRAF V600E mutation in their apparently normal colonic mucosa had each developed hyperplastic polyps, and in some cases additional adenomas. Although these individuals did not present with an additional colorectal carcinoma, the finding of pre-neoplastic lesions is consistent with a common molecular mechanism underlying their phenotype.…”

Prospects and implications of using chromatin insulators in gene therapy and transgenesis

“…We would like to speculate on two possibilities for achieving this: the first one is by using the homologous recombination Cre-Lox system in combination with insulators, and the second option is to drive the insertion event directly into heterochromatin in an attempt to avoid integration in coding sequences. (61,73) Obviously, this strategy would require the presence of insulators in the transgenes, and it would also require some certainty of the effectiveness of the insulator shielding. Clearly, further studies of insulator function in repressive chromatin environments will be necessary to employ such a strategy.…”

“…(61) As a means of combating these negative viral elements, it has recently been demonstrated that the cHS4 insulator is able to block retroviral silencing in a murine stem cell virus (MSCV) vector in transgenic mice. (60) Interestingly, the results obtained with this MSCV retrovirus are in agreement with the data obtained previously in chicken stably transfected cell lines.…”

The biological significance of substrate inhibition: A mechanism with diverse functions

“…Interestingly, DNMT1 is also activated by nearby methylated cytosines on the complementary DNA strand [56][57][58][59][60]. The exact molecular mechanisms by which the inhibition and activation of DNMT1 take place are an area of active investigation.…”