5-Methyl-2''-deoxycytidine in single-stranded DNA can act in cis to signal de novo DNA methylation.

Methylation of cytosine residues in DNA plays an important role in regulating gene expression during vertebrate embryonic development. Conversely, disruption of normal patterns of methylation is common in tumors and occurs early in progression of some human cancers. In vertebrates, it appears that the same DNA methyltransferase maintains preexisting patterns of methylation during DNA replication and carries out de novo methylation to create new methylation patterns. There are several indications that inherent signals in DNA structure can act in vivo to initiate or block de novo methylation in adjacent DNA regions. To identify sequences capable of enhancing de novo methylation of DNA in vitro, we designed a series of oligodeoxyribonucleotide substrates with substrate cytosine residues in different sequence contexts. We obtained evidence that some 5-methylcytosine residues in these single-stranded DNAs can stimulate de novo methylation of adjacent sites by murine DNA 5-cytosine methyltransferase as effectively as 5-methylcytosine residues in double-stranded DNA stimulate maintenance methylation. This suggests that double-stranded DNA may not be the primary natural substrate for de novo methylation and that looped single-stranded structures formed during the normal course of DNA replication or repair serve as "nucleation" sites for de novo methylation of adjacent DNA regions.     

De novo methylation of an embryonic globin gene during normal development is strand specific and spreads from the proximal transcribed region.

The recently discovered de novo methyltransferases DNMT3a and DNMT3b have been shown to be critical to embryonic development. However, at a single gene level, little is known about how the methylation pattern is established during development. The avian embryonic rho-globin gene promoter is completely unmethylated in 4-day-old chicken embryonic erythroid cells, where it is expressed at a high level, and completely methylated in adult erythroid cells, where it is silent. The methylation pattern of the rho-globin gene promoter, proximal transcribed region, and distal transcribed region on both DNA strands was examined during development in chicken erythroid cells. It was found that de novo methylation targets the CpG-dense proximal transcribed region on the coding (top) strand initially, followed by spreading into the 3' region and into the promoter region. Methylation of the template (bottom) strand lags behind that of the coding strand, and complete methylation of both strands occurs only after the gene has been silenced. The results of the study indicate that establishment of the de novo methylation pattern involves strand-specificity and methylation spreading.     

Nucleotide-sequence-specific de novo methylation in a somatic murine cell line.

DNA fragments encoding the mouse steroid 21-hydroxylase (C21 or Cyp21A1) gene are de novo methylated when introduced into the mouse adrenocortical tumor cell line Y1 by DNA-mediated gene transfer. Although CCGG sequences within the C21 gene are de novo methylated, CCGG sites within flanking vector sequences, other mammalian gene sequences driven by the C21 promoter, and the neomycin-resistance gene, which was cotransfected with the C21 gene, do not become methylated. At least two separate signals for de novo methylation are encoded within the gene since three fragments derived from the C21 gene were methylated de novo. Specific de novo methylation of C21-derived sequences does not occur in L cells or Y1 kin8 cells; this suggests that the cellular factors needed for de novo methylation of the C21 gene are not ubiquitous. Most DNA sequences are not de novo methylated when introduced into somatic cells and DNA sequences other than the C21 gene are not de novo methylated when introduced into Y1 cells. Several groups have suggested that de novo methylation occurs in early embryonic cells and that somatic cells strictly maintain their methylation pattern by a semiconservative methyltransferase. Our results suggest that de novo methylation of specific nucleotide sequences can occur in some mammalian somatic cells.     

DNA methylation and silencing of gene expression.

DNA methylation is associated with the silencing of gene expression. The predominant mechanism involves the methylation of DNA and the subsequent recruitment of binding proteins that preferentially recognize methylated DNA. In turn, these proteins associate with histone deacetylase and chromatin remodelling complexes to cause the stabilization of condensed chromatin. Recent studies have indicated that the opposite might also hold; namely, that targeting of methylation might depend on altered chromatin structure. The family of methyltransferases and methyl-binding proteins is expanding and becoming better characterized. This review will focus on the mechanisms of methylation-associated silencing of gene expression.