We have investigated the function and sequence specificity of DNA methylation in the hypermethylated CpG island promoter region of the endogenous human LINE-1 (L1) retrotransposon family. In nontransformed human embryonic fibroblasts, inhibition of DNA methylation with 5-azadeoxycytidine induced a greater than 4-fold increase in transcription from potentially functional L1 elements without increasing the transcription level of the majority of degenerate elements, implicating hypermethylation in the repression of L1 activity. Using bisulfite genomic sequencing to assess the pattern of methylation in a subset of nondegenerate L1 elements, we found 29 sites within a 460-base pair region of the noncoding (top) DNA strand of the L1 promoter in which cytosine methylation was maintained with high efficiency. Of these, 25 were at CG dinucleotides and four were in non-CG sites. When the methylation sites were analyzed for the complementary (bottom) strand, the only highly conserved sites of methylation were in CG dinucleotides. Several of these sites of CG methylation in the bottom (coding) strand were at positions where top (noncoding) strand-derived sequences were unmethylated, suggesting that these sites might be maintained in a hemimethylated state. Hence, there is a subset of human L1 elements in which methylation is efficiently maintained in asymmetric non-CG sites and further that this non-CG methylation may be part of a wider phenomenon involving hemi-methylation at CG dinucleotides. Maintenance of asymmetric methylation at non-CG sites (and possibly at hemi-methylated CG dinucleotides) could be through a novel DNA methyltransferase activity. Alternatively, the promoter region of L1 elements may be induced by factor binding to form some type of secondary structure that presents as a highly efficient substrate for de novo methylation.Five to ten percent of the human genome is derived from one transposable element family, the L1 or LINE-1 family (1, 2), which belongs to the non-LTR retrotransposon class of elements that are spread widely among eukaryotes. Although the majority of human L1 elements are inactive degenerate remnants, some are clearly functional, as de novo insertion of L1 elements have been documented in the germ line of both humans (3-5) and mice (6) as well as in somatic (tumor) cells (7). Nondegenerate full-length mammalian L1 elements are some 6 kilobases in length and contain two evolutionarily conserved open reading frames, the second of which encodes a reverse transcriptase (8, 9) with intrinsic RNase-H and AP endonuclease-like activity (10, 11). The promoter responsible for the full-length L1 transcript has been shown to be located within the 5Ј end of the element and downstream from the transcriptional start site (12). This promoter region also contains a strong binding site for the ubiquitous transcription factor, YY1, which has recently been shown to be the nuclear matrix-associated protein, NMP-1 (13). In contrast to regions normally associated with the nuclear matrix that are high in AT b...
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