Asymmetric Methylation in the Hypermethylated CpG Promoter Region of the Human L1 Retrotransposon

Abstract

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...

Differential Patterns of Methylation of the IFN-γ Promoter at CpG and Non-CpG Sites Underlie Differences in IFN-γ Gene Expression Between Human Neonatal and Adult CD45RO− T Cells

“…In a sequence covering four gene regions, we detected little methylation at these putative CpNpG sites. In another study examining human embryonic fibroblasts, a number of non-CpG methylation sites were identified in the sequence of an L1 retrotransposon (29). The most recent study of importance here was a genome-wide examination of non-CpG methylation in murine embryonic stem cells (26), which showed that methylation occurred at CpA and CpT sites in wildtype embryonic stem cells and different somatic tissues.…”

Antisense Promoter of Human L1 Retrotransposon Drives Transcription of Adjacent Cellular Genes

“…For example, chimeric N10 and N12 transcripts were more abundant in JEG 3 rather than NTera2D1 cells, suggesting more efficient transcription from the L1Hs ASP in the former cell line. It should be emphasized that activation and repression of L1Hs transcription and presumably its antisense transcription depend on the methylation status of the genomic L1Hs (37,39). An interesting aspect of L1Hs transcription is that at least in NTera2D1 cells where sense transcription is activated, ASPdirected transcripts complementary to the sense transcripts of about 500 nt are also produced.…”

Repetitive sequence environment distinguishes housekeeping genes

“…We show that this decrease in Alu concentration is accompanied by an increase in long repeats and repeat tracts, the most abundant of which are LINE-1 elements. Long repeats including LINE-1 elements are normally heavily DNA-methylated (Woodcock et al, 1988;Crowther et al, 1991;Woodcock et al, 1997), a feature of heterochromatin. DNA methylation (and heterochromatin in general) has been reported to suppress homologous recombination (Pàldi et al, 1995;Maloisel and Rossignol, 1998;Schnable et al, 1998;Fu et al, 2002;Yao et al, 2002;Yamada et al, 2004;Myers et al, 2005) as well as transposition (Yoder et al, 1997;Walsh et al, 1998;Hirochika et al, 2000;Robertson, 2001;Bird, 2002;Kato et al, 2003).…”