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

Martha E. Linsenmeyer,William D. Warren,Celine B. Lawler,David M. Woodcock,Judith P. Doherty

doi:10.1074/jbc.272.12.7810

Martha E. Linsenmeyer, William D. Warren + Show 3 more

Open Access

https://doi.org/10.1074/jbc.272.12.7810

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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 hemi-methylated 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.

Highlights

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
In contrast to regions normally associated with the nuclear matrix that are high in AT bases [14], the 5Ј end of the human L1 element is an atypical CpG island in that it is very heavily methylated in DNA from both the human embryonic fibroblast culture used in the experiments reported here and in stimulated normal human peripheral lymphocytes in short term culture [15, 16]
We have employed a sensitive RNase protection assay that allows the quantitation of transcription from a subset of nondegenerate, potentially functional elements in the presence of a high background of transcription from degenerate elements [17] and the bisulfite genomic sequencing technique that generates a positive signal for sites of DNA methylation in individual strands from genomic DNA [18]

Summary

EXPERIMENTAL PROCEDURES

Nontransformed human embryonic fibroblast culture (HEF) and the human teratocarcinoma cell line Ntera2D1 cells were maintained as described previously [17]. PCR amplification of these control sequences was performed as above using the primer corresponding to bases 505– 484 of the L1 consensus in combination with a primer (5ЈGGAATTCGGTGAATTTGAGTTTGGT-3Ј) specific for the flanking plasmid vector DNA sequence. Of 20 cloned sequences amplified from this control plasmid (derived from eight separate modification reactions), a total of three unmodified cytosines were found, indicating an overall cytosine conversion efficiency greater than 99.9% This total excludes cytosine methylation in dcm sites (the inner C of CC(A/T)GG) observed in otherwise fully bisulfite-modified sequences amplified from pA41 plasmid grown in the dcmϩ Escherichia coli host DH12S. PCR amplification was performed with the 505– 484 consensus primer used for both genomic L1 products and pA41 controls in combination with a mixture of two plasmid-specific primers (equal quantities of the primer used above for amplification of unmethylated pA41 in combination with a primer 5Ј-ATAGGGCGAATTCGAGCTCGG-3Ј-specific for the M.SssI methylated form of the flanking plasmid vector DNA sequence).

RESULTS

No of clones

DISCUSSION