Hemimethylation and non-CpG methylation levels in a promoter region of human LINE-1 (L1) repeated elements.
DNA methylation within the promoter region of human LINE1 (L1) transposable elements is important for maintaining transcriptional inactivation and for inhibiting L1 transposition. Determining methylation patterns on the complementary strands of repeated sequences is difficult using standard bisulfite methylation analysis. Evolutionary changes in each repeat and the variations between cells or alleles of the same repeat lead to a heterogeneous population of sequences. Potential sequence biases can arise during analyses that are different for the converted sense and antisense strands. These problems can be avoided with hairpin-bisulfite PCR, a double-stranded PCR method in which complementary strands of individual molecules are attached by a hairpin linker ligated to genomic DNA. Using human L1 elements to study methylation of repeated sequences, (i) we distinguish valid L1 sequences from redundant and contaminant sequences by applying the powerful new method of molecular barcodes, (ii) we resolve a controversy on the level of hemimethylation of L1 sequences in fetal fibroblasts in favor of relatively little hemimethylation, (iii) we report that human L1 sequences in different cell types also have primarily concordant CpG methylation patterns on complementary strands, and (iv) we provide evidence that non-CpG cytosines within the regions analyzed are rarely methylated.
Burden
Alice F
AF
Department of Biology, University of Washington, Seattle, Washington 98195, USA. afb4@u.washington.edu
Manley
Nathan C
NC
Clark
Aaron D
AD
Gartler
Stanley M
SM
Laird
Charles D
CD
Hansen
R Scott
RS
D001483:Base Sequence; D018899:CpG Islands; D003596:Cytosine / Q000737:chemistry; D019175:DNA Methylation; D019143:Evolution, Molecular; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D020084:Long Interspersed Nucleotide Elements; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012689:Sequence Homology, Nucleic Acid; D013447:Sulfites / Q000378:metabolism / Q000494:pharmacology
D016376:Oligonucleotides, Antisense; D013447:Sulfites; D003596:Cytosine