Abstract
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.
Highlights
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 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
Methylation of CpG dyads within the promoter region of human LINE1 (L1)1 transposable elements is important for maintaining transcriptional inactivation and for inhibiting L1 transposition (1–3)
The analysis presented here for methylation patterns of L1 sequences in fibroblasts expands on our previous study (9) in several important ways
Summary
Methylation of CpG dyads within the promoter region of human LINE1 (L1) transposable elements is important for maintaining transcriptional inactivation and for inhibiting L1 transposition (1–3). CpG dyads that are discordant in methylation, i.e. hemimethylated, are expected to occur at low to moderate frequencies (5, 7–9) with the exception of regions undergoing transcriptional silencing or reactivation and transiently during DNA replication (10 –13) Contrary to this expectation, L1 sequences in human fetal fibroblast cultures were reported to be much more methylated on the sense strand than on the antisense strand (11), which would imply high levels of hemimethylation. The primers used for the sense strand might preferentially amplify methylated sequences from a small subclass of L1 sequences, whereas the antisense primers might amplify a different subclass of L1s that are more hypomethylated To overcome these inherent limitations of conventional bisulfite methylation analysis for determining methylation patterns on both strands of L1 repeat elements, we have used a doublestrand PCR method, “hairpin-bisulfite PCR” (9). Hemi- and Non-CpG Methylation Levels in Human L1 Repeats the concordance of CpG methylation on complementary strands in L1 sequences from other human cell types, and (iv) assess the methylation status of non-CpG cytosines within the regions analyzed
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
