Abstract
In vertebrates, DNA methylation-mediated repression of retrotransposons is essential for the maintenance of genomic integrity. In the current study, we developed a technique termed HT-TREBS (High-Throughput Targeted Repeat Element Bisulfite Sequencing). This technique is designed to measure the DNA methylation levels of individual loci of any repeat families with next-generation sequencing approaches. To test the feasibility of HT-TREBS, we analyzed the DNA methylation levels of the IAP LTR family using a set of 12 different genomic DNA isolated from the brain, liver and kidney of 4 one-week-old littermates of the mouse strain C57BL/6N. This technique has successfully generated the CpG methylation data of 5,233 loci common in all the samples, representing more than 80% of the individual loci of the five targeted subtypes of the IAP LTR family. According to the results, approximately 5% of the IAP LTR loci have less than 80% CpG methylation levels with no genomic position preference. Further analyses of the IAP LTR loci also revealed the presence of extensive DNA methylation variations between different tissues and individuals. Overall, these data demonstrate the efficiency and robustness of the new technique, HT-TREBS, and also provide new insights regarding the genome-wide DNA methylation patterns of the IAP LTR repeat elements.
Highlights
About half of the mammalian genome is comprised of repeat elements of different types [1],[2]
HT-TREBS is based on adaptations of the two high throughput bisulfite sequencing techniques: Reduced Representation Bisulfite Sequencing (RRBS) [24] and methylC-Seq [25]
The bisulfite-converted DNA was amplified with PCR using a set of primers: a forward primer binding to the adaptor region and a reverse primer binding to the 24-bp small region of the IAP LTR
Summary
About half of the mammalian genome is comprised of repeat elements of different types [1],[2]. The bulk of these elements are retrotransposons and DNA transposons, making up 42% and 2– 3% of the genome, respectively [3]. The ability of these repeat elements to move to new locations is inhibited by several epigenetic mechanisms of the host genome, including histone modifications and DNA methylation. Recent studies indicated that the small non-coding RNAs, Piwi-interacting RNAs (piRNAs), play a critical role in repressing the transcription of the repeat elements during spermatogenesis [12],[13]
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