High-throughput sequence-based epigenomic analysis of Alu repeats in human cerebellum

Abstract

DNA methylation, the only known covalent modification of mammalian DNA, occurs primarily in CpG dinucleotides. 51% of CpGs in the human genome reside within repeats, and 25% within Alu elements. Despite that, no method has been reported for large-scale ascertainment of CpG methylation in repeats. Here we describe a sequencing-based strategy for parallel determination of the CpG-methylation status of thousands of Alu repeats, and a computation algorithm to design primers that enable their specific amplification from bisulfite converted genomic DNA. Using a single primer pair, we generated amplicons of high sequence complexity, and derived CpG-methylation data from 31 178 Alu elements and their 5′ flanking sequences, altogether representing over 4 Mb of a human cerebellum epigenome. The analysis of the Alu methylome revealed that the methylation level of Alu elements is high in the intronic and intergenic regions, but low in the regions close to transcription start sites. Several hypomethylated Alu elements were identified and their hypomethylated status verified by pyrosequencing. Interestingly, some Alu elements exhibited a strikingly tissue-specific pattern of methylation. We anticipate the amplicons herein described to prove invaluable as epigenome representations, to monitor epigenomic alterations during normal development, in aging and in diseases such as cancer.

Retrotransposon Alu is enriched in the epichromatin of HL-60 cells

“…[23][24][25] Stretches of positioned nucleosomes might facilitate ordering of chromatin fibers at the NE surface. Another structural attribute of Alu elements is their GC-richness and high CpG content (~1/3 of all genomic CpG sites are in Alu, 14 with about 75% of Alu CpG methylated, 26 accounting for ~25% of the total DNA methylation in the human genome 21 ). These methylated DNA sites are clear candidates for the binding of MeCP2, 27 which is associated with heterochromatin formation.…”

The Human Placental Methylome

“…In contrast, average Alu DNA methylation is similar in placenta and fetal tissues using the same technique (Gama-Sosa et al 1983;Price et al 2012). DNA methylation of any individual Alu or L1 element depends on multiple factors such as its location in the genome and age of the element (Szpakowski et al 2009;Xie et al 2009;Price et al 2012). Similarly, DNA methylation of HERV families in placenta show on average reduced, but widely variable, levels across different HERVs (Reiss et al 2007).…”

Epigenetic regulation of transcription and possible functions of mammalian short interspersed elements, SINEs

“…2A). These CpG sites are highly methylated in somatic tissues (Hellmann-Blumberg et al, 1993;Kochanek et al, 1993;Xie et al, 2009), and in vitro experiments have demonstrated that CpG methylation inhibits Pol III transcription from Alu and tRNA genes (Besser et al, 1990;Englander et al, 1993;Kochanek et al, 1993;Liu and Schmid, 1993). DNA methylation likely interferes TFIIIC to bind the A-and B-boxes, thereby Pol III can not be loaded on the promoter.…”