Reduced representation bisulfite sequencing (RRBS) was used to analyze DNA methylation patterns across the mouse brain genome in mice carrying a deletion of the Prader-Willi syndrome imprinting center (PWS-IC) on either the maternally- or paternally-inherited chromosome. Within the ~3.7 Mb imprinted Angelman/Prader-Willi syndrome (AS/PWS) domain, 254 CpG sites were interrogated for changes in methylation due to PWS-IC deletion. Paternally-inherited deletion of the PWS-IC increased methylation levels ~2-fold at each CpG site (compared to wild-type controls) at differentially methylated regions (DMRs) associated with 5' CpG island promoters of paternally-expressed genes; these methylation changes extended, to a variable degree, into the adjacent CpG island shores. Maternal PWS-IC deletion yielded little or no changes in methylation at these DMRs, and methylation of CpG sites outside of promoter DMRs also was unchanged upon maternal or paternal PWS-IC deletion. Using stringent ascertainment criteria, ~750,000 additional CpG sites were also interrogated across the entire mouse genome. This analysis identified 26 loci outside of the imprinted AS/PWS domain showing altered DNA methylation levels of ≥25% upon PWS-IC deletion. Curiously, altered methylation at 9 of these loci was a consequence of maternal PWS-IC deletion (maternal PWS-IC deletion by itself is not known to be associated with a phenotype in either humans or mice), and 10 of these loci exhibited the same changes in methylation irrespective of the parental origin of the PWS-IC deletion. These results suggest that the PWS-IC may affect DNA methylation at these loci by directly interacting with them, or may affect methylation at these loci through indirect downstream effects due to PWS-IC deletion. They further suggest the PWS-IC may have a previously uncharacterized function outside of the imprinted AS/PWS domain.
Keywords: AS, Angelman Syndrome; AS-IC, Angelman Syndrome Imprinting Center; AS-SRO, Angelman Syndrome Shortest Region of deletion Overlap; BGS, Sodium Bisulfite Genomic Sequencing; BISSCA, Bisulfite Sequencing Comparative Analysis; CGI, CpG Island; DH, DNase I Hypersensitive; DMR, Differentially Methylated Region; DNA methylation; EtOH, Ethanol; GO, gene ontology; IC, Imprinting Center; ICR, Imprinting Control Region; IPA, Ingenuity Pathway Analysis ®; PWS, Prader-Willi Syndrome; PWS-IC, Prader-Willi Syndrome Imprinting Center; PWS-SRO, Prader-Willi Syndrome Shortest Region of deletion Overlap; RRBS, Reduced Representation Bisulfite Sequencing; SDS, Sodium Dodecyl Sulfate; SLIM, Sliding Linear Model; TBE, Tris/Borate/EDTA; Tris, Trisaminomethane; UTR, untranslated region; angelman syndrome; genomic imprinting; imprinting center; lncRNA, long non-coding RNA; mat, maternally-inherited allele; pat, paternally-inherited allele; prader-Willi syndrome; reduced representation bisulfite sequencing.