Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming

Abstract

Current DNA methylation assays are limited in the flexibility and efficiency of characterizing a large number of genomic targets. We report a method to specifically capture an arbitrary subset of genomic targets for single-molecule bisulfite sequencing for digital quantification of DNA methylation at single-nucleotide resolution. A set of ~30,000 padlock probes was designed to assess methylation of 66,000 CpG sites within 2,020 CpG islands on human chromosome 12, chromosome 20, and 34 selected regions. To investigate epigenetic differences associated with dedifferentiation, we compared methylation in three human fibroblast lines and eight human pluripotent stem cell lines. Chromosome-wide methylation patterns were similar among all lines studied, but cytosine Correspondence should be addressed to K.Z. (E-mail: kzhang@bioeng.ucsd.edu) or Y.G. (E-mail: ygao@vcu.edu). Accession numbers. All sequence reads and methylation data have been deposited at GEO, with accession number GSE15007.Note: Supplementary information is available on the Nature Biotechnology website. AUTHOR CONTRIBUTIONS NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript methylation was slightly more prevalent in the pluripotent cells than in the fibroblasts. Induced pluripotent stem (iPS) cells appeared to display more methylation than embryonic stem cells. We found 288 regions methylated differently in fibroblasts and pluripotent cells. This targeted approach should be particularly useful for analyzing DNA methylation in large genomes.DNA methylation is a primary epigenetic mechanism for transcriptional regulation during normal development and goes awry in many diseases, including cancers. Genome-scale patterns of DNA methylation have been characterized by microarray hybridization or bisulfite sequencing 1 . Microarray methods have enabled methylation to be quantified at 1,536 discrete CpG sites in the human genome with the GoldenGate assay 2,3 . They have also been coupled with methylated DNA immunoprecipitation or methyl-specific restriction enzyme digestion to quantify relative levels of DNA methylation, although the read-outs of such approaches are only averages of the levels of methylation of multiple adjacent CpG sites [4][5][6] .More recently, next-generation sequencing has enabled absolute quantification of DNA methylation with single-nucleotide resolution on a larger scale than previously possible. These efforts include bisulfite sequencing of PCR amplicons from human tissues and cancer cell lines 7-9 , single-molecule sequencing of reduced representation libraries from mouse embryonic stem cells 10,11 and whole-genome bisulfite sequencing of Arabidopsis thaliana 12,13 . Although whole-genome bisulfite sequencing of a mammalian genome should be technically feasible, the large genome sizes pose a considerable challenge 14 .Selection or enrichment of genomic targets prior to sequencing would substantially reduce sequencing cost. PCR-based target selection is highly specific, but cannot be multiplexed easily for...

Identification of a specific reprogramming-associated epigenetic signature in human induced pluripotent stem cells

“…To gain insight into the epigenetic integrity of hiPSCs, we performed targeted bisulfite sequencing with padlock probes (25,26) to analyze the methylomes of 17 hiPSC lines, their 6 somatic cell…”

Concise Review: Induced Pluripotent Stem Cells Versus Embryonic Stem Cells: Close Enough or Yet Too Far Apart?

“…The DNA methylation pattern is very similar between iPSC and ESC when compared with nonpluripotent lines, such as fibroblasts. However, hierarchical clustering performed on the methylation level of cca 66,000 CpG sites, besides clearly clustering fibroblasts from ESC/iPSC, also distinguishes iPSC from ESC [56]. One analysis on the whole genome scale found 71 differentially methylated regions (DMR) between three iPSC lines and three ESC lines (and 2,179 between fibroblasts and iPSC) [57].…”

Finding the fifth base: Genome-wide sequencing of cytosine methylation

“…2; Garber 2008) include capture of specific sequences by hybridization to DNA molecules on arrays Hodges et al 2007;Okou et al 2007) or bound to beads in solution (Bashiardes et al 2005), with padlock or molecular inversion probes (Nilsson et al 1994;Absalan and Ronaghi 2007;Ball et al 2009;Deng et al 2009), with proteins that bind to methylated DNA , or with an antibody that binds to methylcytosines (MeDIP/mCIP) (Weber et al 2005;Keshet et al 2006;Zhang et al 2006;Penterman et al 2007;Zilberman et al 2007). Recently, Down et al (2008) performed MeDIP with mammalian male gametes followed by sequencing of the immunoprecipitated DNA using the Illumina Genome Analyzer, in a procedure called MeDIP-seq.…”