Abstract
Human embryonic stem cells (hESCs) undergo epigenetic changes in vitro which may compromise function, so an epigenetic pluripotency “signature” would be invaluable for line validation. We assessed Cytosine-phosphate-Guanine Island (CGI) methylation in hESCs by genomic DNA hybridisation to a CGI array, and saw substantial variation in CGI methylation between lines. Comparison of hESC CGI methylation profiles to corresponding somatic tissue data and hESC mRNA expression profiles identified a conserved hESC-specific methylation pattern associated with expressed genes. Transcriptional repressors and activators were over-represented amongst genes whose associated CGIs were methylated or unmethylated specifically in hESCs, respectively. Knockdown of candidate transcriptional regulators (HMGA1, GLIS2, PFDN5) induced differentiation in hESCs, whereas ectopic expression in fibroblasts modulated iPSC colony formation. Chromatin immunoprecipitation confirmed interaction between the candidates and the core pluripotency transcription factor network. We thus identify novel pluripotency genes on the basis of a conserved and distinct epigenetic configuration in human stem cells.
Highlights
The application of human embryonic stem cells to regenerative medicine relies on maintaining appropriate gene expression controlling self renewal or lineage specification in vitro
As a pilot study of the significance of Cytosine-phosphate-Guanine Island (CGI) methylation to pluripotency, 4 human embryonic stem cells (hESCs) lines, differing in provenance, sex, passage number and culture conditions were assessed to identify a conserved pattern of CGI methylation status (Table A in S1 File; summary of hESC lines employed in this study)
To investigate hESC line CGI methylation status, we probed an array of biologically-defined CGIs used previously to study tissue-specific methylation[26] (S3 Table)
Summary
The application of human embryonic stem cells (hESCs) to regenerative medicine relies on maintaining appropriate gene expression controlling self renewal or lineage specification in vitro. Epigenetic modifications of DNA and chromatin control the expression patterns that define cellular identity and function during development and in differentiated tissues [1]. Methylation of cytosine in cytosine-phosphate-guanine (CpG) dinucleotides is an epigenetic mark conferring stability on gene expression states, notably by the establishment of a silent chromatin state [2]. In normal sequence, where CpGs are relatively infrequent (~ 1 per 100 bp), most CpGs are methylated, but, in Cytosine-phosphate-Guanine Islands (CGIs), PLOS ONE | DOI:10.1371/journal.pone.0131102. In normal sequence, where CpGs are relatively infrequent (~ 1 per 100 bp), most CpGs are methylated, but, in Cytosine-phosphate-Guanine Islands (CGIs), PLOS ONE | DOI:10.1371/journal.pone.0131102 July 7, 2015
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