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Novel Human Embryonic Stem Cell Regulators Identified by Conserved and Distinct CpG Island Methylation State.

Pells S, Koutsouraki E, Morfopoulou S, Valencia-Cadavid S, Tomlinson SR, Kalathur R, Futschik ME, De Sousa PA - PLoS ONE (2015)

Bottom Line: Transcriptional repressors and activators were over-represented amongst genes whose associated CGIs were methylated or unmethylated specifically in hESCs, respectively.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.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Regenerative Medicine, School of Clinical Studies, University of Edinburgh, Edinburgh, EH16 4SB, United Kingdom; Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, EH16 4SB, United Kingdom.

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.

No MeSH data available.


Related in: MedlinePlus

Model of the relationship between epigenetically-regulated hESC biomarkers and the pluripotency transcription system.Changes in the expression of an epigenetically-regulated transcriptional regulator (e.g. HMGA1, GLIS2, PFDN5) achieved by siRNA transfection [1], or changes in associated CGI methylation [6], feed through the cellular transcription network, resulting in a reduction in core pluripotency transcription factors (OCT4, NANOG, SOX2, [2]) and differentiation [3]. The core pluripotency transcription factors are permanently downregulated [4], and epigenetic changes to gene promoters, CGIs and other regulatory regions of the genome occur to confer stability on the differentiated phenotype and prevent reversion to pluripotency or quasi-pluripotency [5]. These changes confer permanent changes to the expression of the epigenetically regulated transcriptional regulators [6], and thus stabilise the differentiated phenotype [7].
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pone.0131102.g007: Model of the relationship between epigenetically-regulated hESC biomarkers and the pluripotency transcription system.Changes in the expression of an epigenetically-regulated transcriptional regulator (e.g. HMGA1, GLIS2, PFDN5) achieved by siRNA transfection [1], or changes in associated CGI methylation [6], feed through the cellular transcription network, resulting in a reduction in core pluripotency transcription factors (OCT4, NANOG, SOX2, [2]) and differentiation [3]. The core pluripotency transcription factors are permanently downregulated [4], and epigenetic changes to gene promoters, CGIs and other regulatory regions of the genome occur to confer stability on the differentiated phenotype and prevent reversion to pluripotency or quasi-pluripotency [5]. These changes confer permanent changes to the expression of the epigenetically regulated transcriptional regulators [6], and thus stabilise the differentiated phenotype [7].

Mentions: We confirm a role for GLIS2, HMGA1 and PFDN5, and by inference other genes with an hESC-specific CGI-methylation state, in the maintenance of pluripotency transcription factor expression and pluripotency-associated epigenetic marking (DNA hydroxymethylation). For GLIS2 and PFDN5, and some functions of HMGA1, these roles are novel and suggestive of an epigenetically-defined network of stem cell regulation by genes also expressed in some differentiated cells. We surmised that this network would be controlled in turn by pluripotency-determining factors, and indeed ChIP confirmed the existence of predicted OCT4 binding sites in the promoters of all three genes [25, 37, 38]. Probable binding sites for NANOG and/or SOX2 in the biomarker gene promoters were also identified (data not shown). Our observation of a modulating effect on fibroblast reprogramming (positive for PFDN5, negative for HMGA1 and GLIS2) transfected with established reprogramming factors is consistent with the hypothesis that the biomarkers interact with the core pluripotency factors at some level. The inhibitory effects of HMGA1 and GLIS2 may reflect time- or phase-dependent roles for these factors in reprogramming which our experimental design did not address, as recently described for MBD3 [51], or reflect competing interaction with other factors. HMGA1-induced augmentation of reprogramming was achieved by co-transfection with OCT4, KLF4, SOX2 and L-MYC [48], whereas our study also included LIN28 and a short hairpin RNA directed against p53. Further studies are required to confirm interactions of GLIS2 and PFDN5 with pluripotency transcription factors. Binding sites for GLIS2 are predicted in the promoters of OCT4 and NANOG (data not shown). PFDN5 is known to repress c-MYC activity which regulates genes involved in many processes including cell-cycle control, metabolism, signal transduction, and cell-fate decisions as well as self-renewal (Chappell and Dalton, 2013). HMGA1 binding sites are predicted in the promoters of OCT4 and NANOG (data not shown) and have been shown by chromatin immunoprecipitation in SOX2, LIN28 and c-MYC [48]. The reduction in DNA hydroxymethylation, not accompanied by similar falls in TET gene transcription, following interference with HMGA1, GLIS2, or PFDN5 suggests that these effects are secondary events following disruption of core pluripotency functions. Collectively, our data suggest a model as shown in Fig 7. Downregulation of biomarker expression, either experimentally by RNA interference, or by methylation changes at associated CGIs modulating gene expression, results in downregulation of the core pluripotency transcription network, directly or indirectly. Differentiation is initiated, with the particular lineage decision decided by the presence or absence of secondary transcription regulators, such as epigenetically-regulated factors identified here. Stability of the differentiation process is conferred both by downregulation of pluripotency genes (NANOG, SOX2 and OCT4), and also by epigenetic changes, e.g. OCT4 promoter [52, 53], or methylation changes in CGIs associated with particular lineages. The methylation changes confer stability and heritability on the gene expression changes, and hence on the resulting cellular phenotype.


Novel Human Embryonic Stem Cell Regulators Identified by Conserved and Distinct CpG Island Methylation State.

Pells S, Koutsouraki E, Morfopoulou S, Valencia-Cadavid S, Tomlinson SR, Kalathur R, Futschik ME, De Sousa PA - PLoS ONE (2015)

Model of the relationship between epigenetically-regulated hESC biomarkers and the pluripotency transcription system.Changes in the expression of an epigenetically-regulated transcriptional regulator (e.g. HMGA1, GLIS2, PFDN5) achieved by siRNA transfection [1], or changes in associated CGI methylation [6], feed through the cellular transcription network, resulting in a reduction in core pluripotency transcription factors (OCT4, NANOG, SOX2, [2]) and differentiation [3]. The core pluripotency transcription factors are permanently downregulated [4], and epigenetic changes to gene promoters, CGIs and other regulatory regions of the genome occur to confer stability on the differentiated phenotype and prevent reversion to pluripotency or quasi-pluripotency [5]. These changes confer permanent changes to the expression of the epigenetically regulated transcriptional regulators [6], and thus stabilise the differentiated phenotype [7].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495055&req=5

pone.0131102.g007: Model of the relationship between epigenetically-regulated hESC biomarkers and the pluripotency transcription system.Changes in the expression of an epigenetically-regulated transcriptional regulator (e.g. HMGA1, GLIS2, PFDN5) achieved by siRNA transfection [1], or changes in associated CGI methylation [6], feed through the cellular transcription network, resulting in a reduction in core pluripotency transcription factors (OCT4, NANOG, SOX2, [2]) and differentiation [3]. The core pluripotency transcription factors are permanently downregulated [4], and epigenetic changes to gene promoters, CGIs and other regulatory regions of the genome occur to confer stability on the differentiated phenotype and prevent reversion to pluripotency or quasi-pluripotency [5]. These changes confer permanent changes to the expression of the epigenetically regulated transcriptional regulators [6], and thus stabilise the differentiated phenotype [7].
Mentions: We confirm a role for GLIS2, HMGA1 and PFDN5, and by inference other genes with an hESC-specific CGI-methylation state, in the maintenance of pluripotency transcription factor expression and pluripotency-associated epigenetic marking (DNA hydroxymethylation). For GLIS2 and PFDN5, and some functions of HMGA1, these roles are novel and suggestive of an epigenetically-defined network of stem cell regulation by genes also expressed in some differentiated cells. We surmised that this network would be controlled in turn by pluripotency-determining factors, and indeed ChIP confirmed the existence of predicted OCT4 binding sites in the promoters of all three genes [25, 37, 38]. Probable binding sites for NANOG and/or SOX2 in the biomarker gene promoters were also identified (data not shown). Our observation of a modulating effect on fibroblast reprogramming (positive for PFDN5, negative for HMGA1 and GLIS2) transfected with established reprogramming factors is consistent with the hypothesis that the biomarkers interact with the core pluripotency factors at some level. The inhibitory effects of HMGA1 and GLIS2 may reflect time- or phase-dependent roles for these factors in reprogramming which our experimental design did not address, as recently described for MBD3 [51], or reflect competing interaction with other factors. HMGA1-induced augmentation of reprogramming was achieved by co-transfection with OCT4, KLF4, SOX2 and L-MYC [48], whereas our study also included LIN28 and a short hairpin RNA directed against p53. Further studies are required to confirm interactions of GLIS2 and PFDN5 with pluripotency transcription factors. Binding sites for GLIS2 are predicted in the promoters of OCT4 and NANOG (data not shown). PFDN5 is known to repress c-MYC activity which regulates genes involved in many processes including cell-cycle control, metabolism, signal transduction, and cell-fate decisions as well as self-renewal (Chappell and Dalton, 2013). HMGA1 binding sites are predicted in the promoters of OCT4 and NANOG (data not shown) and have been shown by chromatin immunoprecipitation in SOX2, LIN28 and c-MYC [48]. The reduction in DNA hydroxymethylation, not accompanied by similar falls in TET gene transcription, following interference with HMGA1, GLIS2, or PFDN5 suggests that these effects are secondary events following disruption of core pluripotency functions. Collectively, our data suggest a model as shown in Fig 7. Downregulation of biomarker expression, either experimentally by RNA interference, or by methylation changes at associated CGIs modulating gene expression, results in downregulation of the core pluripotency transcription network, directly or indirectly. Differentiation is initiated, with the particular lineage decision decided by the presence or absence of secondary transcription regulators, such as epigenetically-regulated factors identified here. Stability of the differentiation process is conferred both by downregulation of pluripotency genes (NANOG, SOX2 and OCT4), and also by epigenetic changes, e.g. OCT4 promoter [52, 53], or methylation changes in CGIs associated with particular lineages. The methylation changes confer stability and heritability on the gene expression changes, and hence on the resulting cellular phenotype.

Bottom Line: Transcriptional repressors and activators were over-represented amongst genes whose associated CGIs were methylated or unmethylated specifically in hESCs, respectively.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.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Regenerative Medicine, School of Clinical Studies, University of Edinburgh, Edinburgh, EH16 4SB, United Kingdom; Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh, EH16 4SB, United Kingdom.

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.

No MeSH data available.


Related in: MedlinePlus