Limits...
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

Embryonic lineage preference in hESCs after downregulation of epigenetically-defined biomarkers.Expression at 72 hours after RH1 hESCs were treated with siRNAs indicated. RT-qPCR data showing log10 fold change in expression of the indicated embryonic lineage marker gene. Changes are relative to GAPDH expression, normalised to IDS-NULL-treated RH1 cells. Asterisks indicate levels of statistical significance (unpaired t-test; *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ND: Not Detected, even at 40 cycles of PCR.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4495055&req=5

pone.0131102.g005: Embryonic lineage preference in hESCs after downregulation of epigenetically-defined biomarkers.Expression at 72 hours after RH1 hESCs were treated with siRNAs indicated. RT-qPCR data showing log10 fold change in expression of the indicated embryonic lineage marker gene. Changes are relative to GAPDH expression, normalised to IDS-NULL-treated RH1 cells. Asterisks indicate levels of statistical significance (unpaired t-test; *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ND: Not Detected, even at 40 cycles of PCR.

Mentions: We tested the functional role of three candidate epigenetically-defined hESC biomarkers: the transcriptional activators GLIS2 and HMGA1, and the repressor PFDN5, as all are expressed significantly over background (Table L in S1 File, microarray expression data for GLIS2, HMGA1 and PFDN5). Small Interfering RNA (siRNA) transfection conditions in hESC lines were optimised (S8 Fig). RH1 was selected to represent the cell lines in this study, with results independently confirmed in H9 one of the most commonly-studied hESC lines. HESCs were transfected with Lipofectamine RNAiMAX and siRNA oligonucleotides (Table M in S1 File, sequences of siRNA oligonucleotides used in this study) twice, 24 hours apart, and samples were taken at 48 and 96 hours after the first transfection. An siRNA targeting no human transcript (IDS-NULL, directed against IDS but containing 4 point mutations) was used as a negative control for sample normalisation from the same cell line and time point. An siRNA directed against OCT4 was used as a positive control for effects on pluripotency, and an siRNA directed against YAP1 to control for responses to knockdown of an hESC-expressed gene unnecessary for pluripotency[35]. Response to knockdown of all three genes was similar (RH1, Figs 2–5 and H9, S9–S12 Figs). Target transcript knockdown was significant and strong (typically > 90%) by 24 hours post-siRNA treatment 2 (Fig 2B and S9A Fig). Despite rapid (24 hr.) downregulation of YAP1 to ~10% normal levels, we saw no effect on OCT4, NANOG or SOX2 in RH1 (Fig 2Bi) and minimal effects in H9 (S9Ai Fig). However OCT4 knockdown was accompanied by significant reductions in NANOG and SOX2 (Fig 2Bii and S9Aii Fig). These controls show that our siRNA knockdown system was (1) not “swamping” the cellular RNA degradation system, inducing non-specific effects that perturbed normal phenotype, (2) efficient, producing a measurable target knockdown, and (3) yielded expected effects when targeting genes of known function.


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)

Embryonic lineage preference in hESCs after downregulation of epigenetically-defined biomarkers.Expression at 72 hours after RH1 hESCs were treated with siRNAs indicated. RT-qPCR data showing log10 fold change in expression of the indicated embryonic lineage marker gene. Changes are relative to GAPDH expression, normalised to IDS-NULL-treated RH1 cells. Asterisks indicate levels of statistical significance (unpaired t-test; *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ND: Not Detected, even at 40 cycles of PCR.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131102.g005: Embryonic lineage preference in hESCs after downregulation of epigenetically-defined biomarkers.Expression at 72 hours after RH1 hESCs were treated with siRNAs indicated. RT-qPCR data showing log10 fold change in expression of the indicated embryonic lineage marker gene. Changes are relative to GAPDH expression, normalised to IDS-NULL-treated RH1 cells. Asterisks indicate levels of statistical significance (unpaired t-test; *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). ND: Not Detected, even at 40 cycles of PCR.
Mentions: We tested the functional role of three candidate epigenetically-defined hESC biomarkers: the transcriptional activators GLIS2 and HMGA1, and the repressor PFDN5, as all are expressed significantly over background (Table L in S1 File, microarray expression data for GLIS2, HMGA1 and PFDN5). Small Interfering RNA (siRNA) transfection conditions in hESC lines were optimised (S8 Fig). RH1 was selected to represent the cell lines in this study, with results independently confirmed in H9 one of the most commonly-studied hESC lines. HESCs were transfected with Lipofectamine RNAiMAX and siRNA oligonucleotides (Table M in S1 File, sequences of siRNA oligonucleotides used in this study) twice, 24 hours apart, and samples were taken at 48 and 96 hours after the first transfection. An siRNA targeting no human transcript (IDS-NULL, directed against IDS but containing 4 point mutations) was used as a negative control for sample normalisation from the same cell line and time point. An siRNA directed against OCT4 was used as a positive control for effects on pluripotency, and an siRNA directed against YAP1 to control for responses to knockdown of an hESC-expressed gene unnecessary for pluripotency[35]. Response to knockdown of all three genes was similar (RH1, Figs 2–5 and H9, S9–S12 Figs). Target transcript knockdown was significant and strong (typically > 90%) by 24 hours post-siRNA treatment 2 (Fig 2B and S9A Fig). Despite rapid (24 hr.) downregulation of YAP1 to ~10% normal levels, we saw no effect on OCT4, NANOG or SOX2 in RH1 (Fig 2Bi) and minimal effects in H9 (S9Ai Fig). However OCT4 knockdown was accompanied by significant reductions in NANOG and SOX2 (Fig 2Bii and S9Aii Fig). These controls show that our siRNA knockdown system was (1) not “swamping” the cellular RNA degradation system, inducing non-specific effects that perturbed normal phenotype, (2) efficient, producing a measurable target knockdown, and (3) yielded expected effects when targeting genes of known function.

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