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

Epigenetically-defined hESC biomarkers have a role in pluripotency.(A) Transcriptional activators (Expressed hESC-UnMe-GA-CGIs) and transcriptional repressors (Expressed hESC-Me-GA-CGIs) identified as functionally overrepresented hESC biomarkers. (B, C) Functional testing of transcriptional regulators in RH1 hESCs by siRNA knockdown. (B) RT-qPCR data showing log10 fold change in expression of the siRNA-targeted gene, and associated effects on OCT4, NANOG and SOX2. Changes are relative to GAPDH expression, normalised to RH1 hESCs treated with negative control siRNA IDS-NULL. 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. (C) Immunohistochemistry for NANOG and OCT4 72 hours after siRNA treatment. Scale bar = 100 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131102.g002: Epigenetically-defined hESC biomarkers have a role in pluripotency.(A) Transcriptional activators (Expressed hESC-UnMe-GA-CGIs) and transcriptional repressors (Expressed hESC-Me-GA-CGIs) identified as functionally overrepresented hESC biomarkers. (B, C) Functional testing of transcriptional regulators in RH1 hESCs by siRNA knockdown. (B) RT-qPCR data showing log10 fold change in expression of the siRNA-targeted gene, and associated effects on OCT4, NANOG and SOX2. Changes are relative to GAPDH expression, normalised to RH1 hESCs treated with negative control siRNA IDS-NULL. 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. (C) Immunohistochemistry for NANOG and OCT4 72 hours after siRNA treatment. Scale bar = 100 μm.

Mentions: Correlation of Me-GA-CGIs and UnMe-GA-CGIs (Table D in S1 File) with transcriptome data identified expressed genes with an hESC-specific CGI methylation state (Tables E-G in S1 File). Of 109 gene loci with an hESC-UnMe-GA-CGI, 78 genes with non-redundant Entrez IDs were expressed. Similarly, of 216 gene loci with an hESC-Me-GA-CGI, 169 unique genes were expressed. The subset of CGI-associated genes may be enriched for functional categories, so we first analysed the distribution of genes associated with CGIs on the array, and identified 11357 non-redundant Entrez IDs, about 2/3 (64.3%) of the genes annotated in Gene Ontology (17673 genes). CGI-associated genes were enriched (e.g. transcription factor, developmental process) or depleted (egg. receptors, signal transducers) for various categories (S7 Fig and Table J in S1 File); thus the functional analysis of expressed hESC-methylated or unmethylated genes was compared with the set of CGI-associated genes on the array. These hESC-specific gene sets were tested for enrichment in Gene Ontology categories relative to the proportion expected for CGI-associated genes. Transcriptional activators (GO:0016563) are significantly overrepresented among genes with an hESC-unmethylated CGI (GO:0016563, P < 0.01, FDR = 0.01; Table K in S1 File); similarly related GO categories including transcriptional regulator activity (GO:0030528), transcription factor binding (GO:0008134), DNA binding (GO:0003677) and sequence-specific DNA binding (GO:0043565) are also overrepresented (Fig 2A). Only two genes associated with transcriptional repressor activity (MSX1 and TBX3) have an hESC-UnMe-CGI. For genes with an associated hESC-Me-CGI, only two categories were enriched (FDR < 0.25): phosphoinositide binding (GO: 0035091) and transcription repressor activity (GO: 0016564); P < 0.001, FDR = 0.137 in both cases; Fig 2A).


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)

Epigenetically-defined hESC biomarkers have a role in pluripotency.(A) Transcriptional activators (Expressed hESC-UnMe-GA-CGIs) and transcriptional repressors (Expressed hESC-Me-GA-CGIs) identified as functionally overrepresented hESC biomarkers. (B, C) Functional testing of transcriptional regulators in RH1 hESCs by siRNA knockdown. (B) RT-qPCR data showing log10 fold change in expression of the siRNA-targeted gene, and associated effects on OCT4, NANOG and SOX2. Changes are relative to GAPDH expression, normalised to RH1 hESCs treated with negative control siRNA IDS-NULL. 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. (C) Immunohistochemistry for NANOG and OCT4 72 hours after siRNA treatment. Scale bar = 100 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131102.g002: Epigenetically-defined hESC biomarkers have a role in pluripotency.(A) Transcriptional activators (Expressed hESC-UnMe-GA-CGIs) and transcriptional repressors (Expressed hESC-Me-GA-CGIs) identified as functionally overrepresented hESC biomarkers. (B, C) Functional testing of transcriptional regulators in RH1 hESCs by siRNA knockdown. (B) RT-qPCR data showing log10 fold change in expression of the siRNA-targeted gene, and associated effects on OCT4, NANOG and SOX2. Changes are relative to GAPDH expression, normalised to RH1 hESCs treated with negative control siRNA IDS-NULL. 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. (C) Immunohistochemistry for NANOG and OCT4 72 hours after siRNA treatment. Scale bar = 100 μm.
Mentions: Correlation of Me-GA-CGIs and UnMe-GA-CGIs (Table D in S1 File) with transcriptome data identified expressed genes with an hESC-specific CGI methylation state (Tables E-G in S1 File). Of 109 gene loci with an hESC-UnMe-GA-CGI, 78 genes with non-redundant Entrez IDs were expressed. Similarly, of 216 gene loci with an hESC-Me-GA-CGI, 169 unique genes were expressed. The subset of CGI-associated genes may be enriched for functional categories, so we first analysed the distribution of genes associated with CGIs on the array, and identified 11357 non-redundant Entrez IDs, about 2/3 (64.3%) of the genes annotated in Gene Ontology (17673 genes). CGI-associated genes were enriched (e.g. transcription factor, developmental process) or depleted (egg. receptors, signal transducers) for various categories (S7 Fig and Table J in S1 File); thus the functional analysis of expressed hESC-methylated or unmethylated genes was compared with the set of CGI-associated genes on the array. These hESC-specific gene sets were tested for enrichment in Gene Ontology categories relative to the proportion expected for CGI-associated genes. Transcriptional activators (GO:0016563) are significantly overrepresented among genes with an hESC-unmethylated CGI (GO:0016563, P < 0.01, FDR = 0.01; Table K in S1 File); similarly related GO categories including transcriptional regulator activity (GO:0030528), transcription factor binding (GO:0008134), DNA binding (GO:0003677) and sequence-specific DNA binding (GO:0043565) are also overrepresented (Fig 2A). Only two genes associated with transcriptional repressor activity (MSX1 and TBX3) have an hESC-UnMe-CGI. For genes with an associated hESC-Me-CGI, only two categories were enriched (FDR < 0.25): phosphoinositide binding (GO: 0035091) and transcription repressor activity (GO: 0016564); P < 0.001, FDR = 0.137 in both cases; Fig 2A).

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