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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

Association of candidate epigenetically-defined biomarkers with the core pluripotency network.GLIS2 (A), HMGA1 (B) and PFDN5 (C) all have OCT4 binding sites in their promoter regions. Panels created by Geneprof (geneprof.org) using OCT4 ChIP-seq data from Young and colleagues (unpublished), Kunarso et al. (2010) and Mullen et al. (2011) (GLIS2), Young and colleagues (unpublished) and Mullen et al. (2011) (HMGA1) and Lister et al. (2009) and Mullen et al. (2011) (PFDN5); Y-axis indicates read number from the dataset indicated. (D) OCT4 knockdown in hESCs as described perturbs HMGA1, GLIS2 and PFDN5 transcript levels. Asterisks indicate levels of statistical significance (unpaired t-test; *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). (E) Chromatin immunoprecipitation (ChIP)-qPCR suggests that HMGA1, GLIS2 and PFDN5 expression is regulated by OCT4. Immunoprecipitation of hESC chromatin with an antibody to OCT4, followed by quantitative amplification of gene-specific promoter regions above background (a silent region of the genome) in hESCs. (F) Representative experiment whereby human dermal fibroblasts (HDFs) were transfected with a set of three episomal plasmids expressing OCT4, SOX2, KLF4, L-MYC, LIN28 and a short hairpin RNA directed against p53 (OKSML/hn53) alone (left-most column) or with OKSML/hn53 supplemented with either HMGA1, GLIS2 or PFDN5, or all three, followed by detection of alkaline phosphatase positive colonies (No. AP+ Colonies) representative of pluripotent cells. Error bars indicate standard deviation of the mean; **** indicates the level of statistical significance (P≤0.0001).
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pone.0131102.g006: Association of candidate epigenetically-defined biomarkers with the core pluripotency network.GLIS2 (A), HMGA1 (B) and PFDN5 (C) all have OCT4 binding sites in their promoter regions. Panels created by Geneprof (geneprof.org) using OCT4 ChIP-seq data from Young and colleagues (unpublished), Kunarso et al. (2010) and Mullen et al. (2011) (GLIS2), Young and colleagues (unpublished) and Mullen et al. (2011) (HMGA1) and Lister et al. (2009) and Mullen et al. (2011) (PFDN5); Y-axis indicates read number from the dataset indicated. (D) OCT4 knockdown in hESCs as described perturbs HMGA1, GLIS2 and PFDN5 transcript levels. Asterisks indicate levels of statistical significance (unpaired t-test; *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). (E) Chromatin immunoprecipitation (ChIP)-qPCR suggests that HMGA1, GLIS2 and PFDN5 expression is regulated by OCT4. Immunoprecipitation of hESC chromatin with an antibody to OCT4, followed by quantitative amplification of gene-specific promoter regions above background (a silent region of the genome) in hESCs. (F) Representative experiment whereby human dermal fibroblasts (HDFs) were transfected with a set of three episomal plasmids expressing OCT4, SOX2, KLF4, L-MYC, LIN28 and a short hairpin RNA directed against p53 (OKSML/hn53) alone (left-most column) or with OKSML/hn53 supplemented with either HMGA1, GLIS2 or PFDN5, or all three, followed by detection of alkaline phosphatase positive colonies (No. AP+ Colonies) representative of pluripotent cells. Error bars indicate standard deviation of the mean; **** indicates the level of statistical significance (P≤0.0001).

Mentions: As perturbation of either OCT4 or HMGA1, GLIS2 or PFDN5 (all transcriptional regulators) results in loss of stem cell phenotype, and biomarker knockdown also results in downregulation of OCT4, NANOG and SOX2, a transcriptional connection between the core pluripotency factors and biomarkers is possible. GLIS2, HMGA1 and PFDN5 all apparently contain OCT4 binding sites in their promoters (Fig 6A–6C; [37, 38] R. Young, Whitehead Institute, Cambridge, MA [unpublished dataset], and [25], and also binding sites for other transcription factors which interact with OCT4, NANOG and SOX2, including EOMES, TRIM28, E2F1, CDX2 and ETS1.


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)

Association of candidate epigenetically-defined biomarkers with the core pluripotency network.GLIS2 (A), HMGA1 (B) and PFDN5 (C) all have OCT4 binding sites in their promoter regions. Panels created by Geneprof (geneprof.org) using OCT4 ChIP-seq data from Young and colleagues (unpublished), Kunarso et al. (2010) and Mullen et al. (2011) (GLIS2), Young and colleagues (unpublished) and Mullen et al. (2011) (HMGA1) and Lister et al. (2009) and Mullen et al. (2011) (PFDN5); Y-axis indicates read number from the dataset indicated. (D) OCT4 knockdown in hESCs as described perturbs HMGA1, GLIS2 and PFDN5 transcript levels. Asterisks indicate levels of statistical significance (unpaired t-test; *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). (E) Chromatin immunoprecipitation (ChIP)-qPCR suggests that HMGA1, GLIS2 and PFDN5 expression is regulated by OCT4. Immunoprecipitation of hESC chromatin with an antibody to OCT4, followed by quantitative amplification of gene-specific promoter regions above background (a silent region of the genome) in hESCs. (F) Representative experiment whereby human dermal fibroblasts (HDFs) were transfected with a set of three episomal plasmids expressing OCT4, SOX2, KLF4, L-MYC, LIN28 and a short hairpin RNA directed against p53 (OKSML/hn53) alone (left-most column) or with OKSML/hn53 supplemented with either HMGA1, GLIS2 or PFDN5, or all three, followed by detection of alkaline phosphatase positive colonies (No. AP+ Colonies) representative of pluripotent cells. Error bars indicate standard deviation of the mean; **** indicates the level of statistical significance (P≤0.0001).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131102.g006: Association of candidate epigenetically-defined biomarkers with the core pluripotency network.GLIS2 (A), HMGA1 (B) and PFDN5 (C) all have OCT4 binding sites in their promoter regions. Panels created by Geneprof (geneprof.org) using OCT4 ChIP-seq data from Young and colleagues (unpublished), Kunarso et al. (2010) and Mullen et al. (2011) (GLIS2), Young and colleagues (unpublished) and Mullen et al. (2011) (HMGA1) and Lister et al. (2009) and Mullen et al. (2011) (PFDN5); Y-axis indicates read number from the dataset indicated. (D) OCT4 knockdown in hESCs as described perturbs HMGA1, GLIS2 and PFDN5 transcript levels. Asterisks indicate levels of statistical significance (unpaired t-test; *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001). (E) Chromatin immunoprecipitation (ChIP)-qPCR suggests that HMGA1, GLIS2 and PFDN5 expression is regulated by OCT4. Immunoprecipitation of hESC chromatin with an antibody to OCT4, followed by quantitative amplification of gene-specific promoter regions above background (a silent region of the genome) in hESCs. (F) Representative experiment whereby human dermal fibroblasts (HDFs) were transfected with a set of three episomal plasmids expressing OCT4, SOX2, KLF4, L-MYC, LIN28 and a short hairpin RNA directed against p53 (OKSML/hn53) alone (left-most column) or with OKSML/hn53 supplemented with either HMGA1, GLIS2 or PFDN5, or all three, followed by detection of alkaline phosphatase positive colonies (No. AP+ Colonies) representative of pluripotent cells. Error bars indicate standard deviation of the mean; **** indicates the level of statistical significance (P≤0.0001).
Mentions: As perturbation of either OCT4 or HMGA1, GLIS2 or PFDN5 (all transcriptional regulators) results in loss of stem cell phenotype, and biomarker knockdown also results in downregulation of OCT4, NANOG and SOX2, a transcriptional connection between the core pluripotency factors and biomarkers is possible. GLIS2, HMGA1 and PFDN5 all apparently contain OCT4 binding sites in their promoters (Fig 6A–6C; [37, 38] R. Young, Whitehead Institute, Cambridge, MA [unpublished dataset], and [25], and also binding sites for other transcription factors which interact with OCT4, NANOG and SOX2, including EOMES, TRIM28, E2F1, CDX2 and ETS1.

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