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Esrrb is a direct Nanog target gene that can substitute for Nanog function in pluripotent cells.

Festuccia N, Osorno R, Halbritter F, Karwacki-Neisius V, Navarro P, Colby D, Wong F, Yates A, Tomlinson SR, Chambers I - Cell Stem Cell (2012)

Bottom Line: Moreover, Esrrb can reprogram Nanog(-/-) EpiSCs and can rescue stalled reprogramming in Nanog(-/-) pre-iPSCs.Finally, Esrrb deletion abolishes the defining ability of Nanog to confer LIF-independent ESC self-renewal.These findings are consistent with the functional placement of Esrrb downstream of Nanog.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, Scotland.

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Identification of Nanog Target Genes Including Esrrb(A) Deep-SAGE profile of sorted Nanog-positive (GFP+) and Nanog-negative (GFP–) TNG cells, ESCs with wild-type levels of Nanog expression (RCN(t)) and Nanog−/− ESCs (RCNβH(t)). Genes were ranked according to the expression level and fold difference in expression in TNG+ versus TNG− and RCN(t) versus RCNβH(t); the plot shows the first 250 most upregulated (top) or downregulated (bottom) genes. Colors: yellow, expression above average; blue, below average.(B) Esrrb transcript levels in two cell lines overexpressing Nanog (EF4 and RCN), two cell lines with wild-type Nanog (E14Tg2a and RCN(t)), two Nanog+/− cell lines (TβC44 and RCNβ(t)), and two Nanog−/− cell lines (TβC44Cre6 and RCNβH(t)). Error bars: standard deviation (n = 4).(C) Immunoblot analysis of Esrrb and Nanog levels in the same ESC lines.(D) Immunohistochemical analysis of the intracellular localization of Nanog in ESΔN-NERT cells in response to 1 μM tamoxifen as indicated.(E) Global transcriptional changes after ESΔN-NERT stimulation with tamoxifen as indicated; the Esrrb changes are in red. Mean expression levels in three independent experiments are shown.(F) Venn diagram showing the intersection of significantly upregulated or downregulated genes identified in (E) compared to genes bound by Nanog according to two independent genome-wide ChIP studies.(G) Esrrb pre-mRNA kinetics in ESΔN-NERT cells stimulated with tamoxifen as indicated. Error bars: standard deviation of expression values in three different clones.(H) Chromatin from ESΔN-NERT cells treated with 1 μM tamoxifen for 0 or 24 hr was immunoprecipitated with Nanog or total RNAPolII antibodies. Enrichment relative to the ArpP0 promoter is measured using the primers indicated at Esrrb.Error bars: standard deviation (n = 3); ∗p ≤ 0.05, ∗∗p ≤ 0.01. See also Figures S1 and S2 and Tables S1.1 and 1.2.
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fig1: Identification of Nanog Target Genes Including Esrrb(A) Deep-SAGE profile of sorted Nanog-positive (GFP+) and Nanog-negative (GFP–) TNG cells, ESCs with wild-type levels of Nanog expression (RCN(t)) and Nanog−/− ESCs (RCNβH(t)). Genes were ranked according to the expression level and fold difference in expression in TNG+ versus TNG− and RCN(t) versus RCNβH(t); the plot shows the first 250 most upregulated (top) or downregulated (bottom) genes. Colors: yellow, expression above average; blue, below average.(B) Esrrb transcript levels in two cell lines overexpressing Nanog (EF4 and RCN), two cell lines with wild-type Nanog (E14Tg2a and RCN(t)), two Nanog+/− cell lines (TβC44 and RCNβ(t)), and two Nanog−/− cell lines (TβC44Cre6 and RCNβH(t)). Error bars: standard deviation (n = 4).(C) Immunoblot analysis of Esrrb and Nanog levels in the same ESC lines.(D) Immunohistochemical analysis of the intracellular localization of Nanog in ESΔN-NERT cells in response to 1 μM tamoxifen as indicated.(E) Global transcriptional changes after ESΔN-NERT stimulation with tamoxifen as indicated; the Esrrb changes are in red. Mean expression levels in three independent experiments are shown.(F) Venn diagram showing the intersection of significantly upregulated or downregulated genes identified in (E) compared to genes bound by Nanog according to two independent genome-wide ChIP studies.(G) Esrrb pre-mRNA kinetics in ESΔN-NERT cells stimulated with tamoxifen as indicated. Error bars: standard deviation of expression values in three different clones.(H) Chromatin from ESΔN-NERT cells treated with 1 μM tamoxifen for 0 or 24 hr was immunoprecipitated with Nanog or total RNAPolII antibodies. Enrichment relative to the ArpP0 promoter is measured using the primers indicated at Esrrb.Error bars: standard deviation (n = 3); ∗p ≤ 0.05, ∗∗p ≤ 0.01. See also Figures S1 and S2 and Tables S1.1 and 1.2.

Mentions: To identify genes controlled by Nanog, we compared the transcriptional profiles of ESCs in which GFP has been knocked in to one of the Nanog alleles (TNG cells; Chambers et al., 2007) that were sorted into SSEA1+/GFPhigh and SSEA1+/GFPlow populations, together with Nanog+/+ and Nanog−/− cells (Chambers et al., 2007) (Figure 1A). Good agreement between duplicate samples of Nanog−/− RNA indicated reliable output from the Deep-SAGE protocols. Moreover, broad agreement was observed between both Nanog−/− and Nanog:GFP− as well as between Nanog+/+ and Nanog:GFP+ cells. Of 500 genes showing the greatest change in expression, Esrrb was the transcription factor that showed the closest positive correlations with Nanog and consistent variations in both Nanog:GFP+ versus Nanog:GFP− and wild-type versus Nanog−/− comparisons (fold change ≥1.5), closely followed by Klf4 (Table S1.1). To better understand the role of Esrrb in ESC pluripotency, we further characterized the expression of the Esrrb gene in ESCs and its regulation by Nanog.


Esrrb is a direct Nanog target gene that can substitute for Nanog function in pluripotent cells.

Festuccia N, Osorno R, Halbritter F, Karwacki-Neisius V, Navarro P, Colby D, Wong F, Yates A, Tomlinson SR, Chambers I - Cell Stem Cell (2012)

Identification of Nanog Target Genes Including Esrrb(A) Deep-SAGE profile of sorted Nanog-positive (GFP+) and Nanog-negative (GFP–) TNG cells, ESCs with wild-type levels of Nanog expression (RCN(t)) and Nanog−/− ESCs (RCNβH(t)). Genes were ranked according to the expression level and fold difference in expression in TNG+ versus TNG− and RCN(t) versus RCNβH(t); the plot shows the first 250 most upregulated (top) or downregulated (bottom) genes. Colors: yellow, expression above average; blue, below average.(B) Esrrb transcript levels in two cell lines overexpressing Nanog (EF4 and RCN), two cell lines with wild-type Nanog (E14Tg2a and RCN(t)), two Nanog+/− cell lines (TβC44 and RCNβ(t)), and two Nanog−/− cell lines (TβC44Cre6 and RCNβH(t)). Error bars: standard deviation (n = 4).(C) Immunoblot analysis of Esrrb and Nanog levels in the same ESC lines.(D) Immunohistochemical analysis of the intracellular localization of Nanog in ESΔN-NERT cells in response to 1 μM tamoxifen as indicated.(E) Global transcriptional changes after ESΔN-NERT stimulation with tamoxifen as indicated; the Esrrb changes are in red. Mean expression levels in three independent experiments are shown.(F) Venn diagram showing the intersection of significantly upregulated or downregulated genes identified in (E) compared to genes bound by Nanog according to two independent genome-wide ChIP studies.(G) Esrrb pre-mRNA kinetics in ESΔN-NERT cells stimulated with tamoxifen as indicated. Error bars: standard deviation of expression values in three different clones.(H) Chromatin from ESΔN-NERT cells treated with 1 μM tamoxifen for 0 or 24 hr was immunoprecipitated with Nanog or total RNAPolII antibodies. Enrichment relative to the ArpP0 promoter is measured using the primers indicated at Esrrb.Error bars: standard deviation (n = 3); ∗p ≤ 0.05, ∗∗p ≤ 0.01. See also Figures S1 and S2 and Tables S1.1 and 1.2.
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fig1: Identification of Nanog Target Genes Including Esrrb(A) Deep-SAGE profile of sorted Nanog-positive (GFP+) and Nanog-negative (GFP–) TNG cells, ESCs with wild-type levels of Nanog expression (RCN(t)) and Nanog−/− ESCs (RCNβH(t)). Genes were ranked according to the expression level and fold difference in expression in TNG+ versus TNG− and RCN(t) versus RCNβH(t); the plot shows the first 250 most upregulated (top) or downregulated (bottom) genes. Colors: yellow, expression above average; blue, below average.(B) Esrrb transcript levels in two cell lines overexpressing Nanog (EF4 and RCN), two cell lines with wild-type Nanog (E14Tg2a and RCN(t)), two Nanog+/− cell lines (TβC44 and RCNβ(t)), and two Nanog−/− cell lines (TβC44Cre6 and RCNβH(t)). Error bars: standard deviation (n = 4).(C) Immunoblot analysis of Esrrb and Nanog levels in the same ESC lines.(D) Immunohistochemical analysis of the intracellular localization of Nanog in ESΔN-NERT cells in response to 1 μM tamoxifen as indicated.(E) Global transcriptional changes after ESΔN-NERT stimulation with tamoxifen as indicated; the Esrrb changes are in red. Mean expression levels in three independent experiments are shown.(F) Venn diagram showing the intersection of significantly upregulated or downregulated genes identified in (E) compared to genes bound by Nanog according to two independent genome-wide ChIP studies.(G) Esrrb pre-mRNA kinetics in ESΔN-NERT cells stimulated with tamoxifen as indicated. Error bars: standard deviation of expression values in three different clones.(H) Chromatin from ESΔN-NERT cells treated with 1 μM tamoxifen for 0 or 24 hr was immunoprecipitated with Nanog or total RNAPolII antibodies. Enrichment relative to the ArpP0 promoter is measured using the primers indicated at Esrrb.Error bars: standard deviation (n = 3); ∗p ≤ 0.05, ∗∗p ≤ 0.01. See also Figures S1 and S2 and Tables S1.1 and 1.2.
Mentions: To identify genes controlled by Nanog, we compared the transcriptional profiles of ESCs in which GFP has been knocked in to one of the Nanog alleles (TNG cells; Chambers et al., 2007) that were sorted into SSEA1+/GFPhigh and SSEA1+/GFPlow populations, together with Nanog+/+ and Nanog−/− cells (Chambers et al., 2007) (Figure 1A). Good agreement between duplicate samples of Nanog−/− RNA indicated reliable output from the Deep-SAGE protocols. Moreover, broad agreement was observed between both Nanog−/− and Nanog:GFP− as well as between Nanog+/+ and Nanog:GFP+ cells. Of 500 genes showing the greatest change in expression, Esrrb was the transcription factor that showed the closest positive correlations with Nanog and consistent variations in both Nanog:GFP+ versus Nanog:GFP− and wild-type versus Nanog−/− comparisons (fold change ≥1.5), closely followed by Klf4 (Table S1.1). To better understand the role of Esrrb in ESC pluripotency, we further characterized the expression of the Esrrb gene in ESCs and its regulation by Nanog.

Bottom Line: Moreover, Esrrb can reprogram Nanog(-/-) EpiSCs and can rescue stalled reprogramming in Nanog(-/-) pre-iPSCs.Finally, Esrrb deletion abolishes the defining ability of Nanog to confer LIF-independent ESC self-renewal.These findings are consistent with the functional placement of Esrrb downstream of Nanog.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, 5 Little France Drive, Edinburgh EH16 4UU, Scotland.

Show MeSH
Related in: MedlinePlus