Critical roles of coactivator p300 in mouse embryonic stem cell differentiation and Nanog expression

Abstract

p300 is a well known histone acetyltransferase and coactivator that plays pivotal roles in many physiological processes. Despite extensive research for the functions of p300 in embryogenesis and transcription regulation, its roles in regulating embryonic stem (ES) cell pluripotency are poorly understood. To address this issue, we investigated the self-renewal ability and early differentiation process in both wild-type mouse ES cells and ES cells derived from p300 knock-out (p300(-/-)) mice. We found that p300 ablation did not affect self-renewal capacity overtly when ES cells were maintained under undifferentiated conditions. However, the absence of p300 caused a significantly abnormal expression pattern of germ layer markers when differentiation was induced by embryoid body (EB) formation. Interestingly, the expression level of pluripotency marker Nanog but not Oct4 was markedly lower in EBs from p300(-/-) ES cells compared with that in EBs from wild-type ES cells. Exogenous expression of Nanog rescued abnormal expression of extra-embryonic endoderm marker partially but not mesoderm and ectoderm markers. Furthermore, we demonstrate that p300 was directly involved in modulating Nanog expression. Importantly, epigenetic modification of histone acetylation at the distal regulatory region of Nanog was found to be dependent on the presence of p300, which could contribute to the mechanism of regulating Nanog expression by p300. Collectively, our results show that p300 plays an important role in the differentiation process of ES cells and provide the first evidence for the involvement of p300 in regulating Nanog expression during differentiation, probably through epigenetic modification of histone on Nanog.

FIGURE 1.

The p300 expression pattern during ES cell differentiation. A, mRNA level of p300 during EB formation by qPCR. EBs derived from wild-type ES cells were harvested on days 0, 2, 4, 6, 8, and 10. Data were from three independent experiments. B and C, protein level of p300 during EB formation by Western blot (WB) analysis. Quantitative data and representative results of three independent experiments are shown.

FIGURE 2.

p300 is required for ES cells to differentiate normally in vitro. A, colony formation efficiency of p300^+/+ and p300^-/- ES cells. B, mRNA levels of pluripotency markers Oct4, Nanog, and Sox2 and differentiation markers Gata6, T, and Fgf5 in undifferentiated p300^+/+ and p300^-/- ES cells by qPCR. The mRNA level of each marker in p300^-/- ES cells was divided by that in p300^+/+ ES cells. KO, knock-out; WT, wild-type. C, morphology of EBs derived from p300^+/+ and p300^-/- ES cells. EBs were formed and photographed on day 3 (×200 magnification) and on day 6 (×100 magnification), respectively. D, mRNA levels of germ layer markers in EBs derived from p300^+/+ and p300^-/- ES cells. EBs were harvested every 2 days from days 0 to 10 and subjected to qPCR. Data are shown as mean ± S.D. (n = 10 for Gata6; n = 3 for Fgf5 and T). ^*, p < 0.05; ^**, p < 0.01.

FIGURE 3.

Reduction in the expression level of Nanog in the absence of p300 during ES cell differentiation. A, transcript levels of pluripotency markers Nanog and Oct4 during EB formation from p300^+/+ and p300^-/- ES cells by qPCR. B, protein levels of Nanog and Oct4 during EB formation from p300^+/+ and p300^-/- ES cells. Results from EBs of p300^+/+ and p300^-/- ES cells were quantified and compared. C, representative data of Nanog and Oct4 protein levels during EB formation from p300^+/+ and p300^-/- ES cells by Western blot (WB) analysis. Data are shown as mean ± S.D. (n = 4). ^*, p < 0.05; ^**, p < 0.01.

FIGURE 4.

Overexpression of Nanog partially rescues the phenotype of EBs derived from p300^-/- ES cells. A, verification of exogenous Nanog overexpression in p300^-/- ES cells by Western blot (WB) analysis. The asterisk and arrow indicate exogenous and endogenous Nanog protein, respectively, in p300^-/-+Nanog ES cells (right lane). B, morphology of EBs derived from p300^+/+, p300^-/-, and p300^-/-+Nanog ES cells. EBs of day 5 were photographed at ×200 magnification. C, exogenous Nanog overexpression rescues the abnormal expression of extra-embryonic endoderm markers but not those of other germ layers in EBs from p300^-/-+Nanog ES cells. EBs from p300^+/+, p300^-/-, and p300^-/-+Nanog ES cell lines were harvested every 2 days from days 0 to 10. Differentiation markers of Gata6, Gata4, Ihh, T, and Fgf5 were examined by qPCR. Data are shown as mean ± S.D. (n = 3). ^*, p < 0.05; ^**, p < 0.01.

FIGURE 5.

p300 regulates Nanog expression directly. A, schematic structure of the Nanog 5′ upstream regulatory region and reporter plasmids. Upper panel, the structure of the 5′ upstream regulatory region of Nanog and the positions of the T-Stat3-binding site, distal enhancer (DE), proximal enhancer (PE), and exon 1. Lower panel, structure of the Nanog-4.8kb and Nanog-3.8kb reporter plasmids. B, p300 activates the Nanog reporter dose-dependently. The Nanog-4.8kb reporter was cotransfected with p300 into p300^-/- ES cells cultured in the presence of LIF. C, p300 specifically functions at the 5′ distal regulatory region of Nanog from -4.8 to -3.8 kb. The Nanog-4.8kb and Nanog-3.8kb reporters were cotransfected with p300 into p300^-/- ES cells cultured with LIF, and luciferase (Luc) activities were measured. Data are shown as mean ± S.D. (n = 4 for B and C). ^**, p < 0.01.

FIGURE 6.

Epigenetic modification of histone is involved in the regulation of Nanog expression by p300. A, the direct association of p300 with the distal regulatory region of Nanog in CGR8 ES cells was detected by ChIP assays. p300-associated DNA fragments were detected by qPCR. -Fold enrichment is the relative abundance of the indicated DNA fragments over the glyceraldehyde-3-phosphate dehydrogenase fragment. n = 3. ^*, p < 0.05. DE, distal enhancer; PE, proximal enhancer. B, the acetylation state of histone H3 lysines 9 and 14 was examined in p300^+/+ and p300^-/- ES cells after LIF withdrawal for 2 days by ChIP assays. Acetyl-H3K9 and acetyl-H3K14-associated genomic regions were amplified by qPCR. Data are presented as in A. n = 3. IP, immunoprecipitation; Ab, antibody.