Nanog binds to Smad1 and blocks bone morphogenetic protein-induced differentiation of embryonic stem cells

Abstract

ES cells represent a valuable model for investigating early embryo development and hold promise for future regenerative medicine strategies. The self-renewal of pluripotent mouse ES cells has been shown to require extrinsic stimulation by the bone morphogenetic protein (BMP) and leukemia inhibitory factor signaling pathways and the expression of the transcription factors Oct4 and Nanog. However, the network of interactions among extrinsic and intrinsic determinants of ES cell pluripotency is currently poorly understood. Here, we show that Nanog expression is up-regulated in mouse ES cells by the binding of T (Brachyury) and STAT3 to an enhancer element in the mouse Nanog gene. We further show that Nanog blocks BMP-induced mesoderm differentiation of ES cells by physically interacting with Smad1 and interfering with the recruitment of coactivators to the active Smad transcriptional complexes. Taken together, our findings illustrate the existence of ES cell-specific regulatory networks that underlie the maintenance of ES cell pluripotency and provide mechanistic insights into the role of Nanog in this process.

Fig. 1.

Presence of STAT- and T-binding sites in the mouse Nanog promoter. (A) Schematic representation of the 5′ upstream regulatory region of the mouse Nanog gene. Putative STAT- and T-binding sites are indicated. (B) T binds to the putative T-binding site in the Nanog regulatory region, as shown by pull-down assays. WT and mutated (MUT) versions of double-strand oligonucleotides representing the putative T-binding site were used as probes. Input lysates were also blotted with anti-Myc antibody. (C) Chromatin immunoprecipitation (ChIP) assays for the putative T- and STAT-binding sites in the Nanog regulatory region demonstrate specific binding of T and STAT3 to the regulatory region. (Lower) A PCR amplification of input DNA before immunoprecipitation. (D) LIF-dependent binding of STAT3 to the putative STAT-binding site in the Nanog regulatory region. WT and mutated versions of double-strand oligonucleotides for the putative STAT-binding site were used as probes. Input lysates were also blotted with anti-FLAG antibody.

Fig. 2.

Regulation of Nanog expression by LIF/STAT3 signaling and T. (A and B) Analysis of transcriptional activities of the Nanog regulatory region by luciferase reporter assay in mouse ES cells (1,000 or 400 units/ml LIF). (A) Both −5203Nanog-Luc and −4191Nanog-Luc showed a similar activation with 1,000 units/ml LIF, whereas −5203Nanog-Luc activity was further increased in cultures maintained with 400 units/ml LIF. (B) Both the T- and STAT3-binding sites were required for activation of Nanog EM enhancer activity in ES cells cultured with 400 units/ml LIF. WT indicates base pairs −5203 to −4192. MUT/T, MUT/S, and MUT/TS indicate the mutation in T-, STAT3-, or both T- and STAT3-binding sites, respectively, in the Nanog EM enhancer. Bars show mean ± SD (n = 4). (C) T and STAT3 physically interact inside cells. T and STAT3 were coimmunoprecipitated when STAT3 was activated by LIF. IP, immunoprecipitation.

Fig. 3.

The Nanog EM enhancer is active in EM progenitors. Fluorescent images of T-EGFP and Nanog EM enhancer DsRed2 expression in ES cell colonies formed in culture with 400 units/ml LIF. The coexpression of EGFP with DsRed2 in WT (A), but not when T- and/or STAT3-binding sites are mutated (B–D), indicates that the activity of the Nanog EM enhancer in EM progenitor cells is regulated by T and STAT3. (Scale bar: 10μm.)

Fig. 4.

BMP signaling promotes the mesoderm specification of ES cells. (A) Flow-cytometric analysis of T-positive cells in T-EGFP ES cells cultured for three passages with 400 units/ml LIF under conditions of inhibition (noggin) or activation (BMP2, BMP4, and BMP7) of BMP signaling. Bar shows mean ± SD (n = 4). (B) Flow-cytometric analysis of T-positive cells produced from purified T-positive cells cultured with 400 units/ml LIF under conditions of inhibition or activation of BMP signaling. Inhibition of endogenous BMP signaling by noggin decreased the percentage of T-positive cells at a similar level of Nanog overexpression, whereas BMP activation increased the percentage of T-positive cells. Bar shows mean ± SD (n = 4). (C) RT-PCR analyses in ES cells cultured with 400 units/ml LIF with or without addition of BMP7 show that BMP-dependent Id1 expression is negatively regulated by overexpressing Nanog and enhanced when Nanog function is down-regulated. (D) A reporter construct of −1147Id1-Luc containing the Smad-binding sites, but not −927Id1-Luc, was activated in a BMP-dependent manner in ES cells cultured with 400 units/ml LIF. Nanog and inhibitory Smads (Smad6 and Smad7) down-regulated −1147Id1-Luc activity in a similar manner. Bars show mean ± SD (n = 4).

Fig. 5.

Nanog down-regulates the expression of BMP targets. (A) Overexpression of p300 rescues the down-regulation of −1147Id1-Luc activity induced by Nanog. Bars show mean ± SD (n = 4). (B) The −396T-Luc, but not the −204T-Luc, reporter construct is activated in a BMP-dependent manner in ES cells cultured with 400 units/ml LIF. Nanog and inhibitory Smads (Smad6 and Smad7) down-regulate −396T-Luc activity in a similar manner. The down-regulation of −396T-Luc activity induced by Nanog is rescued by overexpression of p300. Bars show mean ± SD (n = 4). (C) Sequence of the 5′ upstream regulatory region of the mouse T gene. Three putative BMP-responsive Smad-binding sites are indicated by boxes. (D) Schematic representation of the negative feedback mechanism by which Nanog blocks BMP-induced T expression in the presence of LIF/STAT3 signaling. Black arrows depict positive direct transcriptional regulation, gray arrows depict positive posttranslational regulation, and red lines represent inhibitory regulation. See Results for details.