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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Stem Cells. Author manuscript; available in PMC Jan 17, 2012.
Published in final edited form as:
PMCID: PMC3260005
NIHMSID: NIHMS347189

Proteomic Analysis of Sox2-associated Proteins During Early Stages of Mouse Embryonic Stem Cell Differentiation Identifies Sox21 as a Novel Regulator of Stem Cell Fate

Abstract

Small increases in the levels of master regulators, such as Sox2, in embryonic stem cells (ESC) have been shown to promote their differentiation. However, the mechanism by which Sox2 controls the fate of ESC is poorly understood. In this study, we employed Multidimensional Protein Identification Technology and identified >60 nuclear proteins that associate with Sox2 early during ESC differentiation. Gene ontology analysis of Sox2-associated proteins indicates that they participate in a wide range of processes. Equally important, a significant number of the Sox2-associated proteins identified in this study have been shown previously to interact with Oct4, Nanog, Sall4 and Essrb. Moreover, we examined the impact of manipulating the expression of a Sox2-associated protein on the fate of ESC. Using ESC engineered for inducible expression of Sox21, we show that ectopic expression of Sox21 in ESC induces their differentiation into specific cell types, including those that express markers representative of neurectoderm and heart development. Collectively, these studies provide new insights into the range of molecular processes through which Sox2 is likely to influence the fate of ESC, and provide further support for the conclusion that the expression of Sox proteins in ESC must be precisely regulated. Importantly, our studies also argue that Sox2, along with other pluripotency-associated transcription factors, is woven into highly interconnected regulatory networks that function at several levels to control the fate of ESC.

Introduction

Embryonic stem cells (ESC) have the ability to both self-renew and differentiate into cells derived from each of the three embryonic germ layers. The behavior of ESC is controlled by a complex array of gene regulatory networks [1,2]. Over the past decade, the number of transcription factors shown to control these networks has grown significantly. Two transcription factors in particular, Sox2 and Oct4, cooperatively regulate a growing list of genes in ESC known as Sox2:Oct4 target genes, many of which are essential for the self-renewal and pluripotency of ESC [3-7]. Genome-wide DNA binding assays argue that Sox2 and Oct4, along with Nanog, constitute core transcription factors that regulate several thousand genes in ESC [1,2,8,9].

Recent studies have demonstrated that Oct4 and Sox2 function as molecular rheostats, and their levels must be maintained within a narrow range to preserve the self-renewal and pluripotency of ESC. Reduction of Oct4 levels in mouse ESC promotes their differentiation into trophectoderm-like cells, whereas small increases in Oct4 cause ESC to differentiate into cells of endoderm and mesoderm lineages [10]. Similar to Oct4, knockdown of Sox2 causes ESC to differentiate into trophectoderm-like cells [11]. In contrast, elevating the levels of Sox2 (2-fold or less) triggers differentiation of ESC into cells that express markers of ectoderm, mesoderm and trophectoderm, but not endoderm [12]. Importantly, other studies have demonstrated the necessity of achieving the correct levels of Sox2 and Oct4 during the reprogramming of somatic cells into induced pluripotent stem cells [13-15]. Although it is clear that Sox2 and Oct4 levels must be precisely regulated, the mechanisms by which small increases in the levels of Sox2 or Oct4 influence the fate of pluripotent stem cells have not been examined.

In this study, we focused on the identification of the molecular machinery responsible for inducing ES cell differentiation when Sox2 levels are elevated. We performed an unbiased screen of Sox2-associated proteins using Multidimensional Protein Identification Technology (MudPIT), which is a highly sensitive mass spectrometry-based method capable of analyzing complex mixtures of proteins [16,17]. We extended these findings by examining how manipulating the expression of a Sox2-associated protein influences the fate of ESC. For these studies, we focused on Sox21 because it is rapidly induced when Sox2 levels are elevated in ESC [12], and because it has been reported to be a transcriptional repressor that antagonizes Sox2 during neurogenesis [18,19]. Taken together, our studies provide further support for the conclusion that altering the expression of Sox proteins in ESC strongly influences their fate.

Materials and Methods

Cell Culture

Cultivation of i-Sox2-ESC and 293T cells has been described previously [12,20,21]. A2Lox.cre ESC and i-Sox21-ESC clones were maintained in DMEM supplemented with 15% FBS, 100 μM β-mercaptoethanol, and 10 ng/ml LIF on gelatin-coated tissue culture plastic. The procedure for generating i-Sox21-ESC is provided in Supplemental Methods. The cloning efficiency of i-Sox21-ESC was determined as described previously [12]. All cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO2.

Isolation of Flag-Sox2 Protein Complexes and MudPIT Analysis

Nuclear extracts from Doxycycline (Dox)-treated (4 μg/ml of Dox for 24 hours) and untreated i-Sox2-ESC were prepared by dounce homogenization. Details regarding the dounce homogenization, use of anti-Flag M2 affinity beads (Sigma-Aldrich, St. Louis, MO) for the isolation of Flag-tagged proteins, and silver staining of immunoprecipitation eluates are provided in the Supplemental Methods. Proteins isolated from Dox-treated and untreated i-Sox2-ESC were precipitated by adding Tris-HCl (pH 8.5) and 100% trichloroacetic acid (TCA) (Sigma-Aldrich, St. Louis, MO), followed by overnight incubation at 4° C. The protein precipitate was dried by heating at 45°C and analyzed by MudPIT as described in the Supplemental Methods. False discovery rate was determined by the method of Elias and co-workers [22].

Immunoprecipitation Studies in 293T Cells, i-Sox2-ESC, and i-Sox21-ESC

293T cells were transfected by the calcium phosphate precipitation method, as described previously [21]. One day after transfection, nuclear extracts were prepared using NE-PER™ kit reagents (Pierce, Rockford, IL) supplemented with protease inhibitors. Anti-Flag M2 affinity beads were used for immunoprecipitation of Flag-tagged proteins from nuclear extracts. Details regarding the expression plasmids, wash buffers, and western blot analyses are provided in Supplemental Methods. i-Sox2-ESC and i-Sox21-ESC treated for 24 hours with 4 μg/ml and 500 ng/ml of Dox, respectively, were used for immunoprecipitation studies using a Sox2 antibody. Nuclear extracts were prepared using NE-PER™ kit reagents (Pierce, Rockford, IL). Details regarding the immunoprecipitation of Sox2 from i-Sox2-ESC and i-Sox21-ESC are provided in Supplemental Methods.

i-Sox21-ESC Nuclear Extract Preparation and Western Blotting

Nuclear extracts were prepared from untreated and Dox-treated i-Sox21-ESC using the NEPER™ kit (Pierce, Rockford, IL). Protein concentrations of the nuclear extracts were determined using a micro BCA™ protein assay kit (Pierce, Rockford, IL), and equal amounts of protein (30μg) were used for western blot analysis.

RNA Analysis

i-Sox21-ESC were seeded in T-75 flasks at concentrations of 1.8×106 and 0.9×106 cells, and after 24 hours the cells were refed with fresh medium with or without 500 ng/ml Dox for 9 and 48 hours, respectively. RNA was isolated from these cells as described previously [12], except RNA pellets were resuspended in approximately 120 μl HPLC H2O. cDNA synthesis was performed with 0.5 μg of RNA using the AccuScript High Fidelity 1st Strand cDNA Synthesis Kit (Stratagene, La Jolla, CA). Microarray Analysis of i-Sox21- and i-Sox2-ESC was performed as described previously [12]. cDNA synthesized from untreated i-Sox21-ESC and 500 ng/ml Dox-treated i-Sox21-ESC for 9 hours or 48 hours was analyzed using SYBR Green (SuperArray Bioscience Corporation, Federick, MD) quantitative Real-Time polymerase chain reaction (qRTPCR) as described previously [12]. Primers for Nanog, Oct4, Fgf-4, Utf1, Sox2, Sox21, Lefty1, BMP4, Hand1, Sox3, Sox13, Pax6, Brachyury, Vimentin, Gata-4, Gata-6, Sox17, Cdx2, Cdh3, Esx1 have been described previously [12,23]. Sequences of primers for the remainder of the genes analyzed by qRT-PCR are provided in Supplemental Table 1.

Results

Sox2 Associates With Many Nuclear Proteins

To understand the mechanisms by which elevated levels of Sox2 exerts its effects, we performed an unbiased screen to identify the proteins that associate with Sox2 during the early stages of Sox2-mediated ESC differentiation. For this purpose, we employed ESC, referred to as i-Sox2-ESC, in which Sox2 levels are elevated by Dox. A flow diagram is provided as a guide for our screening strategy (Fig. 1A). In brief, nuclear extracts were prepared from untreated i-Sox2-ESC and from i-Sox2-ESC treated with 4 μg/ml of Dox for 24 hours. This concentration of Dox elevates the expression of Flag-Sox2 by ~2-fold relative to endogenous Sox2 [12]. At this early time point, the expression of differentiation markers is just beginning to appear. Flag-Sox2 and its associated proteins in the nuclear extracts were isolated using anti-Flag M2 agarose beads and eluted using 3X Flag peptide. Since the M2 antibody recognizes the Flag epitope, Flag-Sox2 was readily detected in the eluted proteins from Dox-treated i-Sox2-ESC, but not in the coimmunoprecipitation eluate prepared from the control untreated i-Sox2-ESC (Fig. 1B). Silver-staining demonstrated a clear differential in the protein profile between untreated and Dox-treated i-Sox2-ESC (Fig. 1C). Many protein bands were detected in the co-immunoprecipitation eluate from Dox-treated cells, ranging from low molecular weight (25 kD) to high molecular weight (>250 kD). Proteins isolated from untreated i-Sox2-ESC contained a low background, except for one prominent band that was detected in the eluted proteins from both untreated and Dox-treated i-Sox2-ESC. This band is a common contaminant detected routinely in samples prepared using M2 beads [16].

Figure 1
Preparation of samples for MudPIT analyses. (A) Flow chart describing the experimental outline for identification of Sox2-associated proteins from Dox-treated i-Sox2-ESC. Samples processed from untreated i-Sox2-ESC served as a negative control. (B) Western ...

To identify the profile of Sox2-associated proteins in Sox2-immunoprecipitated complexes, we employed MudPIT, a mass spectrometry-based method used to identify proteins present in highly complex mixtures [16,17]. In the present study, three independent MudPIT analyses identified many nuclear proteins that associate with Sox2. The high confidence Sox2-associated proteins are categorized into two main groups. One group consists of proteins detected only in the Dox-treated i-Sox2-ESC in all three MudPIT analyses (Fig. 2A, Supplemental Table 2A). A second group consists of proteins detected in both untreated and Dox-treated i-Sox2-ESC, but enriched in the latter. Only those proteins with enrichment values >9-fold are included in this second group (Fig. 2B, Supplemental Table 2B). The abundance of different Sox2-associated proteins is presented as Normalized Spectral Abundance Factor (NSAF) values, which are used to express relative levels of different proteins in a sample [16,24,25]. Together, there are a total of >60 proteins in these two groups (p <0.05), which include transcription factors (e.g. Sall4, Sox21), repressor proteins (e.g. Mbd3), chromatin architectural proteins (e.g. Banf1), and RNA binding proteins (e.g. Lin28, Musashi homolog 2). The false discovery rate was determined by the method of Elias and co-workers and found to be <0.4% for all three MudPIT analyses [22].

Figure 2Figure 2
Sox2-associated proteins identified by MudPIT analyses. (A) NSAF values of statistically significant (p < 0.05) Sox2-associated proteins identified only in Dox-treated i-Sox2-ESC, in all three MudPIT analyses (see also Supplemental Table 2A). ...

In addition to the two main groups of Sox2-associated proteins described above (Fig. 2A, 2B), our MudPIT analyses identified two other groups of Sox2-associated proteins. One group consists of proteins identified only in the Dox-treated samples in 2 of the 3 MudPIT analyses (Supplemental Table 3A). The second group consists of proteins detected in both the Dox-treated and untreated i-Sox2-ESC with enrichment values >3-fold and <9-fold in the samples from the Dox-treated cells (Supplemental Table 3B).

Validation of Sox2-Associated Proteins

To validate the findings from our MudPIT analyses where Flag-Sox2 was used to isolate Sox2 protein complexes, we initially focused on the pluripotency-associated protein, Sall4, because it had a relatively low enrichment value (~10-fold) among the proteins enriched in the Dox-treated i-Sox2-ESC (Fig. 2B). Thus, it serves as a stringent validation of our MudPIT results. We determined that Sall4 co-immunoprecipitates with Flag-Sox2 from Dox-treated i-Sox2-ESC that express Flag-Sox2, but not from untreated i-Sox2-ESC (Supplemental Fig. 1A). We also determined that Sall4 and Sox2 co-immunoprecipitate when expressed in 293T cells (Supplemental Fig. 1B). In addition, we verified the association of Sox2 with Sox21 and Lin28 by performing immunoprecipitation studies in 293T cells (Supplemental Fig. 1C, 1D). We chose to verify association of Sox2 with Sox21 and Lin28 because the former has the highest NSAF value in our MudPIT analyses, and the latter has important functions in ESC, as well as in reprogramming of somatic cells (Fig. 2A, Supplemental Table 2A) [26,27]. Using ectopically expressed proteins in 293T cells, we determined that Sox2 co-immunoprecipitates with both Sox21 and Lin28 (Supplemental Fig. 1C, 1D). As further validation of the proteins in our samples, we determined that HDAC1, which has an enrichment value of ~3.5 fold (Supplemental Table 3B), is pulled down by Flag-Sox2 from Dox-treated i-Sox2-ESC which express Flag-Sox2, but not from untreated i-Sox2-ESC (Supplemental Fig. 1E). We also determined that Sox2 and HDAC1 can associate with one another in 293T cells (data not shown).

In our MudPIT analyses and co-immunoprecipitation studies described above, we utilized anti-Flag M2 antibody conjugated to agarose beads to immunoprecipitate Flag-tagged proteins. To further validate our MudPIT findings, we tested the ability of a Sox2 antibody to coimmunoprecipitate a select group of Sox2-associated proteins in Dox-treated i-Sox2-ESC. These studies demonstrated that Lin28, Sall4, HDAC1, and HDAC2 co-immunoprecipitate with Sox2 in i-Sox2-ESC (Fig. 3A). Using the same Sox2 antibody for immunoprecipitation, we determined that Sox2 and Sox21 co-immunoprecipitate in Dox-treated i-Sox21-ESC (Fig.3B).

Figure 3
Validation of MudPIT results by co-immunoprecipitation studies in i-Sox2-ESC and i-Sox21-ESC. (A) An antibody against Sox2 was used to co-immunoprecipitate Lin28, Sall4, HDAC1, and HDAC2 in i-Sox2-ESC treated with 4 μg/ml of Dox for 24 hours. ...

To better understand the potential changes in the Sox2-interactome identified during the early stages of ESC differentiation, we examined the protein levels of several Sox2-associated proteins in undifferentiated i-Sox2-ESC and in i-Sox2-ESC induced to differentiate by elevating Sox2 levels for 24 hours. We determined that Sall4, Banf1, HDAC1, and HDAC2 do not change significantly, and Lin28 levels decreased by ~40% when i-Sox2-ESC were induced to differentiate (Supplemental Fig. 2A). We also performed microarray analysis of undifferentiated i-Sox2-ESC and i-Sox2-ESC induced to differentiate by elevating Sox2 levels for 72 hours (Supplemental Fig. 2B). Of the 33 genes encoding Sox2-associated proteins that are represented on the DNA microarray, expression of only 2 genes (Top1 and Xrcc6) changed 2-fold or more at the 72 hour time point compared to undifferentiated ESC (Supplemental Fig. 2B). These results suggest that induction of ESC differentiation by elevated levels of Sox2 does not significantly alter the levels of Sox2-associated proteins when compared to their levels in undifferentiated ESC. Moreover, we have previously shown by DNA microarray analysis that the expression of only a small number of genes at the RNA level (<1%) decrease when Sox2 levels are elevated in ESC [12]. Thus, we suspect that many, if not most, of the Sox2-associated proteins identified in our MudPIT analyses associate with Sox2 in ESC, as well as during the early stages of Sox2-induced differentiation. Nonetheless, the expression of a few genes, including the Sox21 gene, increase when Sox2 levels are elevated [12].

Sox2 and Other ESC Pluripotency Factors are Interconnected at Multiple Levels

Recently, several studies have reported the identification of partner proteins of pluripotency factors, such as Oct4, Nanog, Sall4, and Esrrb, in ESC [28-31]. Integration of proteomics data from several different studies demonstrated a significant overlap between the partner proteins of different pluripotency factors [29]. Therefore, we examined whether Sox2-associated proteins identified in this study are shared by other pluripotency factors in ESC. For this purpose, we generated an integrated interactome of Sox2-associated proteins and the proteins that associate with pluripotency factors Oct4, Nanog, Sall4, and Esrrb (Fig. 4A). Approximately 20% of the Sox2-associated proteins identified in our study have also been shown to interact with either one or several of these pluripotency factors. Additionally, several Sox2-associated proteins, such as Gatad2b, HDAC1, HDAC2, Mta2, and Smarca4, interact with multiple (three or more) pluripotency factors. Among the different pluripotency factor interactomes analyzed, we determined that Oct4 and Sall4 share ~18% and ~12% of the identified Sox2-associated proteins, respectively. Equally important, Nanog, Sall4, and Esrrb were identified as Sox2-associated proteins in our study.

Figure 4Figure 4Figure 4
Integrated protein - protein and transcription factor - gene interactomes of Sox2-associated proteins. (A) Sox2-associated proteins presented in Figure 2 were used to generate the integrated interactome of Sox2-associated proteins and proteins that associate ...

We also examined whether any of the genes encoding Sox2-associated proteins are potential targets of transcription factors Sox2, Oct4, and Nanog in ESC. For this purpose, we utilized recently published genome-wide DNA binding data for Sox2, Oct4, and Nanog in mouse ESC [2,8,9], and constructed a transcription factor-gene interactome for Sox2-associated proteins identified in our study (Fig. 4B). We determined that ~75% of the genes encoding Sox2-associated proteins are bound by Sox2, Oct4, and Nanog in different combinations. Importantly, ~25% of the genes coding for Sox2-associated proteins are bound by all three transcription factors, Sox2, Oct4, and Nanog, and ~22% of the genes are bound by two of the three transcription factors (Sox2 and Oct4, Sox2 and Nanog, or Oct4 and Nanog) (Fig. 4C). Taken together, the combined analysis of the proteomic data and the genome-wide DNA binding data argue that these pluripotency factors not only interact with many of the same proteins, but they also appear to converge to regulate the expression of many of their associated proteins.

Ectopic Expression of Sox21 in ESC Triggers Differentiation

To examine whether Sox2-associated proteins influence the fate of ESC, we focused on Sox21 because its expression is activated within 3 hours after the levels of Sox2 begin to rise in i-Sox2-ESC [12], and because it exhibited the highest NSAF value amongst the Sox2-associated proteins identified in our MudPIT analyses (Fig. 2A). Equally important, Sox21 is a transcriptional repressor that has been reported to antagonize Sox1, Sox2 and Sox3 during neurogenesis [18,19]. Together, these findings suggested that expression of Sox21 in ESC would trigger their differentiation. To test this possibility, we engineered mouse ESC for inducible expression of Flag-Strep-tagged Sox21 (i-Sox21-ESC) using an improved cassette exchange system implemented in the ESC line, A2Lox.cre, and referred to as inducible cassette exchange (ICE) [32]. Similar to the previous A2Lox ESC [33], these cells contain a Tet-responsive promoter in a non-essential region of the X-chromosome (not inactivated during differentiation) and constitutively express the reverse-Tet transactivator (rtTA) due to its location in the Rosa26 locus. Through inducible cassette exchange recombination [32], we generated Dox-inducible i-Sox21-ESC as cre catalyzed its own replacement by the FS-Sox21 cDNA. As a result, after the FS-Sox21 cDNA had been integrated, the addition of Dox allowed the activation of FS-Sox21 expression (Fig. 5A). Multiple clones were isolated, seven of which genotyped positively by PCR (data not shown). All of the i-Sox21-ESC clones exhibited a morphology typical of the parental A2Lox.cre ESC cultured on gelatin-coated dishes in the presence of LIF.

Figure 5Figure 5
Dox-induced expression of Flag-Strep-tagged Sox21 triggers the differentiation of ESC. (A) The parental cell line, A2Lox.cre, was used to generate i-Sox21-ES cells. i-Sox21-ESC constitutively express the reverse-Tet transactivator (rtTA) from the Rosa26 ...

Four i-Sox21-ESC clones were examined for inducible Sox21 protein expression. Each clone was found to express Sox21, and underwent morphological changes in response to Dox (data not shown). Two of the four clones were selected for further study. Western blot analysis demonstrated that both clones exhibited a similar expression of Sox21 when treated with Dox (Fig. 5B, Supplemental Fig. 3). Moreover, Sox21 expression was only detected when i-Sox21-ESC were treated with Dox. Importantly, both clones rapidly underwent differentiation when treated with Dox and the vast majority of the cells exhibited flattened morphology and a large increase in the cytoplasmic to nuclear ratio (Fig. 5C); whereas, the parental cell line, A2Lox.cre, exhibited no morphological changes (Fig. 5D), because it lacks the Sox21 transgene. The level of Sox21 expression responsible for the differentiated phenotype was determined by western blot analysis using nuclear extracts prepared from i-Sox21-ESC and i-Sox2-ESC [12] that had been cultured in the optimal levels of Dox. Under these conditions, Sox2 levels are elevated ~2-fold above that in untreated i-Sox2-ESC [12], and Sox21 expression in i-Sox21-ESC reached between 6% and 18% in clones 1 and 7, respectively, relative to the level of Dox-induced Sox2 (Supplemental Fig. 3). Thus, relatively little ectopic expression of Sox21 in ESC is sufficient to trigger their differentiation, which argues that Sox21 is a potent regulator of ESC fate.

To determine the extent of differentiation induced by ectopic expression of Sox21, we compared the ability of i-Sox21-ESC to form colonies in the absence and presence of Dox. Growth at clonal densities provides a simple method for estimating the self-renewal of ESC. ESC seeded at clonal density give rise primarily to colonies that exhibit an ESC morphology, plus a lower number of mixed colonies containing both ESC and differentiated cells, as well as colonies consisting primarily of differentiated cells [12]. For this study, i-Sox21-ESC were seeded at clonal density for 96 hours in the absence or presence of Dox. We determined that ectopic expression of Sox21 in i-Sox21-ESC reduced their ability to form ESC colonies and greatly increased the number of differentiated colonies formed (Fig. 6A). More specifically, the ability of the untreated i-Sox21-ESC to form ESC colonies decreased from 60-70% (some differentiation due to the low density of the cultured cells) to 4-15% for the Dox-treated i-Sox21-ESC. Upon removal of Dox for 48 hours, the morphology of the differentiated cells does not revert to that of ESC (Fig. 6B). Thus, a low level of ectopic Sox21 expression substantially reduces the ability of the ESC to self-renew and induces their differentiation.

Figure 6
Ectopic expression of Sox21 in ESC disrupts their self-renewal and induces their differentiation. (A) The cloning efficiency of i-Sox21-ESC at a clonal density of 900 cells per 60-mm dish, in the absence or presence of Dox (200 ng/ml), was determined ...

Sox21-Differentiated Cells Primarily Express Cardiac Mesoderm and Neuroectoderm Markers

To identify the cell types formed upon differentiation of the i-Sox21-ESC, we extracted RNA from Dox-treated and untreated i-Sox21-ESC. Initially, RNA was examined using a DNA microarray that contained ~10,000 genes. We determined that ~0.6% of the genes present on the DNA microarray exhibited a change in expression of ~2-fold or greater. More specifically, 37 genes exhibited an increase in expression and 25 genes exhibited a decrease in expression (Supplemental Table 4). The change in expression of only a few genes in response to ectopic expression of Sox21 in ESC suggests that the function of Sox21 is highly specific. Interestingly, these gene changes primarily represented markers expressed by cells of the mesodermal and ectodermal lineage. To verify these results, the expression of a select panel of over 50 genes was examined more closely by qRT-PCR. Importantly, qRT-PCR analysis revealed that many of the Sox2:Oct4 target genes, including Nanog, FGF-4, and Lefty-1, and other genes important for the self-renewal of ESC, such as Dax1 and Sall4 [34,35], exhibited decreased expression after differentiation. However, one splice-variant of Sall4, Sall4-c, turned on with differentation (Fig. 7). We extended our analysis of the Dox-treated i-Sox21-ESC by examining the expression of several essential ESC-associated genes at the protein level using western blot analysis. Although Oct4 levels did not change significantly, we observed reductions in Sox2, Nanog and Sall4 protein of 30%, 40% and 70%, respectively (Supplemental Fig. 4).

Figure 7
Quantitative RT-PCR (qRT-PCR) of cDNA prepared from the RNA of i-Sox21-ESC cultured in the absence or presence of Dox (500 ng/ml) for 48 hours. Results are shown as average differences in cycle threshold (Ct) values from Dox-treated i-Sox21-ESC compared ...

Closer examination of the differentially regulated genes indicated that many are expressed by specific developmental lineages (Fig. 7). Many of the upregulated genes are typically expressed by cells present in the mesoderm, in particular during cardiac development. These include Nkx2.5, Hand1, Mef2c, Gata6, Gata4, HDAC7, HDAC5, Sparc, Sm22a, and Anxa6 [36-44]. We also observed increases in genes expressed by cells from the ectodermal lineage, including: Tapa-1 (CD81-neuron surface marker [45]), Atbf1 (activates NeuroD1 promoter [46]), and neuronal bHLH genes, NeuroD1, Mash1, Hes6, and Hes1 [47-49]. Interestingly, there is also an increase in the expression of Idb2 (a HLH gene also known as ID2), a protein that dimerizes with bHLH proteins and inhibits their DNA binding [50,51]. Additionally, there was a large increase in the expression of Esx1 and Cdh3, which represent trophectodermal cells; however, Cdx2 expression was not detected (Fig. 7). Finally, we examined the initial transcriptional response elicited by Sox21 expression. i-Sox21-ESC were cultured in the absence or presence of Dox for 9 hours before RNA was isolated. qRT-PCR analysis indicated that Sox21 begins to exert its effects soon after it is first expressed (Supplemental Fig. 5). Together, our findings indicate that ectopic expression of Sox21 in ESC disrupts their self-renewal and promotes their differentiation and expression of markers representative of cardiac and neuroectoderm lineages.

Discussion

Significant progress has been made recently toward defining the protein-interaction networks of several pluripotency factors in ESC [28-31]. However, definition of the protein-interaction landscape during the differentiation of ESC has received far less attention. Specifically, identification of protein-interaction networks for pluripotency factors, such as Sox2, Oct4 and Nanog, during differentiation will provide new insights into the mechanisms that control the fate of pluripotent stem cells. Previously, we have shown that a 2-fold increase in the levels of Sox2 induces differentiation of ESC [12]. In the current study, we performed an unbiased proteomic screen of Sox2-associated proteins during the initial period when ESC are induced to differentiate in response to elevated levels of Sox2. Importantly, our study identified >60 Sox2-associated proteins, the vast majority of which are shown for the first time to interact with Sox2. They include: Sox21, Banf1, Lin28, Sall4, Mbd3, Musashi homolog 2, and Replication Proteins A1, A2 and A3 (Fig. 2). Equally important, we extended these findings and demonstrated that ectopic expression of one of the Sox2-associated proteins, Sox21, in ESC disrupts their self-renewal and induces their differentiation.

To gain insights into biological processes performed by Sox2-associated proteins, we initially conducted gene ontology (GO) analysis of Sox2-associated proteins identified in our MudPIT analyses. We determined that Sox2-associated proteins are involved in a wide range of biological processes, which can be grouped into eight broad categories (Supplemental Fig. 6). The top three categories of biological processes represented by Sox2-associated proteins are: chromatin organization and assembly, DNA processing, and post-translational modification. Given the behavior of Sox2 as a master regulator in ESC, it is not surprising that Sox2 interacts with proteins known to have many different functions.

The importance of Sox2-associated proteins identified in our study is highlighted by studies showing that proteins involved in regulation of chromatin architecture, such as polycomb proteins [52,53], components of the NuRD complex [54,55], Swi/Snf family proteins [56,57], and the Tip60-p400 protein complex [58], play essential roles during the growth and differentiation of ESC. Remarkably, Sox2, Oct4 and Nanog have now each been shown to interact with the key components of the NuRD complex (Fig. 4A) [28-31]. Equally interesting is our finding that several Sox2-associated proteins are involved in RNA processing (Supplemental Fig. 6). Recent work by Sampath and co-workers illustrates the importance of post-transcriptional mechanisms in promoting the global increase in protein synthesis when ESC are induced to differentiate [59]. Therefore, it is tempting to speculate that Sox2-associated proteins involved in both chromatin structure and RNA processing play essential roles in controlling the fate of ESC.

To better understand the roles of Sox2-associated proteins, we integrated the Sox2-interactome generated in this study with published interactomes for the pluripotency factors, Oct4, Nanog, Sall4, and Esrrb [28-31]. Importantly, this analysis demonstrates that these pluripotency factors interact with many of the same nuclear proteins (Fig. 4A). Even more surprising are genome-wide binding studies performed with Sox2, Oct4, and Nanog [2,8,9], which show that these transcription factors bind to a large percentage of the genes that code for Sox2-associated proteins (Fig. 4B, 4C). Given the high degree of integration between Sox2, Oct4 and Nanog at both the gene expression level and at the protein interaction level, it is evident that these factors work together at multiple levels to control the fate of ESC. Thus, it is reasonable to speculate that many Sox2-associated proteins will also be found to play key roles in the regulation of ESC. In this regard, it has been shown that Sall4 and Smarca4 (also known as Brg-1) co-occupy the promoters of a subset of genes bound by Sox2, Oct4, and Nanog in ES cells [60,61]. These results suggest that Sox2-associated proteins are integral parts of the core transcription circuitry anchored by Sox2, Oct4, and Nanog, and it is possible that the association of a subset of these proteins with Sox2 is mediated by DNA.

Recent studies have shown that several of the Sox2-associated proteins identified in this study do in fact influence the self-renewal of ESC. Specifically, the Sox2-associated proteins, Sall4 and Lin28 have been shown to influence the self-renewal of ESC [27,34,60,62]; whereas, HDAC1 influences the differentiation, but not the proliferation, of ESC [63]. In this study, we felt it would be instructive to focus on a Sox2-associated protein that is induced when Sox2 levels are increased in i-Sox2-ESC. As discussed earlier, Sox21, which possesses a transrepression domain [19], appeared to be an ideal candidate. Sox21 is rapidly induced when Sox2 levels are elevated in i-Sox2-ESC [12], and Sox21 is the Sox2-associated protein with the highest NSAF value (Fig. 2A). Furthermore, given that Sox21 has been reported to antagonize the action of Sox 1, 2 and 3 during neurogenesis [18], we suspected that Sox21 may influence the function of Sox2 when Sox21 is expressed in i-Sox2-ESC. The studies described in this report demonstrate that expression of Sox21 disrupts ESC self-renewal and induces their differentiation.

Surprisingly, ectopic expression of Sox21 in ESC alters the expression of relatively few genes ~2-fold or greater. However, consistent with the disruption of ESC self-renewal Nanog, FGF-4, Lefty-1, Sall4, Dax1, and Rex1 exhibited decreased expression 48 hours after exposure to Dox (Fig. 7). We also detected decreased protein expression of Sall4, Nanog and Sox2 (decreased by 70%, 40%, and 30%, respectively) (Supplemental Fig. 4). Interestingly, Oct4, at both the RNA and protein level, does not change in response to Sox21-induced differentiation during the time frame examined. The reason for this is unclear; however, the expression of Nr5a2, a positive regulator of Oct4 gene expression [64], and Dax1, a negative regulator of the transcriptional activity of Oct4 [35], both decrease at the RNA level as early as 9 hours after Dox induction of Sox21 (Supplemental Fig. 5).

Interestingly, many of the genes whose expression increases in response to ectopic expression of Sox21 represent markers expressed by cells undergoing neural and cardiac development (Fig. 7). Within the cardiac network induced by Sox21-mediated differentiation, the genes of Hand1 (bHLH protein) and its cofactor Mef2c are regulated by Nkx2.5 [65-67], and these transcription factors activate cardiac-specific genes [65]. We also observed increases in HDAC7 and HDAC5 expression, which regulate cardiomyogenesis by inhibiting Mef2c-dependent transcription [40,41]. Importantly, two increased GATA factor genes, Gata6 and Gata4, are often categorized as endoderm markers; however, GATA factors have been shown to recruit Mef2c to its targets in cardiac myocytes, thereby playing a role in positive regulation of cardiac development [39]. In addition to markers of cardiac development, ectopic expression of a low level of Sox21 in ESC resulted in increased expression of genes that encode bHLH proteins for neural development. This network includes positive regulators NeuroD1, Mash1, and Hes6 and the negative regulators Hes1 and Idb2 (Fig. 7) [47-49,51]. Additionally, Atbf1 may be involved in activating the promoter of NeuroD1 (bHLH protein) and suppressing Nestin expression to allow proper neurogenesis [46]. Collectively, these connections suggest that Sox21 may have a broad effect over bHLH gene regulation in neuroectoderm and heart development.

Although ESC express several Sox proteins besides Sox2, including Sox4, Sox11 and Sox15 [68], it is clear from our studies, and the work of others, that altering the expression of Sox proteins in ESC influences their fate. Furthermore, these studies indicate that different Sox proteins direct ESC to differentiate into distinct cell-types. Ectopic expression of Sox21 (this study) and small increases of Sox2 in ESC [12] promote differentiation into cells that express markers of ectoderm, mesoderm, and trophectoderm (Fig. 7; [12]). Although i-Sox21- and i-Sox2-differentiated cells exhibit similar changes in the expression of a small set of genes (Supplemental Table 5), the phenotypes of these two populations are morphologically distinct and their RNA profiles differ significantly. For example, Sox21-differentiated cells express gene markers for neuronal ectoderm and cardiac mesoderm, but not the trophectoderm gene Cdx2; whereas, Sox2-differentiated cells express Cdx2, retinal genes, and mesodermal markers Vim, Brachyury and Flk-1 (Fig. 7; [12]). Given these results and our finding that ectopic expression of Sox21 can induce ESC differentiation, it is likely that the activation of Sox21 expression, which results from elevating Sox2, contributes, in part, to the gene expression profile observed in Dox-treated i-Sox2-ESC. However, the context of Sox21 expression differs in the two cellular systems. More specifically, the responses to Sox21 expression in the Dox-treated i-Sox21-ESC and i-Sox2-ESC are likely to differ, because other components of the transcriptional machinery are affected differently when Sox2 levels are increased versus when Sox21 is ectopically expressed.

Sox21- and Sox2-directed differentiation also differ from Sox9- and Sox17-directed differentiation of ESC. Ectopic expression of Sox9 in ESC increases mRNA expression of collagen IIA, aggrecan, and Pax1, which are marker genes for chondrogenic differentiation [69]; whereas, ectopic expression of Sox17 in ESC induces their differentiation toward extra-embryonic endoderm [70]. Interestingly, we do not observe the expression of extra-embryonic endoderm markers in our Sox2-induced [12] or Sox21-induced differentiated cells (Fig. 7). Together, these studies highlight the importance of carefully regulating the expression of Sox proteins in ESC. Moreover, these studies suggest that manipulating the levels of Sox proteins, possibly by protein transduction [71] could prove useful in directing the differentiation of pluripotent stem cells to cell populations that are useful clinically.

Conclusion

Our study has identified for the first time >60 nuclear proteins that associate with Sox2, and demonstrates that ectopic expression of Sox21 in ESC disrupts their self-renewal and pluripotency. Importantly, a significant number of the Sox2-associated proteins identified in this study have been shown previously to interact with Oct4, Nanog, Sall4 and Essrb. Surprisingly, Sox2, Oct4 and Nanog are known to associate with a high percentage of genes that code for Sox2-associated proteins. Thus, it is evident that these transcription factors are woven into highly interconnected regulatory networks that function at several levels to control the fate of ESC.

Supplementary Material

Supp Fig S1

Supp Table S1

Supp Table S2a

Supp Table S2b

Supp Table S3a

Supp Table S3b

Supp Table S4a

Supp Table S4b

Supp Table S5

Supp Fig S2a

Supp Fig S2b

Supp Fig S3

Supp Fig S4

Supp Fig S5

Supp Fig S6a

Supp Fig S6b

Supp Material

Acknowledgements

This work was supported by a grant from the National Institutes of Health (GM 080571). JMG and MPW were supported by the Stowers Institute for Medical Research.

Footnotes

Author contribution:

SKM: collection of data, data analysis, manuscript writing, final approval of manuscript

BDO: collection of data, data analysis, manuscript writing, final approval of manuscript

MI: creation of the A2lox.Cre ES cells, final approval of manuscript

JMG: collection of data, data analysis, final approval of manuscript

JLC: collection of data, data analysis, final approval of manuscript

MK: design of A2lox.Cre ES cells, final approval of manuscript

MPW: data analysis and interpretation, financial support, final approval of manuscript

AR: conception and design, financial support, manuscript writing, final approval of manuscript

The authors indicate no potential conflicts of interest.

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