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Keywords:

  • Zfp143;
  • Oct4;
  • Nanog;
  • Embryonic stem cell

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

Identification of regulators governing the maintenance of embryonic stem (ES) cells is crucial to the understanding of ES cell biology. We identified a zinc finger protein, Zfp143, as a novel regulator for self-renewal. Depletion of Zfp143 by RNA interference causes loss of self-renewal of ES cells. Chromatin immunoprecipitation and electrophoretic mobility shift assays show the direct binding of Zfp143 to the Nanog proximal promoter. Knockdown of Zfp143 or mutation of the Zfp143 binding motif significantly downregulates Nanog proximal promoter activity. Importantly, enforced expression of Nanog is able to rescue the Zfp143 knockdown phenotype, indicating that Nanog is one of the key downstream effectors of Zfp143. More interestingly, we further show that Zfp143 regulates Nanog expression through modulation of Oct4 binding. Coimmunoprecipitation experiments revealed that Zfp143 and Oct4 physically interact with each other. This interaction is important because Oct4 binding to the Nanog promoter is promoted by Zfp143. Our study reveals a novel regulator functionally important for the self-renewal of ES cells and provides new insights into the expanded regulatory circuitry that maintains ES cell pluripotency.

Disclosure of potential conflicts of interest is found at the end of this article.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

Author contributions: X.C.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; F.F.: collection and/or assembly of data, data analysis and interpretation, manuscript writing; X.C. and F.F. contributed equally to this work. Y.-C.L.: provision of study material, data analysis and interpretation; H.-H.N.: conception and design, data analysis and interpretation, manuscript writing, final approval of manuscript

Embryonic stem (ES) cells are isolated from the inner cell mass (ICM) of blastocysts at day 3.5 of mouse development [1]. These cells are considered pluripotent, as they exhibit the ability to differentiate into most specialized cell types found in the adult mouse [2, 3]. In culture, they are able to self-renew and expand for an extended period of time. The maintenance of self-renewal is controlled by external signaling pathways such as the leukemia inhibitory factor (LIF)/signal transducer and activator of transcription 3 (STAT3) pathway [4, [5]6] and bone morphogenic protein (BMP) pathway [7] and the intrinsic transcription factors centered around Oct4, Sox2, and Nanog [8]. Recent studies have begun to identify new key regulators such as Esrrb [8, 9], Tbx3 [9], Sall4 [10, [11], [12]13], Zfx [14], Zic3 [15], and Klf2, Klf4, and Klf5 [16]. These transcription factors are preferentially upregulated in the undifferentiated ES cells. Depletion of these factors impairs the ability of ES cells to proliferate or maintain pluripotency.

The POU family transcription factor Oct4, which is encoded by Pou5f1 [17] and specifically expressed in the pluripotent cells of the ICM and epiblast, acts as a gatekeeper to prevent ES cell differentiation. It has been reported to regulate diverse downstream targets by forming heterodimers with Sox2 [SRY-related high-mobility group (HMG) box 2]. Sox2 is an HMG domain-containing transcription factor that has an expression pattern similar to that of Oct4 during mouse preimplantation development [18, [19]20]. Both Oct4- and Sox2-null mice have primary defects in the pluripotent epiblast, and both Oct4- and Sox2-null blastocysts are incapable of giving rise to pluripotent ES cells [17, 21]. Interestingly, the forced expression of Oct4 is able to rescue the pluripotency of Sox2-null ES cells [22]. Another key regulator residing in the same complex with Oct4 [23] is Nanog, an NK-2 class homeobox transcription factor, whose expression is also highly restricted to the ICM and epiblast [24, 25]. Nanog knockout embryos fail to form epiblasts and are mostly composed of disorganized extraembryonic tissue [24, 25]. More recently, it has been shown that although downregulation of Nanog predisposes ES cells toward differentiation, ES cells can, however, self-renew in the complete absence of Nanog [26]. This finding suggests that Nanog plays a major role in stabilizing the “stemness” state of ES cells.

In this study, we report Zfp143 (a selenocysteine tRNA gene transcription-activating factor) as a novel regulator that maintains the undifferentiated state of ES cells by regulating the transcription of Nanog. Depletion of Zfp143 by RNA interference (RNAi) resulted in cellular differentiation and a significant reduction in Nanog expression. Chromatin immunoprecipitation (ChIP) and luciferase assays revealed Zfp143 binding at the proximal promoter region of Nanog. Furthermore, we found that it interacts with Oct4 on this cis-regulatory element of Nanog. Our data extend knowledge of the transcription network in ES cells by integrating Zfp143 as an upstream activator of Nanog, through modulation of Oct4 binding.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

Coimmunoprecipitation

Transfected cells were lysed in cell lysis buffer (50 mM Tris HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, and 10% glycerol with protease inhibitor cocktail) for 1 hour. Whole cell extracts were collected and precleared. Beads coated with Oct4 (sc-8628; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, http://www.scbt.com) or hemagglutinin (HA) (sc-7392, Santa Cruz Technology, Inc.) antibody were incubated with the precleared whole cell extracts at 4°C for overnight. The beads were washed with cell lysis buffer four times. Finally, the beads were boiled in 2× sample buffer for 10 minutes. The eluents were analyzed by either protein staining or Western blot.

ChIP and RNA Expression Analysis

ChIP was performed as described previously [8] with Zfp143 antibody (H00007702-M01; Abnova, Walnut, CA, http://www.abnova.com), Oct4 antibody, HA antibody, Sox2 antibody (sc-17320; Santa Cruz Technology, Inc.), or RNA polymerase II antibody (05-623; Upstate, Charlottesville, VA, http://www.upstate.com). RNA extraction, reverse transcription, and quantitative real-time polymerase chain reaction (PCR) were carried out as described previously [8]. H1 human ES cells were used for retinoic acid (RA)-induced differentiation. The statistical analysis was performed using a t test. The results were considered significant when p < .05 and/or p < .005. Primers used for quantitative real-time PCR are listed in supplemental online Table 1.

Sequential ChIP

Oct4 antibody was cross-linked to protein G-Sepharose beads using dimethylpimelimidate to prevent the leaching of antibody during sodium dodecyl sulfate (SDS) elution. The beads were then incubated with chromatin extracts overnight. Subsequently, the beads were washed and eluted with 1% SDS elution buffer at 37°C for 45 minutes. The eluate was diluted to a final SDS concentration of 0.1% and incubated with fresh antibody-bound beads for the second immunoprecipitation (IP). For the final round of IP, washed beads were eluted with 1% SDS elution buffer at 68°C for 30 minutes. Eluate was decross-linked in the presence of pronase and heated at 68°C for 6 hours, and DNA was purified by phenol-chloroform extraction.

Short Hairpin RNA-Mediated Knockdown

The 19 nucleotides targeted by the small interfering RNAs are GGCAGATGGTGACAATTTA (for Zfp143-1) and GCAGTACGCAGCAAAGGTA (for Zfp143-2). We obtained similar results for the two RNAi constructs for Zfp143 knockdown. The RNAi experiments were carried out as described in [8].

Cell Culture

E14 or D3 mouse ES cells, cultured under feeder-free conditions were maintained in Dulbecco's modified Eagles medium (DMEM) (Gibco, Grand Island, NY, http://www.invitrogen.com), with 15% heat-inactivated ES qualified fetal bovine serum (FBS) (Gibco), 0.055 mM β-mercaptoethanol (Gibco), 2 mM l-glutamine, 0.1 mM minimal essential medium nonessential amino acid, 5,000 units/ml of penicillin/streptomycin, and 1,000 units/ml of LIF (Chemicon, Temecula, CA, http://www.chemicon.com). 293T cells were cultured in DMEM with 10% FBS and maintained at 37°C with 5% CO2.

Glutathione S-Transferase Pulldown Assay

Full-length Zfp143 and various deletion fragments were cloned into pET42b (Novagen, Gibbstown, NJ, http://www.emdbiosciences.com). The plasmids were transformed into BL21 Escherichia coli. The Zfp143 proteins were expressed and purified with glutathione (GSH)-Sepharose beads (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com) followed by nickel-nitrilotriacetic acid (Ni-NTA) beads (Qiagen, Hilden, Germany, http://www1.qiagen.com). The purified proteins were bound to GSH beads and incubated with Oct4-overexpressed cell lysates for 2 hours in 4°C. The beads were washed six times with cell lysis buffer. The eluents were analyzed by Western blot.

Electrophoretic Mobility Shift Assays

The DNA-binding domain of Zfp143 was cloned into pET42b. The plasmid was transformed into BL21 E. coli. The DNA-binding domain of Zfp143 protein was expressed and purified with GSH-Sepharose beads followed by Ni-NTA beads. The purified protein was dialyzed against dialysis buffer (protease inhibitor cocktail: 20 mM HEPES, pH 7.9, 20% glycerol, 100 mM KCl, 0.83 mM EDTA, and 1.66 mM dithiothreitol [DTT]; Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com) at 4°C for 4 hours. The concentration of the protein was measured with a Bradford assay kit (Bio-Rad, Hercules, CA, http://www.bio-rad.com). Double-stranded DNA oligonucleotides (Sigma-Proligo, Singapore, http://www.proligo.com) labeled with biotin at the 5′ termini of the sense strands were annealed with reverse strands in annealing buffer (10 mM Tris-HCl, pH 8.0, 50 mM NaCl, and 1 mM EDTA) and purified with an agarose gel DNA extraction kit (Qiagen). The sense strand sequence is shown in Figure 3A. An electrophoretic mobility shift assays (EMSA) was performed in a 10-μl reaction mixture containing 10 mM HEPES, pH 7.5, 10 mM KCl, 10 mM MgCl2, 1 mM DTT, 1 mM EDTA, 10% glycerol, 3 ng of biotin-labeled oligonucleotide, 1 μg of poly(dI-dC) (Amersham), and 100 ng of recombinant Zfp143 DNA-binding domain protein. Binding reaction mixtures were incubated for 10 minutes at room temperature and then were subjected to electrophoresis on prerun 5% native polyacrylamide gels in 0.5× Tris borate-EDTA buffer. Gels were transferred to Biodyne B nylon membranes (Pierce, Rockford, IL, http://www.piercenet.com) and detected with a LightShift Chemiluminescent EMSA kit (Pierce).

Luciferase Assay

E14 embryonic stem cells were transfected with reporter constructs by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) following the manufacturers protocol. A Renilla luciferase plasmid (pRL-SV40; Promega, Madison, WI, http://www.promega.com) was cotransfected as an internal control. Cells were harvested after 36 h, and the luciferase activity of the cell lysates was measured with the Dual-Luciferase Reporter Assay System (Promega).

Plasmids

The promoter region of murine Nanog was amplified from genomic DNA. Primers for amplification, with restriction sites for cloning purposes indicated in lowercase, were GTCTGTagatctAATGGAAGAGGAAACTCAGATCC (Nanog promoter forward) and CCACACacatgtCAGTGTGATGGCGAGGGAAGGG (Nanog promoter reverse). Products were cloned into pGL3 vector and sequence-verified. For the Nanog proximal promoter luciferase construct containing deletion of the Zfp143 binding site, the sequence CCTCTTTTTGGG was deleted.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

Zfp143 Maintains the Undifferentiated State of ES Cells

As Oct4, Nanog, and several other key regulators of ES cells are predominantly expressed in the ICM, the identification of genes that are preferentially expressed in ICM would be useful to provide a potential list of pluripotency regulators. Yoshikawa et al. [27] have used whole-mount in situ hybridization to identify 48 genes predominantly expressed in ICM. The specific expression patterns suggest their potential roles in regulating ES pluripotency. Zfp143 is one of the 48 genes that show predominant expression in the ICM. In both mouse and human ES cells, Zfp143 is also downregulated upon RA-induced differentiation (supplemental online Fig. 1). To assess the functional role of Zfp143 in ES cells, we depleted endogenous Zfp143 by RNAi. Two short hairpin RNAi constructs targeting different regions of Zfp143 coding sequence were used to ensure that the effects were specific. Both RNAi constructs were effective in reducing the transcript level of Zfp143 compared with empty vector and control luciferase RNAi (Fig. 1B). Strikingly, Zfp143 knockdown cells lost the typical mouse ES colony morphology. Alkaline phosphatase staining of pluripotent ES cells (red color) was reduced dramatically in the Zfp143 knockdown cells, indicative of differentiation (Fig. 1A). RNAi depletion of three other ICM-specific transcripts (Etv5, Mll3, and 4930548G07Rik) [27] did not result in a differentiation phenotype (data not shown). This indicates that not all genes that are preferentially expressed in ICM will be important in the maintenance of ES cells.

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Figure Figure 1.. Zfp143 is required for the maintenance of the undifferentiated state of embryonic stem (ES) cells. (A):Zfp143 knockdown induced ES cell differentiation. Flattened fibroblast-like cells lacking alkaline phosphatase activity were formed when Zfp143 was depleted by RNAi. In empty vector and luciferase short hairpin (shRNA)-transfected cells, normal undifferentiated ES colonies with positive alkaline phosphatase staining (red color staining) were maintained. (B): Quantitative real-time polymerase chain reaction (PCR) analysis of Zfp143 expression after knockdown using two shRNA constructs targeting different regions of the Zfp143 coding sequence. The levels of the transcripts were normalized against control empty vector transfection. (C): Real-time PCR analysis of ES cell-associated gene expression in Zfp143 knockdown ES cells. The levels of the transcripts were normalized against control empty vector transfection. (D): Real-time PCR analysis of lineage-specific marker gene expression in Zfp143 knockdown cells. The levels of the transcripts were normalized against control empty vector transfection. (E): Coexpression of RNAi-resistant Zfp143 could rescue the differentiation phenotype induced by Zfp143 knockdown. RNAi-resistant Zfp143 expression constructs were co-transfected with corresponding Zfp143 RNAi construct into mouse ES cells. Typical colony morphology of ES cells with positive alkaline phosphatase staining (red) was restored. (F): Coexpression of RNAi-resistant Zfp143 rescued the downregulation of Zfp143 upon knockdown by two RNAi constructs. (G): RNAi-resistant Zfp143 rescued the downregulation of Nanog and Esrrb upon Zfp143 knockdown by two RNAi constructs. (H): RNAi-resistant Zfp143 restored the differentiation markers Fgf5 and Cdx2 to the normal ES cell level. Data are presented as the mean ± SEM and are derived from three independent experiments. *, p < .05; **, p < .005. Abbreviations: Luc, luciferase; RNAi, RNA interference.

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To gain insights into the molecular alteration induced by Zfp143 knockdown, the expression of pluripotency and lineage marker genes was analyzed. The expression of Nanog and Esrrb was reduced to 50% and 65%, respectively, relative to the control, whereas the expression of Pou5f1 and Sox2 did not show appreciable changes (Fig. 1C). Primitive ectoderm marker Fgf5 and several trophectoderm markers (Cdx2, Cdh3, and Esx1) were up-regulated (Fig. 1D). The resulting cells after Zfp143 knockdown were likely to be composed of a mixture of different cell types. We repeated the knockdown experiment in another ES cell line (D3 ES cell line) and obtained similar results (supplemental online Fig. 2). To further characterize the Zfp143-depleted ES cells, we analyzed their ability to form colonies in a replating assay. Transfected cells were dissociated with trypsin and replated to allow the ES cells to expand into colonies. Zfp143 knockdown reduced the number of ES cell colony-forming units by approximately 10-fold compared with control knockdown (supplemental online Fig. 3). Importantly, RNAi-immune Zfp143 rescue constructs could significantly restore the colony-forming ability (supplemental online Fig. 3). These results suggest that Zfp143 plays a role in maintaining the self-renewal of ES cells.

To exclude off-target effects of short hairpin RNA (shRNA), we performed rescue experiments. An Zfp143 RNAi-immune construct was made by introducing four silent mutations in the shRNA-targeted region of the Zfp143 open-reading frame. The rescue expression construct was cotransfected with Zfp143 shRNA. The result showed that RNAi-resistant Zfp143 was able to rescue the differentiation phenotype induced by Zfp143 depletion (Fig. 1E). The expression of pluripotency and lineage marker genes was comparable with normal ES cell levels (Fig. 1F–1H). The second RNAi-immnue Zfp143 expression construct was also able to rescue the second shRNA targeting a different region of Zfp143 transcript. All of these data demonstrate that the Zfp143 knockdown phenotype is indeed caused by the Zfp143 RNAi.

Zfp143 Binds to and Regulates Nanog

Next, we investigated how Zfp143 maintains the undifferentiated state of ES cells. As Nanog was down-regulated when Zfp143 was reduced by RNAi, we therefore asked whether Nanog is a direct target of Zfp143. A ChIP assay was performed using a monoclonal antibody raised against Zfp143. The specificity of the antibody was characterized by Western blotting using whole cell lysates transfected with control vector or Zfp143 RNAi constructs (Fig. 2A). Real-time PCR was used to quantify the ChIP-enriched DNA along the Nanog proximal promoter. The result showed that Zfp143 occupied the Nanog proximal promoter (Fig. 2B, 2C) whereas mock glutathione S-transferase (GST) ChIP did not show any significant enrichment in this region (Fig. 2D). In addition, a ChIP assay using HA antibody against ectopically expressed HA-Zfp143 showed the same binding profile at the Nanog proximal promoter in ES cells (Fig. 2E). These data independently confirm that Zfp143 binds to the Nanog proximal promoter in vivo. Interestingly, the profile of Zfp143 binding mirrored that of Oct4 binding at the Nanog proximal promoter (Fig. 2F). Whether Zfp143 and Oct4 co-occupy this region of Nanog or occupy it in a mutually exclusive manner is of interest. To address this issue, we performed a sequential ChIP assay. Chromatin extracts were first immunoprecipitated using the anti-Oct4 antibody. The eluents were then subjected to a second ChIP using the anti-Zfp143 antibody or a control antibody. Further enrichment after the second ChIP indicated that Zfp143 and Oct4 co-occupied the same molecule of DNA (Fig. 2G, OZ). An anti-green fluorescent protein (GFP) antibody used as a control in the second round of ChIP did not show any further enrichment of the Nanog sequence (Fig. 2G, OC). Thus, we concluded that Oct4 and Zfp143 co-occupy the Nanog proximal promoter.

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Figure Figure 2.. Zfp143 and Oct4 co-occupy the Nanog proximal promoter. (A): Specificity of Zfp143 monoclonal antibody. Western blotting analysis of Zfp143 knockdown and control embryonic stem (ES) lysates was performed using anti-Zfp143 monoclonal antibody. β-Actin served as loading control. (B): The locations of the amplified products (black boxes) along the Nanog proximal promoter. (C): Zfp143 binds to the Nanog proximal promoter. A ChIP assay was performed using anti-Zfp143 monoclonal antibody to detect enriched fragments. Fold enrichment is the relative abundance of DNA fragments at the amplified region over a control amplified region. (D): GST antibody was used as mock ChIP control. (E): A 3HA-tagged Zfp143 construct was transiently transfected into ES cells, and chromatin was extracted and subject to ChIP analysis using anti-HA antibody. 3HA-tagged GFP protein served as a mock control. (F): Oct4 binds to the Nanog proximal promoter. A ChIP assay was performed using an anti-Oct4 antibody to detect enriched fragments. (G): Zfp143 and Oct4 co-occupy the Nanog proximal promoter. A sequential ChIP assay was performed using the anti-Oct4 antibody first (O). The eluants were then subjected to a second ChIP assay using an anti-Zfp143 antibody (OZ) or a control antibody (OC). Data are presented as the mean ± SEM and are derived from three independent experiments. *, p < .05; **, p < .005. Abbreviations: bp, base pairs; ChIP, chromatin immunoprecipitation; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin; Luc, luciferase; Mw, molecular weight; RNAi, RNA interference.

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A Zfp143 consensus binding site can be found at the peak region revealed by Zfp143 ChIP (Fig. 2C, 2E). Using an EMSA, we further showed that the DNA-binding domain of Zfp143 could interact with this sequence (Fig. 3A). Furthermore, mutagenesis of the DNA probe revealed that the CCCA sequence, which was reported to be critical for Zfp143 binding [28], was required for this interaction (Fig. 3A). These results showed that Zfp143 directly binds to the Nanog proximal promoter through a conserved binding motif.

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Figure Figure 3.. Zfp143 regulates the Nanog proximal promoter. (A): Zfp143 directly binds to the Nanog proximal promoter region. Electrophoretic mobility shift assays (EMSA) were used to analyze the binding of Zfp143 on the Nanog proximal promoter. The purified recombinant DNA binding domain of Zfp143 was used for EMSAs. An EMSA with the wild-type probe detected a specific Zfp143/DNA complex. The effect of mutation on the Zfp143 DNA-binding domain/DNA complex was also shown. The right panel shows the sequence of the Zfp143 element (shown in red) and corresponding mutation (shown in green) used in this study. (B): The Zfp143 binding site is crucial for the Nanog promoter activity. The Zfp143 binding site was mutated in the Nanog proximal promoter reporter (Mut) and tested for promoter activity in ES cells. (C): Depletion of Zfp143 attenuates Nanog promoter activity. Nanog promoter-Luc reporter or control vector was cotransfected with a Zfp143 RNAi construct into mouse ES cells and the luciferase activities were assayed. All luciferase activities were measured relative to the Renilla luciferase internal control. Data are presented as the mean ± SEM and are derived from three independent experiments. **, p < .005. Abbreviations: Luc, luciferase; Mut, mutated; RNAi, RNA interference; WT, wild type.

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Having established the interaction between Zfp143 and the Nanog proximal promoter, we sought to understand the functional roles of Zfp143 on this promoter. A mutation in the Zfp143 binding motif was introduced into a luciferase reporter construct driven by the Nanog promoter. Reporter assays showed that the mutation reduced Nanog promoter activity (Fig. 3B). To further dissect the functional roles of Zfp143, a Zfp143 RNAi construct was cotransfected with the Nanog proximal promoter driving the luciferase reporter into normal ES cells. Nanog depletion by RNAi was used as a positive control. The depletion of Zfp143 reduced the Nanog proximal promoter activity to the same extent as mutating the Zfp143 motif (Fig. 3C). Taken together, these data demonstrate that Zfp143 directly binds to the Nanog proximal promoter and regulates Nanog expression.

Nanog Is One of the Key Downstream Effectors of Zfp143 for the Maintenance of ES Cells

As Nanog is a direct target regulated by Zfp143, we next investigated whether enforced expression of Nanog will rescue the effects induced by Zfp143 knockdown. To test this hypothesis, Nanog was coexpressed in Zfp143 knockdown ES cells. ES cells cotransfected with the control vector and Zfp143 RNAi construct differentiated, as observed by the loss of ES colony and alkaline phosphatase staining (Fig. 4A). However, ES cells cotransfected with the Nanog expression vector and Zfp143 RNAi construct were able to retain the undifferentiated phenotype of ES cells evidenced by morphology and alkaline phosphatase staining (Fig. 4A, 4B). The depletion of Zfp143 was not affected by the enforced expression of Nanog, excluding the possibility that the rescued phenotype was due to inefficient depletion of Zfp143 (Fig. 4C). We further analyzed the transcripts of pluripotency and lineage markers affected by Zfp143 depletion. With the enforced expression of Nanog in Zfp143 knockdown cells, Fgf5 and Cdx2 were not induced (Fig. 4D). These data suggest that Nanog is one of the key effectors of Zfp143 and can compensate for the depletion of Zfp143 in ES cells.

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Figure Figure 4.. Nanog is a key downstream effector of Zfp143 for maintaining embryonic stem (ES) cells. (A): Enforced expression of Nanog could rescue the differentiation phenotype induced by Zfp143 knockdown. Typical colony morphology of ES cells with positive alkaline phosphatase staining (red) was restored in Zfp143 knockdown cells with enforced expression of Nanog. ES cells were cotransfected with a Nanog expression vector and Zfp143 RNAi construct. The cells were stained for alkaline phosphatase activity, and the morphologies were examined by microscopy. (B): Enforced expression of Nanog rescued the downregulation of Nanog induced by Zfp143 knockdown. (C): Enforced expression of Nanog did not affect Zfp143 knockdown efficiency. (D): Enforced expression of Nanog rescued the upregulation of Fgf5 and Cdx2 induced by Zfp143 knockdown to normal ES cell level. Quantitative real-time polymerase chain reaction was used to determine the expression. Data are presented as the mean ± SEM and derived from three independent experiments. **, p < .005. Abbreviations: Luc, luciferase; RNAi, RNA interference.

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Zfp143 Is a Novel Oct4 Interacting Transcription Factor

Because Oct4 and Zfp143 co-occupy the Nanog proximal promoter and share the same binding pattern, we tested for the potential interaction between the two proteins. Coimmunoprecipitation experiments were performed using ES cell nuclear extracts. Zfp143 was found to coprecipitate with Oct4 (Fig. 5A). The reciprocal coimmunoprecipitation could not be performed as we found that our Zfp143 antibody could not efficiently immunoprecipitate Zfp143 from nuclear extract. Hence, we transfected a construct expressing HA-tagged Zfp143 into ES cells to do the reverse coimmunoprecipitation experiment. Using an anti-HA monoclonal antibody to immunoprecipitate HA-Zfp143, we showed that Oct4 coimmunoprecipitated with HA-Zfp143 (Fig. 5B). Coimmunoprecipitation results obtained in a heterogeneous cell type, 293T cells overexpressing Oct4 and HA-Zfp143, independently confirmed this interaction (Fig. 5C, 5D).

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Figure Figure 5.. Zfp143 is an Oct4 interacting protein. (A): Co-IP using embryonic stem (ES) cell nuclear extracts was performed using anti-Oct4 antibody. Western blotting was performed with anti-Zfp143 antibody. Control IP was performed using an anti-green fluorescent protein (GFP) antibody. The affinity of anti-Oct4 antibody to pull down Oct4 was detected in the lower panel. (B): Reverse co-IP using the ES cell lysates transiently expressing 3HA-tagged Zfp143 was performed using anti-HA antibody. Western blotting was carried out with anti-Oct4 antibody. Control HA IP was performed using ES cell lysates transiently expressing 3HA-tagged GFP. (C): Co-IP using 293T cell lysates overexpressing Oct4 and 3HA-tagged Zfp143 was performed using anti-HA antibody. Western blotting was performed with anti-Oct4 antibody. 293T cell lysates expressing 3HA-tagged GFP and Oct4 served as controls. (D): Reverse co-IP using 293T cell lysates overexpressing Oct4 and 3HA-tagged Zfp143 was performed using an anti-Oct4 antibody to confirm the interaction between Zfp143 and Oct4. Western blotting was performed with anti-HA antibody. 293T cell lysates expressing 3HA-tagged GFP and Oct4 served as controls. (E): Schematic diagram of full-length and truncated forms of Zfp143 protein. (F): A glutathione S-transferase (GST) pulldown assay was performed using GST-tagged Zfp143 proteins and 293T cell lysates overexpressing Oct4. Western blotting was performed with an anti-Oct4 antibody. (G): GST-tagged full length and different truncated forms of Zfp143 proteins. Abbreviations: DBD, DNA-binding domain; FL, full length; GST, glutathione S-transferase; HA, hemagglutinin; IP, immunoprecipitation; Luc, luciferase; Mw, molecular weight.

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To determine the region of Zfp143 that interacts with Oct4, full-length and truncated fragments of Zfp143 were expressed and purified as recombinant GST fusion proteins (Fig. 5E, 5G). These proteins were immobilized onto GSH-Sepharose beads and incubated with extracts harvested from 293T cells overexpressing Oct4. Zfp143 containing only the N-terminal repeats failed to pull down Oct4 (Fig. 5F). However, the fragment containing only the DNA-binding domain of Zfp143 was able to pull down Oct4. This demonstrates that the DNA-binding domain of Zfp143 interacts with Oct4.

Zfp143 Modulates Binding of Oct4 at the Nanog Promoter

We have demonstrated that Zfp143 and Oct4 interact with each other and co-occupy the Nanog proximal promoter to regulate Nanog. However, whether they work independently is not clear. To gain insights into the molecular mechanism of this regulation, we depleted Zfp143 and examined the occupancy of Oct4 at different genomic sites. ES cells transfected with Zfp143 shRNA constructs were cross-linked, and the extracts were used for a ChIP assay. The protein level of Oct4 was not altered by Zfp143 depletion (Fig. 6A). As expected, the binding of Zfp143 on the Nanog proximal promoter was reduced upon depletion of Zfp143 (Fig. 6B). Interestingly, we observed the reduction in Oct4 binding on the same region as well (Fig. 6C) when Sox2 binding was not affected (Fig. 6D). Oct4 occupancy at the Oct4 enhancer, which is not occupied by Zfp143 (data not shown), was, however, not affected (Fig. 6E). To further investigate the relationship between Zfp143 and the basal transcription machinery, we performed ChIP against RNA polymerase II. Consistent with the reduction in the Nanog transcript after Zfp143 depletion, the binding of RNA polymerase II to the Nanog proximal promoter was also significantly reduced (Fig. 6F). These data indicate that Zfp143 controls the binding of Oct4 at the Nanog promoter.

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Figure Figure 6.. The binding of Oct4 to chromatin is dependent on Zfp143. (A):Zfp143 knockdown did not affect the Oct4 protein level. Western blotting analysis of control embryonic stem (ES) lysates and Zfp143 knockdown lysates were performed using anti-Zfp143 monoclonal antibody and anti-Oct4 antibody. β-Actin served as a loading control. (B): Zfp143 binding was reduced upon Zfp143 knockdown. Chromatin extracts from control ES cells or Zfp143 knockdown cells were subjected to ChIP using anti-Zfp143 antibody. (C): Oct4 binding was reduced upon Zfp143 knockdown. Chromatin extracts from control ES cells or Zfp143 knockdown cells were subjected to ChIP using anti-Oct4 antibody. (D): Sox2 binding was not affected upon Zfp143 knockdown. Chromatin extracts from control ES cells or Zfp143 knockdown cells were subjected to ChIP using anti-Sox2 antibody. The primers used to detect ChIP-enriched DNA in (B–D) were the peak pair of primers numbered three in Fig. 2B. (E): Oct4 binding at the Pou5f1 enhancer was not altered upon Zfp143 knockdown. ChIP using chromatin from control ES cells or Zfp143 knockdown cells was performed using anti-Oct4 antibody to detect Oct4 binding at the enhancer region of mouse Pou5f1. (F): RNA polymerase II binding was reduced upon Zfp143 knockdown. Chromatin extracts from control ES cells or Zfp143 knockdown cells were subjected to ChIP using anti-RNA polymerase II antibody. The primers used are shown schematically in the lower panel. Data are presented as the mean ± SEM and are derived from three independent experiments. *, p < .05. Abbreviations: bp, base pairs; ChIP, chromatin immunoprecipitation; Luc, luciferase.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

Our study uncovered the fact that Zfp143 has a novel function in the maintenance of ES cells. Zfp143, also known as STAF, is a zinc finger protein that was first identified as a transcription activator of small nuclear RNA (snRNA) and snRNA-type genes transcribed by RNA polymerase II and III [28, [29]30] with seven-zinc finger repeats for DNA binding in the middle and two transactivation domains at the amino-terminal portion [29]. Zfp143 also plays a key role in basal and tissue-specific expression of transaldolase and regulating the metabolic network controlling cell survival and differentiation [31]. In addition, Zfp143 activates gene expression in response to DNA damage and binds to cisplatin-modified DNA [32]. Zfp143 is also inducible by other DNA-damaging agents such as γ-irradiation, etoposide, and Adriamycin. During mouse early embryogenesis, Zfp143 is expressed predominantly in the ICM [27], which suggests it might be a good candidate for further analysis of its role in preimplantation development and cellular pluripotency.

Differentiation induced by Zfp143 knockdown indicates that it plays a role in the maintenance of ES cells. The depletion of Zfp143 leads to a reduction in the Nanog level, whereas the levels of Pou5f1 and Sox2 are modestly affected. This result suggests that Zfp143 is directly regulating Nanog. Using ChIP assays, we showed that Zfp143 binds to the Nanog proximal promoter and maintains its activity. Enforced expression of Nanog rescued the differentiation induced by Zfp143 depletion, suggesting that Nanog is one of the key effectors of Zfp143. However, we cannot exclude the possibility that there could be other important self-renewal or pluripotency genes directly controlled by Zfp143. Nevertheless, our findings show that Nanog can compensate for the depletion of Zfp143 in ES cells. It is also conceivable that overexpression of Nanog renders the ES cells more resistant to differentiation [26].

The co-occurrence of an octamer and Zfp143 motif (also known as the Staf motif) is often found in the distal sequence element of a large number of RNA polymerase II and III transcribed snRNA-type genes [28]. It has been shown that the trans-activation function of the distal sequence element is mediated essentially by Oct1 and Zfp143 binding at the octamer and Staf motif [28]. Our results here indicate that this octamer/Zfp143 motif regulation model is not restricted to snRNA-type genes only but is also used to control the gene expression of an ES cell-specific gene. Disruption of either motif, Oct4 or Zfp143, as shown by EMSA and luciferase assays, downregulates the transcription of Nanog. It has been shown that addition of an octamer element in the vicinity of a Zfp143 binding site in the Xenopus polymerase II UIb2 and polymerase III U6 genes produced a synergistic effect on transcriptional activation, thus suggesting a functional cooperativity between the two DNA-bound factors. However, the molecular mechanism of how octamer-binding protein and Zfp143 collaborate to control the transcription was not explained. Here, we demonstrate for the first time that Zfp143 interacts with Oct4 through its DNA-binding domain.

Knocking down of Zfp143 significantly reduced Oct4 binding on the Nanog proximal promoter, whereas Oct4 transcription and protein level as well as its binding on other cis-regulatory elements remained unaltered. Our data show that Oct4 binding at the Nanog promoter is dependent on Zfp143. It should be noted that Zfp143 depletion did not lead to a complete loss of Oct4 binding. It is possible that there exists other Zfp143-independent binding of Oct4 at other nearby sites. Although Oct4 and Sox2 heterodimer extensively co-occupies genome-wide targets [8, 33], this study shows that Oct4 can interact with factors other than Sox2 to assist in its binding to chromatin and regulate transcription.

Nanog expression is restricted to pluripotent ES cells and is precisely controlled during mouse embryogenesis. To date, two major cis-regulatory regions have been uncovered (Fig. 7). The first region is an enhancer 5 kilobases upstream of the transcription start site. This site is reported to be bound and positively regulated by STAT3, T (brachyury), the Nanog-Sall4 complex, and Klf transcription factors (Klf2, Klf4, and Klf5) (supplemental online Table 2) [12, 16, 34]. The second important regulatory region is the proximal promoter that is bound and regulated by the Oct4-Sox2 heterodimer and FoxD3 (supplemental online Table 2) [19, 20, 35]. The Oct4-Sox2 complex and FoxD3 directly bind to the Nanog proximal promoter and promote Nanog expression. More recently, a whole genome binding site mapping study revealed that Smad1, Esrrb, and Tcfcp2l1 are also found at the Nanog enhancer and that n-Myc, c-Myc, Zfx, and E2f1 co-occupy the Nanog proximal promoter [36]. Hence, the Nanog cis-regulatory regions are densely bound by a multitude of transcription factors. Such enhancer and promoter architecture may allow for stable and robust expression of Nanog in undifferentiated ES cells. Protein-protein interactions are likely to play a role in stabilizing the transcription factor complexes on chromatin. For example, it is known that Sall4 and Esrrb are found in the Nanog complex [12, 23, 37]. The precise balance between the maintenance of Nanog level and the ability of lineage differentiation requires negative regulators in the transcription network. Several repressors have also been characterized to downregulate Nanog expression. Tcf3, a transcription factor downstream of the Wnt signaling pathway, directly binds to the Nanog promoter and represses its expression. The depletion of Tcf3 delayed ES cell differentiation and upregulated the Nanog protein level [38]. p53, another negative regulator of Nanog, represses Nanog expression after phosphorylation of the Ser315, which is induced upon differentiation [39]. Germ cell nuclear factor [40], an orphan nuclear receptor, mediates Nanog repression upon RA-induced ES cell differentiation by binding to the Nanog promoter and 3′-untranslated region. Other than sequence-specific DNA-binding transcription factors, recent studies have begun to uncover novel pathways involved in the regulation of Nanog. Jmjd2c, a histone demethylase that converts H3K9 trimethylation to dimethylation, has recently been identified to regulate the H3K9Me3 status of Nanog [41]. Specific demethylation of H3K9Me3 by Jmjd2c at the Nanog promoter could inhibit the binding of transcription corepressors such as HP1 and KAP1 and sustain Nanog expression [41]. At the post-transcriptional regulation level, microRNA has been reported to exert functional roles in regulating Nanog expression. For instance, miR-134 was found to specifically attenuate the translation of Nanog [42]. Furthermore, a recent study uncovered a novel mode of regulating Nanog at the post-translational level [43]. Caspase-3 is shown to be involved in cleaving Nanog, and this provides an efficient way to destroy the gene product controlling pluripotency. Altogether, these studies highlight the intricacy in modulating the expression of Nanog through positive and negative regulation at both the transcriptional and post-transcriptional levels.

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Figure Figure 7.. A model depicting the different transcriptional regulators that interact with Nanog cis-regulatory regions. Nanog is regulated by both activators and repressors. The enhancer and promoter regions of Nanog are shown as the blue bar and orange bar, respectively. STAT3, T, Klf transcription factors (Klf2, Klf4, and Klf5), and Nanog-Sall4 complex occupy the Nanog enhancer and activate Nanog transcription. The Oct4-Sox2 complex, FoxD3, and Zfp143-Oct4 complex positively regulate the proximal promoter. Tcf3 and p53 occupy the promoter and exert repressive roles. GCNF binds to two regulatory elements located at 2.5 kb upstream of the transcription start site and 3′-untranslated region to repress Nanog expression upon retinoic acid-induced differentiation. Abbreviations: GCNF, germ cell nerve factor; kb, kilobase.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

We thank Andrew Hutchins, Ching-Aeng Lim, Yuin-Han Loh, Wai-Leong Tam, and Boon-Seng Soh for critical comments on the manuscript. We are grateful to Na-Yu Chia for RNA from H1 ES cells and Katty Kuay for technical assistance. This work was supported by the Agency for Science, Technology and Research (A*STAR) of Singapore and Singapore Stem Cell Consortium grant. X.C. is supported by the Singapore Millennium Foundation scholarship and National University of Singapore graduate scholarship. F.F. is supported by Singapore-MIT alliance scholarship.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References
  10. Supporting Information
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