Genome-wide analysis reveals Sall4 to be a major regulator of pluripotency in murine-embryonic stem cells

Abstract

Embryonic stem cells have potential utility in regenerative medicine because of their pluripotent characteristics. Sall4, a zinc-finger transcription factor, is expressed very early in embryonic development with Oct4 and Nanog, two well-characterized pluripotency regulators. Sall4 plays an important role in governing the fate of stem cells through transcriptional regulation of both Oct4 and Nanog. By using chromatin immunoprecipitation coupled to microarray hybridization (ChIP-on-chip), we have mapped global gene targets of Sall4 to further investigate regulatory processes in W4 mouse ES cells. A total of 3,223 genes were identified that were bound by the Sall4 protein on duplicate assays with high confidence, and many of these have major functions in developmental and regulatory pathways. Sall4 bound approximately twice as many annotated genes within promoter regions as Nanog and approximately four times as many as Oct4. Immunoprecipitation revealed a heteromeric protein complex(es) between Sall4, Oct4, and Nanog, consistent with binding site co-occupancies. Decreasing Sall4 expression in W4 ES cells decreases the expression levels of Oct4, Sox2, c-Myc, and Klf4, four proteins capable of reprogramming somatic cells to an induced pluripotent state. Further, Sall4 bound many genes that are regulated in part by chromatin-based epigenetic events mediated by polycomb-repressive complexes and bivalent domains. This suggests that Sall4 plays a diverse role in regulating stem cell pluripotency during early embryonic development through integration of transcriptional and epigenetic controls.

Sall4 is a zinc-finger transcription factor that was originally cloned based on sequence homology to Drosophila spalt (sal) (1–3). In Drosophila, sal is a homeotic gene essential in the development of posterior-head and anterior-tail segments (4). Human SALL4 mutations are associated with the Duane-radial ray syndrome (Okihiro syndrome), a human autosomal-dominant disease involving multiple organ defects (3, 5, 6). Sall4 homozygous knockout mice die at an early embryonic stage (7, 8). Our group and others have recently shown that mouse Sall4 plays an essential role in maintaining the self-renewal and pluripotent properties of ES cells and in governing the fate of the inner-cell mass through transcriptional modulation of Oct4 (also known as Pou5f1) and Nanog (8–10).

ES cells are derived from the inner cell mass of the developing embryo, and ES-cell pluripotency is regulated in part by Oct4, Sox2, and Nanog, as well as through 2 polycomb-repressive complexes (PRCs) (11, 12). Sall4 is expressed by cells of the early embryo, exhibiting an expression pattern similar to Oct4 (8, 9). In recent studies, Sall4 has also been used as part of a gene signature for pluripotency and an enhancer for somatic cell reprogramming (13, 14). However, the complete mechanism whereby Sall4 controls pluripotency and differentiation in ES cells is unknown. The studies reported here demonstrate that Sall4 interacts with core transcription factors, genes in multiple signal transduction pathways, and genes relating to epigenetic processes associated with PRCs as well as bivalent histone methylations. These observations suggest that Sall4 is an essential regulator of cell pluripotency and differentiation.

Results

Sall4 Is a Major Transcriptional Regulator in ES Cells.

A growing body of evidence has shown that Sall4 plays a vital role in maintaining ES cell pluripotency and in governing ES cell-fate decisions (9, 10, 14, 15). This prompted us to investigate the global downstream targets of Sall4 in mouse ES cells. By using a duplicate set of ChIP-on-chip assays, we performed a global analysis of Sall4 binding sites in the mouse ES-cell line W4. This cell line was chosen because it was previously used to generate a conditional Sall4 knockout ES-cell line (9). The majority of transcription factor binding sites in humans are known to occur ≈1–2 kb of the transcription start site (15). Thus, promoter tiling arrays (NimbleGen, build MM8) spanning 2.5 kb of promoter regions (2 kb upstream and 500 bp downstream from the transcription start site) were selected for hybridization to chromatin-immunoprecipitated DNA obtained by using an affinity-purified anti-Sall4 antibody (16).

Successful ChIP assays critically depend on the specificity of the antibody used. Therefore, we rigorously characterized the antibody used in these immunoprecipitation assays. First, western blot analysis was used to compare the Sall4 antibody preparation with a commercially available anti-HA antibody to demonstrate specificity for either WT Sall4 or a Sall4-HA fusion protein. Initially, in mouse fibroblast cells transfected with Sall4-HA, we were able to detect the fusion protein by using an anti-HA antibody, whereas in untransfected fibroblast cells, no Sall4 band was detected [supporting information (SI) Fig. S1A, Lanes 0 and 1]. Although no expression was observed in fibroblasts, experiments in W4 ES cells were able to detect expression of endogenous Sall4 [Lanes 2 and 3 (14)]. The endogenous band observed in ES cells was also successfully absorbed (Lanes 4 and 5).

Next we sought to determine whether our antibody was applicable in ChIP experiments. ChIP-PCR of DNA fragments obtained by using the anti-Sall4 antibody was able to detect enrichment of the peaks identified by the ChIP-on-chip assay. By using heterozygous Sall4 ES cells overexpressing Sall4-HA, immunoprecipitation of the HA-tag identified 88% (23:26) of the genes identified by the anti-Sall4 antibody (Fig. S1B). This suggests that our anti-Sall4 antibody is both sensitive and specific for the Sall4 protein when used in immunoprecipitation. We have also used this antibody for immunohistochemistry to detect Sall4 protein in different tissue samples (Fig. S1C) and for flow cytometry to identify cell populations corresponding to leukemic blasts in patient bone marrow samples that uniquely express Sall4 [Fig. S1D (16)].

Following binding site determination by NimbleGen, the ChIP-on-chip duplicate assays identified roughly 5,200 Sall4-bound genes in array 1, and 4,400 Sall4-bound genes in array 2. The overall false discovery rate was <0.20. Comparison of the data from arrays 1 and 2 showed that 3,223 gene promoters gave positive hybridization signals on both arrays. Of the 1,000 genes exhibiting the most intense hybridization signals on array 2, 947 were also positive on array 1. When only the top 200 genes were considered, the concordance rate was 98.5%. In contrast, when the 800 lowest intensity signals on each array were analyzed, only 37.2% (array 1) and 52.6% (array 2) of the signals were concordant in both assays. Therefore, we selected only the 3,223 genes that were positive on both arrays for further analysis.

We next validated a subset of the putative Sall4 binding sites by using a ChIP PCR strategy. A total of 55 genes were interrogated. Primer pairs were prepared for a randomly selected set of hybridization positive genes with varying degrees of signal intensity. If a selected gene did not initially produce an amplicon level above background, a new primer set was designed 200–300 bases distal to the first primer site, and the quantitative real-time PCR (Q-RT-PCR) assay was repeated. In some cases, a third primer set was used before designating that gene to be a false positive. In addition, ChIP-PCR using primers located adjacent to true positive loci were shown to give negative amplification results, further demonstrating the specificity of Sall4 binding-site identification. Based on the Q-RT-PCR data (52:55 positive), we concluded that ≈94.5% of the 3,223 genes common to both arrays are true positive SALL4 binding sites in the mouse ES cell line (Fig. S2).

The full list of the 3,223 Sall4-bound genes and their respective array hybridization data can be found within the supplemental data (Dataset S1) and on the gene expression omnibus (GEO) as accession number GSE11305. The number of promoter sequences binding Sall4 is quite high, but other transcription factor proteins, such as E2f1, have been reported to bind over 5,000 gene promoters (17). Myc has recently been reported to bind a similar number of genes in mouse ES cells (18).

We then sought to determine the distribution of Sall4 binding sites within the mapped regions of the genome by using DAVID (19). Analysis of over-represented annotations for promoter regions bound by Sall4 revealed significant representation of a broad variety of genes that may be important for stem cell functions (Fig. 1A). These include developmental genes and genes necessary for signal transduction and other regulatory processes. Further classification of the developmental genes revealed over-representation of genes associated with organ development, pattern specification, and brain development (Fig. 1B). Sall4 bound to promoter regions of 11 members of the Hox gene family, and 42 other homeobox or homeobox-like genes (Table S1). The binding of Sall4 to promoter regions of vital developmental genes and others that govern ES cell fate support the phenotypic consequence of Sall4 reduction in ES cells. This also suggests that Sall4 plays a vital role in ES cells that may be similar to Oct4 and Nanog. This hypothesis is supported by 3 lines of evidence: (a) Sall4 is expressed very early in the developing embryo and is subsequently down-regulated in most differentiated tissues, (b) both over- and under-expression of Sall4 cause ES-cell differentiation, demonstrating the necessity for tight regulation of Sall4 expression, and (c) the finding that Sall4 modulates expression of both Oct4 and Nanog (9, 10). However, the magnitude of the Sall4 transcriptional network is quite striking and suggests that Sall4 may play a central role in embryonic development.

Fig. 1.

Sall4 is a major regulator in mouse ES cells. (A) Sall4 bound to promoters that over-represent a broad classification of GO annotations. These included various potential regulatory and developmental annotations. Analysis was done with DAVID, and the x axis represents the gene number. (B) Further classification of developmentally important genes over-represented in the Sall4 binding pool. For the organ development annotation, the over-representation was insignificant (P < 0.074) but notable. The x axis represents the gene number. P values are inset following each bar and were calculated by using Fisher's Exact Test based on over-representation in comparison to the genome. (C) Sall4 binds promoter regions belonging to a variety of pathways that have definitive roles in development, suggesting that Sall4 may control a wide variety of developmental processes. Listed genes are only representative of the Sall4-bound genes in each pathway. P values for this analysis are not presented because of the low number of genes within each pathway. Classification was done by using Ingenuity Pathway Analysis (www.ingenuity.com).

Sall4 Targets Important Signals That Control ES-Cell Differentiation and Lineage Specification.

Numerous signaling pathways play important roles in maintaining pluripotency during embryogenesis. For example, the Wnt signaling pathway has important roles in embryogenesis and cancer (20–23). TGF-β signaling is necessary to maintain ES cell pluripotency, and PTEN signaling plays important roles in the maintenance of hematopoetic stem cell self-renewal. Fig. 1C shows the number of genes bound by Sall4 within several developmentally important pathways and examples of the genes bound within each pathway. This suggests that Sall4 may play a broad role in regulation of ES-cell pluripotency through interactions with key signaling pathways.

Magnitude of the Sall4 Transcriptional Network in ES Cells.

Recently, ChIP-on-chip studies have been performed on the gatekeeper genes, Oct4 and Nanog. This enabled us to compare genes bound by Oct4 and Nanog with those bound by Sall4. Interestingly, ChIP-on-chip assays showed that Oct4 had 783 promoter binding sites, whereas Nanog had 1,284 binding sites within the mouse genome (18). The ChIP-on-chip data presented here revealed that Sall4 bound ≈3,200 gene promoters. Given the similar expression patterns of the transcription factors Sall4, Oct4, and Nanog, this is remarkable. These observations suggest that Sall4 may play a similar, but broader role in regulating ES-cell properties. However, the roles of each in vivo are not completely understood.

Interaction and Co-Occupation of Sall4 with Oct4 and Nanog in ES Cells.

Wu et al. elegantly demonstrated that Sall4 and Nanog form a regulatory complex in ES cells (10). Liang et al. (24) recently showed that Sall4 forms a complex (or complexes) with both Oct4 and Nanog by using mass spectrometry and immunoprecipitation of endogenous proteins. We have confirmed these observations by immunoprecipitation experiments using ES cells transiently transfected with Sall4-HA. Western blotting detected an overexpression of Sall4-HA protein by both anti-HA (Fig. 2A) and anti-Sall4 antibodies (data not shown). Immunoprecipitation with an anti-HA antibody produced a unique endogenous 45-kDa protein, Oct4, in the precipitate. By contrast, an IgG-negative control failed to generate the Oct4 band in the same extract, indicating a specific Sall4–Oct4 interaction. By using the same method, the Sall4–Nanog interaction was confirmed in the same anti-HA-pulldown precipitate (Fig. 2A).

Fig. 2.

Coimmunoprecipitation and co-occupancy of Sall4, Oct4, and Nanog. (A) Transient transfection of W4 ES cells with a Sall4-HA construct exhibited protein expression detected by both anti-HA and anti-Sall4 antibodies (the latter data not shown) in the cell extract (left). Oct4 and Nanog are detected by using respective antibodies in the whole ES cell extract (input). Immunoprecipitation of Sall4-HA with an anti-HA antibody revealed both Oct4 and Nanog bands, whereas immunoprecipitation with an IgG antibody detected neither protein. (B) Venn diagram showing the overlapping target genes of Sall4, Oct4, and Nanog as determined by ChIP-Chip and ChIP-PET experiments. These complexes may function in the regulation of stem cell pluripotency.

Because these transcription factors physically interact, one would expect them to colocalize to some of the same gene promoters (25). A gene bound by any two of these proteins we will refer to as “co-occupied”. However, Oct4 and Sall4 co-occupied only 92 common genes representing 12% of genes bound by Oct4. Similarly, Sall4 binding was identified at 198 Nanog target genes, representing only 15% of Nanog's bound genes (Fig. 2B). This suggests that Sall4–Oct4 and Sall4–Nanog interactions may form functional complexes only at select promoter regions (9, 10). There are only 45 genes that are co-occupied by Oct4, Sall4, and Nanog (Table S2). However, this group includes developmentally important genes, such as Dkk1, Msx2, Fbxl10, and Epc1.

Sall4^+/− ES Cells Exhibit Decreased Expression of iPS Genes.

Recent studies have shown that ectopic expression of Oct4, Sox2, Klf4, and c-Myc is capable of reprogramming somatic cells to confer a pluripotent state, termed induced pluripotent stem (iPS) cells (26). It has previously been demonstrated that Sall4 binds to Oct4 and regulates its expression (9). We show here that the Sall4 protein binds to the promoter regions of Oct4, c-Myc, Sox2, and Klf4 through ChIP-PCR (Fig. 3A).

Fig. 3.

Decreased expression of iPS genes in Sall4^+/− ES cells. Ectopic expression of 4 key transcription factors, Oct4, Sox2, c-Myc, and Klf4, produces iPS cells. (A) Sall4 binds to promoter regions of Oct4, Sox2, c-Myc, and Klf4 as shown by ChIP-PCR. (B) Following adenovirus induced removal of 1 Sall4 allele, expression of all 4 transcription factors is decreased as measured by Q-RT-PCR. The Sall4/Gapdh ratio in control cells was set at 1. The values are the mean of triplicate reactions, and the bars indicate SD.

To determine the relationship between binding and Sall4 function, we used Q-RT-PCR to measure mRNA levels from Sall4^+/− ES cells. Expression levels of all 4 transcription factors decreased in Sall4^+/− ES cells indicating that Sall4 plays an activating role on these genes (Fig. 3B). Because Sall4 is not expressed in the majority of differentiated tissues including fibroblasts, this suggests that exogenous expression of Sall4 may play a role in reprogramming somatic cells to confer a pluripotent state. This hypothesis has recently been supported by others (27).

Sall4 Binds to Genes Associated with H3K27 Methylation Domains as well as to Target Genes of PRC1 and PRC2.

Numerous studies have implicated epigenetic modifications as a means for regulating stem cell pluripotency (28–30). We have previously shown that Bmi-1, a polycomb group member, is a downstream target of SALL4 (31), thus, we focused on other polycomb-associated genes for analysis. Although various covalent modifications can influence chromatin remodeling, here we investigate the combined roles of Sall4, methylation of histone 3 on lysine 27 (H3K27), and PRCs. PRCs are key modulators of stem-cell pluripotency and consist of 2 distinct groups (11, 12). PRC1 consists of >10 proteins, including Bmi1, Rnf2, PhcI, and the HPC proteins, whereas PRC2 contains Ezh2, Eed, Suz12, and RbAp46:48 (32). Representative ChIP-on-chip assays have been performed for PRC1 genes Rnf2 and Phc1, and PRC2 group members Suz12 and Eed (11). PRCs maintain ES-cell pluripotency by facilitating H3K27 methylation, a modification that represses gene expression (11). The majority of H3K27 trimethylation and PRC binding sites are ≈1 kb of the transcription start site, and both are frequently present on gene promoters. However, not all H3K27 methylated domains are associated with PRCs. Thus, Sall4 may bind and potentially regulate expression of a subset of genes associated with H3K27 methylated domains. Binding of Sall4 to Genes Associated with Histone Methylations (GAHMs) occurred at 17% (422:2557) of previously identified H3K27 methylation domains within 1 kb of annotated-transcription start sites of mouse ES cells. We expect PRCs to be associated with some of the Sall4-bound GAHMs. Sall4, PRC1, and PRC2 co-occupied 160 GAHMs (Fig. 4A). There were also GAHMs co-occupied by Sall4 and one of the polycomb proteins, with 29 and 69 bound by PRC1 and PRC2, respectively. Interestingly, Sall4 bound 164 GAHMs that were not occupied by either PRC1 or PRC2. To determine the function of this subset of genes, we categorized them based on overrepresentation by using DAVID. As expected, GAHM-H3K27 and PRCs had extremely significant roles in development (P < 0.001). Surprisingly, GAHM-H3K27 and Sall4 also had notable roles in development (P < 0.07; Fig. 4B). This reveals a system in which regulation of GAHM-H3K27 may be controlled by dynamic involvement of both Sall4 and PRCs.

Fig. 4.

The role of Sall4 in H3K27 methylation regulation (A) Sall4 binds to 422 GAHM that are methylated at H3K27. Some of the GAHMs are also bound by PRC1 (Rnf2, Phc1) and PRC2 (Suz12, Eed). One hundred sixty-four of these genes are associated with Sall4, PRC1, and PRC2 (inner orange circle) with 69 PRC2-bound genes and 29 PRC1-bound genes also bound by Sall4 (outer orange circle). However, Sall4 binds 164 of GAHM-H3K27 that do not bind the polycomb group proteins (outer gray circle). (B) Two hundred fifty-eight genes are bound by one or more polycomb group protein(s) and Sall4. Of these, 81 have developmental functions that display significantly over-represented (P < 0.001) binding to genes associated with various developmental processes (orange). Binding of Sall4 to GAHM-H3K27, but neither PRC1 nor PRC2 occurs for 164 genes, with 23 genes having developmental processes that are notable but not statistically significant (P < 0.07; white). This suggests that two or more mechanisms may interact to regulate cell fate through histone methylation H3K27. The x axis represents the gene number. These GO annotations are not mutually exclusive, and P values were determined by using Fisher's Exact Test.

Many Sall4 Targets Harbor Bivalent Domains.

It has been reported that dual epigenetic markers, coined “bivalent domains”, consisting of methylations at H3K27 and at histone 3 on lysine 4 (H3K4), exist for a large set of developmental genes within Highly Conserved Noncoding Elements (HCNEs) (33). ES-cell pluripotency is hypothesized to be maintained, in part, through a balance of H3K4 gene activation and H3K27 gene repression at these bivalent domains (33). To explore the role that Sall4 may play in this epigenetic mechanism, Sall4 binding sites were compared with bivalent domains identified within HCNEs. We found that Sall4 co-occupied 27% (37:135) of Genes Associated with Bivalent Histone Methylations (GABHMs), including 11 Hox gene family members (Fig. 5, Table S3). In contrast, Oct4 and Nanog each bind only 12% of GABHMs (Fig. S3). Surprisingly, there are no genes that are bound by Sall4, Oct4, and Nanog. Only 11 of the GABHMs are co-occupied by any 2 proteins, suggesting that Sall4, Oct4, and Nanog may play independent roles in methylation regulation. Further, these 3 transcription factors account for binding to only 39% of identified bivalent domains. It remains to be determined what other genes emerge as further epigenetic regulators.

Fig. 5.

Sall4 target genes are associated with bivalent domains. Venn diagram displaying the GAHMs bound by Sall4 within HCNEs. Notably, the majority of Sall4-bound genes within characterized HCNEs are marked by bivalent histone methylation domains including a cluster of homeobox genes (see Table S3).

Discussion

We have shown that Sall4 binds ≈3,200 genes within their promoter regions in mouse ES cells. An analogous ChIP-chip assay preformed by using chromatin-precipitated DNA obtained by using Oct4 and Nanog antibodies revealed 783 and 1,284 bound genes, respectively. Given the similar gene expression patterns of Sall4 and Oct4, the magnitude of Sall4 binding is remarkable. Although extensive functional studies need to be done on Sall4, Oct4, and Nanog, it appears that the role of Sall4 may be significant in determining stem cell fate.

Sall4, Oct4, and Nanog have been shown to form heteromeric protein complexes that may regulate ES-cell gene expression in complex ways. Transient combinatorial binding of Sall4, Oct4, and Nanog may determine cell fate with different combinations of these proteins controlling different aspects of pluripotency. Although trimeric protein complexes may exist, there are relatively few genes bound by all 3 transcription factors. The Sall4–Oct4 complex binds genes that have statistically significant roles in some developmental processes associated with stem cell activities (P < 0.05). In contrast, although Sall4–Nanog complexes bind genes that have similar developmental and transcriptional functions, this dimeric protein combination also binds genes important for organ development and pattern specification at statistically significant frequencies (P < 0.05; Fig. S4 and Table S4). This data suggests that the binding of these 3 transcription factors at select promoter regions may dynamically control transcription required for the stem cell state, although it is likely that many other proteins also play important roles.

Another interesting observation is that down-regulation of Sall4 also causes down-regulation of Oct4, Sox2, Klf4, and c-Myc, 4 genes that induce reprogramming of somatic cells to induced pluripotent stem cells. This suggests a mechanism by which Sall4 could be a key regulator for the reprogramming process. This interpretation was recently supported by Wong et al., who used cell fusion to demonstrate that Sall4 can enhance somatic cell reprogramming (27). Nevertheless, the importance of Sall4 in somatic cell reprogramming and the role it plays in regulating this gene quartet remain to be determined.

Interestingly, recent work by Dr. Austin Smith's group has suggested that inhibiting a cell's intrinsic signaling pathways is sufficient to prevent differentiation (23). These pathways include those that proceed through mitogen-activated protein kinases (ERK1/2) and glycogen synthase kinases (GSK3). Work from our lab and from others suggests that Sall4 may interact with Stat3, and preliminary data indicate that Stat3 is an upstream regulator of Sall4. This would implicate Sall4 in this complex regulatory loop. How Sall4 interacts with other microenvironmental signals is unknown at this time.

Important questions remain to be answered regarding evidence for an Oct4–Sall4–Nanog complex and the regulatory role that it may play in ES cell pluripotency maintenance. Further, evidence presented here indicates that Sall4 may play an important role in regulating chromatin remodeling. This connects the independent processes of transcriptional regulation and epigenetic regulation and may provide insights into an integrated control process involved in determining stem cell fate.

Materials and Methods

Cell Culture.

Embryonic stem cells from the W4 mouse cell line (Gene Targeting Core Facility, University of Iowa) were cultured with irradiated mouse embryonic fibroblast feeders or under feeder-free conditions as described previously (9). For W4 clone EA231, Sall4^+/− ES cells were cultured with the antibiotic G418 at a concentration of 125 μg/ml.

ChIP-on-chip Assays.

A complete ChIP-on-chip assay protocol was provided by NimbleGen Systems, Inc. In brief, W4 ES cells were cross-linked with formaldehyde and lysed, and then the DNA was sheared by sonication. A sonication regime consisting of 8 pulses lasting 20 seconds each were used with 90 seconds in-between spent on ice. The Misonix Sonicator 3000 was used at power level 3.5 for the sonication procedure. Following immunoprecipitation with an affinity-purified anti-Sall4 antibody (16), ChIP-purified DNA was blunt-ended, ligated to linkers, and subjected to low-cycle PCR amplification. Resultant ChIP-DNA was then hybridized to duplicate promoter tiling arrays (RefSeq arrays, build MM8) each containing 19,457 promoter annotations produced by NimbleGen. Design of the mouse promoter array is a single array containing 2.5 kb of each RefSeq promoter region. The promoter region is covered by 50–75 mer probes at roughly 100-bp spacing dependent on the sequence composition of the region. The arrays were hybridized and the data extracted according to NimbleGen standard procedures. Data extraction was done by using NimbleScan, which searches for 4 or more probes above a specified cutoff value ranging from 90–15% using a 500-bp sliding window. The cutoff value is a percentage of the hypothetical maximum determined by using the mean plus 6 standard deviations and is decreased in 1% increments from 90–15%. The data are then randomized 20 times to evaluate the possibility of false positives, and each peak is assigned a false discovery rate based on this randomization. Confirmation of the predicted binding sites was performed by using ChIP-PCR analysis of the amplicons applied to the arrays (Fig. S1A). Negative control primers were designed adjacent to Sall4-bound peaks (Fig. S2B).

Coimmunoprecipitation and Western Blotting.

For Oct4–Sall4 and Nanog–Sall4 interactions, plasmid pcDNA3/Sall4-HA was transfected into W4 ES cells to express the Sall4-HA protein by using Lipofectamine 2000 reagent (Invitrogen). Coimmunoprecipitations were performed following the Catch and Release v2.0 High Throughput Immunoprecipitation Assay Kit (Upstate) as recommended. For western blots, the membrane was incubated with Oct-3:4 (H-134), Nanog (M-149) (both from Santa Cruz Biotechnology, Inc), or Sall4 antibodies at a 1:300 dilution at 4 °C overnight. Detection was done by using SuperSignal West Pico solutions (Pierce).