Inhibition of master transcription factors in pluripotent cells induces early stage differentiation Debojyoti Dea, Myong-Ho Jeonga, Young-Eun Leema, Dmitri I. Svergunb, David E. Wemmerc, Jong-Sun Kanga,1, Kyeong Kyu Kima,1, and Sung-Hou Kimc,1 a

Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea; European Molecular Biology Laboratory, Hamburg Outstation, 22603 Hamburg, Germany; and cDepartment of Chemistry, University of California, Berkeley, CA 94720 b

Contributed by Sung-Hou Kim, December 18, 2013 (sent for review October 10, 2013)

The potential for pluripotent cells to differentiate into diverse specialized cell types has given much hope to the field of regenerative medicine. Nevertheless, the low efficiency of cell commitment has been a major bottleneck in this field. Here we provide a strategy to enhance the efficiency of early differentiation of pluripotent cells. We hypothesized that the initial phase of differentiation can be enhanced if the transcriptional activity of master regulators of stemness is suppressed, blocking the formation of functional transcriptomes. However, an obstacle is the lack of an efficient strategy to block protein–protein interactions. In this work, we take advantage of the biochemical property of seventeen kilodalton protein (Skp), a bacterial molecular chaperone that binds directly to sex determining region Y-box 2 (Sox2). The small angle X-ray scattering analyses provided a low resolution model of the complex and suggested that the transactivation domain of Sox2 is probably wrapped in a cleft on Skp trimer. Upon the transduction of Skp into pluripotent cells, the transcriptional activity of Sox2 was inhibited and the expression of Sox2 and octamer-binding transcription factor 4 was reduced, which resulted in the expression of early differentiation markers and appearance of early neuronal and cardiac progenitors. These results suggest that the initial stage of differentiation can be accelerated by inhibiting master transcription factors of stemness. This strategy can possibly be applied to increase the efficiency of stem cell differentiation into various cell types and also provides a clue to understanding the mechanism of early differentiation.

S

tem cells have enormous potential to differentiate into various specialized cell types and have provided important clues to understand the process of organism development (1). With respect to its therapeutic potential, recent years have seen a vast expansion in this field as it holds much promise for regenerative medicine (2). Based on the ability to generate various cell types, stem cells are broadly classified into pluripotent embryonic stem (ES) cells and multipotent adult stem cells. Despite the enormous prospective of ES cells, a primary hurdle lies in the efficiency of commitment to specific cell types as well as the rejection of transplanted differentiated cells. On the other hand, limited potency and supply of adult stem cells restricts their practical applicability. The generation of induced pluripotent stem cells (iPSCs) of autologous origin has renewed hope for circumventing these issues to some extent (3). To guide the process of cell differentiation in vitro, various approaches based on chemical (4) or genetic alterations (5) have been used. However, the precise molecular targets of these chemical agents are still obscure, which often hinders the optimization of the differentiation protocols. Viral-based genetic alteration of stem cells is also problematic due to safety issues. Moreover, another challenge is the efficiency of commitment into desired cell types. Hence for the therapeutic use of stem cells, nonviral approaches with specific targets must be developed to improve the efficacy, safety, and reliability. Cellular differentiation is a multistep process involving major phases, including early progenitor generation and precursor commitment followed by terminal specification

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and differentiation. Previous investigations have established that stem cells are tightly regulated by the interplay of a few transcription factors (6, 7), which are termed “master stemness regulators.” It has been stated that these transcription factors regulate several hundred genes essential for stemness within the stem cells, and thus they function as fate determinants (8). These factors have certain features in common. They consist of a basic DNA binding domain and transactivation domains (9, 10). These transactivation domains are necessary to interact with several other cofactors (9, 11), both in stem cells and in early progenitor lineages, and cooperate to form a functional transcriptome. The spatiotemporal variability with respect to their presence can regulate the cell fate differentially. It has also been reported that these factors are tightly controlled by feedback circuits that regulate themselves as well as each other (12), and their abundance determines the commitment of each germ layer and possibly tunes the further development process (13, 14). Therefore, it can be hypothesized that a functional inhibition of these factors could result in the termination of stemness and the initiation of differentiation. To pursue this hypothesis, it is required to deter these stemness factors from a functional transcriptome. Whereas enzymes can be inhibited by small inhibitory molecules that block the catalytic site, transcription factors cannot be effectively modulated because their functions are mediated by the protein–protein interaction with large surface area. Although sporadic attempts have been made to inhibit protein–protein interaction via specific antibodies against targets on the cell surface (15, 16), it is still a challenge to Significance Though the potential of stem cells to differentiate into diverse specialized cell types has given much hope to the field of regenerative medicine, low efficiency of commitment is still a major obstacle to practical application. We hypothesized that initial differentiation can be enhanced if the transcriptional activity of core stemness regulators is suppressed. By taking advantage of a sex determining region Y-box 2 (Sox2) interacting protein from heterologous origin, we proved that the inhibition of transcriptional activity of Sox2 resulted in the expression of early differentiation markers and appearance of early neuronal and cardiac progenitors. This strategy can possibly be applied to induce efficient differentiation of stem cells and provide a clue to understanding the mechanism of early differentiation. Author contributions: D.D., Y.-E.L., D.I.S., J.-S.K., K.K.K., and S.-H.K. designed research; D.D., M.-H.J., Y.-E.L., D.I.S., and K.K.K. performed research; K.K.K. and S.-H.K. contributed new reagents/analytic tools; D.D., M.-H.J., Y.-E.L., D.I.S., D.E.W., J.-S.K., K.K.K., and S.-H.K. analyzed data; and D.D., D.I.S., D.E.W., J.-S.K., K.K.K., and S.-H.K. wrote the paper. The authors declare no conflict of interest. 1

To whom correspondence may be addressed. E-mail: [email protected], [email protected], or [email protected].

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1323386111/-/DCSupplemental.

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Results Small Angle X-Ray Scattering Study of Sox2–Skp Complex. To characterize the binding mode of Skp to Sox2, the solution structure of full-length Sox2 in complex with Skp was analyzed by SAXS. The experimental data recorded at different concentrations (Table S1 and Fig. S1) displayed a moderate concentration effect at the smallest angles. Such an effect is typical for systems with slightly attractive interactions between dissolved macromolecules, and the data were extrapolated to infinite dilution following standard procedures. The molecular mass (MM) of the solute (84 ± 10 kDa), determined from the forward scattering, agreed well with the calculated MM of the Sox2:Skp complex at 1:3 molar ratio (81 kDa), suggesting that one Skp trimer binds to one Sox2 molecule. The tight mode of binding was further corroborated by the fact that the data from different concentrations can be superimposed within experimental errors beyond the smallest angles (Fig. S1A). The absence of concentration-dependent changes allows one to confidently rule out the dissociation of complex under tested concentrations, which is consistent with the results of size exclusion chromatography, which depicts coelution of Sox2 and trimeric Skp (Fig. S1B). The experimental radius of gyration Rg extrapolated to zero concentration (35 ± 1 Å, see Fig. S1C) and the maximum size Dmax = 130 ± 10 Å points to an elongated particle shape. These values significantly exceed the parameters (Rg = 31 Å, Dmax = 100 Å) calculated from the high-resolution model of Skp trimer alone (Protein Data Bank, PDB no. 1SG2; ref. 18), and the scattering pattern computed from the model shows considerable deviations from the experimental data, with discrepancy χ = 4.1 (Fig. 1C, blue line). The low-resolution shape of the particle displays a bulkier part, compatible in size with the Skp trimer and a protuberance (presumably due to the Sox moiety) (Fig. 1A). From the exclusion volume of the model (160,000 ± 10,000 Å3) calculated by the ATSAS program package (19), the molecular mass of 80 ± 10 kDa is estimated, which further confirms the stoichiometry of the complex. The high-resolution structure of Skp trimer is docked into the bulkier portion of the ab initio shape in such a way that the C-terminal part faces the protuberance. The exclusion volume appears, however, too small to accommodate the entire Sox2 (317 residues), suggesting that a portion of Sox2 might enter the internal cavity of the cup-like Skp trimer. Indeed, this cavity is known to provide a space to capture substrate proteins, thereby stabilizing substrates and facilitating their folding (20). To further rationalize this observation, molecular modeling of Sox2:Skp complex was performed by the program BUNCH (21), De et al.

Fig. 1. SAXS models of the Sox2:Skp complex in solution. (A) Superposition of the ab initio envelope of Sox2:Skp complex (transparent beads, an average of 20 DAMMIF runs) with the ribbon representation of the crystallographic Skp trimer (red) and the BUNCH model of Sox2 (cyan balls). (B) The model represented in A is rotated 90° counterclockwise along the vertical axis. (C) Comparison of the experimental SAXS data (black dots) with the scattering computed from Skp trimer alone (blue line) and the Skp:Sox2 model (dashed red line).

using the trimeric structure of Skp and a hybrid model of Sox2. The latter contained the high-resolution crystal structure of the high mobility group (HMG) domain (PDB no. 1GT0; ref. 22) flanked by transactivation domains of 38 (N-terminal) and 199 (C-terminal) residues. Given that the DNA binding activity of intact Sox2 was not affected by Skp binding (17), the HMG domain is expected to be freely exposed from the complex. This domain was initially fitted into the protuberance of the ab inito envelope in the starting model. The termini, unstructured in free Sox2, were represented by chains of dummy residues, randomly generated in the initial model and subsequently folded by BUNCH to best fit the experimental data. Multiple runs of the program were performed, and the presented model (Fig. 1 A and B) is compatible with the ab initio shape and yields a very good fit with discrepancy of χ = 1.3 to the experimental data (Fig. 1C, red line). Interestingly, models from repeated reconstructions of the unstructured region of Sox2 using different random generations consistently displayed significant portions of the transactivation domain positioned inside the trimeric cavity of Skp. Moreover, the Rg of the reconstructed Sox2:Skp models were always around 36 ± 2 Å. A random chain of 317 residues (like the full-length Skp) is expected to have an Rg of about 60 Å, and a random chain of 237 residues (like the transactivation domain alone) would have had an Rg of about 51 Å according to the calculation by Kohn et al. (23). The SAXS data therefore indicate that Skp has an essential degree of folding in the complex. Overall, SAXS results lend experimental support to the hypothesis that Sox2 is stabilized by the complex formation with Skp, whereby the unstructured transactivation domain of Sox2 is likely to be located inside the trimeric cavity of Skp. To further corroborate the proposed binding mode of Sox2 to Skp, we performed limited proteolysis of Sox2:Skp complex, assuming that the concealed portion of Sox2 is protected from proteolytic cleavage. By mass spectrometry analyses of the major proteolytic fragments of Sox2, we figured out that the HMG domain is cleaved by chymotrypsin, but most of the C-terminal part remains undigested when Sox2 forms a complex with Skp (Fig. S2). In addition, by the analyses of complex formation of various deletion mutants of Sox2 with Skp, we confirmed that the interaction interface is largely provided from the C-terminal part of Sox2, including linker 2 and transactivation domain 2 (Fig. S3 D–F). The N-terminal parts, including HMG domain, linker 1, and transactivation domain 1, have weak or no interaction with Skp as they failed to coelute with Skp (Fig. S3). These results consistently support that the C-terminal part of Sox2 is largely involved in Skp binding by lurking inside the Skp trimer. Skp Transduction, Visualization, and Its Association with Sox2 in Pluripotent Stem Cells. Several reports suggested immense im-

portance of the transactivation domain of Sox2 in the formation of functional transcriptomes (9–11). The SAXS results with Sox2:Skp complex indicates that Sox2 interacts with Skp via its PNAS | February 4, 2014 | vol. 111 | no. 5 | 1779

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inhibit the interaction between transcription factors and coactivators to tweak their function. Recently, we found that Escherichia coli molecular chaperon seventeen kilodalton protein (Skp) coelutes with sex determining region Y-box 2 (Sox2) during the purification process of recombinant Sox2, and the complex form of Sox2 with Skp still retained its putative DNA binding activity (17). This led us to assume that the transactivation domain of Sox2, not the DNA binding domain mediates the interaction with Skp. Therefore, we hypothesized that at the molecular level, a Skp trimer may shield the majority of the transactivation domain, whereas the DNA binding domain remains exposed. This led us to hypothesize that Skp could block interaction between transactivation domains of Sox2 and its transcriptome, inducing early differentiation by inhibiting the expression of the stemness genes. In this study, we have investigated the binding mode of Skp with Sox2 by small angle X-ray scattering (SAXS) and examined the effect of Skp transduction in mouse pluripotent stem cells. We found that Skp interacts with Sox2 and enhances the efficiency to differentiate into three germ layers during embryoid body formation as well as early neuronal and cardiomyocyte progenitors. Therefore, in this study, we show that stem cell differentiation can be induced by suppressing stemness transcription factor(s), providing insight to the mechanisms regulating early stages of differentiation and offering unique strategies to improve stem cell differentiation.

transactivation domain. Therefore, we hypothesize that the accessibility of Sox2 to other coactivators in the transcriptome can be limited if Skp is available in cellular contexts. In these cases, Skp would restrict the effective formation of the complete transcriptome and possibly compromise the transcriptional activity of Sox2. Skp was shown to bind to Sox2 in vitro (17), so is expected to interact with Sox2 in cells in a similar way. Furthermore, as the expression of core stemness transcription factors is regulated via feedback circuits, we hypothesize that the suppression of Sox2 may also restrict the expression of other stemness transcription factors and their target genes, which could eventually suppress the stem cell phenotype and induce the differentiation. To test our hypothesis, we engineered the Skp protein by inserting a nuclear localization signal and a membrane-penetrating TAT peptide (24) at the N terminus (Fig. 2A). His and HA tags were also added for the purpose of purification and detection (Fig. 2A). The Skp construct was expressed in E. coli (TAT-Skp; Fig. 2B, Left), and the purified protein was FITC labeled for visualization (FITC-TAT-Skp; Fig. 2B, Left). We then transduced TAT-Skp into mouse teratocarcinoma P19 cells, an excellent model of pluripotency, which is maintained by a core circuitry of stemness transcription factors including Sox2 and amenable to differentiate into various lineages upon induction (25, 26). The protein band corresponding to TAT-Skp was only observed in transduced cells (Fig. 2B, Right). TAT-Skp appeared to diffuse throughout the cells including nuclei, not being localized in any particular organelle (Fig. 2C). Next we examined whether the transduced Skp was capable of physically associating with Sox2. After transduction of TAT-Skp, endogenous Sox2 was immunoprecipitated with goat anti-Sox2 antibody. The pull-down product was Western blotted using an anti-His antibody. Skp was shown to coimmunoprecipitate with Sox2 but not with nonspecific goat IgG, indicating physical

Fig. 2. Transduction construct of TAT-Skp and delivery to P19 cells. (A) Schema of TAT-Skp transduction construct containing 6× His tag (HIS), TAT peptide, HA epitope, and nuclear localization signal (NLS). (B, Left) The purity of TAT-Skp was confirmed by Coomassie blue staining and FITC labeling of TAT-Skp was visualized by UV. (Right) Transduction of TAT-Skp into P19 cells was confirmed by Western blotting using anti-His antibody. TATSkp band was detected in transduced P19 cells (+), but not observed in control (−). (C) Cellular localization of Skp probed by confocal microscopy. P19 cells transduced with FITC-TAT-Skp were visualized by UV (green) and by immunostaining with mouse anti-His antibody (red, Alexa 633). Nuclei stained with DAPI are depicted in blue. (Scale bar, 10 μm.) (D) Mouse P19 cells were transduced with His-tagged TAT-Skp. Cell lysates were immunoprecipitated (IP) with anti-Sox2 antibody, and then blotted with anti-His antibody [Western blot (WB)]. IP product was compared with 10% input. Goat IgG was used as a negative control. Black lines denote the splicing points, and the unspliced gel picture is added in Fig. S4.

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Fig. 3. Effect of Skp transduction on stemness transcription factors. (A) P19 cells were transduced with various amounts of TAT-Skp. After 12 h, these cells were transfected with Fbx15 promoter–luciferase reporter construct. The reporter activity was measured after 48 h. The data represent the average from three independent experiments. (B) P19 cells were incubated with various amounts of TAT-Skp for 48 h and immunoblotted using antibodies against Sox2 or Oct4. Band intensities of both Sox2 and Oct4 were normalized to the intensity of β-actin band, and numbers below the band indicate the relative ratio to the untreated control.

interaction between Skp and Sox2 in vivo (Fig. 2D), consistent with the in vitro binding (17). Effect of Skp Transduction on Stemness. The transcriptional activity of Sox2 was measured in a reporter assay where a functional luciferase gene is driven by a minimal promoter of Fbx15 containing juxtaposed Sox2 and octamer-binding transcription factor 4 (Oct4) binding sites (Fbx15-Luc). We observed a gradual decrease in luciferase activity in response to increasing doses of TAT-Skp transduced into P19 cells. With the highest dose, the reporter activity was decreased up to 43% in nontransduced cells (Fig. 3A). This result suggested that Skp binding to the Sox2 transactivation domain inhibits Sox2 function by limiting the access of transcription machinery. The effect of Skp transduction on core stemness transcription factor protein levels was analyzed by checking the expression levels of Sox2 and Oct4 proteins in the TATSkp transduced P19 cells. Because Sox2 and Oct4 factors are also needed in the lineage commitment in the later stages, it is required to suppress these factors only in the initial phase. Therefore, we treated the P19 cells with TAT-Skp for a period of 48 h and subsequently analyzed the effect of Skp on these stemness factors. The level of Oct4 was diminished when the concentration of Skp was higher than 20 μg/mL; Sox2 also appeared to be reduced by Skp (Fig. 3B). These results suggest that Skp directly inhibits the transcription activity of Sox2, which eventually depresses Oct4 via a mutual feedback mechanism. Based on these results, Skp concentration of 35 μg/mL was used in additional differentiation experiments. Effect of Skp Transduction on Cell Differentiation. We further evaluated the effect of Skp transduction on the initial stages of stem cell differentiation. This process was monitored in mouse P19 and ES cells by examining the transcript levels of representative differentiation markers in each germ layer and lineage markers (Fig. 4). Mouse P19 cells were transduced with FITClabeled TAT-Skp and FITC-positive cells were sorted after 48 h of incubation (Fig. S5). Then sorted cells were grown in suspension for 3 d to make them form embryoid bodies and cultured in a monolayer condition for an additional 6 d. From this approach, we confirmed that P19 cells showed enhanced differentiation ability when transduced with TAT-Skp, as determined by the expression of transcripts of following representative marker genes: β-tubulin III as a pan neuronal marker; Gata4 as a mesodermal marker; and Nkx2.5, cTNT, αSMA, Flk1, and Isl-1 as cardiac differentiation markers (Fig. 4A). Similar results were obtained when TAT-Skp was transduced into the suspension culture of P19 cells instead of preincubation and sorting, possibly De et al.

because the transduction efficiency was near 80%, as estimated by the counting of FITC-positive cells (Fig. S5). Therefore, the sorting step was not included for further transduction experiments. To further verify the effect of Skp, mouse ES cells were grown as a monolayer with leukemia inhibitory factor (LIF), and then LIF was depleted to induce differentiation. TAT-Skp was simultaneously added to the LIF-free media for transduction. In Skp-transduced cells, the expression of three germ layer markers was enhanced, indicating the induction of early differentiation. It was interesting that the differential expression of these transcripts was more prominent in the early stages i.e., on day 3 (Fig. 4B), but their expression levels were virtually the same on day 6 of differentiation. Hence the Skp-driven differentiation induction appears to affect only the early stage of commitment (day 3) to a greater extent, possibly because the expression levels of differentiation markers reached saturation in both Skp-treated and control cells in the later stage. Next, the extent of differential lineage commitment in Skptransduced and control cells was visualized and quantitated by pursuing the early neuronal and cardiac progenitor commitments. Neuronal differentiation of P19 cells, cultured for 3 d as embryoid bodies in the presence of TAT-Skp and for an additional De et al.

3 d as a monolayer without TAT-Skp treatment, was analyzed by immunostaining with anti-Pax6 and anti–β-tubulin III antibodies. Pax6 is an early neuronal marker expressed at earlier time points than β-tubulin III. We observed considerable increase in Pax6positive cells subsequent to TAT-Skp transduction either by microscopic observation (Fig. 4C, Upper Right) or quantification (Fig. 4C, Upper Left). This result proves that the transduced cells have started neurogenesis earlier than the control. Consistently, the number of cells that express β-tubulin III with conspicuous neuronal structures was higher in Skp-transduced culture compared with control culture (Fig. 4C, Upper Left). The lineage commitment to neuronal cells was quantified by counting the number of β-tubulin III-positive cells among DAPI-stained cells. Whereas β-tubulin III-positive cells were less than 10% in the control culture, the proportion of β-tubulin III-positive cells was increased to about 60% upon Skp transduction (Fig. 4C, Upper Left). Moreover, the neurites generated in the Skp-transduced cells were much more elongated, with length ranging from 600 to 800 μm, whereas neurites of control cells were 200–300 μm long (Fig. 4C, Lower Left). Because the elongated neurite length is characteristic of more mature neurons, this observation suggests that the Skp-transduced cells maturate earlier than control. To PNAS | February 4, 2014 | vol. 111 | no. 5 | 1781

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Fig. 4. Effect of Skp transduction on differentiation. (A) Mouse P19 cells incubated with FITC-TAT-Skp for 48 h. The FITC-positive cells were grown as embryoid bodies for a span of 3 d, followed by monolayer culture for an additional 6 d without further transduction. The transcript levels of lineage markers were analyzed by semiquantitative RT-PCR from the cells on days 3, 6, and 9. (B) Mouse ES cells were grown in monolayer culture for a span of 6 d after withdrawing LIF. Cells harvested on days 3 and 6 were analyzed for the expression of early markers by semiquantitative RT-PCR. The band intensities in both A and B were normalized with respect to corresponding Gapdh transcript levels and represented graphically. (C) Mouse P19 cells were grown in suspension in the presence (+) or absence (−) of TAT-Skp for a span of 3 d followed by monolayer culture for an additional 3 d without any further Skp treatment. Cells were harvested on day 6 and immunostained using anti-Pax6 and anti–β-tubulin III antibodies (Upper Left). (Scale bar, 50 μm in Pax6 and 100 μm in β-tubulin III.) The number of Pax6 and β-tubulin III positive cells were counted within five randomly selected areas and averaged for quantification of the differentiation (Upper Right). To analyze neurogenesis, transcript levels of two proneural genes Mash1 and Neurogenin1 at day 3 and day 6 were quantitated by quantitative realtime PCR (Lower Left), and the length of the neurites was measured manually (Lower Right). (D) P19 cells prepared as described in Fig. 4C were harvested on day 4 for Gata4 and day 6 for Desmin and MHC to examine their expression by immunostaining (Left). (Scale bar, 100 μm in the pictures of Desmin and Gata4 and 50 μm in MHC picture.) The Inset depicts an enlarged view of the area marked in the red box. The number of Desmin-, Gata4-, and MHC-positive cells were counted for quantification of the cardiac lineage (Right). (E) Mouse embryonic stem cells were treated in the same way as P19 cells. The harvested cells were immunostained to probe the expression of cardiac markers, Desmin, MHC, and Nkx2.5 (Left). (Scale bar, 100 μm.) An enlarged view is inserted in the MHC and Nkx2.5 costaining figures. The numbers of MHC-, Nkx2.5-, and Desmin-positive cells were counted (Right).

further analyze the neurogenesis, we assessed the transcript levels of two proneuronal genes Mash1 and Neurogenin1. In agreement with other data, transcripts of both Mash1 and Neurogenin1 gene were shown to be highly expressed in Skp-transduced cells (Fig. 4C, Lower Right). When we analyzed the cardiac differentiation capacity in Skptransduced cells, results were similar to those for expression of the neuronal marker. The control cells showed low levels of Desmin, Gata4, or MHC immunopositivity, whereas Skp-transduced cultures had a significantly higher number of the Desmin, Gata4, or MHC-positive cells (Fig. 4D). These data are further confirmed by the finding that Skp-tranduced ES cells displayed stronger immunopositivity for cardiac lineage markers, MHC, Nkx2.5, and Desmin (Fig. 4E). In both P19 and mouse ES cells, a larger number of early progenitors were induced at an earlier time point in the case of Skp transduction. This observation unambiguously prompts that Skp increases the efficiency of lineage commitment on a quantitative scale. In another aspect, as seen from the longer length of neurites, a state of more maturated differentiation was also prompted, which reinforces that Skp accelerates the early progenitor commitment. Discussion Here we report a nongenetic approach to specifically modulate a pluripotency by blocking its interaction with other cofactors, and thereby proving that the inhibition of stemness can be beneficial to induce differentiation. Initially, we hypothesized that the initial phases of differentiation can be modulated by suppressing the transcriptional activity of a master transcription regulator such as Sox2. However, the practical obstacle was how to inhibit the protein–protein interaction of these transcription factors. In this study, this problem was overcome by the transduction of a heterologous protein, Skp, which is known to bind Sox2 (17). Our SAXS analysis suggests that the transactivation domain of Sox2 is buried at a gap formed by Skp subunits (Fig. 1), which leads to the hypothesis that Skp can inhibit Sox2 function by blocking the interaction of the transactivation domain of Sox2 in the transcriptome. Transduced Skp was shown to physically associate with Sox2 in mouse P19 cells. It also inhibited the transcription activity of Sox2 as confirmed by decreased reporter activity (Fig. 3A), which is possibly because the bound Skp prevents Sox2 from binding to transcription machinery. Furthermore, the level of Oct4, which is a direct transcription target of Sox2, was also controlled. Our hypothesis was further corroborated by the fact that Skp transduction accelerated the differentiation program of mouse teratocarcinoma P19 and embryonic stem cells (Fig. 4) as proven by increases in the expression of three germ layer markers in the early stage of the differentiation process and the number of early cardiac or neuronal progenitor cells (Fig. 4). These observations suggested that Skp transduction and the following inhibition of stemness transcription factors enhance the efficiency of stem cell differentiation. This enhancing effect was also well demonstrated when the lengths of early neurites produced from Skp-transduced and nontransduced cells were compared (Fig. 4C). Current results can be summarized as a working model of Skp-induced acceleration of early differentiation (Fig. 5). For the sake of simplicity, we broadly categorized the process of differentiation into early embryoid body phase and late attachment phase. The expression levels of differentiation markers or the number of differentiated cells are depicted in the schema with hypothetical trajectories, solid and dotted lines red for Skp-transduced and control cells, respectively. By inhibiting the function of Sox2, and subsequently Oct4 during days 0–3 of differentiation, notable differences were made in the early differentiation phase, manifested by the early expression of representative markers of three germ layers as well as the early lineage markers of cardiac and neuronal types. Thereafter, Skp transduction increased the number of cells expressing early differentiation markers, which is most evident in the number of cells expressing cardiac or neuronal markers at day 6. Consequently, the difference in the 1782 | www.pnas.org/cgi/doi/10.1073/pnas.1323386111

Fig. 5. Schematic model of summarizing the P19 cell differentiation by TATSkp transduction. Each curve is approximated by the expression level of markers representing the early progenitor cells or the number of neuronal and cardiac progenitor cells in the Skp-transduced (solid line) and control (dotted line) P19 cells.

expression level of the selected markers in Skp-treated and control cells was most prominent at 3–6 d, but gradually reduced until day 9 when expression levels of many markers were saturated. However, some markers such as BryT, Gata4, Nkx2.5, and Flk1 still showed differential levels on day 9. The markers chosen in the current study are representative of early phase of differentiation (27, 28), and thus observed differences were somewhat more pronounced at earlier time points. Nonetheless, in principle, it can be said that the modulation of master regulators of stemness can contribute to higher efficiency of differentiation. One of the most important bottlenecks in the field of regenerative medicine is the low efficiency of commitment as well as the complicacy (29). Furthermore, the targets of many chemical agents enhancing the process of commitment remain unclear (30), which hinders the fine tuning of the differentiation process. To overcome these bottlenecks, it is necessary to develop a way to modulate the most important regulator(s) for stemness and differentiation, which will enable us to control the differentiation process more precisely and efficiently. In these aspects, our study provides an example to overcome these problems. We demonstrated that Skp transduction reduced the time requirement for the initiation of cell fate change from stemness to differentiation by suppressing the stemness transcription factors and accelerated differentiation into different germ layers, which is the initial step of most committed differentiation. As a result, our approach can enhance the efficiency of differentiation in the early stage, which could be supplemented with next steps of a specific lineage commitment program. The current approach has advantages in terms of safety, because it is free of dangers posed by genetic integration. We also demonstrated that TAT-Skp can be transduced into pluoripotent stem cells with high efficiency (Fig. S5), suggesting that incorporation of TAT peptide can be used to deliver macromolecules of therapeutic potential. Several successes were reported describing the applicability of TAT to mediate delivery of cargo (31), but, it has not yet been tried to transduce any macromolecule to realize its inhibitory effect on transcription factors and thereby modulate cell fate. Hence we suggest that there are further opportunities to use similar strategies for blocking protein–protein interaction using large molecules. Another possible application of this strategy could be the field of cancer therapy. Recently it has been reported that most advanced grade tumors contain a rare population of cancer stem De et al.

Materials and Methods Sample Preparation. Skp and Sox2 proteins were prepared as described previously with minor modification (17). Details of cloning, proteins purification, and FITC labeling are described in SI Materials and Methods.

samples at solute concentrations 1.0, 1.4, 2.0, 3.8, and 4.8 mg/mL on a Pilatus 1M detector (Dectris). At the sample–detector distance of 2.7 m and wavelength λ = 1.5 Å, the range of momentum transfer 0.01 < s < 0.5 Å–1 was covered (s = 4π sinθ/λ, where 2θ is the scattering angle). No radiation damage effects were detected by comparison of successive 15-s exposures of the solute. Data processing and modeling procedures are described in SI Materials and Methods. Cell Biology Experiments. Cell culture, transduction, reporter assay, immunoprecipitation, Western blotting, fluorescence-activated cell sorting, and immunocytochemistry are described in SI Materials and Methods. Differentiation of Pluripotent Cells. P19 cells and mouse embryonic stem cells were grown as suspension embryoid bodies on the bacterial culture dishes for 3 d in the presence or absence of TAT-Skp at 35 μg/mL concentration. After 3 d, cells were transferred to attachment grade cell culture dishes and grown for an additional 3 d unless mentioned otherwise. To confirm the differentiation pluripotent cells, the transcripts of the differentiation makers were analyzed by semiquantitative or quantitative RT-PCR. For these purposes, mouse ES cells were grown as monolayers for a span of 6 d without leukemia inhibitory factor to induce differentiation. Early differentiation lineages were confirmed by confocal microscopy after immunostaining using antibodies against marker proteins. The details are described in SI Materials and Methods.

Small Angle X-Ray Scattering. SAXS data were collected at the X33 beamline (35) of the EMBL (Deutsches Elektronen Synchrotron) at 4 °C using protein

ACKNOWLEDGMENTS. This work was supported by the Korea Research Foundation (KRF-2008-220-C00040) and the National Research Foundation (NRF-2012M3A9C6049939 and 2011-0028878) of Korea.

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BIOCHEMISTRY

cells, which comprises the tumor initiating properties. It has also been said that these cells show high expression of various stemness genes including Sox2 and Oct4. Hence our strategy could provide a beneficial tool to initiate differentiation of these cells, thereby providing an avenue for alternative cancer therapy. In addition, our results contribute to understanding the mechanism of the early differentiation process and the significance of timely expression or balance of master transcription factors during differentiation. Complete deletion of these factors may not suffice to achieve any fruitful differentiation outcome as these factors not only control stemness but also have multiple roles in differentiation (32, 33). For example, the knock-in null Sox2 allele caused serious defects in neurogenesis in mouse (34). In this regard, it appears that protein transduction is suitable for this purpose, because transduced Skp was shown to transiently suppress stemness transcription factors until it was degraded in cells. Taking these observations together, we believe that our study has opened up unique avenues to reveal the role of transcription factors in differentiation, which may hold the key for future engineering of stem cells.