Inhibition of Master Transcription Factors in Pluripotent Cells Induces Early Stage Differentiation

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

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

Contributed by Sung-Hou Kim, December 18, 2013 (sent for review October 10, 2013) The potential for pluripotent cells to differentiate into diverse spe- and differentiation. Previous investigations have established cialized cell types has given much hope to the field of regenerative that stem cells are tightly regulated by the interplay of a few medicine. Nevertheless, the low efficiency of cell commitment has transcription factors (6, 7), which are termed “master stem- been a major bottleneck in this field. Here we provide a strategy to ness regulators.” It has been stated that these transcription enhance the efficiency of early differentiation of pluripotent cells. factors regulate several hundred genes essential for stemness We hypothesized that the initial phase of differentiation can be within the stem cells, and thus they function as fate determi- enhanced if the transcriptional activity of master regulators of nants (8). These factors have certain features in common. They stemness is suppressed, blocking the formation of functional tran- consist of a basic DNA binding domain and transactivation scriptomes. However, an obstacle is the lack of an efficient strategy domains (9, 10). These transactivation domains are necessary to to block protein–protein interactions. In this work, we take advan- interact with several other cofactors (9, 11), both in stem cells tage of the biochemical property of seventeen kilodalton protein and in early progenitor lineages, and cooperate to form a func- (Skp), a bacterial molecular chaperone that binds directly to sex determining region Y-box 2 (Sox2). The small angle X-ray scattering tional transcriptome. The spatiotemporal variability with respect analyses provided a low resolution model of the complex and to their presence can regulate the cell fate differentially. It has

suggested that the transactivation domain of Sox2 is probably also been reported that these factors are tightly controlled by BIOCHEMISTRY wrapped in a cleft on Skp trimer. Upon the transduction of Skp into feedback circuits that regulate themselves as well as each other pluripotent cells, the transcriptional activity of Sox2 was inhibited (12), and their abundance determines the commitment of each and the expression of Sox2 and octamer-binding transcription fac- germ layer and possibly tunes the further development process tor 4 was reduced, which resulted in the expression of early dif- (13, 14). Therefore, it can be hypothesized that a functional in- ferentiation markers and appearance of early neuronal and cardiac hibition of these factors could result in the termination of progenitors. These results suggest that the initial stage of differ- stemness and the initiation of differentiation. To pursue this entiation can be accelerated by inhibiting master transcription fac- hypothesis, it is required to deter these stemness factors from tors of stemness. This strategy can possibly be applied to increase a functional transcriptome. Whereas enzymes can be inhibited by the efficiency of stem cell differentiation into various cell types small inhibitory molecules that block the catalytic site, tran- and also provides a clue to understanding the mechanism of early scription factors cannot be effectively modulated because their differentiation. functions are mediated by the protein–protein interaction with large surface area. Although sporadic attempts have been made tem cells have enormous potential to differentiate into var- to inhibit protein–protein interaction via specific antibodies Sious specialized cell types and have provided important clues against targets on the cell surface (15, 16), it is still a challenge to to understand the process of organism development (1). With respect to its therapeutic potential, recent years have seen a vast Significance expansion in this field as it holds much promise for regenerative medicine (2). Based on the ability to generate various cell types, Though the potential of stem cells to differentiate into diverse stem cells are broadly classified into pluripotent embryonic stem specialized cell types has given much hope to the field of re- (ES) cells and multipotent adult stem cells. Despite the enormous generative medicine, low efficiency of commitment is still a ma- prospective of ES cells, a primary hurdle lies in the efficiency jor obstacle to practical application. We hypothesized that initial of commitment to specific cell types as well as the rejection of differentiation can be enhanced if the transcriptional activity of transplanted differentiated cells. On the other hand, limited core stemness regulators is suppressed. By taking advantage of potency and supply of adult stem cells restricts their practical a sex determining region Y-box 2 (Sox2) interacting protein from applicability. The generation of induced pluripotent stem cells heterologous origin, we proved that the inhibition of transcrip- (iPSCs) of autologous origin has renewed hope for circumventing tional activity of Sox2 resulted in the expression of early dif- these issues to some extent (3). To guide the process of cell differentiation markers and appearance of early neuronal and ferentiation in vitro, various approaches based on chemical (4) or cardiac progenitors. This strategy can possibly be applied to in- genetic alterations (5) have been used. However, the precise duce efficient differentiation of stem cells and provide a clue to molecular targets of these chemical agents are still obscure, understanding the mechanism of early differentiation. which often hinders the optimization of the differentiation pro- tocols. Viral-based genetic alteration of stem cells is also prob- Author contributions: D.D., Y.-E.L., D.I.S., J.-S.K., K.K.K., and S.-H.K. designed research; D.D., lematic due to safety issues. Moreover, another challenge is the 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. efficiency of commitment into desired cell types. Hence for the analyzed data; and D.D., D.I.S., D.E.W., J.-S.K., K.K.K., and S.-H.K. wrote the paper. therapeutic use of stem cells, nonviral approaches with specific The authors declare no conflict of interest. targets must be developed to improve the efficacy, safety, and 1 To whom correspondence may be addressed. E-mail: [email protected], reliability. Cellular differentiation is a multistep process in- [email protected], or [email protected]. volving major phases, including early progenitor generation This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and precursor commitment followed by terminal specification 1073/pnas.1323386111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1323386111 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 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 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 aver- exposed. This led us to hypothesize that Skp could block interaction age of 20 DAMMIF runs) with the ribbon representation of the crystallo- between transactivation domains of Sox2 and its transcriptome, graphic Skp trimer (red) and the BUNCH model of Sox2 (cyan balls). (B)The inducing early differentiation by inhibiting the expression of the model represented in A is rotated 90° counterclockwise along the vertical stemness genes. In this study, we have investigated the binding axis. (C) Comparison of the experimental SAXS data (black dots) with the mode of Skp with Sox2 by small angle X-ray scattering (SAXS) scattering computed from Skp trimer alone (blue line) and the Skp:Sox2 and examined the effect of Skp transduction in mouse pluripotent model (dashed red line). 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 using the trimeric structure of Skp and a hybrid model of Sox2. progenitors. Therefore, in this study, we show that stem cell dif- The latter contained the high-resolution crystal structure of the ferentiation can be induced by suppressing stemness transcription high mobility group (HMG) domain (PDB no. 1GT0; ref. 22) factor(s), providing insight to the mechanisms regulating early flanked by transactivation domains of 38 (N-terminal) and 199 stages of differentiation and offering unique strategies to improve (C-terminal) residues. Given that the DNA binding activity of stem cell differentiation. intact Sox2 was not affected by Skp binding (17), the HMG domain is expected to be freely exposed from the complex. This Results domain was initially fitted into the protuberance of the ab inito Small Angle X-Ray Scattering Study of Sox2–Skp Complex. To char- envelope in the starting model. The termini, unstructured in free acterize the binding mode of Skp to Sox2, the solution structure Sox2, were represented by chains of dummy residues, randomly of full-length Sox2 in complex with Skp was analyzed by SAXS. generated in the initial model and subsequently folded by The experimental data recorded at different concentrations BUNCH to best fit the experimental data. Multiple runs of the (Table S1 and Fig. S1) displayed a moderate concentration effect program were performed, and the presented model (Fig. 1 A and at the smallest angles. Such an effect is typical for systems with B) is compatible with the ab initio shape and yields a very good slightly attractive interactions between dissolved macromolecules, fit with discrepancy of χ = 1.3 to the experimental data (Fig. 1C, and the data were extrapolated to infinite dilution following red line). Interestingly, models from repeated reconstructions of standard procedures. The molecular mass (MM) of the solute the unstructured region of Sox2 using different random gen- (84 ± 10 kDa), determined from the forward scattering, agreed erations consistently displayed significant portions of the trans- well with the calculated MM of the Sox2:Skp complex at 1:3 activation domain positioned inside the trimeric cavity of Skp. molar ratio (81 kDa), suggesting that one Skp trimer binds to Moreover, the Rg of the reconstructed Sox2:Skp models were one Sox2 molecule. The tight mode of binding was further cor- always around 36 ± 2 Å. A random chain of 317 residues (like roborated by the fact that the data from different concentrations the full-length Skp) is expected to have an Rg of about 60 Å, and can be superimposed within experimental errors beyond the a random chain of 237 residues (like the transactivation domain smallest angles (Fig. S1A). The absence of concentration-depen- alone) would have had an Rg of about 51 Å according to the dent changes allows one to confidently rule out the dissociation calculation by Kohn et al. (23). The SAXS data therefore in- of complex under tested concentrations, which is consistent with dicate that Skp has an essential degree of folding in the complex. the results of size exclusion chromatography, which depicts Overall, SAXS results lend experimental support to the hy- coelution of Sox2 and trimeric Skp (Fig. S1B). The experimental pothesis that Sox2 is stabilized by the complex formation with radius of gyration Rg extrapolated to zero concentration (35 ± Skp, whereby the unstructured transactivation domain of Sox2 is 1Å,seeFig. S1C) and the maximum size Dmax = 130 ± 10 Å points likely to be located inside the trimeric cavity of Skp. to an elongated particle shape. These values significantly exceed To further corroborate the proposed binding mode of Sox2 the parameters (Rg = 31 Å, Dmax = 100 Å) calculated from the to Skp, we performed limited proteolysis of Sox2:Skp complex, high-resolution model of Skp trimer alone (Protein Data Bank, assuming that the concealed portion of Sox2 is protected from PDB no. 1SG2; ref. 18), and the scattering pattern computed proteolytic cleavage. By mass spectrometry analyses of the major from the model shows considerable deviations from the experi- proteolytic fragments of Sox2, we figured out that the HMG mental data, with discrepancy χ = 4.1 (Fig. 1C, blue line). domain is cleaved by chymotrypsin, but most of the C-terminal The low-resolution shape of the particle displays a bulkier part remains undigested when Sox2 forms a complex with Skp part, compatible in size with the Skp trimer and a protuberance (Fig. S2). In addition, by the analyses of complex formation of (presumably due to the Sox moiety) (Fig. 1A). From the exclu- various deletion mutants of Sox2 with Skp, we confirmed that the sion volume of the model (160,000 ± 10,000 Å3) calculated by interaction interface is largely provided from the C-terminal part the ATSAS program package (19), the molecular mass of 80 ± of Sox2, including linker 2 and transactivation domain 2 (Fig. S3 10 kDa is estimated, which further confirms the stoichiometry D–F). The N-terminal parts, including HMG domain, linker 1, of the complex. The high-resolution structure of Skp trimer is and transactivation domain 1, have weak or no interaction with docked into the bulkier portion of the ab initio shape in such Skp as they failed to coelute with Skp (Fig. S3). These results a way that the C-terminal part faces the protuberance. The ex- consistently support that the C-terminal part of Sox2 is largely clusion volume appears, however, too small to accommodate the involved in Skp binding by lurking inside the Skp trimer. entire Sox2 (317 residues), suggesting that a portion of Sox2 might enter the internal cavity of the cup-like Skp trimer. Indeed, this Skp Transduction, Visualization, and Its Association with Sox2 in cavity is known to provide a space to capture substrate proteins, Pluripotent Stem Cells. Several reports suggested immense im- thereby stabilizing substrates and facilitating their folding (20). portance of the transactivation domain of Sox2 in the formation To further rationalize this observation, molecular modeling of of functional transcriptomes (9–11). The SAXS results with Sox2:Skp complex was performed by the program BUNCH (21), Sox2:Skp complex indicates that Sox2 interacts with Skp via its

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1323386111 De et al. Downloaded by guest on September 29, 2021 transactivation domain. Therefore, we hypothesize that the ac- cessibility 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. Further- more, as the expression of core stemness transcription factors is regulated via feedback circuits, we hypothesize that the suppres- sion 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-pene- Fig. 3. Effect of Skp transduction on stemness transcription factors. (A) P19 trating TAT peptide (24) at the N terminus (Fig. 2A). His and cells were transduced with various amounts of TAT-Skp. After 12 h, these HA tags were also added for the purpose of purification and cells were transfected with Fbx15 promoter–luciferase reporter construct. detection (Fig. 2A). The Skp construct was expressed in E. coli The reporter activity was measured after 48 h. The data represent the av- (TAT-Skp; Fig. 2B, Left), and the purified protein was FITC erage from three independent experiments. (B) P19 cells were incubated labeled for visualization (FITC-TAT-Skp; Fig. 2B, Left). We 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 then transduced TAT-Skp into mouse teratocarcinoma P19 cells, β an excellent model of pluripotency, which is maintained by a core normalized to the intensity of -actin band, and numbers below the band indicate the relative ratio to the untreated control. 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 interaction between Skp and Sox2 in vivo (Fig. 2D), consistent observed in transduced cells (Fig. 2B, Right). TAT-Skp appeared with the in vitro binding (17). to diffuse throughout the cells including nuclei, not being lo- calized in any particular organelle (Fig. 2C). Effect of Skp Transduction on Stemness. The transcriptional activity Next we examined whether the transduced Skp was capable of

of Sox2 was measured in a reporter assay where a functional BIOCHEMISTRY physically associating with Sox2. After transduction of TAT-Skp, luciferase gene is driven by a minimal promoter of Fbx15 con- endogenous Sox2 was immunoprecipitated with goat anti-Sox2 taining juxtaposed Sox2 and octamer-binding transcription factor antibody. The pull-down product was Western blotted using an 4 (Oct4) binding sites (Fbx15-Luc). We observed a gradual decrease anti-His antibody. Skp was shown to coimmunoprecipitate with in luciferase activity in response to increasing doses of TAT-Skp Sox2 but not with nonspecific goat IgG, indicating physical 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 TAT- Skp 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 Fig. 2. Transduction construct of TAT-Skp and delivery to P19 cells. (A) P19 and ES cells by examining the transcript levels of repre- Schema of TAT-Skp transduction construct containing 6× His tag (HIS), TAT sentative differentiation markers in each germ layer and lineage peptide, HA epitope, and nuclear localization signal (NLS). (B, Left) The markers (Fig. 4). Mouse P19 cells were transduced with FITC- purity of TAT-Skp was confirmed by Coomassie blue staining and FITC la- labeled TAT-Skp and FITC-positive cells were sorted after 48 h beling of TAT-Skp was visualized by UV. (Right) Transduction of TAT-Skp into of incubation (Fig. S5). Then sorted cells were grown in sus- P19 cells was confirmed by Western blotting using anti-His antibody. TAT- pension for 3 d to make them form embryoid bodies and cultured Skp band was detected in transduced P19 cells (+), but not observed in − in a monolayer condition for an additional 6 d. From this ap- control ( ). (C) Cellular localization of Skp probed by confocal microscopy. proach, we confirmed that P19 cells showed enhanced differen- P19 cells transduced with FITC-TAT-Skp were visualized by UV (green) and by immunostaining with mouse anti-His antibody (red, Alexa 633). Nuclei tiation ability when transduced with TAT-Skp, as determined by μ the expression of transcripts of following representative marker stained with DAPI are depicted in blue. (Scale bar, 10 m.) (D) Mouse P19 β cells were transduced with His-tagged TAT-Skp. Cell lysates were immuno- genes: -tubulin III as a pan neuronal marker; Gata4 as a me- precipitated (IP) with anti-Sox2 antibody, and then blotted with anti-His sodermal marker; and Nkx2.5, cTNT, αSMA, Flk1, and Isl-1 as antibody [Western blot (WB)]. IP product was compared with 10% input. cardiac differentiation markers (Fig. 4A). Similar results were Goat IgG was used as a negative control. Black lines denote the splicing obtained when TAT-Skp was transduced into the suspension points, and the unspliced gel picture is added in Fig. S4. culture of P19 cells instead of preincubation and sorting, possibly

De et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 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 μminβ-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 real- time 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).

because the transduction efficiency was near 80%, as estimated 3 d as a monolayer without TAT-Skp treatment, was analyzed by by the counting of FITC-positive cells (Fig. S5). Therefore, the immunostaining with anti-Pax6 and anti–β-tubulin III antibodies. sorting step was not included for further transduction experiments. Pax6 is an early neuronal marker expressed at earlier time points To further verify the effect of Skp, mouse ES cells were grown than β-tubulin III. We observed considerable increase in Pax6- as a monolayer with leukemia inhibitory factor (LIF), and then positive cells subsequent to TAT-Skp transduction either by LIF was depleted to induce differentiation. TAT-Skp was si- microscopic observation (Fig. 4C, Upper Right) or quantification multaneously added to the LIF-free media for transduction. In (Fig. 4C, Upper Left). This result proves that the transduced cells Skp-transduced cells, the expression of three germ layer markers have started neurogenesis earlier than the control. Consistently, was enhanced, indicating the induction of early differentiation. It the number of cells that express β-tubulin III with conspicuous was interesting that the differential expression of these tran- neuronal structures was higher in Skp-transduced culture com- scripts was more prominent in the early stages i.e., on day 3 (Fig. pared with control culture (Fig. 4C, Upper Left). The lineage 4B), but their expression levels were virtually the same on day commitment to neuronal cells was quantified by counting the 6 of differentiation. Hence the Skp-driven differentiation in- number of β-tubulin III-positive cells among DAPI-stained cells. duction appears to affect only the early stage of commitment Whereas β-tubulin III-positive cells were less than 10% in the (day 3) to a greater extent, possibly because the expression levels control culture, the proportion of β-tubulin III-positive cells was of differentiation markers reached saturation in both Skp-trea- increased to about 60% upon Skp transduction (Fig. 4C, Upper ted and control cells in the later stage. Left). Moreover, the neurites generated in the Skp-transduced Next, the extent of differential lineage commitment in Skp- cells were much more elongated, with length ranging from 600 to transduced and control cells was visualized and quantitated by 800 μm, whereas neurites of control cells were 200–300 μm long pursuing the early neuronal and cardiac progenitor commit- (Fig. 4C, Lower Left). Because the elongated neurite length is ments. Neuronal differentiation of P19 cells, cultured for 3 d as characteristic of more mature neurons, this observation suggests embryoid bodies in the presence of TAT-Skp and for an additional that the Skp-transduced cells maturate earlier than control. To

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1323386111 De et al. Downloaded by guest on September 29, 2021 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 Skp- transduced 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 matu- rated differentiation was also prompted, which reinforces that Skp accelerates the early progenitor commitment. Discussion Fig. 5. Schematic model of summarizing the P19 cell differentiation by TAT- Here we report a nongenetic approach to specifically modulate Skp transduction. Each curve is approximated by the expression level of a pluripotency by blocking its interaction with other cofactors, markers representing the early progenitor cells or the number of neuronal and thereby proving that the inhibition of stemness can be and cardiac progenitor cells in the Skp-transduced (solid line) and control beneficial to induce differentiation. Initially, we hypothesized (dotted line) P19 cells. that the initial phases of differentiation can be modulated by

suppressing the transcriptional activity of a master transcription BIOCHEMISTRY regulator such as Sox2. However, the practical obstacle was how expression level of the selected markers in Skp-treated and to inhibit the protein–protein interaction of these transcription control cells was most prominent at 3–6 d, but gradually reduced factors. In this study, this problem was overcome by the trans- until day 9 when expression levels of many markers were satu- duction of a heterologous protein, Skp, which is known to bind rated. However, some markers such as BryT, Gata4, Nkx2.5, and Sox2 (17). Our SAXS analysis suggests that the transactivation Flk1 still showed differential levels on day 9. The markers chosen domain of Sox2 is buried at a gap formed by Skp subunits (Fig. in the current study are representative of early phase of differ- 1), which leads to the hypothesis that Skp can inhibit Sox2 entiation (27, 28), and thus observed differences were somewhat function by blocking the interaction of the transactivation do- more pronounced at earlier time points. Nonetheless, in princi- main of Sox2 in the transcriptome. Transduced Skp was shown ple, it can be said that the modulation of master regulators of to physically associate with Sox2 in mouse P19 cells. It also stemness can contribute to higher efficiency of differentiation. inhibited the transcription activity of Sox2 as confirmed by de- One of the most important bottlenecks in the field of re- creased reporter activity (Fig. 3A), which is possibly because the generative medicine is the low efficiency of commitment as well bound Skp prevents Sox2 from binding to transcription ma- as the complicacy (29). Furthermore, the targets of many chemical chinery. Furthermore, the level of Oct4, which is a direct tran- agents enhancing the process of commitment remain unclear scription target of Sox2, was also controlled. Our hypothesis was (30), which hinders the fine tuning of the differentiation process. further corroborated by the fact that Skp transduction acceler- To overcome these bottlenecks, it is necessary to develop a way ated the differentiation program of mouse teratocarcinoma P19 to modulate the most important regulator(s) for stemness and and embryonic stem cells (Fig. 4) as proven by increases in the differentiation, which will enable us to control the differentiation expression of three germ layer markers in the early stage of the process more precisely and efficiently. In these aspects, our study differentiation process and the number of early cardiac or neu- provides an example to overcome these problems. We demon- ronal progenitor cells (Fig. 4). These observations suggested that strated that Skp transduction reduced the time requirement for Skp transduction and the following inhibition of stemness tran- the initiation of cell fate change from stemness to differentiation scription factors enhance the efficiency of stem cell differentia- by suppressing the stemness transcription factors and accelerated tion. This enhancing effect was also well demonstrated when the differentiation into different germ layers, which is the initial step lengths of early neurites produced from Skp-transduced and of most committed differentiation. As a result, our approach can nontransduced cells were compared (Fig. 4C). Current results enhance the efficiency of differentiation in the early stage, which can be summarized as a working model of Skp-induced accel- could be supplemented with next steps of a specific lineage eration of early differentiation (Fig. 5). For the sake of sim- commitment program. The current approach has advantages in plicity, we broadly categorized the process of differentiation into terms of safety, because it is free of dangers posed by genetic early embryoid body phase and late attachment phase. The ex- integration. We also demonstrated that TAT-Skp can be trans- pression levels of differentiation markers or the number of dif- duced into pluoripotent stem cells with high efficiency (Fig. S5), ferentiated cells are depicted in the schema with hypothetical suggesting that incorporation of TAT peptide can be used to trajectories, solid and dotted lines red for Skp-transduced and deliver macromolecules of therapeutic potential. Several suc- control cells, respectively. By inhibiting the function of Sox2, and cesses were reported describing the applicability of TAT to subsequently Oct4 during days 0–3 of differentiation, notable mediate delivery of cargo (31), but, it has not yet been tried to differences were made in the early differentiation phase, man- transduce any macromolecule to realize its inhibitory effect on ifested by the early expression of representative markers of three transcription factors and thereby modulate cell fate. Hence we germ layers as well as the early lineage markers of cardiac and suggest that there are further opportunities to use similar strat- neuronal types. Thereafter, Skp transduction increased the egies for blocking protein–protein interaction using large mole- number of cells expressing early differentiation markers, which is cules. Another possible application of this strategy could be the most evident in the number of cells expressing cardiac or neu- field of cancer therapy. Recently it has been reported that most ronal markers at day 6. Consequently, the difference in the advanced grade tumors contain a rare population of cancer stem

De et al. PNAS Early Edition | 5of6 Downloaded by guest on September 29, 2021 cells, which comprises the tumor initiating properties. It has also samples at solute concentrations 1.0, 1.4, 2.0, 3.8, and 4.8 mg/mL on a Pilatus been said that these cells show high expression of various 1M detector (Dectris). At the sample–detector distance of 2.7 m and wave- – stemness genes including Sox2 and Oct4. Hence our strategy length λ = 1.5 Å, the range of momentum transfer 0.01 < s < 0.5 Å 1 was could provide a beneficial tool to initiate differentiation of these covered (s = 4π sinθ/λ, where 2θ is the scattering angle). No radiation damage cells, thereby providing an avenue for alternative cancer therapy. effects were detected by comparison of successive 15-s exposures of the In addition, our results contribute to understanding the mecha- solute. Data processing and modeling procedures are described in SI Mate- nism of the early differentiation process and the significance rials and Methods. of timely expression or balance of master transcription factors during differentiation. Complete deletion of these factors may Cell Biology Experiments. Cell culture, transduction, reporter assay, immu- not suffice to achieve any fruitful differentiation outcome as noprecipitation, Western blotting, fluorescence-activated cell sorting, and these factors not only control stemness but also have multiple immunocytochemistry are described in SI Materials and Methods. roles in differentiation (32, 33). For example, the knock-in null Sox2 allele caused serious defects in neurogenesis in mouse (34). Differentiation of Pluripotent Cells. P19 cells and mouse embryonic stem cells In this regard, it appears that protein transduction is suitable for were grown as suspension embryoid bodies on the bacterial culture dishes for this purpose, because transduced Skp was shown to transiently 3 d in the presence or absence of TAT-Skp at 35 μg/mL concentration. After suppress stemness transcription factors until it was degraded in 3 d, cells were transferred to attachment grade cell culture dishes and grown cells. Taking these observations together, we believe that our for an additional 3 d unless mentioned otherwise. To confirm the differ- study has opened up unique avenues to reveal the role of tran- entiation pluripotent cells, the transcripts of the differentiation makers were scription factors in differentiation, which may hold the key for analyzed by semiquantitative or quantitative RT-PCR. For these purposes, future engineering of stem cells. mouse ES cells were grown as monolayers for a span of 6 d without leukemia inhibitory factor to induce differentiation. Early differentiation lineages Materials and Methods were confirmed by confocal microscopy after immunostaining using anti- Sample Preparation. Skp and Sox2 proteins were prepared as described bodies against marker proteins. The details are described in SI Materials previously with minor modification (17). Details of cloning, proteins purifi- and Methods. cation, and FITC labeling are described in SI Materials and Methods. ACKNOWLEDGMENTS. This work was supported by the Korea Research Small Angle X-Ray Scattering. SAXS data were collected at the X33 beamline Foundation (KRF-2008-220-C00040) and the National Research Foundation (35) of the EMBL (Deutsches Elektronen Synchrotron) at 4 °C using protein (NRF-2012M3A9C6049939 and 2011-0028878) of Korea.

1. Keller G (2005) Embryonic stem cell differentiation: Emergence of a new era in bi- 18. Korndörfer IP, Dommel MK, Skerra A (2004) Structure of the periplasmic chaperone ology and medicine. Genes Dev 19(10):1129–1155. Skp suggests functional similarity with cytosolic chaperones despite differing archi- 2. Riazi AM, Kwon SY, Stanford WL (2009) Stem cell sources for regenerative medicine. tecture. Nat Struct Mol Biol 11(10):1015–1020. Methods Mol Biol 482:55–90. 19. Petoukhov MV, et al. (2012) New developments in the ATSAS program package for 3. Emborg ME, et al. (2013) Induced pluripotent stem cell-derived neural cells survive small-angle scattering data analysis. J Appl Cryst 45:342–350. and mature in the nonhuman primate brain. Cell Rep 3(3):646–650. 20. Walton TA, Sousa MC (2004) Crystal structure of Skp, a prefoldin-like chaperone that 4. Xu T, Zhang M, Laurent T, Xie M, Ding S (2013) Concise review: Chemical approaches protects soluble and membrane proteins from aggregation. Mol Cell 15(3):367–374. for modulating lineage-specific stem cells and progenitors. Stem Cells Transl Med 2(5): 21. Petoukhov MV, Svergun DI (2005) Global rigid body modeling of macromolecular 355–361. complexes against small-angle scattering data. Biophys J 89(2):1237–1250. 5. Giudice A, Trounson A (2008) Genetic modification of human embryonic stem cells for 22. Reményi A, et al. (2003) Crystal structure of a POU/HMG/DNA ternary complex sug- derivation of target cells. Cell Stem Cell 2(5):422–433. gests differential assembly of Oct4 and Sox2 on two enhancers. Genes Dev 17(16): 6. Chambers I, Tomlinson SR (2009) The transcriptional foundation of pluripotency. 2048–2059. Development 136(14):2311–2322. 23. Kohn JE, et al. (2004) Random-coil behavior and the dimensions of chemically un- 7. Kashyap V, et al. (2009) Regulation of stem cell pluripotency and differentiation in- folded proteins. Proc Natl Acad Sci USA 101(34):12491–12496. volves a mutual regulatory circuit of the NANOG, OCT4, and SOX2 pluripotency 24. Schwarze SR, Ho A, Vocero-Akbani A, Dowdy SF (1999) In vivo protein transduction: transcription factors with polycomb repressive complexes and stem cell microRNAs. Delivery of a biologically active protein into the mouse. Science 285(5433):1569–1572. Stem Cells Dev 18(7):1093–1108. 25. Chambers I, Smith A (2004) Self-renewal of teratocarcinoma and embryonic stem 8. Sharov AA, et al. (2008) Identification of Pou5f1, Sox2, and Nanog downstream target cells. Oncogene 23(43):7150–7160. genes with statistical confidence by applying a novel algorithm to time course micro- 26. van der Heyden MA, Defize LH (2003) Twenty one years of P19 cells: What an em- array and genome-wide chromatin immunoprecipitation data. BMC Genomics 9:269. bryonal carcinoma cell line taught us about cardiomyocyte differentiation. Cardiovasc 9. Nowling TK, Johnson LR, Wiebe MS, Rizzino A (2000) Identification of the trans- Res 58(2):292–302. activation domain of the transcription factor Sox-2 and an associated co-activator. 27. Moretti A, et al. (2006) Multipotent embryonic isl1+ progenitor cells lead to cardiac, J Biol Chem 275(6):3810–3818. smooth muscle, and endothelial cell diversification. Cell 127(6):1151–1165. 10. Pan GJ, Pei DQ (2003) Identification of two distinct transactivation domains in the 28. Zahir T, et al. (2009) Neural stem/progenitor cells differentiate in vitro to neurons by pluripotency sustaining factor nanog. Cell Res 13(6):499–502. the combined action of dibutyryl cAMP and interferon-gamma. Stem Cells Dev 18(10): 11. Lim HY, et al. (2009) Implication of human OCT4 transactivation domains for self- 1423–1432. regulatory transcription. Biochem Biophys Res Commun 385(2):148–153. 29. Mummery CL, et al. (2012) Differentiation of human embryonic stem cells and in- 12. Macarthur BD, Ma’ayan A, Lemischka IR (2009) Systems biology of stem cell fate and duced pluripotent stem cells to cardiomyocytes: a methods overview. Circ Res 111(3): cellular reprogramming. Nat Rev Mol Cell Biol 10(10):672–681. 344–358. 13. Niwa H, Miyazaki J, Smith AG (2000) Quantitative expression of Oct-3/4 defines dif- 30. Sachinidis A, et al. (2003) Cardiac specific differentiation of mouse embryonic stem ferentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 24(4):372–376. cells. Cardiovasc Res 58(2):278–291. 14. Wang Z, Oron E, Nelson B, Razis S, Ivanova N (2012) Distinct lineage specification roles 31. Gump JM, Dowdy SF (2007) TAT transduction: The molecular mechanism and thera- for NANOG, OCT4, and SOX2 in human embryonic stem cells. Cell Stem Cell 10(4): peutic prospects. Trends Mol Med 13(10):443–448. 440–454. 32. Hay DC, Sutherland L, Clark J, Burdon T (2004) Oct-4 knockdown induces similar 15. Verma SK, et al. (2009) Antibodies against refolded recombinant envelope protein patterns of endoderm and trophoblast differentiation markers in human and mouse (domain III) of Japanese encephalitis virus inhibit the JEV infection to Porcine Stable embryonic stem cells. Stem Cells 22(2):225–235. Kidney cells. Protein Pept Lett 16(11):1334–1341. 33. Kishi M, et al. (2000) Requirement of Sox2-mediated signaling for differentiation of 16. Guevara Patiño JA, Holder AA, McBride JS, Blackman MJ (1997) Antibodies that in- early Xenopus neuroectoderm. Development 127(4):791–800. hibit malaria merozoite surface protein-1 processing and erythrocyte invasion are 34. Ferri AL, et al. (2004) Sox2 deficiency causes neurodegeneration and impaired neu- blocked by naturally acquired human antibodies. J Exp Med 186(10):1689–1699. rogenesis in the adult mouse brain. Development 131(15):3805–3819. 17. Ha SC, Pereira JH, Jeong JH, Huh JH, Kim SH (2009) Purification of human transcrip- 35. Blanchet CE, et al. (2012) Instrumental setup for high-throughput small- and wide- tion factors Nanog and Sox2, each in complex with Skp, an Escherichia coli periplasmic angle solution scattering at the X33 beamline of EMBL Hamburg. J Appl Cryst 45: chaperone. Protein Expr Purif 67(2):164–168. 489–495.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1323386111 De et al. Downloaded by guest on September 29, 2021