Identification of a Small Molecule That Turns ON the Pluripotency

Letters pubs.acs.org/acschemicalbiology

Identification of a Small Molecule That Turns ON the Pluripotency Gene Circuitry in Human Fibroblasts Ganesh N. Pandian,†,∇ Shinsuke Sato,†,∇ Chandran Anandhakumar,‡ Junichi Taniguchi,‡ Kazuhiro Takashima,‡ Junetha Syed,‡ Le Han,‡,§ Abhijit Saha, Toshikazu Bando, Hiroki Nagase,∥,# and Hiroshi Sugiyama*,†,‡ †

Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Sakyo, Kyoto 606-8502, Japan Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo, Kyoto 606-8501, Japan § Shanghai Key Laboratory of New Drug Design, State Key Laboratory of Bioreactor Engineering, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China ∥ Division of Cancer Genetics, Department of Advanced Medical Science, Nihon University School of Medicine, Tokyo 173-8610, Japan # Division of Cancer Genetics, Chiba Cancer Center, Research Institute, 666-2 Nitona-cho, Chuo-ku, Chiba-shi, Chiba 260-8717, Japan ‡

S Supporting Information *

ABSTRACT: A nontransgenic approach to reprogram mouse somatic cells into induced pluripotent stem cells using only small molecules got achieved to propose a potential clinicalfriendly cellular reprogramming strategy. Consequently, the screening and identification of small molecules capable of inducing pluripotency genes in human cells are increasingly a focus of research. Because cellular reprogramming is multifactorial in nature, there is a need for versatile small molecules capable of modulating the complicated gene networks associated with pluripotency. We have developed a targeting small molecule called SAHA-PIP comprising the histone deacetylase inhibitor SAHA and the sequence-specific DNA binding pyrrole-imidazole polyamides for modulating distinct gene networks. Here, we report the identification of a SAHA-PIP termed I ̀ that could trigger genome-wide epigenetic reprogramming and turn ON the typically conserved core pluripotency gene network. Through independent lines of evidence, we report for the first time a synthetic small molecule inducer that target and activate the OCT-3/4 regulated pluripotency genes in human dermal fibroblasts.

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factors in terms of both sequence selectivity and bioactivity at various levels. Previously, we showed targeted hyperacetylation in the promoter region of the p16 tumor suppressor gene by using a novel small molecule called SAHA-PIP.9 SAHA-PIPs contain both the epigenetically active SAHA and the hairpin pyrroleimidazole polyamides (PIPs), which are capable of differentially binding predesigned DNA sequences according to the binding rule. Because chromatin remodeling gets associated with pluripotency, we screened a library of 16 SAHA-PIPs (A to P) that could target a unique six-base-pair sequence and identified the SAHA-PIPs that could differentially activate pluripotency genes in mouse embryonic fibroblasts (MEFs).10 Subsequently, we synthesized an advanced SAHA-PIP library (Q to Φ) with improved recognition of GC rich sequences by

rtificial induction of pluripotency in somatic cells through transcriptional reprogramming has revolutionized the conventional viewpoint that the fate of specialized cells is unchangeable.1 Sequential and/or parallel modifications in chromatin-modifying enzymes such as histone deacetylases (HDACs) can turn transcriptional machinery ON and OFF at the right time and place to regulate cell fate.2,3 The epigenome is inherently flexible and can be modulated through pharmacological interventions. 4 Chromatin-modifying HDAC1 regulates pluripotency and lineage-specific transcriptional networks.5 Small molecule inhibitors such as sodium butyrate, valproic acid and suberoylanilide hydroxamic acid (SAHA) exhibit potent inhibitory activity against HDAC1mediated somatic cell reprogramming by inducing changes in global gene expression profiles and epigenetic states.6−8 However, these effectors artificially alter the epigenome in a sequence-independent manner. Hence, there is a demand for small molecules that are able to mimic natural transcription © XXXX American Chemical Society

Received: February 14, 2014 Accepted: October 24, 2014

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dx.doi.org/10.1021/cb500724t | ACS Chem. Biol. XXXX, XXX, XXX−XXX

ACS Chemical Biology

Letters

Figure 1. Genome-wide gene analysis demonstrates the remarkable ability of I to switch ON the core pluripotency gene circuitry in human dermal fibroblasts. (a) Chemical structures of SAHA-PIP I and the control K. Microarray scatter plots showing the global gene expression profile of HDFs treated with (b) I, (c) SAHA, and (d) K. The horizontal axis and vertical axis represents the expression profile of HDFs treated with the effectors and iPSCs, respectively. 5-fold induction is kept as the cutoff for remarkable effect. Red color indicates core pluripotency genes. I altered 33 core pluripotency genes. On the other hand, SAHA and K altered 3 and 1 core pluripotency genes, respectively. (e) A dendrogram generated based on the unsupervised hierarchical clustering analysis of the representative core pluripotency genes induced in biological triplicate samples of SAHA (SAHA-1, -2, and -3) and SAHA-PIP I (I-1, -2, and -3) treated fibroblasts. Biological replicates of 201B7-iPS- cells are provided as a control. Nonfunctional SAHA-PIP I lacking SAHA moiety (Pre-I) was used as the negative control to verify the importance of the SAHA moiety.

placing imidazole at different positions in the top arm of the compounds. A SAHA-PIP (δ) induced multiple pluripotency genes and initiated cellular reprogramming in MEFs by altering the global gene expression profile.11 Screening studies to analyze the effects of the 32 SAHA-PIP variants (A to Φ or 1 to 32) on genome-wide gene expression in human cells showed that individual SAHA-PIPs triggered transcriptional activation of different gene networks.12 In the above-mentioned proof-ofconcept study, SAHA-PIPs dramatically induced the expression of silent developmental genes, which suggests the possibility of

rewiring of the transcriptional machinery by varying the pyrrole-imidazole content in the top or bottom arms of SAHA-PIP. The epigenome of each cell type is unique, and in accordance with our prediction, δ did not activate pluripotency genes in human fibroblasts. Given the potential applications of a small molecule that can activate pluripotency gene in human cells, we carried out screening studies. We describe here a specific SAHA-PIP that can remarkably turn ON core pluripotency gene circuitry in human fibroblasts. B



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Figure 2. SAHA-PIP I specifically induces OCT-3/4 pathway genes by triggering transcriptionally permissive chromatin. (a) Analysis of upstream regulators in I-treated HDFs revealed the activation of critical pluripotency genes belonging to the OCT-3/4 pathway (results derived from the summary of data obtained from biological triplicates). QRT-PCR analysis to evaluate the endogenous expression of (b) OCT-3/4, (c) SOX2, (d) NANOG, (e) REX1, (f) CDH1, (g) LIN28, (h) SALL4, (i) EPCAM, and (j) DPPA4 in the effector (1 μM) treated HDFs. The dark blue bar represents the expression profile of the hit SAHA-PIP I, while the light blue bar represents that of the SAHA-PIP K. Orange and red bars represent the expression profile of nonfunctional SAHA-PIP I possessing a methyl ester in the SAHA moiety (Pre-I) and SAHA that were employed at the same concentration as the other SAHA-PIPs. The gray bar represents the expression profile of 0.1% DMSO treated cells, which is used as an internal standard that corresponds to 1-fold. Each bar represents the mean ± SD from 24 wells (8 biological replicates). The statistical significance was determined by the t test and p < 0.05 is considered to be significant.

First, we compared the global gene expression profile (≥2fold up/down-regulated) in individual SAHA-PIP-treated cells to that of 253G1-iPS cells (Figure 1 of the Supporting Information). All data were obtained using a SurePrint G3 Human GE v2 8 × 60K Microarray (Agilent Technologies,

U.S.A.). Scatter plot analysis showed that among the 32 SAHAPIPs, a specific SAHA-PIP called I notably up-regulated many genes found in 253G1-iPS cells (Figure 1b of the Supporting Information, Panel I). Ingenuity pathway analysis (IPA) revealed that I significantly (P < 0.002) induced a set of C



Letters

ingly, I did not activate miR-367, a key microRNA previously shown to activate Oct-3/4 expression (Figure 1e, MIR-367). A compound called Pre-I lacking a functional SAHA moiety was synthesized to verify the effect of PIP (Figure 4 of Supporting Information). A heat map of the microarray data generated from Pre-I treated HDFs showed that simple binding of PIP is insufficient to induce pluripotency gene expression and has a different expression profile. No notable induction of any pluripotency genes was observed (Figure 1e, Pre-I). The six-base-pair sequence recognizing I activate an overwhelming number of pluripotency genes, and hence, identifying a specific mechanism is not straightforward. Analysis of the upstream effectors suggests that I specifically targets OCT-3/4 and activates a set of critical pluripotency genes and epithelial markers (Figure 2a). To confirm this result, we carried out qRT-PCR studies after isolating RNA from HDFs treated for 48 h with the purified effectors (SAHA, K and I) at a final concentration of 1 μM, as previously described.12 Pre-I was used as a control to evaluate the role of SAHA in pluripotency gene activation. Consistent with the microarray data, I significantly (P = 0.008) induced OCT-3/4 by approximately 20-fold (Figure 2b, dark blue bar) when compared to SAHA (Figure 2b, red bar). Likewise, I alone significantly (P < 0.05) induced the pluripotency genes SOX2, NANOG, REX1, LIN28, CDH1, SALL4, and EPCAM (Figure 2c−i, dark blue bars) Pre-I could not induce these pluripotency genes, which confirms the need for a functional SAHA moiety for bioactivity (Figure 2b−i, orange bars). SAHA alone did not induce the endogenous expression of standard pluripotency genes (Figure 2b−i, red bars), which suggests an essential role for PIPs. Because the germ cell-specific SAHA-PIP K did not upregulate any of these genes, recognition of PIP is likely to play a major role in targeted transcriptional activation. A similar pattern was observed with DPPA4 and I alone induced the endogenous expression of this key pluripotency gene (Figure 2j). Neither I nor any other effectors induced KLF4 and c-MYC, which belong to a separate regulatory network (Figure 5 of the Supporting Information). Dose-dependence studies validated 1 μM as the effective concentration of I, but no significant effect on the induction pattern of OCT-3/4, SOX2, and NANOG and KLF4 was observed by increasing the concentration (Figure 6 of the Supporting Information). At 1 μM, I, K, and Pre-I displayed no cytotoxic effects at 48 h, so the gene expression profile was not influenced by cytotoxicity12 (Figure 7 of the Supporting Information). The microarray analysis and RT-PCR results suggests OCT-3/4 as the probable target of I. The binding affinity of I toward the human OCT-3/4 promoter sequence was evaluated using a surface plasmon resonance assay as described in Methods.18 Based on our previous report on germ cell gene switching,13 K and the human PIWIL2 promoter sequence were chosen as the control SAHA-PIP and region, respectively. Consistent with the gene expression profile, I showed higher binding affinity toward its corresponding OCT-3/4 promoter specific sequence than the mismatch PIWIL2 sequence (Figure 8b and c of the Supporting Information). In contrast, K had a higher binding affinity toward PIWIL2 and not OCT-3/4 (Figure 8d and e of the Supporting Information). The rates of association (Ka) and dissociation (Kd) and the dissociation constants (KD) shown in Table 4 of Supporting Information substantiate the differential binding affinities of I and K. Consistent with the in vitro binding association studies, chromatin immunoprecipitation (ChIP) PCR studies of the human OCT-3/4 promoter

genes belonging to the OCT4-mediated embryonic stem cell pluripotency pathway (Figure 2a of the Supporting Information). No pluripotency-related pathways were observed in SAHA-treated HDFs (Figure 2b of the Supporting Information). Additionally, I alone and not SAHA, significantly induced several genes associated with cellular development, embryonic development, cellular morphology, and cell function and maintenance with remarkable P-scores (>35) (Table 1 of the Supporting Information). To validate the results, we compared the global gene expression profiles of I-treated HDFs with the profiles of K-treated HDFs. It is important to note that K has a similar chemical architecture to I but different sequence recognition specificity (Figure 1a). The purified SAHA-PIPs (Figure 3 of the Supporting Information) were used at a final concentration of 1 μM for 48 h, as described previously.12 Scatter plot analysis of altered genes (≥5-fold up/downregulated (vs DMSO)) in I-treated HDFs versus 253G1-iPS cells revealed that I but not the controls (K and SAHA) could remarkably induce multiple iPS genes, including 33 core pluripotency genes (Figure 1b−d) and (Table 2 of the Supporting Information). Recently. K was shown to activate a germ-cell-specific PIWI pathway comprised of genes such as PIWIL2.13 Because many iPS genes were activated in I-treated HDFs, we assumed that, like PIWI-pathway-targeting K, I may be targeting the OCT4-regulated core pluripotency gene network comprised of 345 intertwined genes.14 The gene expression profile of I shared approximately 90% of its ncRNAs with pluripotent stem cell lines. The noncoding RNAs (ncRNAs) representing the dark matter of the genome is known to be expressed in a development-specific manner.1 Interestingly, most ncRNAs expressed in pluripotent stem cell lines were also activated by I but not by the SAHA- and Ktreated HDFs (Table 3 of the Supporting Information). To confirm the remarkable ability of SAHA-PIP I to distinctively activate pluripotency genes, we carried out microarray analysis using a Human Gene 2.1 ST Array Strip (Affymetrix, USA). A heat map generated by the gene expression data (biological triplicates) obtained from SAHAand I-treated HDFs and the data (biological duplicates) obtained from 201B7-iPS-cells confirmed that I induces key pluripotency genes in HDFs and generates an expression profile that is comparable to an established pluripotent cell line (Figure 1e). In particular, the core pluripotency marker genes including POU5F1, SOX2, NANOG, CDH1, SALL4, EPCAM, ZFP42, and DPPA4 were notably induced in biological triplicate samples of I-treated HDFs (Figure 1e, I-1, I-2, and I-3). SAHA-treated HDFs did not show these effects, which confirms that in just 48 h I could induce a large number of major pluripotency genes that are typically expressed in the later stages of reprogramming. The human embryonic stem-cell−specific cell-cycle−regulating microRNAs (miRNAs) including the miR-302 and miR-372 families have been shown to promote reprogramming of human fibroblasts into iPS cells by facilitating mesenchymal-epithelialtransition (MET) phases.15,16 Anokye-Danso et al. showed that the enforced expression of miR302/367 clusters could rapidly reprogram human somatic cells to iPS cells.17 Synthetic strategies to develop small molecules capable of specifically activating essential cell fate regulating microRNAs are in increasing demand. Surprisingly, I but not SAHA notably induced the miR-302 family (Figure 1e, MIR-302A-D), which suggests the possibility of developing SAHA-PIPs as tools to selectively activate cell fate regulating microRNAs. InterestD



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Figure 3. Hyper acetylation in the promoter and transcribed regions of OCT-3/4-regulated pluripotency genes in SAHA-PIP I treated HDFs. Chromatin immunoprecipitation analysis were carried out after immunoprecipitation with H3K9ac and H3K14ac antibody. The amount of promoter sequence of (a) OCT-3/4, (b) SOX2, (c) NANOG and sequences corresponding to enhancer region in (d) SOX2 in the coprecipitated DNAs was determined by quantitative PCR. Enrichment fold in acetylation is calculated by normalizing the data against input DNA and by normalizing the enrichment with IgG antibody. Each bar represents the mean ± SD from 12 wells. ChIP-Seq analysis of (e) OCT-3/4, (f) NANOG, (g) LIN28B, (h) SALL4, (i) SOX2, and (j) ACTB regions was performed with H3K14ac antibody as described in the Methods given in the Supporting Information. The blue line indicates the statistical significance of the obtained MACS peaks.

sequence suggested that neither SAHA nor K but only I resulted in enrichment in the acetylation level of both H3K9 and H3K14 (Figure 3a). The pattern observed in the model oligonucleotide duplex can be extrapolated to the corresponding sequence within the chromatin.

Similarly, with I but not the controls a notable increase in the enrichment of acetylation was observed in the promoter region of SOX2, NANOG and the enhancer region of SOX2 containing OCT-NANOG binding sites (Figure 3b−d). Therefore, I alone triggers the conferral of epigenetic marks to transcriptionally permissive chromatin and induces core pluripotency genes in E



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Table 1. Differentially Expressed Genes in the Top Ten Pathways in I-Treated HDFs pathway role of Oct4 in mammalian embryonic stem cell pluripotency transcriptional regulatory network in embryonic stem cells embryonic stem cell differentiation into cardiac lineages agranulocyte adhesion and diapedesis Sertoli cell−Sertoli cell junction signaling epithelial adherens junction signaling leukocyte extravasation signaling signaling by Rho family GTPases factors promoting cardiogenesis in vertebrates Wnt/β-catenin signaling

P-value 2.57 4.68 4.79 6.92 7.94 8.91 1.35 1.38 1.55 1.95

× × × × × × × × × ×

genes

10−08 10−04 10−04 10−03 10−03 10−03 10−02 10−02 10−02 10−02

SOX2↑, NANOG↑, SPP1↑, JARID2↑, IGF2BP1↑, SALL4↑, POU5F1↑ SOX2↑, NANOG↑, ZIC3↑, POU5F1↑ SOX2↑, NANOG↑, POU5F1↑ EZR↑, PODXL↑, CLDN7↑, CLDN6↑, ACTA1↑ F11R↑, CDH1↑, SORBS1↑, CLDN7↑, CLDN6↑, ACTA1↑, TUBB2B↑ CDH2↑, CDH1↑, SORBS1↑, ACVR2B↑, ACVR1C↓, ACTA1↑, TUBB2B↑ F11R↑, EZR↑, MMP13↓, CLDN7↑, CLDN6↑, PRKCZ↑, ACTA1↑ RND2↑, CDH1↑, SEPT3↑, CDH3↑, EZR↑, GNA14↑, PRKCZ↑, ACTA1↑ CER1↑, CDC6↑, ACVR2B↑, ACVR1C↓, PRKCZ↑ SOX2↑, CDH1↑, ACVR2B↑, ACVR1C↓, POU5F1↑

and barriers in the reprogramming of human fibroblasts into pluripotent stem cells.25 Histone modifications orchestrated by a hypothetical histone code is believed to be the key to the embryonic development.26 Site-specific chromatin modifiers could potentially overcome epigenetic memory, which is a major bottleneck that is hampering the clinical translation of cellular reprogramming strategies.27 The development of multitarget small molecules such as SAHA-PIP may concomitantly modulate developmental genes and precisely modulate cell fate while using fewer effectors. Despite similar chemical architecture, K and I can specifically upregulate the typically conserved germ cell and pluripotency genes, respectively, and open up the possibility of de- and/or trans- differentiating human somatic cells. Because SAHA-PIP has the potential to induce sequence-specific pluripotent stem cell lines, it is beyond the scope of this report to study its ability to enhance the efficacy of current reprogramming protocols, which follow a completely different mechanism. Because none of the chemicals employed in this study enhanced the efficacy of I to match the expression profile of pluripotent stem cell lines, an alternative strategy such as using gene-suppressing PIP conjugates may be required. Interestingly, nerve cells that differentiated from partially reprogrammed epithelial-like cells display superior gliogenic differentiation than those grown from completely reprogrammed iPSCs, which suggests that complete reprogramming is not always essential for efficient neuroregeneration.28 Because I-treated cells partially acquire pluripotent properties, it may have potential for use in such studies. PIPs can bind to methylated sequences and have demonstrated higher sequence specificity than the natural binding proteins.29 Chromatin remodeling is not a secluded phenomenon and factors such as cell permeability, accessibility and stochastic epigenetic modifications should be considered when developing SAHA-PIP. The first ever OCT-3/4-activating small molecule identified in this screening study may be further developed to coax somatic cells into pluripotent stem cells and may also facilitate the logical design of SAHA-PIP. Functional analysis suggests the presence of a six-base-pair binding site recognized by SAHA-PIP in critical pluripotency gene OCT-3/4. Thus, it is now possible to construct SAHA-PIPs that specifically target OCT-3/4 and/or another pluripotency genes by expanding the recognition sequence of I through systematically altering the pyrrole/imidazole content. Such a complex feat can be achieved by developing PIPs capable of recognizing larger nucleotide sequences (≥16 bp).30 The modification of SAHA into SAHAPIP alters its specificity toward different HDAC enzymes.31 Therefore, different bioactive effectors can be conjugated with PIPs for additional versatile applications.32,33

the OCT-3/4 pathway. ChIP-Seq analysis further confirmed that I induces hyperacetylation in the transcribed regions of OCT-3/4, NANOG, LIN28B, SALL4, and SOX2, and a relatively higher H3K14Ac occupancy was observed in Itreated cells than in DMSO-treated cells (Figure 3e−i). Conversely, the ubiquitously expressed ACTBL2 and GAPDH did not show any effect (Figure 9b of Supporting Information and Figure 3j). In accordance with the gene expression profile, KLF4 showed a relatively lower acetylation profile (Figure 9a of Supporting Information). The phase in which mesenchymal cells transit to epithelial cells (MET) is a bottleneck during the reprogramming of the somatic genome.19 Cell-to-cell interactions that occur during the transition to the epithelial cells can establish and maintain pluripotency. As shown in the pathway analysis, I significantly induced several marker genes associated with adhesion and tight junction signaling (Table 1) and notably induced the MET-promoting miR-302 (Figure 1e). QRT-PCR analysis validated the microarray data and I alone significantly (P < 0.05) induced the MET markers (Figure 2f and i). It is reasonable to assume that I may initiate cellular reprogramming by shifting HDFs from a somatic to an epithelial state. However, I has relatively lower induction values in the pluripotent stem cell lines, which could be attributed to HDFs that are elite or stochastically resistant to reprogramming.20 We also evaluated the effect of I on the morphology of HDFs by treating them over the timeline as shown in Figure 10a of the Supporting Information. We used a chemical cocktail (CC) comprised of the inhibitors of differentiation signals: TGF-β and GSK-β,21 pifithrin-α (p53 inhibitor), and PS-48 (protein kinase activator).22 After 2 weeks, little or no cells tested positive for alkaline phosphatase (AP), which is one of the essential markers associated with cellular reprogramming (Figure 10b of the Supporting Information). However, after 3 weeks I-treated cells and I+CC-treated cells were AP+ (Figure 10c of the Supporting Information). Although CC increased the number of AP+ cells with an induction efficiency of 0.06% ± 0.03%, CC-alone-treated cells were AP−. However, prolonged incubation of I showed only a marginal increase in the induction of pluripotency gene expression (Figure 11 of the Supporting Information). Recently, cellular reprogramming of mouse embryonic fibroblasts into iPS cells was achieved using only a cocktail of seven small molecules.23 However, the requirement of several small molecules and the time taken to achieve completely reprogrammed cell lines are a major concern. Chemically induced human pluripotent stem cells are yet to be achieved and at least one factor, such as OCT-3/4, is still required.24 Chromatin-modifying enzymes may turn as both facilitators F



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ASSOCIATED CONTENT

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(2) Barrero, M. J., Boué, S., and Izpisúa Belmonte, J. C. (2010) Epigenetic mechanisms that regulate cell identity. Cell Stem Cell 7, 565−570. (3) Iizuka, M., and Smith, M. M. (2003) Functional consequences of histone modifications. Curr. Opin. Genet. Dev. 13, 154−160. (4) Pandian, G. N., and Sugiyama, H. (2012) Strategies to modulate heritable epigenetic defects in cellular machinery: Lessons from nature. Pharmaceuticals 6, 1−24. (5) Kidder, B. L., and Palmer, S. (2012) HDAC1 regulates pluripotency and lineage specific transcriptional networks in embryonic and trophoblast stem cells. Nucleic Acids Res. 40, 2925− 2939. (6) Hochedlinger, K., and Plath, K. (2009) Epigenetic reprogramming and induced pluripotency. Development 136, 509−523. (7) Huangfu, D., Osafune, K., Maehr, R., Guo, W., Eijkelenboom, A., Chen, S., Muhlestein, W., and Melton, D. A. (2008) Induction of pluripotent stem cells from primary human fibroblasts with only Oct4 and Sox2. Nat. Biotechnol. 26, 1269−1275. (8) Mali, P., Chou, B. K., Yen, J., Ye, Z., Zou, J., Dowey, S., Brodsky, R. A., Ohm, J. E., Yu, W., Baylin, S. B., Yusa, K., Bradley, A., Meyers, D. J., Mukherjee, C., Cole, P. A., and Cheng, L. (2010) Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotencyassociated genes. Stem Cells 28, 713−720. (9) Ohtsuki, A., Kimura, M. T., Minoshima, M., Suzuki, T., Ikeda, M., Bando, T., Nagase, H., Shinohara, K., and Sugiyama, H. (2009) Synthesis and properties of PI polyamide−SAHA conjugate. Tetrahedron Lett. 50, 7288−7292. (10) Pandian, G. N., Shinohara, K., Ohtsuki, A., Nakano, Y., Masafumi, M., Bando, T., Nagase, H., Yamada, Y., Watanabe, A., Terada, N., Sato, S., Morinaga, H., and Sugiyama, H. (2011) Synthetic small molecules for epigenetic activation of pluripotency genes in mouse embryonic fibroblasts. ChemBioChem 12, 2822−2828. (11) Pandian, G. N., Nakano, Y., Sato, S., Morinaga, H., Bando, T., Nagase, H., and Sugiyama, H. (2012) A synthetic small molecule for rapid induction of multiple pluripotency genes in mouse embryonic fibroblasts. Sci. Rep. 2, 544. (12) Pandian, G. N., Taniguchi, J., Junetha, S., Sato, S., Han, L., Saha, A., AnandhaKumar, C., Bando, T., Nagase, H., Vaijayanthi, T., Taylor, R. D., and Sugiyama, H. (2014) Distinct DNA-based epigenetic switches trigger transcriptional activation of silent genes in human dermal fibroblasts. Sci. Rep. 4, 3843. (13) Han, L., Pandian, G. N., Junetha, S., Sato, S., Anandhakumar, C., Taniguchi, J., Saha, A., Bando, T., Nagase, H., and Sugiyama, H. (2013) A synthetic small molecule for targeted transcriptional activation of germ cell genes in a human somatic cell. Angew. Chem., Int. Ed. 52, 13410−13413. (14) Loh, Y. H., Wu, Q., Chew, J. L., Vega, V. B., Zhang, W., Chen, X., Bourque, G., George, J., Leong, B., Liu, J., Wong, K. Y., Sung, K. W., Lee, C. W., Zhao, X. D., Chiu, K. P., Lipovich, L., Kuznetsov, V. A., Robson, P., Stanton, L. W., Wei, C. L., Ruan, Y., Lim, B., and Ng, H. H. (2006) The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat. Genet. 38, 431−440. (15) Subramanyam, D., Lamouille, S., Judson, R. L., Liu, J. Y., Bucay, N., Derynck, R., and Blelloch, R. (2011) Multiple targets of miR-302 and miR-372 promote reprogramming of human fibroblasts to induced pluripotent stem cells. Nat. Biotechnol. 29, 443−448. (16) Lee, M. R., Prasain, N., Chae, H. D., Kim, Y. J., Mantel, C., Yoder, M. C., and Broxmeyer, H. E. (2013) Epigenetic regulation of NANOG by miR-302 cluster-MBD2 completes induced pluripotent stem cell reprogramming. Stem Cells 31, 666−681. (17) Anokye-Danso, F., Trivedi, C. M., Juhr, D., Gupta, M., Cui, Z., Tian, Y., Zhang, Y., Yang, W., Gruber, P. J., Epstein, J. A., and Morrisey, E. E. (2011) Highly efficient miRNA-mediated reprogramming of mouse and human somatic cells to pluripotency. Cell Stem Cell 8, 376−388. (18) Morinaga, H., Bando, T., Takagaki, T., Yamamoto, M., Hashiya, K., and Sugiyama, H. (2012) Cysteine cyclic pyrrole-imidazole

Synthesis and Purification of SAHA-PIP Conjugates. The synthesis and characterization of 4-(8-methoxy-8-oxooctanamido) benzoic acid and its subsequent conversion to SAHA-PIP conjugates were performed on a PSSM-8 peptide synthesizer (Shimadzu, Kyoto) as previously described.12 The purities of the all of the SAHA-PIP conjugates were checked by HPLC (elution with trifluoroacetic acid and a 0−100% acetonitrile linear gradient (0−40 min) at a flow rate of 1.0 mL min−1 under 254 nm). The 1H NMR of SAHA-PIP I ̀ is as follows, (600 MHz, [D6] DMSO): δ = 10.38 (s, 1H), 10.32 (s, 1H), 10.26 (s, 1H), 10.06 (s, 1H), 9.93 (s, 1H), 9.92 (s, 1H), 9.90 (s, 2H), 9.84 (s, 1H), 9.41 (s, 1H), 9.25 (brs, 1H), 8.36 (brt, 1H), 8.15 (brt, 1H), 8.09 (brt, 1H), 8.01 (brt, 1H), 7.76 (d, J = 8.9 Hz, 2H), 7.64 (d, J = 8.9 Hz, 2H), 7.63 (s, 1H), 7.58 (s, 1H), 7.29 (s, 1H), 7.22 (s, 2H), 7.20 (s, 1H), 7.17 (s, 2H), 7.07 (s, 2H), 7.04 (s, 1H), 6.95 (s, 1H), 6.94 (s, 1H), 6.89 (s, 1H), 4.02 (s, 3H), 4.01 (s, 3H), 3.85 (s, 3H), 3.84 (s, 3H), 3.83 (s, 6H), 3.82 (s, 6H), 3.33 (m, 4H), 3.24 (m, 4H), 3.08 (m, 2H), 2.79 (s, 3H), 2.78 (s, 3H), 2.47 (brt, 2H), 2.43 (t, J = 7.6 Hz, 2H), 2.35 (t, J = 7.6 Hz, 2H), 2.30 (t, J = 7.6 Hz, 2H), 2.28 (t, J = 7.6 Hz, 2H), 1.93 (t, J = 7.6 Hz, 2H), 1.84 (m, 2H), 1.79 (m, 2H), 1.56 (m, 2H), 1.48 (m, 2H), 1.26 (m, 2H); ESI-TOF-MS(positive) m/z calculated for C76H97N25O152+ [M+2H]2+ 799.87; observed 799.91. Surface Plasmon Resonance Analysis. SPR analyses were performed using a BIACORE X instrument as previously described.18 Biotinylated DNAs were purchased from JBioS (Tokyo, Japan). The matching sequence to confirm the binding specificity of SAHA-PIP I to the human OCT-3/4 promoter region is as follows: 5′-Biotin-GCG CCT TCC TTC CCC TTT TGG GGA AGG AAG GCG C-3′. The mismatch sequence was chosen from the human PIWIL2 promoter region to validate the SAHA-PIP I binding specificity and is as follows: 5′-Biotin-CGT CCT TTC CAG CAG TTT TCT GCT GGA AAG GAC G-3′. To measure the rates of association (ka) and dissociation (kd) and dissociation constant (KD), data processing was performed with a fitting model using the BIAevaluation 4.1 program as described previously.18

S Supporting Information *

Supplementary methods, Tables 1−5, and Figures 1−11. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-75-753-4002. Fax: 81-75-753-3670. E-mail: hs@ kuchem.kyoto-u.ac.jp. Author Contributions ∇

G.N.P. and S.S. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan. The World Premier International Research Centre Initiative, MEXT, JAPAN, supports the iCeMS. We thank Nagase Science and Technology foundation for their support. We thank the challenging exploratory grant and Grants-in-aid for Young Scientists-B for support to G.N.P.

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REFERENCES

(1) Wu, Y. L., Pandian, G. N., Ding, Y. P., Zhang, W., Tanaka, Y., and Sugiyama, H. (2013) Clinical grade iPS cells: Need for versatile small molecules and optimal cell sources. Chem. Biol. 20, 1311−1322. G



Letters

polyamide for sequence-specific recognition in the DNA minor groove. J. Am. Chem. Soc. 133, 18924−18930. (19) Samavarchi-Tehrani, P., Golipour, A., David, L., Sung, H. K., Beyer, T. A., Datti, A., Woltjen, K., Nagy, A., and Wrana, J. L. (2010) Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. Cell Stem Cell 7, 64−77. (20) Wakao, S., Kitada, M., Kuroda, Y., Shigemoto, T., Matsuse, D., Akashi, H., Tanimura, Y., Tsuchiyama, K., Kikuchi, T., Goda, M., Nakahata, T., Fujiyoshi, Y., and Dezawa, M. (2011) Multilineagedifferentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts. Proc. Natl. Acad. Sci. U.S.A. 108, 9875−9880. (21) Zhu, S., Li, W., Zhou, H., Wei, W., Ambasudhan, R., Lin, T., Kim, J., Zhang, K., and Ding, S. (2010) Reprogramming of human primary somatic cells by OCT4 and chemical compounds. Cell Stem Cell 7, 651−655. (22) Li, W., Jiang, K., and Ding, S. (2012) Concise review: A chemical approach to control cell fate and function. Stem Cells 30, 61− 68. (23) Hou, P., Li, Y., Zhang, X., Liu, C., Guan, J., Li, H., Zhao, T., Ye, J., Yang, W., Liu, K., Ge, J., Xu, J., Zhang, Q., Zhao, Y., and Deng, H. (2013) Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science 341, 651−654. (24) Masuda, S., Wu, J., Hishida, T., Pandian, G. N., Sugiyama, H., and Izpisua Belmonte, J. C. (2013) Chemically induced pluripotent stem cells (CiPSCs): A transgene-free approach. J. Mol. Cell Biol. 5, 354−355. (25) Onder, T. T., Kara, N., Cherry, A., Sinha, A. U., Zhu, N., Bernt, K. M., Cahan, P., Marcarci, B. O., Unternaehrer, J., Gupta, P. B., Lander, E. S., Armstrong, S. A., and Daley, G. Q. (2012) Chromatinmodifying enzymes as modulators of reprogramming. Nature 483, 598−602. (26) Margueron, R., Trojer, P., and Reinberg, D. (2005) The key to development: Interpreting the histone code? Curr. Opin. Genet. Dev. 15, 163−176. (27) Pandian, G. N., and Sugiyama, H. (2012) Programmable genetic switches to control transcriptional machinery of pluripotency. Biotechnol. J. 7, 798−809. (28) Matsui, T., Takano, M., Yoshida, K., Ono, S., Fujisaki, C., Matsuzaki, Y., Toyama, Y., Nakamura, M., Okano, H., and Akamatsu, W. (2012) Neural stem cells directly differentiated from partially reprogrammed fibroblasts rapidly acquire gliogenic competency. Stem Cells 30, 1109−1119. (29) Carlson, C. D., Warren, C. L., Hauschild, K. E., Ozers, M. S., Qadir, N., Bhimsaria, D., Lee, Y., Cerrina, F., and Ansari, A. Z. (2010) Specificity landscapes of DNA binding molecules elucidate biological function. Proc. Natl. Acad. Sci. U.S.A. 107, 4544−4549. (30) Saha, A., Pandian, G. N., Sato, S., Taniguchi, J., Hashiya, K., Bando, T., and Sugiyama, H. (2013) Synthesis and biological evaluation of a targeted DNA-binding transcriptional activator with HDAC8 inhibitory activity. Bioorg. Med. Chem. 21, 4201−4209. (31) Trauger, J. W., Baird, E. E., and Dervan, P. B. (1998) Recognition of 16 base pairs in the minor groove of DNA by a pyrrole−imidazole polyamide dimer. J. Am. Chem. Soc. 120, 3534− 3535. (32) Vaijayanthi, T., Bando, T., Hashiya, K., Pandian, G. N., and Sugiyama, H. (2013) Design of a new fluorescent probe: Pyrrole/ imidazole hairpin polyamides with pyrene conjugation at their γ-turn. Bioorg. Med. Chem. 21, 852−855. (33) Vaijayanthi, T., Bando, T., Pandian, G. N., and Sugiyama, H. (2012) Progress and prospects of pyrrole-imidazole polyamide− fluorophore conjugates as sequence-selective DNA probes. ChemBioChem 13, 2170−2185.

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