Abstract
Terminally differentiated cells can be reprogrammed to pluripotency by the forced expression of Oct4, Sox2, Klf4 and c-Myc1,2. However, it remains unknown how this leads to the multitude of epigenetic changes observed during the reprogramming process. Interestingly, Oct4 is the only factor that cannot be replaced by other members of the same family to induce pluripotency3,4,5. To understand the unique role of Oct4 in reprogramming, we determined the structure of its POU domain bound to DNA. We show that the linker between the two DNA-binding domains is structured as an α-helix and exposed to the protein’s surface, in contrast to the unstructured linker of Oct1. Point mutations in this α-helix alter or abolish the reprogramming activity of Oct4, but do not affect its other fundamental properties. On the basis of mass spectrometry studies of the interactome of wild-type and mutant Oct4, we propose that the linker functions as a protein–protein interaction interface and plays a crucial role during reprogramming by recruiting key epigenetic players to Oct4 target genes. Thus, we provide molecular insights to explain how Oct4 contributes to the reprogramming process.
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References
Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).
Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).
Shi, Y. et al. Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. Cell Stem Cell 3, 568–574 (2008).
Nakagawa, M. et al. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat. Biotechnol. 26, 101–106 (2008).
Feng, B. et al. Reprogramming of fibroblasts into induced pluripotent stem cells with orphan nuclear receptor Esrrb. Nat. Cell Biol. 11, 197–203 (2009).
Klemm, J. D., Rould, M. A., Aurora, R., Herr, W. & Pabo, C. O. Crystal structure of the Oct-1 POU domain bound to an octamer site: DNA recognition with tethered DNA-binding modules. Cell 77, 21–32 (1994).
Reményi, A. et al. Differential dimer activities of the transcription factor Oct-1 by DNA-induced interface swapping. Mol. Cell 8, 569–580 (2001).
Reményi, A. et al. Crystal structure of a POU/HMG/DNA ternary complex suggests differential assembly of Oct4 and Sox2 on two enhancers. Genes Dev. 17, 2048–2059 (2003).
Williams, D. C. Jr, Cai, M. & Clore, G. M. Molecular basis for synergistic transcriptional activation by Oct1 and Sox2 revealed from the solution structure of the 42-kDa Oct1.Sox2.Hoxb1–DNA ternary transcription factor complex. J. Biol. Chem. 279, 1449–1457 (2004).
Jauch, R., Choo, S. H., Ng, C. K. & Kolatkar, P. R. Crystal structure of the dimeric Oct6 (POU3f1) POU domain bound to palindromic MORE DNA. Proteins 79, 674–677 (2011).
Nishimoto, M. et al. Oct-3/4 maintains the proliferative embryonic stem cell state via specific binding to a variant octamer sequence in the regulatory region of the UTF1 locus. Mol. Cell Biol. 25, 5084–5094 (2005).
Pesce, M., Gross, M. K. & Schöler, H. R. In line with our ancestors: Oct-4 and the mammalian germ. Bioessays 20, 722–732 (1998).
Kehler, J. et al. Oct4 is required for primordial germ cell survival. EMBO Rep. 5, 1078–1083 (2004).
Klemm, J. D. & Pabo, C. O. Oct-1 POU domain-DNA interactions: cooperative binding of isolated subdomains and effects of covalent linkage. Genes Dev. 10, 27–36 (1996).
Van den Berg, D. L. et al. An Oct4-centered protein interaction network in embryonic stem cells. Cell Stem. Cell 6, 369–381 (2010).
Pardo, M. et al. An expanded Oct4 interaction network: implications for stem cell biology, development, and disease. Cell Stem Cell 6, 382–395 (2010).
Ding, J., Xu, H., Faiola, F., Ma’ayan, A. & Wang, J. Oct4 links multiple epigenetic pathways to the pluripotency network. Cell Res. 22, 155–167 (2012).
Singhal, N. et al. Chromatin-remodeling components of the BAF complex facilitate reprogramming. Cell 141, 943–955 (2010).
Trotter, K. W. & Archer, T. K. The BRG1 transcriptional coregulator. Nucl. Recept. Signal. 6, e004 (2008).
Hu, G. & Wade, P. A. NuRD and pluripotency: a complex balancing act. Cell Stem Cell 10, 497–503 (2012).
Rai, K. et al. DNA demethylation in zebrafish involves the coupling of a deaminase, a glycosylase, and gadd45. Cell 135, 1201–1212 (2008).
Bhutani, N. et al. Reprogramming towards pluripotency requires AID-dependent DNA demethylation. Nature 463, 1042–1047 (2010).
Niwa, H., Miyazaki, J. & Smith, A. G. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. 24, 372–376 (2000).
Adachi, K. & Schöler, H. R. Directing reprogramming to pluripotency by transcription factors. Curr. Opin. Genet. Dev. 22, 416–422 (2012).
Schöler, H. R., Balling, R., Hatzopoulos, A. K., Suzuki, N. & Gruss, P. Octamer binding proteins confer transcriptional activity in early mouse embryogenesis. EMBO J. 8, 2551–2557 (1989).
Mueller-Dieckmann, J. The open-access high-throughput crystallization facility at EMBL Hamburg. Acta Crystallogr. D 62, 1446–1452 (2006).
Kabsch, W. Evaluation of single-crystal X-ray diffraction data from a position-sensitive detector. J. Appl. Crystallogr. 21, 916–924 (1988).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Murshudov, G. N., Vagin, A. A. & Dodson, E. J. Refinement of macromolecularstructures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).
Sauter, P. & Matthias, P. Coactivator OBF-1 makes selective contacts with both the POU-specific domain and the POU homeodomain and acts as a molecular clamp on DNA. Mol. Cell Biol. 18, 7397–7409 (1998).
Tomilin, A. et al. Synergism with the coactivator OBF-1 (OCA-B, BOB-1) is mediated by a specific POU dimer configuration. Cell 103, 853–864 (2000).
Kim, J. B. et al. Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature 454, 646–650 (2008).
Wisniewski, J. R., Zougman, A., Nagaraj, N. & Mann, M. Universal sample preparation method for proteome analysis. Nat. Methods 6, 359–362 (2009).
Rappsilber, J., Ishihama, Y. & Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 75, 663–670 (2003).
Sali, A. & Blundell, T. L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 234, 779–815 (1993).
Fiser, A., Do, R. K. & Sali, A. Modeling of loops in protein structures. Protein Sci. 9, 1753–1773 (2000).
Acknowledgements
X-ray diffraction experiments were performed on the ID23-1 beamline at the European Synchrotron Radiation Facility (ESRF), Grenoble, and X12 and X13 at EMBL Hamburg, DESY. We are grateful to E. Fioravanti of the ESRF for providing assistance in using beamline ID23-1. We thank G. Bourenkov for invaluable assistance in crystallographic problem resolution and data collection. We thank A. Nolte for help with mass spectrometry measurements. We also thank R. Grindberg for critically reading, and A. Malapetsas and J. Bruder for editing the manuscript. We acknowledge the financial support from DFG (SPP1356 Pluripotency and Cellular Reprogramming priority program) AJ DFG SI 1695/1-2 and AJ DFG CO 975/1.
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D.E. and J.V. performed the experiments and wrote the manuscript; J.V., M.R.G. and V.P. collected and analysed the crystallographic data; V.C. performed the comparative modelling experiments and wrote the manuscript; H.v.B. performed the cycloheximide experiments; H.C.A.D. performed the MS experiments; M.J.A-B. analysed the proteomic data sets; D.H. performed the rescue experiments; and C.N. assessed the heterodimer formation ability of Oct4 and Sox2. All authors commented on the manuscript; and R.J., M.W. and H.R.S. as co-senior authors supervised the project and assisted in writing the manuscript.
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Esch, D., Vahokoski, J., Groves, M. et al. A unique Oct4 interface is crucial for reprogramming to pluripotency. Nat Cell Biol 15, 295–301 (2013). https://doi.org/10.1038/ncb2680
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DOI: https://doi.org/10.1038/ncb2680
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