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Synergistic binding of transcription factors to cell-specific enhancers programs motor neuron identity

Abstract

Efficient transcriptional programming promises to open new frontiers in regenerative medicine. However, mechanisms by which programming factors transform cell fate are unknown, preventing more rational selection of factors to generate desirable cell types. Three transcription factors, Ngn2, Isl1 and Lhx3, were sufficient to program rapidly and efficiently spinal motor neuron identity when expressed in differentiating mouse embryonic stem cells. Replacement of Lhx3 by Phox2a led to specification of cranial, rather than spinal, motor neurons. Chromatin immunoprecipitation–sequencing analysis of Isl1, Lhx3 and Phox2a binding sites revealed that the two cell fates were programmed by the recruitment of Isl1-Lhx3 and Isl1-Phox2a complexes to distinct genomic locations characterized by a unique grammar of homeodomain binding motifs. Our findings suggest that synergistic interactions among transcription factors determine the specificity of their recruitment to cell type–specific binding sites and illustrate how a single transcription factor can be repurposed to program different cell types.

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Figure 1: NIL and NIP transcription factors program spinal and cranial motor neurons, respectively.
Figure 2: NIL and NIP induce different transcriptomes.
Figure 3: Induced iNIL cells bypass progenitor stages.
Figure 4: Context-specific Isl1 genome association in iNIL and iNIP cells correlates with differential gene expression.
Figure 5: Sequence motifs occupied by Isl1 in iNIL and iNIP cells.

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References

  1. Mann, R.S. & Carroll, S.B. Molecular mechanisms of selector gene function and evolution. Curr. Opin. Genet. Dev. 12, 592–600 (2002).

    Article  CAS  Google Scholar 

  2. Tapscott, S.J. et al. MyoD1: a nuclear phosphoprotein requiring a Myc homology region to convert fibroblasts to myoblasts. Science 242, 405–411 (1988).

    Article  CAS  Google Scholar 

  3. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006).

    Article  CAS  Google Scholar 

  4. Pfisterer, U. et al. Direct conversion of human fibroblasts to dopaminergic neurons. Proc. Natl. Acad. Sci. USA 108, 10343–10348 (2011).

    Article  CAS  Google Scholar 

  5. Sekiya, S. & Suzuki, A. Direct conversion of mouse fibroblasts to hepatocyte-like cells by defined factors. Nature 475, 390–393 (2011).

    Article  CAS  Google Scholar 

  6. Son, E.Y. et al. Conversion of mouse and human fibroblasts into functional spinal motor neurons. Cell Stem Cell 9, 205–218 (2011).

    Article  CAS  Google Scholar 

  7. Zhou, Q., Brown, J., Kanarek, A., Rajagopal, J. & Melton, D.A. In vivo reprogramming of adult pancreatic exocrine cells to beta-cells. Nature 455, 627–632 (2008).

    Article  CAS  Google Scholar 

  8. Peter, I.S. & Davidson, E.H. Evolution of gene regulatory networks controlling body plan development. Cell 144, 970–985 (2011).

    Article  CAS  Google Scholar 

  9. Slattery, M. et al. Cofactor binding evokes latent differences in DNA binding specificity between Hox proteins. Cell 147, 1270–1282 (2011).

    Article  CAS  Google Scholar 

  10. Jessell, T.M. Neuronal specification in the spinal cord: inductive signals and transcriptional codes. Nat. Rev. Genet. 1, 20–29 (2000).

    Article  CAS  Google Scholar 

  11. Song, M.R. et al. T-Box transcription factor Tbx20 regulates a genetic program for cranial motor neuron cell body migration. Development 133, 4945–4955 (2006).

    Article  CAS  Google Scholar 

  12. Lee, S.K. & Pfaff, S.L. Synchronization of neurogenesis and motor neuron specification by direct coupling of bHLH and homeodomain transcription factors. Neuron 38, 731–745 (2003).

    Article  CAS  Google Scholar 

  13. Hester, M.E. et al. Rapid and efficient generation of functional motor neurons from human pluripotent stem cells using gene delivered transcription factor codes. Mol. Ther. 19, 1905–1912 (2011).

    Article  CAS  Google Scholar 

  14. Lee, S. et al. Fusion protein Isl1-Lhx3 specifies motor neuron fate by inducing motor neuron genes and concomitantly suppressing the interneuron programs. Proc. Natl. Acad. Sci. USA 109, 3383–3388 (2012).

    Article  CAS  Google Scholar 

  15. Thaler, J.P., Lee, S.K., Jurata, L.W., Gill, G.N. & Pfaff, S.L. LIM factor Lhx3 contributes to the specification of motor neuron and interneuron identity through cell type–specific protein-protein interactions. Cell 110, 237–249 (2002).

    Article  CAS  Google Scholar 

  16. Sharma, K. et al. LIM homeodomain factors Lhx3 and Lhx4 assign subtype identities for motor neurons. Cell 95, 817–828 (1998).

    Article  CAS  Google Scholar 

  17. Pfaff, S.L., Mendelsohn, M., Stewart, C.L., Edlund, T. & Jessell, T.M. Requirement for LIM homeobox gene Isl1 in motor neuron generation reveals a motor neuron-dependent step in interneuron differentiation. Cell 84, 309–320 (1996).

    Article  CAS  Google Scholar 

  18. Hirsch, M.R., Glover, J.C., Dufour, H.D., Brunet, J.F. & Goridis, C. Forced expression of Phox2 homeodomain transcription factors induces a branchio-visceromotor axonal phenotype. Dev. Biol. 303, 687–702 (2007).

    Article  CAS  Google Scholar 

  19. Pattyn, A., Morin, X., Cremer, H., Goridis, C. & Brunet, J.F. Expression and interactions of the two closely related homeobox genes Phox2a and Phox2b during neurogenesis. Development 124, 4065–4075 (1997).

    CAS  Google Scholar 

  20. Coppola, E., Pattyn, A., Guthrie, S.C., Goridis, C. & Studer, M. Reciprocal gene replacements reveal unique functions for Phox2 genes during neural differentiation. EMBO J. 24, 4392–4403 (2005).

    Article  CAS  Google Scholar 

  21. Mazzoni, E.O. et al. Embryonic stem cell–based mapping of developmental transcriptional programs. Nat. Methods 8, 1056–1058 (2011).

    Article  CAS  Google Scholar 

  22. Novitch, B.G., Chen, A.I. & Jessell, T.M. Coordinate regulation of motor neuron subtype identity and pan-neuronal properties by the bHLH repressor Olig2. Neuron 31, 773–789 (2001).

    Article  CAS  Google Scholar 

  23. Mizuguchi, R. et al. Combinatorial roles of olig2 and neurogenin2 in the coordinated induction of pan-neuronal and subtype-specific properties of motoneurons. Neuron 31, 757–771 (2001).

    Article  CAS  Google Scholar 

  24. Hasan, K.B., Agarwala, S. & Ragsdale, C.W. PHOX2A regulation of oculomotor complex nucleogenesis. Development 137, 1205–1213 (2010).

    Article  CAS  Google Scholar 

  25. Miles, G.B. et al. Functional properties of motoneurons derived from mouse embryonic stem cells. J. Neurosci. 24, 7848–7858 (2004).

    Article  CAS  Google Scholar 

  26. Gao, B.X. & Ziskind-Conhaim, L. Development of ionic currents underlying changes in action potential waveforms in rat spinal motoneurons. J. Neurophysiol. 80, 3047–3061 (1998).

    Article  CAS  Google Scholar 

  27. Wichterle, H., Lieberam, I., Porter, J.A. & Jessell, T.M. Directed differentiation of embryonic stem cells into motor neurons. Cell 110, 385–397 (2002).

    Article  CAS  Google Scholar 

  28. Wichterle, H., Peljto, M. & Nedelec, S. Xenotransplantation of embryonic stem cell–derived motor neurons into the developing chick spinal cord. Methods Mol. Biol. 482, 171–183 (2009).

    Article  CAS  Google Scholar 

  29. Dillon, A.K. et al. Molecular control of spinal accessory motor neuron/axon development in the mouse spinal cord. J. Neurosci. 25, 10119–10130 (2005).

    Article  CAS  Google Scholar 

  30. Holmes, G. & Niswander, L. Expression of slit-2 and slit-3 during chick development. Dev. Dyn. 222, 301–307 (2001).

    Article  CAS  Google Scholar 

  31. Brunet, J.F. & Pattyn, A. Phox2 genes: from patterning to connectivity. Curr. Opin. Genet. Dev. 12, 435–440 (2002).

    Article  CAS  Google Scholar 

  32. Grillet, N., Dubreuil, V., Dufour, H.D. & Brunet, J.F. Dynamic expression of RGS4 in the developing nervous system and regulation by the neural type–specific transcription factor Phox2b. J. Neurosci. 23, 10613–10621 (2003).

    Article  CAS  Google Scholar 

  33. Moore, R.Y. Cranial motor neurons contain either galanin or calcitonin gene–related peptide like immunoreactivity. J. Comp. Neurol. 282, 512–522 (1989).

    Article  CAS  Google Scholar 

  34. Mahony, S. et al. Ligand-dependent dynamics of retinoic acid receptor binding during early neurogenesis. Genome Biol. 12, R2 (2011).

    Article  CAS  Google Scholar 

  35. Shen, Y. et al. A map of the cis-regulatory sequences in the mouse genome. Nature 488, 116–120 (2012).

    Article  CAS  Google Scholar 

  36. Berger, M.F. et al. Variation in homeodomain DNA binding revealed by high-resolution analysis of sequence preferences. Cell 133, 1266–1276 (2008).

    Article  CAS  Google Scholar 

  37. Vierbuchen, T. et al. Direct conversion of fibroblasts to functional neurons by defined factors. Nature 463, 1035–1041 (2010).

    Article  CAS  Google Scholar 

  38. Kim, J., Chu, J., Shen, X., Wang, J. & Orkin, S.H. An extended transcriptional network for pluripotency of embryonic stem cells. Cell 132, 1049–1061 (2008).

    Article  CAS  Google Scholar 

  39. Boyer, L.A. et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956 (2005).

    Article  CAS  Google Scholar 

  40. Kim, J. et al. A Myc network accounts for similarities between embryonic stem and cancer cell transcription programs. Cell 143, 313–324 (2010).

    Article  CAS  Google Scholar 

  41. Dasen, J.S. & Jessell, T.M. Hox networks and the origins of motor neuron diversity. Curr. Top. Dev. Biol. 88, 169–200 (2009).

    Article  CAS  Google Scholar 

  42. Mazzoni, E.O. et al. Saltatory remodeling of Hox chromatin in response to rostro-caudal patterning signals. Nat. Neurosci. (in the press).

  43. Li, X.J. et al. Specification of motoneurons from human embryonic stem cells. Nat. Biotechnol. 23, 215–221 (2005).

    Article  Google Scholar 

  44. Perrier, A.L. et al. Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc. Natl. Acad. Sci. USA 101, 12543–12548 (2004).

    Article  CAS  Google Scholar 

  45. Boulting, G.L. et al. A functionally characterized test set of human induced pluripotent stem cells. Nat. Biotechnol. 29, 279–286 (2011).

    Article  CAS  Google Scholar 

  46. Sanges, R., Cordero, F. & Calogero, R.A. oneChannelGUI: a graphical interface to Bioconductor tools, designed for life scientists who are not familiar with R language. Bioinformatics 23, 3406–3408 (2007).

    Article  CAS  Google Scholar 

  47. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  Google Scholar 

  48. Guo, Y. et al. Discovering homotypic binding events at high spatial resolution. Bioinformatics 26, 3028–3034 (2010).

    Article  CAS  Google Scholar 

  49. Robinson, M.D., McCarthy, D.J. & Smyth, G.K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010).

    Article  CAS  Google Scholar 

  50. Albuquerque, C., Joseph, D.J., Choudhury, P. & MacDermott, A.B. Dissection, plating, and maintenance of cortical astrocyte cultures. Cold Spring Harbor Protoc. 8, pdb prot5273 (2009).

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Acknowledgements

We would like to thank I. Lieberam (King's College) for assistance introducing the Hb9-GFP transgene, members of the Wichterle laboratory for helpful comments, S. Brenner-Morton and T. Jessell (Columbia University) for sharing clones of Isl monoclonal antibodies, J.-F. Brunet for Phox2a and Phox2b antibodies, and I. Schieren for technical assistance with flow cytometry. E.O.M. receives funding from the Damon Runyon Cancer Research Foundation (DRG-1937-07). Personnel and work were supported by the Project ALS foundation and US National Institutes of Health grants P01 NS055923 (D.K.G. and H.W.) and R01 NS078097 (H.W.).

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Authors

Contributions

E.O.M., S.M., D.K.G. and H.W. conceived the experiments, analyzed the data and wrote the manuscript. E.O.M. generated and validated inducible cell lines and performed the majority of experiments. S.M. performed all of the computational and statistical analyses of genomic, expression and sequencing data. M.C. performed motor neuron quantifications, synapse analysis and co-immunoprecipitation experiments. C.A.M. assisted with ChIP-seq experiments. S.N. performed axon pathfinding analysis. D.J.W. performed electrophysiological recordings. D.A. assisted with the analysis of single gene inducible lines and motor neuron induction.

Corresponding authors

Correspondence to Esteban O Mazzoni, David K Gifford or Hynek Wichterle.

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The authors declare no competing financial interests.

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Mazzoni, E., Mahony, S., Closser, M. et al. Synergistic binding of transcription factors to cell-specific enhancers programs motor neuron identity. Nat Neurosci 16, 1219–1227 (2013). https://doi.org/10.1038/nn.3467

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