title: The phosphorylation status of Ascl1 is a key determinant of neuronal differentiation and maturation in vivo and in vitro.
abstract: Generation of neurons from patient fibroblasts using a combination of developmentally defined transcription factors has great potential in disease modelling, as well as ultimately for use in regeneration and repair. However, generation of physiologically mature neurons in vitro remains problematic. Here we demonstrate the cell-cycle-dependent phosphorylation of a key reprogramming transcription factor, Ascl1, on multiple serine-proline sites. This multisite phosphorylation is a crucial regulator of the ability of Ascl1 to drive neuronal differentiation and maturation in vivo in the developing embryo; a phosphomutant form of Ascl1 shows substantially enhanced neuronal induction activity in Xenopus embryos. Mechanistically, we see that this un(der)phosphorylated Ascl1 is resistant to inhibition by both cyclin-dependent kinase activity and Notch signalling, both of which normally limit its neurogenic potential. Ascl1 is a central component of reprogramming transcription factor cocktails to generate neurons from human fibroblasts; the use of phosphomutant Ascl1 in place of the wild-type protein significantly promotes neuronal maturity after human fibroblast reprogramming in vitro. These results demonstrate that cell-cycle-dependent post-translational modification of proneural proteins directly regulates neuronal differentiation in vivo during development, and that this regulatory mechanism can be harnessed to promote maturation of neurons obtained by transdifferentiation of human cells in vitro.
authors.affiliation: University of Cambridge, Department of Oncology, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 0XZ, UK John van Geest Centre for Brain Repair, University of Cambridge, Forvie Site, Robinson Way, Cambridge CB2 0PY, UK.
authors.forename: Fahad R
authors.initials: FR
authors.lastname: Ali
authors.affiliation: University of Cambridge, Department of Oncology, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 0XZ, UK.
authors.forename: Kevin
authors.initials: K
authors.lastname: Cheng
authors.affiliation: Gurdon Institute, Department of Biochemistry and Cambridge Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.
authors.forename: Peter
authors.initials: P
authors.lastname: Kirwan
authors.affiliation: John van Geest Centre for Brain Repair, University of Cambridge, Forvie Site, Robinson Way, Cambridge CB2 0PY, UK.
authors.forename: Su
authors.initials: S
authors.lastname: Metcalfe
authors.affiliation: Gurdon Institute, Department of Biochemistry and Cambridge Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK.
authors.forename: Frederick J
authors.initials: FJ
authors.lastname: Livesey
authors.affiliation: John van Geest Centre for Brain Repair, University of Cambridge, Forvie Site, Robinson Way, Cambridge CB2 0PY, UK.
authors.forename: Roger A
authors.initials: RA
authors.lastname: Barker
authors.affiliation: University of Cambridge, Department of Oncology, Hutchison/MRC Research Centre, Hills Road, Cambridge CB2 0XZ, UK ap113@cam.ac.uk.
authors.forename: Anna
authors.initials: A
authors.lastname: Philpott
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000378:metabolism*; D018929:Cell Culture Techniques; D002453:Cell Cycle; D002460:Cell Line; D054338:Cell Transdifferentiation / Q000502:physiology; D005347:Fibroblasts / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D057809:HEK293 Cells; D006801:Humans; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D055495:Neurogenesis; D009474:Neurons / Q000166:cytology* / Q000378:metabolism; D010766:Phosphorylation; D011392:Proline / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051880:Receptors, Notch / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D029867:Xenopus Proteins / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D009419:Nerve Tissue Proteins; D011392:Proline; D051880:Receptors, Notch; D012694:Serine; D029867:Xenopus Proteins; C080088:ascl1 protein, Xenopus
keywords: Ascl1; Neurogenesis; Phosphorylation; Transdifferentiation