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The phosphorylation status of Ascl1 is a key determinant of neuronal differentiation and maturation in vivo and in vitro

Ali, Fahad R; Cheng, Kevin; Kirwan, Peter; Metcalfe, Su; Livesey, Frederick J; Barker, Roger A LU and Philpott, Anna (2014) In Development 141(11). p.24-2216
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... (More)

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.

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published
keywords
Animals, Basic Helix-Loop-Helix Transcription Factors, Cell Culture Techniques, Cell Cycle, Cell Line, Cell Transdifferentiation, Fibroblasts, Gene Expression Regulation, Developmental, HEK293 Cells, Humans, Nerve Tissue Proteins, Neurogenesis, Neurons, Phosphorylation, Proline, Protein Processing, Post-Translational, Receptors, Notch, Serine, Signal Transduction, Xenopus Proteins, Xenopus laevis, Journal Article, Research Support, Non-U.S. Gov't
in
Development
volume
141
issue
11
pages
9 pages
publisher
Society for International Development
external identifiers
  • scopus:84901447700
ISSN
1477-9129
DOI
10.1242/dev.106377
language
English
LU publication?
no
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a7af6d39-005e-40bc-a3f4-dc03fc7e9388
date added to LUP
2016-11-24 15:09:37
date last changed
2017-10-22 05:22:29
@article{a7af6d39-005e-40bc-a3f4-dc03fc7e9388,
  abstract     = {<p>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.</p>},
  author       = {Ali, Fahad R and Cheng, Kevin and Kirwan, Peter and Metcalfe, Su and Livesey, Frederick J and Barker, Roger A and Philpott, Anna},
  issn         = {1477-9129},
  keyword      = {Animals,Basic Helix-Loop-Helix Transcription Factors,Cell Culture Techniques,Cell Cycle,Cell Line,Cell Transdifferentiation,Fibroblasts,Gene Expression Regulation, Developmental,HEK293 Cells,Humans,Nerve Tissue Proteins,Neurogenesis,Neurons,Phosphorylation,Proline,Protein Processing, Post-Translational,Receptors, Notch,Serine,Signal Transduction,Xenopus Proteins,Xenopus laevis,Journal Article,Research Support, Non-U.S. Gov't},
  language     = {eng},
  number       = {11},
  pages        = {24--2216},
  publisher    = {Society for International Development},
  series       = {Development},
  title        = {The phosphorylation status of Ascl1 is a key determinant of neuronal differentiation and maturation in vivo and in vitro},
  url          = {http://dx.doi.org/10.1242/dev.106377},
  volume       = {141},
  year         = {2014},
}