MyoD phosphorylation on multiple C terminal sites regulates myogenic conversion activity.
MyoD is a master regulator of myogenesis with a potent ability to redirect the cell fate of even terminally differentiated cells. Hence, enhancing the activity of MyoD is an important step to maximising its potential utility for in vitro disease modelling and cell replacement therapies. We have previously shown that the reprogramming activity of several neurogenic bHLH proteins can be substantially enhanced by inhibiting their multi-site phosphorylation by proline-directed kinases. Here we have used Xenopus embryos as an in vivo developmental and reprogramming system to investigate the multi-site phospho-regulation of MyoD during muscle differentiation. We show that, in addition to modification of a previously well-characterised site, Serine 200, MyoD is phosphorylated on multiple additional serine/threonine sites during primary myogenesis. Through mutational analysis, we derive an optimally active phospho-mutant form of MyoD that has a dramatically enhanced ability to drive myogenic reprogramming in vivo. Mechanistically, this is achieved through increased protein stability and enhanced chromatin association. Therefore, multi-site phospho-regulation of class II bHLH proteins is conserved across cell lineages and germ layers, and manipulation of phosphorylation of these key regulators may have further potential for enhancing mammalian cell reprogramming.
Department of Oncology, University of Cambridge, Hutchison/MRC Research Centre, Cambridge Biomedical Campus, Cambridge CB2 0XZ, UK; Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR, UK; Peterhouse, University of Cambridge, Trumpington Street, Cambridge, CB2 1RD, UK. Electronic address: ljah2@cam.ac.uk.
Laura J A
LJ
Hardwick
Department of Oncology, University of Cambridge, Hutchison/MRC Research Centre, Cambridge Biomedical Campus, Cambridge CB2 0XZ, UK; Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR, UK. Electronic address: jdd24@cam.ac.uk.
John D
JD
Davies
Department of Oncology, University of Cambridge, Hutchison/MRC Research Centre, Cambridge Biomedical Campus, Cambridge CB2 0XZ, UK; Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR, UK. Electronic address: ap113@cam.ac.uk.
Anna
A
Philpott
D000818:Animals; D001665:Binding Sites; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D051379:Mice; D024510:Muscle Development / Q000502:physiology*; D017570:MyoD Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D032446:Myoblasts / Q000166:cytology* / Q000502:physiology*; D010766:Phosphorylation; D011485:Protein Binding
D017570:MyoD Protein; C057363:MyoD1 myogenic differentiation protein
MyoD; Myogenesis; Phosphorylation; Reprogramming; Xenopus; bHLH