Multi-site phosphorylation controls the neurogenic and myogenic activity of E47.

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Hardwick, Laura JA 
Davies, John D 

The superfamily of basic-Helix-Loop-Helix (bHLH) transcription factors influence cell fate in all three embryonic germ layers, and the tissue-specific class II factors have received prominent attention for their potent ability to direct differentiation during development and in cellular reprogramming. The activity of many class II bHLH proteins driving differentiation, and the inhibitory class VI bHLH factor Hes1, is controlled by phosphorylation on multiple sites by Cyclin-dependent kinases (Cdks). As class II proteins are generally thought to be active through hetero-dimerisation with the ubiquitously expressed class I E proteins, regulation of class I transcription factors such as E47 may influence the activity of multiple tissue-specific bHLH proteins. Using differentiation of nerve and muscle in Xenopus frog embryos as a model system, we set out to explore whether with the ubiquitously expressed class I E protein E47 that hetero-dimerises with Class II bHLHs to control their activity, is also regulated by multi-site phosphorylation. We demonstrate that E47 can be readily phosphorylated by Cdks on multiple sites in vitro, while ectopically-expressed E47 exists in multiple phosphorylated forms in Xenopus embryos. Preventing multi-site phosphorylation using a phospho-mutant version of E47 enhances the neurogenic and myogenic activity of three different class II bHLH reprogramming factors, and also when E47 acts in hetero-dimerisation with endogenous proteins. Mechanistically, unlike phospho-regulation of class II bHLH factors, we find that preventing phosphorylation of E47 increases the amount of chromatin-bound E47 protein but without affecting its overall protein stability. Thus, multi-site phosphorylation is a conserved regulatory mechanism across the bHLH superfamily that can be manipulated to enhance cellular differentiation.

E47, Myogenesis, Neurogenesis, Reprogramming, Xenopus, bHLH, Animals, Mice, Muscle Development, Mutation, Neurogenesis, Phosphorylation, Transcription Factor 3, Xenopus Proteins, Xenopus laevis
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Biochem Biophys Res Commun
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Elsevier BV
Medical Research Council (MR/K018329/1)
Medical Research Council (MR/L021129/1)
Medical Research Council (MC_PC_12009)
This work was supported by Medical Research Council Research Grants MR/L021129/1 and MR/K018329 and AP receives core funding from Wellcome and MRC at the Wellcome-MRC Cambridge Stem Cell Institute. LH is supported by a Peterhouse Research Fellowship.