Hardwick LJ et al. (2016), MyoD phosphorylation on multiple C terminal sit...

XB-ART-52700

Biochem Biophys Res Commun 2016 Dec 02;4811-2:97-103. doi: 10.1016/j.bbrc.2016.11.009.

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MyoD phosphorylation on multiple C terminal sites regulates myogenic conversion activity.

Hardwick LJ , Davies JD , Philpott A .

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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.

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Species referenced: Xenopus
Genes referenced: myod1

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	Fig. 1. MyoD is phospho-regulated on sites in addition to S200 during primary myogenesis. (A) Western blot analysis of protein extracts from stage 12.5 embryos injected with 200 pg mRNA encoding HA-tagged WT or S200A-MyoD. Samples were incubated with or without lambda protein phosphatase. Tubulin provided a loading control. (B) Schematic representation of WT mouse MyoD protein and phospho-mutant variants, showing approximate locations of SP/TP sites that are mutated to AP in each. (C–E) Embryos were unilaterally injected at the 2-cell stage with 100 pg mRNA encoding the respective MyoD constructs. At stage 18, embryos were assayed by RT-qPCR for Myosin Heavy Chain expression (C [n = 3]) or by ISH with semi-quantitative scoring for Muscle Actin expression (D [n = 52–73]) as described in the methods. Representative embryos are shown in (E); injected side to the right. * = (p < 0.05); = (p < 0.025); * = (p < 0.0125).
	Fig. 2. Mutational analysis shows regulatory activity of S200 and additional C terminal phosphorylation sites. Schematic representation of phospho-mutant constructs with mutation of cumulative (A) or individual (D) sites. Two-cell stage embryos were unilaterally injected with 100 pg mRNA of the respective MyoD construct and assayed at stage 18 for expression of Muscle Actin by ISH: (B) Cumulative mutant series [n = 68–74] with representative images shown in (C). (D) Single site mutants [n = 52–83]. NS = Not significant; * = (p < 0.05); = (p < 0.025); * = (p < 0.0125).
	Fig. 3. Maximal myogenic activity is achieved by mutation of multiple C terminal sites. (A) Schematic representation of phospho-mutant constructs. (B) Two-cell stage embryos were unilaterally injected with 100 pg mRNA of the respective MyoD construct and assayed at stage 18 as before [n = 32–41] with representative images shown in (C). NS = Not significant; * = (p < 0.05); = (p < 0.025); * = (p < 0.0125).
	Fig. 4. CT 5T/S-A MyoD shows enhanced protein stability and enhanced chromatin binding relative to both WT and S200A MyoD. Embryos were injected with 200 pg mRNA encoding HA-tagged MyoD constructs as indicated, and western blot analysis was performed on whole embryo extracts at stage 12.5 (A) or cross linking and cytoplasmic/chromatin extracts at stage 13 (B). MyoD protein density was quantified relative to tubulin loading control for whole embryo extracts (C) and cytoplasmic fractions (D), or relative to histone H3 for chromatin fractions (D). Mean values are shown from independent duplicate samples with standard error of the mean.
	Supplementary Fig. 1Protein sequence alignment of human, mouse and Xenopus MyoD. The bHLH domain is underlined and SP/TP sites are highlighted in red. A consensus line is also shown below the alignment to indicate the degree of conservation at each position: Residues may be identical (*), strongly conserved (:) or weakly conserved (.).

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	Fig. 1. MyoD is phospho-regulated on sites in addition to S200 during primary myogenesis. (A) Western blot analysis of protein extracts from stage 12.5 embryos injected with 200 pg mRNA encoding HA-tagged WT or S200A-MyoD. Samples were incubated with or without lambda protein phosphatase. Tubulin provided a loading control. (B) Schematic representation of WT mouse MyoD protein and phospho-mutant variants, showing approximate locations of SP/TP sites that are mutated to AP in each. (C–E) Embryos were unilaterally injected at the 2-cell stage with 100 pg mRNA encoding the respective MyoD constructs. At stage 18, embryos were assayed by RT-qPCR for Myosin Heavy Chain expression (C [n = 3]) or by ISH with semi-quantitative scoring for Muscle Actin expression (D [n = 52–73]) as described in the methods. Representative embryos are shown in (E); injected side to the right. * = (p < 0.05); = (p < 0.025); * = (p < 0.0125).
	Fig. 2. Mutational analysis shows regulatory activity of S200 and additional C terminal phosphorylation sites. Schematic representation of phospho-mutant constructs with mutation of cumulative (A) or individual (D) sites. Two-cell stage embryos were unilaterally injected with 100 pg mRNA of the respective MyoD construct and assayed at stage 18 for expression of Muscle Actin by ISH: (B) Cumulative mutant series [n = 68–74] with representative images shown in (C). (D) Single site mutants [n = 52–83]. NS = Not significant; * = (p < 0.05); = (p < 0.025); * = (p < 0.0125).
	Fig. 3. Maximal myogenic activity is achieved by mutation of multiple C terminal sites. (A) Schematic representation of phospho-mutant constructs. (B) Two-cell stage embryos were unilaterally injected with 100 pg mRNA of the respective MyoD construct and assayed at stage 18 as before [n = 32–41] with representative images shown in (C). NS = Not significant; * = (p < 0.05); = (p < 0.025); * = (p < 0.0125).
	Fig. 4. CT 5T/S-A MyoD shows enhanced protein stability and enhanced chromatin binding relative to both WT and S200A MyoD. Embryos were injected with 200 pg mRNA encoding HA-tagged MyoD constructs as indicated, and western blot analysis was performed on whole embryo extracts at stage 12.5 (A) or cross linking and cytoplasmic/chromatin extracts at stage 13 (B). MyoD protein density was quantified relative to tubulin loading control for whole embryo extracts (C) and cytoplasmic fractions (D), or relative to histone H3 for chromatin fractions (D). Mean values are shown from independent duplicate samples with standard error of the mean.
	Supplementary Fig. 1Protein sequence alignment of human, mouse and Xenopus MyoD. The bHLH domain is underlined and SP/TP sites are highlighted in red. A consensus line is also shown below the alignment to indicate the degree of conservation at each position: Residues may be identical (*), strongly conserved (:) or weakly conserved (.).