Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C083

Oral Communications

Multi-site phosphorylation of bHLH transcription factors regulates neural differentiation in Xenopus

L. Hardwick1, A. Philpott1

1. University of Cambridge, Cambridge, United Kingdom.

  • Figure 1: (A) Schematics of WT NeuroD4 and a sequential phospho-mutant series indicating the SP or TP sites that are mutated to AP. (B) Semi-quantitative scoring of neural-β-tubulin as described in (4); embryos injected with mRNA encoding constructs; (N=59-77 embryos per category from 3 independent experiments).

  • Figure 2: (A) Western blot analysis from whole embryo lysates over-expressing equivalent amounts of WT and phospho-mutant NeuroD4 mRNA; N=3. (B) Western blot of embryos over-expressing WT and phospho-mutant NeuroD4 with cross-linking prior to fractionation as described in (4); N=2.

Throughout embryogenesis there is a critical balance between progenitor cell proliferation and subsequent differentiation; this balance is also pivotal when deranged in diseases like cancer, and to promote differentiation in cellular reprogramming for regenerative medicine (1). During neurogenesis, basic Helix Loop Helix (bHLH) proneural transcription factors act in cascades to drive and coordinate the various stages of neuronal differentiation, yet the same master regulators at the top of the cascade can also critically promote progenitor maintenance to ensure formation of a nervous system of the correct size, shape and integrity. How are these opposing roles coordinated? Proneural proteins Neurogenin2, Ascl1 and NeuroD4 contain conserved Serine/Threonine-Proline (S/T-P) sites that can be targeted by proline directed kinases such as cell-cycle-associated cyclin-dependent kinases (cdks); thus coordinating the activity of the cell cycle and differentiation machinery. Using neurogenesis assays in developing Xenopus embryos, the Philpott Lab has established a multi-site phospho-regulation model whereby phosphorylation on S/T-P sites by cdks or other proline-directed kinases, directly inhibits proneural activity to drive differentiation (2-4). Using NeuroD4 as an example, the wild-type (WT) gene was cloned into a plasmid vector and the serine or threonine residues mutated to alanine (S/T-A) by site-directed mutagenesis to prevent phosphorylation, creating a range of single and sequential site mutant constructs; the latter shown in Figure 1A. mRNA was transcribed in vitro and injected into two cell stage Xenopus embryos and neurogenesis was assayed in neurula stage embryos by qPCR and in situ hybridisation for expression of neural-β-tubulin as described in (4). For proneural proteins, this multi-site phosphorylation acts like a rheostat, with proneural activity sensitively regulated by the number of phosphorylation events (Figure 1B). Mechanistically, western blot analysis of over-expressed WT and phospho-mutant NeuroD4 demonstrates that the dephosphorylated protein has enhanced protein stability (Figure 2A). Additionally, by cross-linking the embryos prior to fractionation to extract cytoplasmic and nuclear fractions, even correcting for total protein level the dephosphorylated protein displays a greater association for chromatin (Figure 2B). Together these properties enable the dephosphorylated protein to drive the expression of differentiation genes that require epigenetic remodelling prior to activation (5). Multi-site phosphorylation enables coordination of differentiation activity within the cellular protein kinase environment during development. Additionally, manipulation of proneural protein phospho-status can dramatically enhance neurogenesis for cellular reprogramming (3).

Where applicable, experiments conform with Society ethical requirements