Multi-site phospho-regulation of proneural transcription factors controls proliferation versus differentiation in development and reprogramming

Abstract

During development of the nervous system, it is essential to co-ordinate the processes of proliferation and differentiation. Basic helix-loop-helix transcription factors play a central role in controlling neuronal differentiation and maturation as well as being components of the combinatorial code that determines neuronal identity. We have recently shown that the ability of the proneural proteins Ngn2 and Ascl1 to drive neuronal differentiation is inhibited by cyclin dependent kinase-mediated multi-site phosphorylation. This limits downstream target promoter dwell time, thus demonstrating a direct mechanistic regulatory link between the cell cycle and differentiation machinery.Proneural proteins are key components of transcription factor cocktails that can bring about the direct reprogramming of human fibroblasts into neurons. Building on our observations demonstrating that phospho-mutant proneural proteins show an enhanced ability to drive neuronal differentiation in vivo, we see that replacing wild-type with phospho-mutant proneural proteins in fibroblast reprogramming cocktails significantly enhances the axonal outgrowth, branching and electrophysiological maturity of the neurons generated. A model is presented here that can explain the enhanced ability of dephosphorylated proneural proteins to drive neuronal differentiation, and some unanswered questions in this emerging area are highlighted.

Keywords: Xenopus; cell cycle; cyclin-dependent kinase; development; differentiation; phosphorylation; proneural; reprogramming; transcription factor.

Figure 1.

(A) Overexpression of wild-type Ngn2 induces limited ectopic neurons in the Xenopus neural plate and ectoderm on the injected side of the embryo, while 9S-A Ngn2 (phospho-mutant Ngn2) shows considerably greater activity, producing extensive ectopic neurons (arrows) on the flank of the embryo. Taken from ref. (B) Brn2, Ascl1, MyT1L and NeuroD together can reprogram human lung fibroblasts into neurons, stained here in green with neural β- tubulin. When wild-type Ascl1 is replaced by S-A Ascl1 (phospho-mutant Ascl1), neurons show significantly increased axonal outgrowth and branching. Taken from ref.

Figure 2.

Model illustrating why phosphorylated Ngn2, found when cdk kinase levels are high, favors progenitor maintenance. Conversely, dephosphorylation of Ngn2 that occurs when cdk levels drop and cdk inhibitors increase, favors differentiation. Taken from ref.

Figure 3.

Model illustrating how changing promoter dwell time by bHLH transcription factors by altering their phospho-status would have differing effects on promoter activation depending on their epigenetic availability (described in more detail in the text).