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During development of the central nervous system, the transition from progenitor maintenance to differentiation is directly triggered by a lengthening of the cell cycle that occurs as development progresses. However, the mechanistic basis of this regulation is unknown. The proneural transcription factor Neurogenin 2 (Ngn2) acts as a master regulator of neuronal differentiation. Here, we demonstrate that Ngn2 is phosphorylated on multiple serine-proline sites in response to rising cyclin-dependent kinase (cdk) levels. This multi-site phosphorylation results in quantitative inhibition of the ability of Ngn2 to induce neurogenesis in vivo and in vitro. Mechanistically, multi-site phosphorylation inhibits binding of Ngn2 to E box DNA, and inhibition of DNA binding depends on the number of phosphorylation sites available, quantitatively controlling promoter occupancy in a rheostat-like manner. Neuronal differentiation driven by a mutant of Ngn2 that cannot be phosphorylated by cdks is no longer inhibited by elevated cdk kinase levels. Additionally, phosphomutant Ngn2-driven neuronal differentiation shows a reduced requirement for the presence of cdk inhibitors. From these results, we propose a model whereby multi-site cdk-dependent phosphorylation of Ngn2 interprets cdk levels to control neuronal differentiation in response to cell cycle lengthening during development.
Fig. 3. Mutation of phosphorylation sites promotes Ngn2 activity. (A) Xenopus embryos were injected (left side) in one of two cells with either 5 or 20 pg mRNA as indicated, fixed at stage 15 and subject to in situ hybridisation for neural β-tubulin. The number of embryos scored was 39-90 per condition. (B) qPCR analysis of neural β-tubulin in stage 15 Xenopus embryos injected at the one-cell stage with 20 pg xNgn2 or 9S-A xNgn2 mRNA. Average fold increase in neural β-tubulin mRNA expression is shown normalised to GFP-injected control (mean ± s.e.m.; *, P≤0.05). (C) Mouse P19 cells transfected with mNgn2 or 9S-A mNgn2 with GFP were fixed 24 hours after transfection and stained for expression of neuron-specific βIII-tubulin (TuJ1) (red), quantitating TuJ1+ among GFP+ cells (mean ± s.e.m.). (D) qPCR analysis of βIII-tubulin in P19 cells 24 hours following transfection with mNgn2 and 9S-A mNgn2. Average fold increase in mRNA expression is shown normalised to housekeeping gene expression (mean ± s.e.m.; ***, P≤0.005).
Fig. 6. 9S-A xNgn2 is resistant to cyclin A2/cdk2-mediated suppression of neurogenesis in vivo. (A) Xenopus embryos were injected (left side) in one of two cells with 20 pg xNgn2 or 9S-A xNgn2 mRNA, together with 500 pg cyclin A2 and cdk2 mRNA, assaying for expression of neural β-tubulin at stage 15 by in situ hybridisation. (B) Semi-quantitative analysis of in situ hybridisation data. The number of embryos scored was 48-86 per condition. Neurogenesis was enhanced (+3, +2, +1), the same as (0) or reduced (–1, –2, –3) compared with the uninjected side (see Fig. S8 in the supplementary material).
Fig. 7. 9S-A xNgn2 does not require the cdk inhibitor Xic1 for activity. (A) Xenopus embryos were injected (left side) in one of four cells (dorsally targeted) with 20 pg mRNA as indicated, together with 20 ng of either control (a,c,e) or Xic1 (b,d,f) morpholino, fixed at stage 15 and subject to in situ hybridisation for neural β-tubulin expression. (B) Semi-quantitative analysis of in situ hybridisation data. The number of embryos scored was 43-59 per condition. Neurogenesis was enhanced (+3, +2, +1), the same as (0) or reduced (–1, –2, –3) compared with the uninjected side (see Fig. S8 in the supplementary material for examples of the scoring method).
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