Cell cycle-regulated multi-site phosphorylation of Neurogenin 2 coordinates cell cycling with differentiation during neurogenesis

Abstract

SUMMARYDuring 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 cyclindependent 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 cdkdependent phosphorylation of Ngn2 interprets cdk levels to control neuronal differentiation in response to cell cycle lengthening during development.

Molecular ties between the cell cycle and differentiation in embryonic stem cells

“…Indeed, many terminal cell systems couple cell cycle inhibition with differentiation (1), and many potent transcription factors interact with cell cycle components in ways similar to MyoD, including Ngn2, Pdx1, Smad3, Mitf, Runx2, PU.1, Hnf4α, and C/EBPβ (17)(18)(19)(20)(21)(22)(23)(24). Whereas experimental validation and extension of our findings with MyoD to other lineages remains to be Fig.…”

How to keep proliferative neural stem/progenitor cells

“…There are many other reports showing that cell cycle regulators may function as cell fate determinants by a role independent of cell cycle regulation. 20,21,43,44 Furthermore, another detailed analysis suggests that not only G 1 -phase but also S-phase is correlated with the differentiation state of NSPCs. 38 Thus, the physiological functions of cyclin D2 in aspects of fate determination in vivo still remain to be elucidated.…”

“…This cascade of events progresses the cell cycle from G 1 -to S-phase. 15,16 It is well known that neuronal progenitor cell fate can be affected by cell cycle regulators including cyclins, [17][18][19] Cdks 20 and their inhibitors. [21][22][23][24] In the developing central nervous system (CNS), mRNA of cyclin D2 shows a unique localization, to the surface of the neural tube, not seen for other cyclins.…”

Retinoic acid signaling and neuronal differentiation

“…p27 xic1 , a Xenopus Cip/Kip protein, is required for primary neurogenesis [120][121][122] and functions by CDK inhibition which prevents phosphorylation and promotes stabilization of the Neurogenin protein [120,123]. Recently, p27 xic1 , was shown to be phosphorylated by atypical protein kinase C (aPKC), which prevents CDK inhibition, shortens the G1 phase of neural progenitors, and encourages their proliferation [124].…”