Department of Oncology, University of Cambridge, Hutchison/MRC Research Centre, Cambridge Biomedical Campus, Cambridge, CB2 0XZ, UK; Peterhouse, University of Cambridge, Trumpington Street, Cambridge, CB2 1RD, UK.
Department of Oncology, University of Cambridge, Hutchison/MRC Research Centre, Cambridge Biomedical Campus, Cambridge, CB2 0XZ, UK; Wellcome Trust/MRC Cambridge Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR, UK.
Biochem Biophys Res Commun. 2019 Feb 5;509(2):557-563. doi: 10.1016/j.bbrc.2018.12.135. Epub 2018 Dec 29.
The processes of cell proliferation and differentiation are intimately linked during embryogenesis, and the superfamily of (basic) Helix-Loop-Helix (bHLH) transcription factors play critical roles in these events. For example, neuronal differentiation is promoted by class II bHLH proneural proteins such as Ngn2 and Ascl1, while class VI Hes proteins act to restrain differentiation and promote progenitor maintenance. We have previously described multi-site phosphorylation as a key regulator of tissue specific class II bHLH proteins in all three embryonic germ layers, and this enables coordination of differentiation with the cell cycle. Hes1 homologues also show analogous conserved proline directed kinase sites. Here we have used formation of Xenopus primary neurons to investigate the effects of xHes1 multi-site phosphorylation on both endogenous and ectopic proneural protein-induced neurogenesis. We find that xHes1 is phosphorylated in vivo, and preventing phosphorylation on three conserved SP/TP sites in the N terminus of the protein enhances xHes1 protein stability and repressor activity. Mechanistically, compared to wild-type xHes1, phospho-mutant xHes1 exhibits greater repression of Ngn2 transcription as well as producing a greater reduction in Ngn2 protein stability and chromatin binding. We propose that cell cycle dependent phosphorylation of class VI Hes proteins may act alongside similar regulation of class II bHLH proneural proteins to co-ordinate their activity.
细胞增殖和分化的过程在胚胎发生过程中密切相关，（基本）螺旋-环-螺旋（bHLH）转录因子超家族在这些事件中发挥着关键作用。例如，神经元分化由 II 类 bHLH 神经前体蛋白如 Ngn2 和 Ascl1 促进，而 VI 类 Hes 蛋白则抑制分化并促进祖细胞维持。我们之前描述了多部位磷酸化作为所有三个胚胎胚层中组织特异性 II 类 bHLH 蛋白的关键调节剂，这使得分化与细胞周期协调一致。Hes1 同源物也显示出类似的保守脯氨酸定向激酶位点。在这里，我们使用形成的非洲爪蟾原代神经元来研究 xHes1 多部位磷酸化对内源性和异位神经前体蛋白诱导的神经发生的影响。我们发现 xHes1 在体内被磷酸化，并且阻止蛋白质 N 端三个保守的 SP/TP 位点的磷酸化增强了 xHes1 蛋白的稳定性和抑制子活性。从机制上讲，与野生型 xHes1 相比，磷酸化突变体 xHes1 对 Ngn2 转录的抑制作用更大，并且导致 Ngn2 蛋白稳定性和染色质结合的降低更大。我们提出，细胞周期依赖性 VI 类 Hes 蛋白的磷酸化可能与类似的 II 类 bHLH 神经前体蛋白的调节一起协调它们的活性。
