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 Mar 26;511(1):111-116. doi: 10.1016/j.bbrc.2019.02.045. Epub 2019 Feb 14.
The superfamily of basic-Helix-Loop-Helix (bHLH) transcription factors influence cell fate in all three embryonic germ layers, and the tissue-specific class II factors have received prominent attention for their potent ability to direct differentiation during development and in cellular reprogramming. The activity of many class II bHLH proteins driving differentiation, and the inhibitory class VI bHLH factor Hes1, is controlled by phosphorylation on multiple sites by Cyclin-dependent kinases (Cdks). As class II proteins are generally thought to be active through hetero-dimerisation with the ubiquitously expressed class I E proteins, regulation of class I transcription factors such as E47 may influence the activity of multiple tissue-specific bHLH proteins. Using differentiation of nerve and muscle in Xenopus frog embryos as a model system, we set out to explore whether with the ubiquitously expressed class I E protein E47 that hetero-dimerises with Class II bHLHs to control their activity, is also regulated by multi-site phosphorylation. We demonstrate that E47 can be readily phosphorylated by Cdks on multiple sites in vitro, while ectopically-expressed E47 exists in multiple phosphorylated forms in Xenopus embryos. Preventing multi-site phosphorylation using a phospho-mutant version of E47 enhances the neurogenic and myogenic activity of three different class II bHLH reprogramming factors, and also when E47 acts in hetero-dimerisation with endogenous proteins. Mechanistically, unlike phospho-regulation of class II bHLH factors, we find that preventing phosphorylation of E47 increases the amount of chromatin-bound E47 protein but without affecting its overall protein stability. Thus, multi-site phosphorylation is a conserved regulatory mechanism across the bHLH superfamily that can be manipulated to enhance cellular differentiation.
碱性螺旋-环-螺旋（bHLH）转录因子超家族影响所有三个胚胎胚层的细胞命运，组织特异性的 II 类因子因其在发育和细胞重编程过程中指导分化的强大能力而受到广泛关注。许多驱动分化的 II 类 bHLH 蛋白的活性，以及抑制性的 VI 类 bHLH 因子 Hes1 的活性，都受到细胞周期蛋白依赖性激酶（Cdks）在多个位点的磷酸化调控。由于通常认为 II 类蛋白通过与普遍表达的 I 类 E 蛋白异二聚化而具有活性，因此 I 类转录因子（如 E47）的调节可能会影响多种组织特异性 bHLH 蛋白的活性。我们使用非洲爪蟾胚胎的神经和肌肉分化作为模型系统，旨在探索普遍表达的 I 类 E 蛋白 E47 是否也通过多位点磷酸化来调节与 II 类 bHLH 蛋白的异二聚化来控制其活性。我们证明 E47 可以在体外被 Cdk 快速磷酸化多个位点，而在非洲爪蟾胚胎中外源表达的 E47 存在多种磷酸化形式。使用 E47 的磷酸化突变体版本防止多位点磷酸化，增强了三种不同的 II 类 bHLH 重编程因子的神经生成和肌生成活性，并且当 E47 与内源性蛋白异二聚化时也是如此。从机制上讲，与 II 类 bHLH 因子的磷酸化调节不同，我们发现，防止 E47 的磷酸化增加了染色质结合的 E47 蛋白的量，但不影响其整体蛋白稳定性。因此，多位点磷酸化是 bHLH 超家族的一种保守调节机制，可以通过这种机制来增强细胞分化。
