Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502.
Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8502; AMED-CREST, 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan.
J Biol Chem. 2019 Jan 4;294(1):195-209. doi: 10.1074/jbc.RA118.004086. Epub 2018 Nov 8.
Lineage specification of the three germ layers occurs during early embryogenesis and is critical for normal development. The nucleosome remodeling and deacetylase (NuRD) complex is a repressive chromatin modifier that plays a role in lineage commitment. However, the role of chromodomain helicase DNA-binding protein 4 (CHD4), one of the core subunits of the NuRD complex, in neural lineage commitment is poorly understood. Here, we report that the CHD4/NuRD complex plays a critical role in neural differentiation of mouse embryonic stem cells (ESCs). We found that RNAi-mediated knockdown suppresses neural differentiation, as did knockdown of methyl-CpG-binding domain protein , another NuRD subunit. and knockdowns similarly affected changes in global gene expression during neural differentiation and up-regulated several mesendodermal genes. However, inhibition of mesendodermal genes by knocking out the master regulators of mesendodermal lineages, and through a CRISPR/Cas9 approach could not restore the impaired neural differentiation caused by the knockdown, suggesting that CHD4 controls neural differentiation by not repressing other lineage differentiation processes. Notably, knockdown increased the acetylation levels of p53, resulting in increased protein levels of p53. Double knockdown of and restored the neural differentiation rate. Furthermore, overexpression of BCL2, a downstream factor of p53, partially rescued the impaired neural differentiation caused by the knockdown. Our findings reveal that the CHD4/NuRD complex regulates neural differentiation of ESCs by down-regulating p53.
三个胚层的谱系特化发生在胚胎早期，对正常发育至关重要。核小体重塑和去乙酰化酶（NuRD）复合物是一种抑制性染色质修饰物，在谱系决定中发挥作用。然而，NuRD 复合物的核心亚基之一 chromodomain helicase DNA-binding protein 4（CHD4）在神经谱系决定中的作用知之甚少。在这里，我们报告 CHD4/NuRD 复合物在小鼠胚胎干细胞（ESCs）的神经分化中发挥关键作用。我们发现，RNAi 介导的敲低抑制神经分化，甲基-CpG 结合域蛋白的敲低也是如此，另一个 NuRD 亚基。和同样影响神经分化过程中的全局基因表达变化，并上调几个中胚层-内胚层基因。然而，通过 CRISPR/Cas9 方法敲除中胚层-内胚层谱系的主调控因子和，不能恢复由敲低引起的受损的神经分化，表明 CHD4 通过不抑制其他谱系分化过程来控制神经分化。值得注意的是，敲低增加了 p53 的乙酰化水平，导致 p53 蛋白水平增加。和的双敲低恢复了神经分化率。此外，p53 的下游因子 BCL2 的过表达部分挽救了由敲低引起的受损的神经分化。我们的研究结果表明，CHD4/NuRD 复合物通过下调 p53 来调节 ESCs 的神经分化。
