Ascl1与HDAC1的相互作用在启动脊椎动物神经元分化中的机制研究

Ascl1在体内能调控多种神经元的分化，在体外能将非神经类体细胞转分化成神经元。但是，Ascl1如何激活一些基因、关闭另一些基因，从而使得细胞能够从一种状态过渡到另一种状态的机制还不明确。我们的前期结果发现，Ascl1 N-和C-末端结构域对Ascl1在启动神经元分化中均有调控作用，并需要HDAC活性参与。Ascl1通过N-末端招募HDAC1。然而，对于不同功能结构域、HDAC如何影响Ascl1的编程及重编程活性等问题，目前没有系统研究。我们计划以非洲爪蛙胚胎为主要模式系统，通过研究不同结构域缺失后Ascl1在全基因组上结合位点的差异，不同HDAC活性条件下Ascl1选择染色质靶点组蛋白修饰的差异，来揭示Ascl1如何选择靶点、改变靶基因调控区域的染色质状态，从而启动神经元分化；同时将在哺乳类细胞中验证相关发现。预期结果将有助于优化Ascl1介导的转分化实验方案。

Ascl1 has been implicated in neural cell fate specification and differentiation in vivo; Ascl1 can also reprogram non-neural somatic cells into functional neurons in vitro. However, the molecular mechanisms underlying Ascl1-regulated cell fate transition remain elusive. Findings from our previous study using Xenopus model organism reveal that the N-terminal domain of Ascl1 is required for Ascl1 to repress mesendodermal cell fate in an HDAC-dependent manner. Ascl1 can recruit HDAC1 through the N-terminal domain of Ascl1. Deletion of the N-terminal domain also compromises the activity of Ascl1 in inducing neuronal gene expression, indicating that the N-terminal domain-mediated transcriptional repression is important for Ascl1-promoted neuronal differentiation. Removal of the C-terminal domain does not affect Ascl1 to repress mesendoderm genes, but totally abolishes its neural-related activity. In addition, application of HDAC inhibitors significantly down-regulates the expression of neuronal marker gene in early Xenopus embryos, and blocks Ascl1-induced expression of neuronal marker gene in the cultured endodermal explants. .These data collectively suggest that further dissection of the function of the N- and C-domain will facilitate to underpin the mechanisms of Ascl1-mediated cell fate transition. Therefore, we plan to perform RNA-seq analyses using Xenopus ectodermal and endodermal explants, then to determine how the genome-wide target selection of Ascl1 may be changed by deleting either the N- or the C-terminal domain. Furthermore, we will perform ChIP-seq experiments under the conditions that the activity of HDAC is increased by overexpressing HDAC1 or decreased by applying HDAC inhibitors in the Xenopus ectodermal explants, then determine how Ascl1 may choose different chromatin targets when HDAC activity is altered. We will also test the functional significance of the N-terminal domain of Ascl1 and the HDAC activity during Ascl1-peomoted neuronal differentiation in the cultured mammalian embryonal carcinoma P19 cells. In addition, we plan to perform CoIP-MS/MS experiments and identify novel epigenetic regulators associated with Ascl1. .Expected results from this proposal will help us to better understand how the N- and C-domains control the genome-wide target selection by Ascl1, how HDAC1 regulates Ascl1 to access its chromatin targets, and how Ascl1 may work together with HDAC1 and other epigenetic regulators to promote cell fate transition. Our results will also help to optimize the protocols of Ascl1-mediated reprogramming of neuronal cell fate in vitro.