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 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.

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。

胚胎发育中细胞谱系命运决定是一个逐渐特化的过程，Ascl1在神经细胞细胞命运决定中发挥多种作用，本课题的前期工作表明 Ascl1在囊胚期和原肠胚期具有组织胚胎前体细胞分化成中内胚层命运的活性，并且发现Ascl1通过与辅助抑制因子HDAC1相互作用阻遏中内胚层基因的表达，本课题重点关注Ascl1与HDAC1的相互作用机制及其在神经发育中的功能。本课题试图从4个不同角度回答这一问题：1）通过过表达实验，详细分析Ascl1的氨基端和羧基端进行结构域功能的关联性，揭示Ascl1调控神经元相关基因表达的机制。2）通过敲降和小分子干扰等方案揭示HDAC1在神经决定和分化中的作用。3）结合过表达和敲降实验，验证Ascl1-HDAC1在神经基因表达调控中的作用。4）结合多种技术方案，分析Ascl1转录活性调控上下游分子机制。取得如下重要结果：鉴定出Ascl1与HDAC1相互作用的保守基序；确定Ascl1 C-末端的转录刺激活性；通过IP-MS技术方案获得Ascl1相互作用蛋白；揭示HDAC1在神经决定和分化中的重要作用；鉴定了Ascl1关键靶基因Msi1。这些研究结果进一步阐明了Ascl1 与HDAC1互作的分子机制，揭示了细胞谱系决定转录因子Ascl1如何与组蛋白修饰酶HDAC1互作进而影响细胞命运的选择与分化的新机理。课题执行期间发表标注研究论文4篇，其中与课题直接相关2篇，间接相关2篇。