泛素连接酶RNF220在神经系统图式形成中的功能与作用机制

The patterning of vertebrate nervous system during early development is governed by a group of signaling pathways, which control the regional expression of a panel of transcription factors. Sonic hedgehog (Shh) signaling through Gli is responsible for the patterning of the ventral neural tube and the Hox genes play crucial roles in the patterning of the hindbrain and spinal cord along the anterior-posterior axis. RNF220 is a RING-finger E3 ubiquitin ligase specifically expressed in the ventral part of the developing mid-hindbrain and spinal cord in both Xenopus and mouse. RNF220 knockout mouse pups were neonatal lethal. Interestingly, a group of Hox genes became abnormally activated in the hindbrainin of RNF220-/- mouse embryos, suggesting general disorder of hindbrain patterning. We have evidence that RNF220 might work through SMCHD1/EED to affect the function of the PRC2 complex, which is required to repress Hox gene expression. We also observed abnormal patterning of the ventral spinal cord in RNF220-/- mouse embryos, supporting a role of RNF220 in Shh signaling, which might be mediated by Gli proteins. In this project, we will analyze in detail the structural and functional defects of the RNF220-/- mouse hindbrain and spinal cord, and dissect the mechanisms involved in the regulation of Hox gene expression and Shh signaling.

脊椎动物神经系统的早期发育受到多种信号通路的调控，通过调节其下游转录因子的区域特异性表达，调控神经组织的区域分化。Shh/Gli信号主要参与神经管腹侧神经的分化，而Hox基因在后脑与脊髓的前后轴分化中具有关键作用。我们的前期研究表明，泛素连接酶RNF220的缺失会导致小鼠发育后期中后脑Hox基因的表达紊乱，脊髓腹侧神经的分化异常。初步分析表明RNF220可能通过对SMCHD1/PRC2组分EED和Gli的调节，参与对Hox基因表达和Shh信号的调控。本项目拟细致分析RNF220缺失导致的神经发育表型，并通过分子细胞生物学、遗传学方法研究RNF220在Hox基因表达与Shh信号调控的功能与作用机制。本研究对于揭示Hox基因表达和Shh信号调控的新机制将具有重要贡献。

RNF220是本课题组鉴定的一个泛素连接酶。本项目结合基因敲除模型，利用分子与细胞生物学等技术，系统研究了RNF220及其辅助因子ZC4H2在神经早期发育等过程中的功能与作用机制。结果发现，RNF220与ZC4H2通过靶向Gli蛋白的K63类型的泛素化修饰调控腹侧神经管Shh/Gli的浓度梯度并影响神经图式形成；在小脑早期发育过程中，RNF220可以通过调控表观因子EED蛋白的稳定性维持Shh信号的高活性，促进颗粒祖细胞的增殖；在蓝斑核的发育中，RNF220通过促进转录因子Phox2a/2b的单泛素化修饰参与后脑去甲肾上腺素能神经元的分化发育。在脊髓中，RNF220可通过调控TDP43蛋白的稳定性参与调节运动神经元的功能与存活。此外，还发现RNF220可通过STAT1调控干扰素信号通路。我们还解析了RNF220通过WDR5调控后脑Hox基因表达的新机制及其潜在功能。相关研究已在Cell Reports、Development等国际主流期刊发表SCI论文7篇，培养硕、博士研究生4名， 完成了预定目标。