Znhit1基因在Lgr5小肠干细胞命运决定中的作用及其机制

The balance between the self-renewal and differentiation of Lgr5+ intestinal stem cells, which locate at the bottom of the intestinal crypts, is critical for the intestinal epithelium homeostasis. Recent studies revealed the functions of several signal pathways in Lgr5+ intestinal stem cell fate determination, however, the mechanism of how these signal pathways integrate at transcription level, and how they cooperate with critical transcription regulating events, such as chromosome remodeling, to determine the fate of Lgr5+ intestinal stem cells is currently unclear. In our preliminary research, we generated Znhit1 gene conditional knockout mice and found that gut-epithelium-specific knockout of Znhit1 leads to failure of Lgr5+ intestinal stem cell postnatal specification and loss of crypt function. Besides, inducible knockout of Znhit1 in adult intestinal epithelium ablates Lgr5+ intestinal stem cells and disrupts intestinal homeostasis. In this proposal, we plan to investigate the detailed mechanism of how Znhit1 controls Lgr5+ intestinal stem cell specification and maintenance. We will examine whether Znhit1 and Znhit1-mediated H2A.Z incorporation can determine the fate of Lgr5+ intestinal stem cells through regulating the expression of critical stemness-related genes, or altering the activity of critical signals. The expected results will provide new insights into the fate determination of Lgr5+ intestinal stem cells and lay a foundation for its applications in the regenerative medicine.

位于小肠腺窝底部的Lgr5小肠干细胞是小肠上皮组织稳态维持的关键。现对其命运决定机制的研究多局限于探讨单条信号通路的功能，这些关键信号通路如何在转录层面整合，并与表观遗传、染色质重构等重要转录调控事件相互协作，来决定Lgr5小肠干细胞的命运并不清楚。在前期工作中，申请人制作了Znhit1基因条件敲除小鼠，并发现Znhit1在肠上皮的特异性敲除导致出生后Lgr5小肠干细胞谱系定向失败和小肠陷窝功能缺陷；同时，在成体小肠上皮诱导敲除该基因可导致Lgr5小肠干细胞缺失和小肠上皮稳态的紊乱。此项目将在此基础上，详细阐述Znhit1调控小肠干细胞谱系定向和干性维持的分子机制，探究Znhit1及其介导的组蛋白变异体H2A.Z整合能否通过改变关键干性基因的表达水平，或调控关键信号通路活性，来决定小肠干细胞命运。预期成果将为全面了解Lgr5小肠干细胞的命运决定方式，进而将其利用到再生医学领域提供重要基础。

位于小肠腺窝底部的Lgr5小肠干细胞是小肠上皮组织稳态维持的关键。现对其命运决定机制的研究多局限于探讨单条信号通路的功能，这些关键信号通路如何在转录层面整合，并与表观遗传、染色质重构等重要转录调控事件相互协作，来决定Lgr5小肠干细胞的命运并不清楚。在前期工作中，申请人制作了Znhit1基因条件敲除小鼠，并发现Znhit1在肠上皮的特异性敲除导致出生后Lgr5小肠干细胞谱系定向失败和小肠陷窝功能缺陷；同时，在成体小肠上皮诱导敲除该基因可导致Lgr5小肠干细胞缺失和小肠上皮稳态的紊乱。我们的研究揭示了Znhit1作为染色质重塑因子通过介导组蛋白变体H2A.Z在Lgr5+干细胞命运决定基因TSS上的整合调控Lgr5+小肠成体干细胞谱系和干性维持，参与小肠上皮稳态维持；进一步明确了染色质重塑在造血干细胞命运决定与造血稳态中的关键作用。此外，通过开发一种实现胆管类器官高效基因操纵的体系，证明了AAV-DJ在胆管类器官中进行基因操纵的实用性和可靠性。