Ascl2 acts as an R-spondin/Wnt responsive switch to control stemness in intestinal crypts [ChIP-seq]

Wnt signals control three functions of intestinal crypts: maintenance of Lgr5 stem cells, proliferation of transit-amplifying daughters and formation of Paneth cells. Here, we study how the Wnt effector β-catenin/Tcf4 cooperates with the Wnt-activated transcription factor Ascl2 to control a stem cell transcription program. DNA elements that are co-occupied and synergistically regulated by Ascl2 and Tcf4 specifically map to stem cell genes. In vitro, Tcf4-/- mini-guts are rescued by Ascl2 expression, while Ascl2-/- organoids are rescued by Wnt signaling. A direct auto-activatory loop leads to an on/off expression pattern of Ascl2 with a threshold that depends on the previous state. Wnt/R-spondin1 activates this loop. This mechanism interprets Wnt levels in crypts and translates this continuous signal into a discrete Ascl2 “on” or “off” decision. In turn Ascl2, together with β-catenin/Tcf, activates stem cell genes. Thus, Ascl2 forms a transcriptional 'stemness switch' that is both Wnt-responsive and Wnt-dependent Examination of Tcf4, B-catenin and Ascl2 DNA occupancy in murine intestinal organoids and human colorectal cancer cell lines *** Original raw files unavailable due to loss during backup ***

Wnt信号（Wnt signals）调控肠隐窝的三项核心功能：维持Lgr5干细胞（Lgr5 stem cells）的稳态、促进转运扩增子代细胞增殖，以及潘氏细胞（Paneth cells）的生成。本研究探讨了Wnt效应蛋白β-连环蛋白/Tcf4（β-catenin/Tcf4）与Wnt激活的转录因子Ascl2如何协同调控干细胞转录程序。分析显示，Ascl2与Tcf4共同占据并协同调控的DNA元件，特异性富集于干细胞基因的调控区域。体外实验证实，Tcf4基因敲除（Tcf4-/-）的微型肠类器官（mini-guts）可通过过表达Ascl2得以挽救；而Ascl2基因敲除（Ascl2-/-）的类器官则可通过激活Wnt信号通路实现修复。本研究发现存在一条直接的自动激活环路，该环路使Ascl2的表达呈现严格的“开/关”模式，其激活阈值依赖于细胞的先前状态；Wnt/R-spondin1可直接激活该环路。此机制可将肠隐窝内的连续Wnt信号水平解读为离散的Ascl2“开启”或“关闭”细胞命运决策。反之，Ascl2与β-连环蛋白/Tcf协同激活干细胞相关基因。综上，Ascl2构成了一个兼具Wnt信号响应性与依赖性的转录“干性开关（stemness switch）”。研究人员还在小鼠肠类器官与人类结直肠癌细胞系中，检测了Tcf4、β-连环蛋白（β-catenin）与Ascl2的DNA结合位点。*** 原始数据文件因备份丢失无法获取 ***