Cell and Developmental Biology, University of Dundee, Dundee, Scotland, UK.
Centre for Gene Regulation and Expression, University of Dundee, Dundee, Scotland, UK
J Cell Biol. 2018 May 7;217(5):1667-1685. doi: 10.1083/jcb.201708023. Epub 2018 Mar 29.
During late mitosis and the early G phase, the origins of replication are licensed by binding to double hexamers of MCM2-7. In this study, we investigated how licensing and proliferative commitment are coupled in the epithelium of the small intestine. We developed a method for identifying cells in intact tissue containing DNA-bound MCM2-7. Interphase cells above the transit-amplifying compartment had no DNA-bound MCM2-7, but still expressed the MCM2-7 protein, suggesting that licensing is inhibited immediately upon differentiation. Strikingly, we found most proliferative Lgr5 stem cells are in an unlicensed state. This suggests that the elongated cell-cycle of intestinal stem cells is caused by an increased G length, characterized by dormant periods with unlicensed origins. Significantly, the unlicensed state is lost in -mutant epithelium, which lacks a functional restriction point, causing licensing immediately upon G entry. We propose that the unlicensed G phase of intestinal stem cells creates a temporal window when proliferative fate decisions can be made.
在有丝分裂后期和早期 G1 期，复制起点通过与 MCM2-7 的双六聚体结合而被许可。在这项研究中，我们研究了复制许可和增殖承诺如何在小肠上皮中偶联。我们开发了一种在含有 DNA 结合的 MCM2-7 的完整组织中鉴定细胞的方法。位于过渡扩增隔室上方的有丝分裂期细胞没有 DNA 结合的 MCM2-7，但仍表达 MCM2-7 蛋白，表明分化后立即抑制了许可。引人注目的是，我们发现大多数增殖的 Lgr5 干细胞处于未许可状态。这表明，肠干细胞的延长细胞周期是由于 G 期长度增加所致，其特征是具有休眠期的无许可起点。重要的是，在缺乏功能限制点的 - 突变上皮中，未许可状态丢失，导致 G1 进入时立即许可。我们提出，肠干细胞的未许可 G 期为增殖命运决策创造了一个时间窗口。
