Characterization of novel E3 ubiquitin ligases that are enriched in LGR5-positive intestinal stem cells and niche. (360G-Wellcome-101241_B_13_Z)

In the intestine, LGR5-positive (LGR5+) crypt base columnar cells comprise a well-defined adult stem cell population. FACS isolation of LGR5-positive intestinal stem cells have facilitated the expression profiling of LGR5+ stem cells, which established stem cell-specific genes of the adult intestine. Amongst are two homologous E3 ubiquitin ligases, RNF43 and ZNRF3, the inactivation of which resulted in unrestricted expansion of intestinal stem cells and formation of adenomas due to hyper-activat ion of Wnt signalling. This has started uncovering the importance of E3 ligases in regulating intestinal stem cells. Besides RNF43 and ZNRF3, additional uncharacterized E3 ubiquitin ligases are enriched in intestinal stem cells. By using cell-surface marker CD24 to sort and profile Paneth cells, novel additional E3 ubiquitin ligases were also detected in these niche cells. These novel E3 ubiquitin ligases are the focus of this research plan, since it is anticipated that they play an important ro le in stem cells, niche cells and their interaction. The plan includes: 1) Functional screening of candidate E3s using primary 3D intestinal organoid culture; 2) Identifying target proteins of candidate E3s using surface proteome analysis and IP-MS; and 3) Validating the role of candidate E3s using mouse genetics in vivo.