To investigate the role of NSUNs/DUBs on epithelial homeostasis, at the first step, I designed and performed a loss-of-function screening to target all genes of the NSUNs and DUBs in mouse intestinal organoids based on the CRISPR/Cas9 technology. In order to detect effects on stem cell numbers, I used the state-of-the-art genetic engineering and organoid manipulation techniques to generate an organoid line with all Lgr5-expressing stem cells marked by eGFP. In addition, a more efficient gRNA library was designed and constructed. With these two tools, a pilot screen for DUBs was completed. Secondly, gene expression of DUBs and NSUNs at RNA level was analysed on mouse intestinal organoids, human keratinocytes, several adult mouse organs and mouse embryos at various developmental stages. Subsequently, generation of two conditional knockout mouse models for the most promising DUBs were commenced for further physiological studies.
Homeostasis is tightly controlled by well-characterized signalling pathways (e.g. Wnt, Notch, EGF, BMP, Hippo, and Hedgehog pathways) and mis-regulation of these signalling pathways often leads to hyperplasia or loss of stem cells. Wnt signalling constitutes the key pathway to maintain stem cell fate and drive proliferation of stem cells. Although Wnt signalling pathway is well known to play pivotal roles in intestinal stem cell proliferation, so far, all studies mostly addressed the effects of Wnt signalling through transcriptional activation by ß-catenin. In this project, I focused on the new branch of Wnt signalling pathway, that is transcription independent and regulates protein abundance, the Wnt-dependent stabilization of proteins signalling pathway (termed Wnt/STOP signalling). In mouse intestinal organoids, the phenomenon of Wnt/STOP signalling pathway was detected and DUBs as potential Wnt/STOP signalling pathway targets were identified.
