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The role of Ubiquitin System on the homeostatic control of stem cell maintenance and differentiation in crypt stem cells

Periodic Reporting for period 1 - IntestineUb (The role of Ubiquitin System on the homeostatic control of stem cell maintenance and differentiation in crypt stem cells)

Période du rapport: 2016-01-01 au 2017-12-31

The epithelium of small intestine and skin is continuously exposed to environmental assault and therefore requires regular and fast self-renewal, which is fuelled by the adult stem cells (SCs). Tight regulation of the adult SC number is required to sustain an adequately sized population for optimal tissue homeostasis. Mis-regulation of homeostasis results in either hyperplasia or loss of SCs. Although transcriptional regulation of SC homeostasis regarding multipotency and differentiation has been extensively studied, only little is known about post-transcriptional and post-translational modifications. A pivotal mechanism of post-transcriptional modification is RNA methylation by RNA methyltransferase like the NSUN family; a key mechanism of post-translational modification is ubiquitination by the ubiquitin system (US) E1, E2, E3 ubiquitin ligase and deubiquitinase (DUBs). The role of NSUNs and DUBs in adult SC homeostasis control is poorly understood.
The in vitro primary 3D intestinal organoid culture system recapitulates in vivo epithelium organization, is amenable to essentially all experimental technologies that have been developed for cell lines, and therefore serves as a novel, robust and powerful model system. Taking advantage of the intestinal organoids and the well-established human keratinocytes culture, this project aims to investigate the role of NSUNs and DUBs on the homeostatic control of stem cell maintenance and differentiation in intestine and skin adult stem cells by: 1. Screening of NSUNs/DUBs required for stem cell number regulation; 2. Characterization of the expression pattern and sub-cellular localization of candidate NSUNs/ DUBs; 3. Examining the effect of candidate NSUNs/DUBs in known signalling pathways involved in the homeostatic control; 4. Elucidating the physiological role of NSUNs/DUBs with conditional knockout mouse models.
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.
Dissecting SC regulatory pathways is fundamental to understanding how perturbed homeostasis causes diseases and cancer. Despite of research on SC regulation for decades, very little is known about the function of NSUNs/DUBs in epithelial SC homeostasis. The originality and innovative nature of this project is to bring in the prominent biochemical aspect of NSUNs/DUBs to the critical and fundamental physiology, the epithelial SC homeostasis. Notably, with the power of state-of-the-art techniques on organoids manipulation and CRISPR/Cas9-mediated gene engineering this proposal is aimed to screen with all NSUNs/DUBs genes and this global characterization of the NSUNs/DUBs will open the way for future studies that focus on specific NSUNs/DUBs enzymes or its substrates.
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