The role of CRL4 ubiquitin ligase complex in homeostasis and regeneration of gastro intestinal tract

• Thesis title in Czech: Úloha CRL4 ubiquitin-ligázového komplexu při homeostáze a regeneraci gastro-intestinálního traktu
• Thesis title in English: The role of CRL4 ubiquitin ligase complex in homeostasis and regeneration of gastro intestinal tract
• Key words: ubiquitin-ligáza, gastrointestinální trakt, regenerace, knockoutní myš
• English key words: ubiquitin ligase, gastrointestinal tract, regeneration, knockout mouse
• Academic year of topic announcement: 2016/2017
• Thesis type: dissertation
• Thesis language: angličtina
• Department: Department of Genetics and Microbiology (31-140)
• Supervisor: Mgr. Jan Procházka, Ph.D.
• Author: hidden - assigned and confirmed by the Study Dept.
• Date of registration: 21.10.2016
• Date of assignment: 24.10.2016
• Confirmed by Study dept. on: 24.10.2016
• Date of electronic submission: 29.08.2023
• Date of proceeded defence: 06.12.2023
• Opponents: Mgr. Lucie Janečková, Ph.D.
• Advisors: doc. RNDr. Radislav Sedláček, Ph.D.

Preliminary scope of work in English

CRL4 ubiquitin ligase complex is a part of important regulatory machinery. Significant component of the CRL4 complex is Cul4a protein (with homolog Cul4b), which belongs to the cullin family of E3 ubiquitin ligase proteins. Normal function of Cul4a/b is a key of genome stability, chromatin remodeling both adequate cell cycle and cell migration. CRL4 is ubiquitination agent for DNA repair proteins, which activated by certain types of DNA damage, and contribute to avoid genome mutations. Nevertheless, CRL4 is a target for different damaging agents, which leads to pathology development. The striking example is an action of the teratogenic agent thalidomide.
It was found that aberrant expression of the Cul4a gene is a cause of many tumor types, while mutations of the Cul4b gene are causally associated with human X-linked mental pathologies [1]. Herewith tumor progression linked to Cul4a overexpression with activation of cell cycle due to reduction the steady-state levels of the CDK inhibitor. Other hand, its action is important for cell surviving and activation tissue regeneration processes.
Thereby, in this project planned study will be the role of CRL4 ubiquitin ligase complex in homeostasis and regeneration of GIT epithelium. For identification and lineage tracing of Cul4a expressing cell population I will be prepare of Cul4a^-creERT2 transgenic mouse model. At this stage I will use CRISPR/Cas9 technology for genome modification and homologues recombination for knock in creERT2 into Cul4a locus.
Our next point of interest will gut physiology. Particularly, our plan is characterize the role of Cul4a/b family genes in regenerative processes of GIT epithelium. Ablation of Cul4a/b expression in vivo by conditional inactivation (with use of Cul4a^flox and Cul4b^flox transgenic mice) of both genes specifically in intestine epithelium should provide us knowledge about CRL4 role in gut stability, inflammatory processes and other damages. Molecular cloning, in situ hybridization, qPCR expression profiling and immunostaining methods would be relevant approaches at this stage. Further I will use mouse models for colorectal carcinoma and assess the role of Cul4a expressing cells during carcinoma initiation and progression, this part of my work will mainly rely on lineage tracing experiments and analysis of clonal behavior of Cul4a progeny in tumor by Confetti clonal reporter.