Abstract
Le nombre de caractères doit être compris entre 1700 caractères et 4000 caractères.Les résumés déposés ici doivent être identique aux résumés présents dans votre thèse.Gene expression is regulated by many chromatin factors including the Mediator complex. This multiprotein complex transmits the signal from enhancer sequences to the transcriptional machinery on promoters, and is composed of four modules. The kinase module contains CDK8 (Cyclin-dependant kinase 8), or CDK19 of the same CDK subfamily. These kinases modulate many transcriptional pathways. Furthermore, CDK8 has been ascribed various roles in cancers, which are highly variable depending on the context (pro- or anti-tumor effects). But since one kinase can replace the other in the kinase module, we do not yet understand the specific and redundant functions of these kinases. The objective of my thesis was to characterize the role of both Mediator kinases, on cell proliferation, gene expression and the response to cellular stress.After the generation of MEFs (Murine Embryonic Fibroblasts) knocked out for Cdk8, Cdk19 and Cdk8/19 by inducible Cre-Lox recombination and CRISPR-Cas9 approaches, I observed a reduction in the growth of populations of Cdk8/19 KO MEFs. Cell viability was not affected, but the loss of Cdk8/19 increased the expression of the cell cycle inhibitor p21. In addition, the loss of CDK8 and CDK19 hindered the exit of cells from quiescence induced by serum starvation.I also tested putative CDK8/19 inhibitors in vitro. To this end, I characterized their effect on CDK8/19 by analysing a model substrate, the phosphorylation of serine 727 of STAT1. Treatment of MEF with a specific inhibitor led to a reduced growth.The transcriptional effect of Cdk8/19 knockout or inhibition was then investigated. Given that the Mediator complexis essential for transcription the transcriptome was surprisingly little affected. The redundancy of CDK8 and CDK19 was clearly visible, while their combined inhibition had a distinct effect from their KO. Among the genes deregulated by both inhibition and loss of Cdk8/19, many are p53 targets, such as the cell cycle inhibitors Cdkn1a, Cdkn1b and Cdkn1c. UV-C response genes were also deregulated, and I focused on this novel finding.I found that loss, but not inhibition, of Cdk8/19 sensitizes MEFs to UV-C-induced apoptosis. In addition, in irradiated Cdk8/19 KO MEFs, p53 stabilisation occurs more rapidly, and depends on PML protein. While CDK8 was diffuse throughout the nucleus PML nuclear bodies were localized in areas with higher signal. Surprisingly, depletion of p53 and PML by siRNA further increased UV-C-induced apoptosis in Cdk8/19 KO MEFs. It therefore seems that the stabilization of p53 via PML limits apoptosis in mutant Cdk8/19 cells, perhaps by blocking the cell cycle. Finally, there was an increase in DNA damage in Cdk8/19 KO MEFs.Thus, my results reveal substantial redundancy between CDK8 and CDK19, as well as differences between inhibition and gene knockout. Moreover, my work suggests that these kinases, in which are dispensable in basal conditions, are essential for the cellular response to stress.