Rôle de TFiH dans la régulation de l'expression des gènes par divers récepte nucléaires – iHREGULATOR

1- Scientific background and objectives TFIIH is a multiprotein complex involved in both transcription as well as DNA repair. Mutations in its two largest subunits (XPD and XPB helicases) yield the rare autosomal recessive disorders, such as Xeroderma Pigmentosum (XP) and Trichothiodystrophy (TTD), initially defined as DNA repair syndromes. These patients have severe clinical features, such as mental retardation, skeletal abnormalities, dwarfism and skin cancers. The clinical complexity of these syndromes cannot be explained solely on the basis of a DNA repair defect but may also involve transcription deficiencies. Indeed, we observed in cells derived from patients bearing different mutations in the xpd gene a deficiency of the ligand-dependent transactivation mediated by several hormonal nuclear receptors (NR), such as Vitamin D Receptor (VDR), Peroxisome Proliferator Activated Receptors (PPAR) and Thyroid hormone Receptors (TR). Moreover, their transactivation defect is associated with a lower capability of TFIIH to phosphorylate these receptors via its cdk7 kinase subunit. However, the role of this phosphorylation is unknown. The aim of our project is thus: - to elucidate the role of the phosphorylation by cdk7 of several activators including NR. - to analyze in a physiological context how the defect of transactivation mediated by NR might affect the development of the individual.

2- Description of project, methodology Our project will be focused on the link between TFIIH and VDR, PPARs or TR, which are known to be involved in the skeleton architecture, the energy homeostasis and the neurological development, processes affected in XP and TTD patients. These receptors will be used as models to analyze whether their phosphorylation by cdk7 promotes the recruitment and/or the release of other transcriptionnal factors on the promoter of target genes. Unphosphorylated or constitutively mutated forms of these NR will be used in immunoprecitpitation assays. In parallel, stable cell lines overexpressing wild-type or mutated NR on their phosphorylated site will be generated. We will then study in these cells by quantitative RT-PCR the expression of specific target genes after treatment with ligand for each NR. Post-translational modifications, recruitment and association of NR with promoters and co-factors (such as co-repressors, co-activators, transcription machinery, TFIIH) will be analyzed by using various technical approaches, such as ChIP assays, IP methods, confocal microscopy. The consequences of phospho/dephosphorylation of NR will be analyzed in functional tests, such as in vitro transcription assays that we would like to set up. In parallel, we will use TTD mice model to analyze in a physiological context the incidence of transactivation defect by NR, such as TR in the brain. This work first requires to study the development of the disease in this trangenic mice. Our objective is to applied 3D high resolution X-ray computed tomography (in collaboration with IRCAD) on TTD animals to follow individually the temporal and spatial outcome of target organs (adipose tisues, liver,…). Robotized biopsies will be also performed to isolate small cores of each tissue of interest for histological and molecular analyses (RT-PCR, Western Blot, ChIP,…). Finally, the in vivo action of TFIIH on NR activity will be analyzed in TFIIH-invalided mice after infection with the Rift Valley Fever Virus (RVFV). Indeed, this bunyavirus is known to specifically inhibit TFIIH activity through its NSs protein in the liver and the brain. We will thus be able to study the blockage of TFIIH through NSs protein and its consequences on transactivation by NR in RVFV infected mice. Histopathological analysis will be realized and NR target genes expression, PPARs, TR, TFIIH, NSs or co-factors recruitment will be investigated by RT-PCR and ChIP methods in the liver and the brain.

3- Expected results This project will permit to better understand the molecular action of NR and their transcriptional partners. We will thus be able to determine : - whether TFIIH, via its kinase activity, affects the NR activity and also the activators in general, according to a common mechanism. - whether the TFIIH action on the transactivation by NR only occurs during specific steps of development and/or in tissue-specific manner. - how the phosphorylation affects in a selective way the expression of specific target genes. In fine, we will be able to establish a link between transcriptional defect and the emergence of clinical features observed in patients, like the lipodystrophy and the neurological disorders. To understand how these factors act in mammalian cells remains a formidable challenge and our group possess all the expertise and the tools to fulfill such a project. Obviously the information that we will obtained from these studies will help in the future to correct or to attenuate the phenotypes of the XP/TTD patients.