Transcription and Human Genetic Disorders – TransDis

In order to perform this project, we will particularly study transgenic mouse models harboring mutations in the TFIIH complex (already available in our lab) and the Mediator. Indeed, transgenic mice bearing mutations in the Mediator complex (Med12/R961W, Med17/L371P, Med23/R617Q) are currently being generated. Such models will allow us to perform gene expression analyses (quantitative RT-PCR, microarrays) and immunoprecipitation (ChIP) assays in combination with macroscopic, histological (H&E staining, oil-Rd-O staining…) and biochemical (immunoprecitpitation, western blots, protein purifications…) assays.
In parallel, several molecular and biochemicals approaches will be performed from cell lines isolated from patients. In particular, these cellular models will allow us to investigate how the NER factors influence the transcription, and more particularly the formation of chromatin loop, histones modifications as well as the CpG demethylation around active promoters. In particular, Chromatin Immunoprecipitations and Chromatin Conformation Capture will be performed to study the chromatin loop formation. Moreover, a number of cell lines isolated from patients will allow us to generate human Induced Pluripotent Stem (hiPS) cells that will allow us to study in a cell-specific context the incidence of mutations in the different factors of interest. The development of the hiPS should be considered as a Task per se, according to the fact that this newfangled cellular technology should be implemented in our laboratory.

The project has been initiated 6 months ago. We particularly focused our attention on the generation of hiPS and the new transgenic mouse models (harbouring mutations in the Mediator). In parallel, experiments have been undertaken from mice bearing TFIIH mutations and cell lines isolated from patients. Our first results suggest that the bone metabolism in TFIIH mutated mice is differently affected depending to the nature of the mutations. Moreover, our studies from cells mutated in NER factors revealed that XPC is strongly implicated in the chromatin loop formation around active promoters. Finally, we observed that transcription is differently affected in cells bearing various mutations in the Mediator complex.

We will continue the project (...), with a special attention to the development of the hiPS and the new transgenic animals. In parallel, various experimental approaches (mass spectrometry, RT-PCR, ChIP, 3C ...) will be undertaken, according to the Task Calendar proposed in our project.

To date, neither publications nor patents have been produced.

The aim of our project is to improve our understanding of the mechanisms regulating the expression of protein coding genes. We initially focused our attention on the transcription/repair factor TFIIH and studied its role in both mechanisms. Mutations in this complex yield rare autosomal recessive disorders, such as Trichothiodystrophy (TTD) and Xeroderma Pigmentosum (XP, sometimes associated to Cockayne syndrome, XP/CS). These diseases, also found in patients bearing mutations in other repair factors (the nucleotide excision repair NER factors XPC, XPA, RPA, XPG, XPF, CSB), were initially defined as DNA repair syndromes. However, the clinical complexity of the patients cannot be explained solely on the basis of a DNA repair defect and may also involve transcription deficiencies. In line with this hypothesis, we recently observed that mutations in TFIIH as well as in NER factors disrupt the transactivation mediated by hormonal nuclear receptors (NR), revealing an unexpected function of the NER factors during transcription. Apart from NER factors, other genetic disorders result from mutations in other transcriptional partners of TFIIH, such as the Mediator. Indeed, mutations in one of the Mediator subunits lead to different diseases, such as Opitz-Kaveggia syndrome (related in particular to the MED12/R961W mutation), cerebellar atrophy (found in patients bearing the MED17/L371P mutation) or nonsyndromic intellectual disability (associated to MED23/R617Q mutation). These observations prompt us to study the molecular mechanism of the Mediator-associated disorders that will help us to better understand how gene expression is regulated at the transcription initiation level.
We would like in the next years:
- To better understand at a molecular level the roles played during transcription by TFIIH, the Mediator and more particularly the NER factors.
- To unveil how mutations in either TFIIH or one of its partners (such as the NER factors and the Mediator) disrupt the transcription and lead to various syndromes.
Several experiments will be performed to define at a molecular level the role of the different factors of interest. In this exciting study with potential impact on human health, in addition to conventional molecular biology technics, different mouse models will be monitored. Moreover, and since limited number of mouse model are available at the present time, innovative approaches will be initiated to generate human Induced Pluripotent Stem (hiPS) cells from a number of mutated cell lines isolated from patients. This will help us to analyze in a specific cellular context the incidence of numerous mutations in either TFIIH, the NER factors or the Mediator, leading to a better understanding of the heterogeneity of the phenotypes depending on the mutations. In fine, this work will allow us to define the aetiology of several transcription diseases resulting from mutations in TFIIH and some of its partners. With a long-term perspective in mind, the information obtained from these studies will help to correct or to attenuate the phenotypes of the patients.