A tissue specific study of the functional engagement of the transcription/repair factor TFIIH – FreTNet

To answer the questions above mentioned, we combined in vivo approaches with more traditional methods and biochemical techniques. We a mouse model that expresses endogenously a protein belonging to the TFIIH complex fused with a fluorescent protein (the green flourescent protein). This mouse model is a unique tool and a source of cells and tissues containing the TFIIH complex fluorescent. With this tool, we can measure TFIIH molecular dynamics in the whole organism, but we can also isolate TFIIH by immunoprecipitation techniques and then disclose new partners.

During those 18 months of project we were able to:
- Disclose a new partner of the TFIIH complex, the protein ELL and we studied its cellular function.
- Find a new role of TFIIH in the genesis and maturation of ribosomal RNA.
- Put in evidence the molecular mechanism of TFIIH mutants that causes breaks in the genome after UV irradiation.

We will in the future study in more detail the effects of a reduced amount of TFIIH on its activities in transcription and repair and the effect of hormones on its activity

Generation of DNA single-strand displacement by compromised nucleotide excision repair. Camille Godon, Sophie Mourgues, Julie Nonnekens, Fréderic Coin, Wim Vermeulen, Pierre-Olivier Mari, Giuseppina Giglia-Mari. EMBO Journal (in publication)

The basal transcription/repair factor TFIIH is a 10 sub-unit complex essential for RNA polymerase II (RNAP2) transcription initiation and NER. In these processes TFIIH acts as a DNA helix opener (by the enzymatic activity of XPB and XPD helicases), required for promoter escape of RNAP2 in transcription initiation, and to set the stage for strand incision within NER.

Biochemical and genetic studies have provided valuable insights into the mechanism of action of TFIIH in repair and transcription, however, a gap of fundamental knowledge exists between the well-known biochemical mechanisms involved in transcription initiation and DNA repair and the actual molecular mechanism of these processes occurring in the whole organism. Because of this gap and despite almost two decades of structural, biochemical and cellular studies devoted to understand these fundamental cellular processes, many questions remain unanswered:

1 How does TFIIH acts in post-mitotic cells?
2 What is the function of TFIIH in RNAP1 transcription?
3 How mutations in TFIIH affect differentially the trans-activation of hormone dependent genes?
4 How mutations in TFIIH lead to different and distinct human diseases?
5 How can we explain the decreased steady state level of TFIIH in TTD cells?

Our aim is to answer the aforementioned open questions in order to improve our fundamental knowledge of TFIIH cellular engagements and our understanding on the relation genotype/phenotype of TFIIH-related diseases.

To this aim, partner 1 has generated a knock-in mouse model that expresses from its endogenous locus a fluorescently tagged TFIIH subunit. This new model system will be used within this Network to answer the aforementioned questions. Our proposal will make use of cellular and molecular assays, sophisticated photo-bleaching techniques, and biochemical approaches. In parallel, Partner 2 unveiled, in TTD mutant mouse model, hormonal dependant transcription defects that are responsible of some of the neurological defects of TTD patients.

Our project will bring together two French research groups focused on the study of TFIIH functions, interactions and regulations. The strength of this consortium is based on the full complementary approaches and tools used to answer the same scientific questions. Our synergic potential will be fundamental to fully comprehend the intricacies of TFIIH cellular roles and disclose novel concepts on fundamental cellular processes such as transcription and DNA repair.