Structure, function and dynamics of the transcription factor TFIIH – TFIIH-Complexes

In eukaryotes, the multi-protein complex TFIIH plays a key role in both the regulation of gene expression and maintenance of genome integrity. Transcription initiation by RNA polymerase II requires the cooperative assembly of more than 40 proteins, among which the 10 subunits of general transcription factor TFIIH. It harbours three enzymatic activities and can be resolved into two functional and structural entities: the 6-subunits core-TFIIH with the XPB helicase and the 3-subunits Cdk Activated Kinase complex (CAK), which also exists as a free complex with a distinct function. The XPD helicase bridges core-TFIIH to CAK. Initially identified as basal transcription factor, TFIIH also participates in the transactivation of several hormone-dependent genes by phosphorylating nuclear receptors and plays a key role in nucleotide excision repair (NER) for opening DNA at damaged sites and recruitment of additional repair factors.

Although discovered more than two decades ago, insights into structural details of TFIIH, its supramolecular organization and interaction with partner molecules are still limited. This proposal aims at increasing our knowledge in the field by applying an array of biochemical, biophysical, structural and live cell imaging techniques to provide integrated structural and functional information from the cellular to the atomic level. Our project pursues the following four objectives:

- Investigate the molecular architecture of TFIIH combining a systematic dissection of the protein-protein interaction network from recombinant proteins with labelling strategies (chemical crosslinking and hydrogen/deuterium exchange) coupled with mass spectrometry. This will provide precise positional information on a high order multi-protein complex and will guide engineering of optimized binary or ternary sub-complexes for X-ray or NMR studies.

- Detail the molecular structure of TFIIH complexes using biophysical approaches and cryo-electron microscopy (cryo-EM). We will focus on complexes that contain stably bound partner molecules such as the endonuclease XPG which cooperates with TFIIH in the context of NER and stabilizes association between core-TFIIH, XPD and CAK: (i) In vitro biochemical and biophysical characterization of TFIIH/XPG complexes in transcription and NER, (ii) High resolution cryo-EM structures of core-TFIIH and Holo-TFIIH complexes.

- Study the cross-talk between CAK, XPD helicase and core-TFIIH. Based on the structure of archeal homologues, we have shown that the XPD ARCH domain is critical for recruitment of CAK to core-TFIIH and have performed preliminary characterization of the only known XPD mutation in the ARCH domain giving rise to Trichothiodystrophy. These results have identified the domain ARCH as a potential regulator of the XPD helicase activity and as a molecular switch from DNA repair to transcription. We will further investigate the CAK/XPD interface, by mass spectrometry studies and mutational approaches to identify residues critical for the association between CAK and XPD and analyse the impact of mutations in the ARCH domain of XPD for TFIIH activity in vivo and in vitro.

- Live cell imaging techniques will provide insights into the spatiotemporal cellular distribution of TFIIH, recruitment during transcription activation and repair, and interaction with partners, RNA Polymerase II for example. Relying on bleaching techniques and single molecule approaches, we will analyze the cytoplasmic and nuclear mobility of TFIIH components. Cell lines containing an artificial locus that allows fluorescent imaging of transcription will be used to monitor the recruitment of TFIIH components to the transcriptional machinery present on the activated genes.

This project will provide original data on regulatory mechanisms underlying transcription and DNA repair in eukaryotes. In relation with human health our results will improve our understanding of molecular defects associated with mutations in TFIIH.