Décryptage des réseaux de régulation de la transcription par l'ARN polymérase III en réponse au stress ou pendant la différenciation – RegPolStress

Deciphering the regulatory pathways of RNA polymerase III transcription in response to stress or during differentiation How do cells adjust their growth rate to physiological changes or environmental variations, especially under stress conditions? What are the processes in control of their proliferation rate, particularly during the anarchic growth of tumoral cells? Nowdays, it seems well established that the regulation of RNA polymerase III (Pol III) transcription is a key feature in this process. In all eukaryotic organisms, a strong correlation is observed between the arrest of cell growth and a decrease in Pol III (and Pol I) transcriptional activity. Similarly, anomalous high levels of RNA are synthesized by Pol III in tumoral cells. Within this project, we aim to decipher the regulatory networks and the molecular mechanisms governing, in vivo, the adaptation of Pol III transcription in response to stress in the yeast S. cerevisiae and during the differentiation of higher eukaryotic cells. For many years, we have contributed substantially to the knowledge of the molecular mechanisms driving the synthesis of RNA by Pol III. Although much more complex in higher eukaryotes, the basal Pol III transcription machinery appears generally highly conserved throughout evolution both in its structural and functional aspects. The only two Pol III transcriptional regulators identified in yeast to date (Maf1 and Sub1/PC4) are proteins that have been highly conserved during evolution, suggesting that the networks implicated in the regulation of Pol III transcription are similarly conserved. It therefore appears of utmost importance to coordinate the research performed in yeast and man and, on the other hand, to complement the functional analysis of Pol III transcription and its regulation with structural studies that will bring us the three-dimensional vision of these processes. For these reasons, our project is composed of two main aims: 1- The deciphering of Pol III regulatory networks in yeast cells growing under stress conditions or in differentiating human cells. We were the first team succeeding in identifying and characterising Maf1, a yeast protein repressing specifically Pol III transcription under stress conditions. More recently, we have observed that Sub1, the yeast homolog of human coactivator PC4, allows a fast resumption of Pol III transcription upon exit from stationary phase. Regulatory cascades that lead to the activation of Pol III transcription through Sub1/PC4, as well as those leading to transcriptional repression during human cell differentiation remain totally unknown. This research project therefore envisages several approaches relying either on classical techniques or more recently emerging technologies; our goal being a complete description of the regulatory networks from signal sensing to the modulation of transcriptional activity. We aim at (i) identifying novel regulators involved in stress responses in yeast or differentiating human cells, (ii) unravelling the molecular mechanisms and the enzymes implicated in the inactivation-activation cycles of the regulators, (iii) defining the DNA and protein targets of the regulators within the basal transcription machinery and (iv) understanding how these regulators modify the activity of the transcriptional machinery. Finally, transgenic mice deprived of selected regulators will be produced in order to determine whether affecting the Pol III transcriptional control favours the emergence of tumours. 2- The elucidation of the structural basis of Pol III transcription and its regulation. Recently, we have obtained the crystallographic structure of a part (~30%) of the multi-protein transcription factor TFIIIC and structural studies on Pol III using crystallography and electron microscopy have already started. The 3D crystallographic structure of Maf1, followed by that of other regulators, will strongly strengthen our view of the Pol III transcriptional system and its regulation. Detailed structural information of Pol III regulators will help to understand better the molecular mechanisms underlying their functioning. Furthermore, structural information on Pol III regulators will open the possibility to interfere with their activity in a directed way and might ultimately help in the design of small molecules interfering with their function. The making of this project requires the coordinated efforts of three teams whose scientific expertise and research fields are highly complementary. In the past, these laboratories have demonstrated their ability to fruitfully collaborate, thus reinforcing the relevance of their cooperation. The funding of our network is therefore mandatory to reach the presented scientific goals; it will allow national teams, often producing pioneering work in the field of transcriptional regulation, to remain at the cutting edge of international competition.