Despite many efforts undertaken for decades to describe the various nuclear processes involving DNA transactions at the molecular level, these investigations have rarely taken into account the complexity of all the processes occurring at given loci in vivo. This complexity could be exemplified with class III genes (like 5S rRNA or tRNA genes) transcribed by RNA polymerase (Pol) III. By now, all components necessary for basal Pol III transcription have been identified in the yeast Saccharomyces cerevisiae. On the other hand, the signalling pathways that modulate Pol III transcription activity in response to physiological changes or when cells are grown under a variety of stress conditions are poorly studied. There are only little information about how all processes take place within the chromatin context and how the distinct pathways (RNA modification, transport, DNA repair, DNA replication) interact to regulate Pol III transcription. To obtain a global view of all the phenomenon that occur on class III genes, it would be very important to characterize all the actors that are present i.e. bound. Very recently, a new approach called PICh (“Proteomic of isolated chromatin segment” described by Partner 2 (J. Déjardin) has been used to extensively identify proteins associated to human telomeres in vivo. The aim of our project is to adapt the PICh technique, based on chromatin hybridization with a modified DNA probe, to yeast chromatin to be able to identify exhaustively the proteins associated with distinct class III genes (protein set) and to determine the proteins responsible for the regulation of class III gene expression. A prerequisite of this project is to set up the PICh methodology in the yeast Saccharomyces cerevisiae. Since 5S rRNA or tRNA genes are highly occupied by the Pol III transcription machinery and are present in several copies in the yeast genome, we believe that class III genes are well adapted for PICh. The project will start with the identification of the proteins associated with the yeast highly repeated 5S rRNA genes. Prior to working with chromatin samples to hybridize, development of optimal probes that colocalize in vivo with Pol III will be required. Analysis and control experiments performed during the set up of PICh to 5S rRNA genes will help to define the resolution of the technique, another possible limit of the method when the yeast compact genome is studied. We propose then to identify the human 5S rRNA genes protein set as well as the polypeptides bound to different selected yeast tRNA genes. The comparative study of the protein set associated to distinct class III genes in both organisms will help to identify new factors involved in class III gene transcription and to determine those that are phylogenetically conserved. To reveal stress-specific Pol III transcription regulatory networks, we plan to characterize the protein composition of 5S rRNA genes in both human and yeast cells after treatment with 4NQO (a UV mimetic agent) or rapamycin (a macrocyclic polyketide that mimics nutrient limitation), two drugs that have been shown to repress Pol III transcription. To validate the whole approach, a functional characterization of the role of selected proteins in Pol III transcription and regulation will be investigated, using a classical range of in vivo and in vitro experiments (genetic approaches, in vivo metabolic labelling, ChIP, in vitro transcription, EMSA).