Application of PICh for the characterization of the RNA Polymerase III transcription regulome – PIChClassIII

We propose to exhaustively characterize the proteins bound to genes transcribed by RNA polymerase III, adapting the PICh technique successfully developed by our partner for the telomeric region of human cells. This complex technique could be divided in four different steps: chromatin preparation, hybridization, purification and mass spectrometry analysis. First, cells are strongly cross-linked with formaldehyde in order to preserve protein-protein and protein-DNA interactions occurring in the cells. Then, chromatin is sheared using a combination of sonication and enzymatic digestion to obtain large quantities of small size soluble DNA fragments. Second, chromatin is hybridized with oligos containing modified nucleic acid (LNA). The hybridization conditions have been optimized by the addition of specific additives. Systematic comparison of magnetic bead from different suppliers was performed and has allowed determining the best beads adapted to our need. (binding capacity, specificity, price…) The next steps will consist to scale up PICh to higher amount of cells to be able to determine the set of proteins bound to 5S ribosomal DNA after mass spectrometry analysis. The comparison of the proteins set revealed upon different growth conditions or in different organisms will allow determining the stress.

We have adapted the technique developed by our partner to specifically capture the telomeric region of human cells, to the purification of 5S ribosomal DNA in baker’s yeast and in human cells. To obtain this result, various steps of this complex protocol have been optimized. We have been able to set up conditions for the preparation of a yeast chromatin adapted to our need, after a strong in vivo cross-link that may preserve protein-protein and protein-DNA interactions. The protocol for the preparation of human chromatin has also been largely modified. We optimized the hybridization step by the use of additives in our buffers.

RNA polymerase III transcription regulation plays a major role on the coordination of cell growth rate due to physiological or environmental changes. The level of RNA produced by RNA polymerase III is abnormally higher in tumour cells. The goal of this project is to exhaustively identify proteins able to bind to genes transcribed by RNA polymerase III. The characterization of these proteins should reveal new regulations pathways and therefore should permit to better understand the deregulation processes.

Presentation of preliminary results to international scientific congresses

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).