Architectural Role of RNA Polymerase III Promoters and Associated Factors in Shaping and Organising the Human Genome (360G-Wellcome-200818_Z_16_Z)

In higher eukaryotes chromosomes are subdivided in defined globular units, named topological associated domains (TADs). TADs borders have been found enriched with tRNA genes and short interspersed element retrotransposons, both RNA Polymerase (Pol) III transcriptional units. In order to obtain a mechanistical understanding of how RNA Pol III loci contribute to the spatial organization of the human genome, we will exploit a "library" of recombinant human RNA Pol III factors generated in my laboratory in order to reconstitute genomic architectural binding sites and to understand the molecular basis of the specific recruitment of complexes involved in structural maintenance of chromosomes (condensins and cohesin) and architectural DNA-binding factors (CTCF and ZNF143). Combining cryo-electron microscopy and x-ray crystallography will enable us to unravel the structures and functions of these complexes and to understand how they cooperate at a molecular level. Integrating structural data with biochemical and functional analysis in vitro and in living cells will allow us to obtain mechanistic models. The output of this research will shed light on how eukaryotic chromosomes are structurally organized and how the spatial organization of the genome impact on fundamental nuclear processes, such as gene transcription.