Architectural role of general transcription factor TFIIIC in the human genome
The three-dimensional organization of the genome, and its transcriptional states has been only recently identified as a paramount player in the development and progression of cancer. In eukaryotes the genetic information encoded in the linear polymeric chain of DNA is dynamically organised into multiple organised structures, from nucleosomes to chromatin fibres and, finally, to large chromosomal domains localised in specific areas within the nucleus. The spatial organization of the eukaryotic genome is not only important to compact the genetic information into a small and compartmentalised environment but also facilitates the coordination of several essential cellular processes, such as gene transcription, DNA replication, DNA repair and mitosis. Recent developments in high-throughput assays capable of inferring the spatial relationships of genomic loci (ie. 3C-based approaches, such as Hi-C, ChIA-PET, 5C) have enabled the detection of short- and long-distance chromatin contacts, revealing that association of linearly distant genomic loci are not randomly distributed and resulting in detailed maps of the 3D spatial organization of the eukaryotic genome. Chromosomes are subdivided into well- defined, globular regions of chromatin, named topologically associated domains (TADs), which establish few long- distance contacts with other TADs. TAD boundaries have been found enriched in tRNA genes and short interspersed element (SINE) retrotransposons, both RNA Polymerase (Pol) III transcriptional units, suggesting that components of the RNA Pol III transcription apparatus may have a role in establishing the topological structure of the genome. Accordingly, several studies in yeast, Drosophila, mouse and human cells established TFIIIC, an RNA Pol III transcription factor which binds tRNA genes and many non-transcribed regions known as extra TFIIIC loci (ETC), as an evolutionary conserved "architectural protein" of the eukaryotic genome. TFIIIC often colocalizes with the cohesin and condensin complexes and with the vertebrate specific CCCTC-binding factor (CTCF), a key architectural protein involved in establishing chromatin contacts genome-wide. These higher-order architectural macromolecular hubs define the boundaries and strength of TADs, as well as facilitating or debilitating intra-TAD contacts which impact on gene expression.
During the course of this PhD, the student will have the opportunity to become proficient in all aspects of modern cryo-EM, from the production of recombinant complexes in insect cells, purification and assembly of stable protein complexes that will be biochemically and structurally characterized through cryo-EM (acces to high end Titan Krios available. Furthermore, using a multidisciplinary approach the student will be involved in collaborations aimed at defining the interaction partners of human TFIIIC bound at specific chromatin loci and defining the genome-wide role of TFIIIC in genome topology.
In summary, the project will capitalise on the state-of-the-art facilities for recombinant protein production and structural determination using Cryo-EM and a multidisciplinary approach to unravel the paramount role of human TFIIIC in genome organisation.