Transcription and processing of ribosomal RNA genes (rRNA) are intimately linked to processes that control cell growth and proliferaton. Expression of rRNA is regulated by multiple signals such as nutrient availability, growth factors and cellular stress. Not surprising, increasing evidence suggest that large changes in the up-regulation of rRNA synthesis may be one of the most important signatures of cancer cells. Transcription Terminator Factor (TTF-I) is an essential factor in rRNA transcription that also determines the overall architecture of the rDNA promoter. TTF-I orchestrates the epigenetic state of both active and silenced genes through the recruitment of different set of chromatin remodelers and modifiers. In active rRNA genes, TTF-I binds to the terminator T0 site and recruits Cockayne Sindrome Protein B (CSB) that in an ATP dependent reaction modifies the position of nucleosomes in the promoter and activates transcription of the gene. In addition, TTF-I can recruit the Nucleolus Remodeler complex (NoRC) to have the opposite effect in transcription and promotes rRNA gene silencing. Although many details about the general features of each mechanism are known, a mechanistic view of the role of TTF-I in the epigenetic regulation of rRNA genes at the atomic level is lacking. In this new R21 we proposed to develop a system to understand: 1) How TTF-I binds chromatin templates and the structural changes occurring after this initial binding and 2) How TTF-I recruits CSB and the changes that occur upon recruitment in the position of the nucleosomes. We will use a combination of functiona assays to map the interactions of TTF-I and CSB and a hybrid structural approach combining X-ray crystallography, single-particle reconstruction electron microscopy (EM) and small-angle x-ray scattering (SAXS). In addition to obtaining for the first time a detailed viw of the remodeling complexes and the changes leading to the activation of rRNA genes, the system developed in this application will lead us into future studies of gene silencing by th recruitment of the NoRC complex and the protein complexes that regulate the cellular abundance of TTF-I such as its interaction with tumor suppressors ARFp19 and MDM2. This work will be in close collaboration with Dr. Montserrat Samso that will provide expertise in single-particle reconstruction EM and Dr. Deepak Bastia that will provide technical help in the biochemical assays.
The epigenetic control of gene expression and the large-scale chromatin organization of rRNA genes have direct implications in cell proliferation and the influence the mutational rates of cancer cells. A mechanistic molecular view of these processes is a requirement for the development of novel therapeutics and will provide new tools in a new emerging front that targets the downregulation of rRNA genes for the treatment of cancer.
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