GLORI · Global analysis of R-loop structures (RNA-DNA hybrids) by advanced microscopic and genetic approaches

Genetic alterations from point mutations to chromosome rearrangements are usually associated with pathological disorders. Unraveling the origin of such events was greatly enhanced by the discovery of cis-acting factors (e.g. chromosomal fragile sites, G quadruplexes)behaving as chromosomal hotspots for genomic instability, but a detailed mechanistic understanding of their mode of action has remained elusive. Most current models of genomic instability incorporate R-loops - nucleic acid structures in which an RNA strand displaces onestrand of a duplex DNA molecule for a limited length - as immediate cause of gene rearrangements. The present application is based on prior observations of the host laboratory on previously unrecognized ribonucleoprotein particles containing RNA-DNA hybrids, being positioned at loop-size intervals in the chromatin of lower and higher eukaryotic cells (Szekvolgyi L et al., PNAS, 2007). The molecular identity and the cell biological significance of the revealed structures are unknown. The goal of this project is to unravel the molecular structure and epigenetic landscape of the regularly spaced ribonucleoprotein complexes containing RNA-DNA hybrids previously described by our laboratory, with a special emphasis on their connection to chromatin loop domain formation and genetic recombination. We expect that our work plan - proposing the global analysis of R-loop structures using a range of cell biophysical and genetic approaches in the genetically tractable model organism S cerevisiae as well as in various human cell lines - will yield novel data relevant to the rules that govern not only normal chromatin architecture but also an RNA-DNA hybrid-mediated pathological scenario leading to chromosomalrearrangements.