DNA:RNA hybrids or R-loops are formed by pairing of transcripts with a complementary DNA strand. R-loops form at transcriptional pause sites, termination regions of genes and at repetitive regions. Aberrant accumulation of R-loops can lead to DNA damage and genome instability, promoting carcinogenesis. Our interest in R-loops was prompted by our studies on the TGS1 hypermethylase, which controls the biogenesis of small nuclear RNAs (snRNAs), the fidelity of splicing and transcription termination in human cells. We also found that TGS1 and Topoisomerase 2 (Top2) physically interact in Drosophila and that their loss causes R-loop accumulation and cell death in the eye imaginal discs. TGS1 and Top2 are overexpressed in tumors with poor prognosis and may therefore protect cancer cells against excessive R-loops that would cause crisis.
We plan to explore the role of TGS1 in R-loop formation and genome stability, by determining the effects of TGS1 depletion on genome-wide transcription patterns in both HeLa cells and Drosophila using both Illumina and Nanopore sequencing. We will also map R-loops in both the human and fly genome and screen for modifiers of the Tgs1-dependent phenotype in Drosophila. Finally, we plan to characterize additional Drosophila genes involved in transcription and whose deficiency in embryos results in DNA damage to explore whether these lesions correlate with R-loop accumulation.
Our experiments should elucidate whether the genes that prevent co-transcriptional stress also prevent R-loop formation and DNA damage, and provide insight into the cascade of events that lead to aberrant accumulation of R-loops.
