Cis- and trans-acting factors controlling R-loop-dependent genome instability – CisTransRloop

To achieve these different aims, the consortium combines unique and complementary skills in molecular biology (R-loop detection, ChIP-seq, genetic instability assays), genomics (systematic screens in budding yeast, whole genome sequencing) and microscopy (live imaging of the loci of interest).

By interfering with R-loop formation in different mutant situations, we have found that R-loop forming loci are repositioned in the nucleus in a R-loop-dependent manner. We have combined candidate approaches and systematic screens to investigate the molecular mechanisms at play, further revealing that R-loop accumulation per se, but not R-loop dependent damage, is the main trigger for repositioning. Potential molecular interactions underlying this process are under characterization.
To further decipher how R-loops contribute to genome mutability and identify the cis-acting genomic features impacting this process, we have also initiated the mutational profiling of R-loop-accumulating mutants.
Finally, in order to get a complete depiction of the pathways preventing R-loop formation, we have used dedicated genetic screens to identify the genes preventing R-loop accumulation and R-loop-dependent genetic instability. These approaches have pinpointed novel factors preventing R-loop formation or modulating their processing, which are currently under investigation.

At this stage, the project has generated novel tools and original datasets regarding our understanding of R-loop metabolism and transcription-dependent genetic instability. The realization of this project is thereby expected to shed light on this emerging field of research, and to provide clues for the understanding of the multiple pathological situations to which R-loops have been associated.

3 presentations at national and international conferences.

Several threats endanger the tight equilibrium between genome maintenance and expression that guarantees nuclear homeostasis. Among them, the formation of R-loops through hybridization of nascent mRNAs onto their DNA templates has emerged as a major cause of transcription-associated genetic instability. However, the molecular mechanisms involved in R-loop sensing and processing remain largely undiscovered. In particular, the cis- and trans-acting factors that influence the fate of R-loops in space and time remain to be deciphered. Based on unique preliminary results and approaches gathered by our consortium, this project will explore the impact of R-loops on genome dynamics, using mainly budding yeast as a model system.

1. We will investigate how R-loops affect the spatial organization of the genome.
We have found that R-loop forming loci are repositioned in the nucleus in a R-loop-dependent manner. We will identify the molecular mechanisms at play and investigate their impact on R-loop genotoxicity. For this purpose, we will perform genome-wide analyses of gene localization and R-loop levels. We will further interfere with R-loop accumulation and analyze the consequences for gene localization, using microscopy and biochemical approaches. Finally, we will artificially modulate the localization of R-loop-forming loci and evaluate the impact on R-loop-associated genetic instability.

2. We will decipher how R-loops contribute to genome mutability and molecular evolution.
To understand why only certain R-loops are genotoxic, we will profile the mutations they trigger through genome-wide analyses in R-loop-accumulating mutants. The genomic features that determine the genotoxic potential of R-loops will be further identified through correlation analyses and validated using specific R-loop-forming reporters.

3. We will identify which trans-acting factors modulate these processes and impinge on R-loop-dependent genetic instability.
We will use innovative genetic screens in an attempt to systematically identify the cellular factors that contribute to R-loop-dependent processes, and characterize their mode of action using dedicated molecular and biochemical assays. Their conservation will be analyzed in the frame of a collaboration.

To achieve these different aims, this consortium combines unique and complementary skills in molecular biology, genomics and microscopy. The realization of this project is thereby expected to shed light on this emerging field of research, and to provide clues for the understanding of the multiple pathological situations to which R-loops have been associated.