Project 2. Regulatory interplay between R-loops and transcription.

Transcription by RNA polymerase II (RNAPII) is essential to all cellular processes and hence to the adaptive response of cells to internal and external stimuli. The interplay between R-loops and transcription remains incompletely understood: R-loops have been reported to play a regulatory role in transcription initiation and termination. In addition, accumulation of R-loops during transcription can lead to genomic instability and multiple mechanisms have been proposed for R-loop-associated genomic instability. We developed RNA-DNA Proximity Proteomics to map the R-loop proximal proteome in human cells using quantitative mass spectrometry. We implicated different cellular proteins in R-loop regulation and identified a role of the tumor suppressor DDX41 in opposing R-loop and double-strand DNA break (DSB) accumulation in gene promoters. DDX41 is enriched in promoter regions in vivo and can unwind RNA-DNA hybrids in vitro. R-loop accumulation upon loss of DDX41 is accompanied by replication stress, an increase in the formation of DSBs and transcriptome changes associated with the inflammatory response. Germline loss-of-function mutations in DDX41 lead to predisposition to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) in adulthood. We proposed that R-loop accumulation and genomic instability-associated inflammatory response contribute to the development of familial AML with mutated DDX41.

Figure 1. Tumor suppressor DDX41 opposes R-loop accumulation in promoters of active genes. Pathogenic DDX41 variants in AML display impaired RNA–DNA hybrid unwinding activity, leading to the accumulation of R-loops at promoters. Accumulation of R-loops in promoters results in increased replication stress, DSBs, and inflammatory signaling.

In this project, we aim to investigate the role of DDX41 in counteracting transcription-associated DSBs in human HSCs). To this end, we will optimize the genomics approaches for mapping R-loops and proteomics for mapping R-loop-regulating proteins in hematopoietic stem cells, and we will develop a new method for robust R-loop detection using a probe based on the hybrid-binding domain of RNaseH1.