DS0405 -

Interplays between Genome Maintenance and Pervasive Transcription – GeMaPer

Submission summary

The occurrence of transcription outside of annotated regions (pervasive transcription) is a universal feature of prokaryotic and eukaryotic transcriptomes. Because DNA is a crowded environment where many different machineries co-exist to read, replicate and maintain the information stored, controlling and coordinating DNA-associated events is essential to avoid potentially disruptive interferences.
In this proposal we aim at addressing the interplays between pervasive transcription and the machineries involved in the replication and the maintenance of the genome by associating our complementary expertise in these fields. This project stems from many preliminary, independent, observations that justify combining efforts to address several timely and challenging questions.
During the S phase, replication initiates at many sites, with different efficiencies and timings. Our preliminary results suggest that the context of transcription around origins influences the efficiency and timing of replication. The first task of this proposal aims at studying genomewide the crosstalks between pervasive transcription and initiation of replication. We will alter the pervasive transcriptome with several complementary strategies and analyze at the same time the landscape of replication initiation and the distribution of RNAPII genomewide. We will be able to associate many different alterations in the transcriptional landscape with alteration in the replication program and establish precise and reliable correlative connections.
The second task stems from intriguing preliminary results indicating that replication forks activate the DNA replication checkpoint when they progress through transcribed genes, possibly because of collisions between DNA and RNA polymerases. However, such a replication stress is transient and does not occur in mid- or late-replicating regions. We postulate that early collisions activate the DNA replication checkpoint, which transiently turns off transcription to allow passage of the replication fork later on. This exciting possibility will be explored by directly assessing the occurrence of stalled replication forks near early origins and the expected pausing of RNAPII in these regions. The transient nature of this phenomenon and its dependence on the checkpoint will be addressed. Finally, we will investigate the mechanism by which these replication-transcription conflicts are resolved and search for checkpoint targets involved in this process.
The third task of this proposal concerns a novel and unexpected role for the Mre11-Rad50-Xrs2 (MRX) complex in transcription termination. MRX is a key component of the response to DNA damage. We have found that MRX mutants have striking defects in transcription termination at sites of action of the Nrd1-Nab3-Sen1 (NNS) complex, a key actor of the pathway that controls pervasive transcription. Importantly, MRX subunits markedly localize at regions of transcription termination, supporting a direct role that is distinct from its canonical role in DNA repair. We will join efforts to characterize termination defects genomewide and will address the mechanisms underlying these defects. We will experimentally challenge the provocative hypothesis that MRX components slow down the progression of the RNA polymerase by binding to the DNA in the termination region and/or by promoting the formation of nucleosome-free domains that might be bound by factors that induce pausing of the elongation complex.
In summary we propose to address many important questions concerning the coordination between different and essential machineries that concur in maintaining a stable expression, and a faithful transmission of the genome. Many of the proposed approaches are challenging, but certainly justified by the interest and the originality of the questions addressed. We believe we are in the position of successfully facing these challenges, both in terms of expertise and methodological know how.

Project coordination

Domenico Libri (Institut Jacques Monod)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

IJM Institut Jacques Monod
IGH Institut Génétique Humaine

Help of the ANR 390,916 euros
Beginning and duration of the scientific project: December 2016 - 42 Months

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