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R-loops as a major modulator of transcription-associated recombination and chromatin dynamics

Periodic Reporting for period 4 - TARLOOP (R-loops as a major modulator of transcription-associated recombination and chromatin dynamics)

Reporting period: 2020-06-01 to 2021-11-30

Coordination of DNA replication with DNA-damage sensing, repair and cell cycle progression ensures genome integrity during cell divisions, thus preventing mutations and DNA rearrangements. Such events may be harmful for the cell and the organism, and are usually associated with pathological disorders, including cancer. Understanding the factors and mechanisms that can compromise the stability of the genome is a key question in Biology
One important type of genome instability is that associated with transcription. Transcription-associated genome instability (TAGIN) is in large part due to collisions between transcription and replication, but increasing evidence indicate that R loops are a major determinant This is of particular relevance, provided our recent observation that tumor suppressor BRCA2 gene and the Fanconi Anemia repair pathway are involved in R loop prevention/resolution.
The project pursues a global analysis of functions and elements that control TAGIN in eukaryotes to define the mechanisms of R loop accumulation and the physiological relevance of these structures in chromatin dynamics and genome integrity. The goal is to identify the DNA sequences and genes controlling R loop formation and removal. The project relies on a multidisciplinary approach using the model organism Saccharomyces cerevisiae and different human cell lines to identify:
a) the proteins and mechanism that actively works in the formation and prevention of intermediates responsible for TAGIN and R loop-mediated genome instability;
b) the role of chromatin and histone modifications in TAGIN, whether or not R loop-mediated, and
c) how trans and cis elements control R loops and their role in replication fork impairment, double-strand break (DSB) formation, chromatin structure and mRNP biogenesis and export.
Since RNA-DNA hybrids have the potential to be a natural source of genome instability in cancer, we believe that they cannot only be a potential diagnostic tool in cancer, but they could also be used as a target in cancer therapies.
Conclusions
We have been able to decipher the differential function of a number of factors in R-loop prevention and resolution that depends on cell cycle stage and the way R-loops themselves are formed, among other possible elements. Our results confirm a varied and complex origin and resolution of R-loops and confirm that they are a threat to genome integrity by compromising replication fork progression.
Using the citidine deaminase AID as a tool we performed a screening of viable yeast strains deleted in genes with nuclear functions for AID-dependent hyper-recombination. We identified a component of the nuclear pore basket, as a gene involved in preventing R loop accumulation and a DNA helicase, whose role in R loop formation and replication stress was then characterized in depth.
In addition, we performed the following siRNA screenings to search human genes with a role in R loop metabolism: i) the human Druggable Genome siRNA library (4795 siRNAs) by H2AX foci Immunofluorescence (IF) in U2OS stable cell lines for the inducible expression of AID constructed for this study; ii) specific siRNA libraries against the DNA damage response (DDR) genes (240 siRNAs), and nuclear DNA/RNA metabolism related genes (77 siRNAs) in HeLa cells via IF with an anti-DNA-RNA antibody. We validated the positive candidates by DNA-RNA immunoprecipitation (DRIP) assays, selecting 15, including the mitochondrial degradosome subunit or the endonuclease Dicer. This study has led to a new role of the mitochondrial degradosome in preventing harmful R loop formation at mtDNA plus a new role of DDR functions involved in replication stress, including ATR, the 9-1-1 complex and postreplicative repair factors. Other factors with a putative role in R loop processing are being explored by proteomic approaches by expressing a GFP protein fused to RNaseH hybrid-binding domain (HB-GFP) in HEK293 cells.
With respect to functions specifically related to chromatin, we completed the screening and analysis of the Non-Essential Histone H3 & H4 mutant collection of S. cerevisiae for mutants that increased R loops by ectopically expressing the human AID. We identified specific histone mutants that facilitate R loops without causing genomic instability, proving for the first time that R loops are not deleterious per se. The different behavior does not depend on the R loop size but on chromatin modifications such as H3S10-P.
In human cells, we provided evidence that R loop protection by RNA binding proteins may also be promoted by chromatin remodeling. We found that human THO interacts with the Sin3A histone deacetylase complex to suppress R loops, DNA damage, and replication impairment.
Our work supports a role of the SWI/SNF chromatin remodeler complex in helping resolve R-loop-mediated T-R conflicts

We found some RNA helicases as protectors of R loop-mediated instability: UAP56/DDX39B that localizes to active chromatin and prevents the accumulation of RNA–DNA hybrids over the entire genome; and DDX5 that interacts with BRAC2 and promotes resolution of R-loops at DNA breaks facilitating DNA repair.
We analyzed replication fork block and DSB hotspots in yeast R loop-accumulating strains, using hpr1- and sen1- degron strains to induce degradation of Hpr1 and Sen1. Depletion of either protein causes genome-wide analysis of R loops, H3S10P and H2AP distribution that are different, defining DSB hotspots coincident with R loop sites at different cell cycle stages.
Dissemination
Results have been published in top journals (Mol. Cell 2017, PNAS 2017, EMBO J. 2017, Nat Commun. 2018, Genes Dev 2018, EMBO Rep 2019, Cell Rep 2019, Genes Dev 2019, Nature Comm. 2019, Genes Dev 2020, PLoS Genet 2020; EMBO J 2021; Nature Genetics 2021; Nat Commun. 2021 (3 papers); eLife 2021; Nat Commun. 2021) and presented in conferences at top international meetings (Gordon Res. Conf., FASEB, EMBO, NAITO and others) and Universities (Beijing, Busan, Madrid, Viena, Paris, etc).
The new candidates found as protectors of R loop-mediated instability cover nuclear metabolic processes, including RNA metabolism, DDR or chromatin remodeling. As we complete this initial characterization, which include the biochemical characterization in vitro of some RNA biogenesis factors to bind, modulate and/or unwind DNA-RNA hybrids, we focused our work on those factors that gave us clues about the mechanisms by which not only R loops are formed and stabilized. We selected representative factors of different biological categories (mRNA biogenesis and chromatin remodeling) to carry out a genome-wide analysis of R loops in K562 human cell lines depleted of these factors
A parallel analysis of cis elements contributing to R loop formation was undertaken to define putative hotspots regions for R loops. We developed a genome-wide approach for R loop mapping taking advantage of the in vivo action of the B-cell specific enzyme AID. For that purpose, we have used a plasmid containing a mutated version of AID that is being used in R loop accumulating strain, to carry out up genome-wide sequencing.
We characterized members of the C. elegans TREX2/THSC complex, involved in mRNA biogenesis at the nuclear periphery to get inside into its role on genome instability.
After genetic, molecular and genome-wide analysis, our work has allowed us to understand how eukaryotic cell prevents R loop accumulation and associated genome instability.
Detection of cytoplasmic RNA-DNA hybrids in HeLa cells by Immunofluorescence with S9.6 mAb.