#	Pagina
attuale pagina	/open-h2020/projects/198088/results.html

Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - TARLOOP (R-loops as a major modulator of transcription-associated recombination and chromatin dynamics)

Teaser

Summary

Coordination of DNA replication with DNA-damage sensing, repair and cell cycle progression ensures with high probability 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 premature aging, various cancer predispositions and inherited diseases. Understanding the factors and mechanisms responsible for high levels of recombination that can compromise the stability of the genome is a key question in Molecular Biology and Biomedicine.
One important type of genome instability is that associated with transcription. Transcription of a DNA sequence increases its frequency of recombination. Different studies suggest that transcription-associated recombination is in large part due to collisions between transcription and replication, but increasing evidence indicate that R loops, formed by a DNA-RNA hybrid and a displaced single-stranded DNA, may be a major determinant of genome instability. 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. Thus, R loops may represent a major potential source of tumorigenesis. Consistently, studies on transcription-associated instability and on R loops have received a great attention in recent years.
The present project pursues global analysis of functions and elements acting in trans and cis that control TAR in eukaryotes with the aim at understanding the mechanisms of R loop accumulation and the physiological relevance of these structures in chromatin dynamics and genome integrity. The goal is to understand the mechanisms of R loop dynamics by identifying the functions and elements acting in cis and trans, that is, the DNA sequences and genes controlling R loop formation and removal. The long-term objective of the proposal is to decipher the mechanisms by which R loops modulate chromatin dynamics and genome instability, as a potential source of cancer.

The project relies on a multidisciplinary approach using the model organism Saccharomyces cerevisiae and different human cell lines with the following general objectives:
a) to identify the proteins and mechanism that actively works in the formation and prevention of intermediates responsible for transcription-associated recombination and R loop-mediated recombination;
b) to define the role of chromatin and histone modifications in transcription-associated recombination, whether or not R loop-mediated, and
c) to define how trans and cis elements control R loops and their role in replication fork impairment, double-strand break 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. Our study should contribute to such long-term objectives.

Work performed

One main objective was a genome-wide identification of functions controlling the formation of DNA-RNA hybrids, structures called R loops in Saccharomyces cerevisiae and human cells. 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) Screening of 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) Screening of specific siRNA libraries against 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, having at this moment 15 candidates, including the mitochondrial degradosome subunit or the endonuclease Dicer which we are characterizing in depth. We are performing analysis of R loop-dependent DNA breakage and replication impairment to define the role of these genes in R loop dynamics. This study 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 and protein IP and mass spectrometry. We are currently evaluating the candidates for its potential effect on genome integrity associated to RNA metabolism.

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 of these R loops does not depend on the R loop size but on the ability to trigger 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 transient chromatin remodeling. We found that human THO interacts with the Sin3A histone deacetylase complex to suppress co-transcriptional R loops, DNA damage, and replication impairment.

To analyze replication fork block and DSB hotspots in yeast R loop-accumulating strains, another relevant objective, we constructed hpr1- and sen1- degron strains to induce degradation of Hpr1 and Sen1 proteins in an accurate manner. Depletion of either protein causes genome instability and a quick accumulation of R loops and chromatin marks associated with DNA damage and DNA compaction (H2A-P and H3S10-P, respectively). We are now performing genome-wide analysis of R loops, H3S10P and H2AP distribution aiming at defining DSB hotspots and this coincidence degree with R loop-accumulation sites.

Results have been published in top journals (Mol. Cell 2017, Genes Dev 2018, PNAS 2017, EMBO J. 2017, Nat Commun. 2018) and presented in conferences at top international meetings (Gordon Res. Conf., FASEB, EMBO, NAITO and others) and Universities (Beijing, Busan, Madrid, Viena, Paris, etc).

Final results

The new candidates found as protectors of R loop-mediated instability cover nuclear metabolic processes, including RNA metabolism, DNA damage Response (DDR) or chromatin remodeling and should allow us to be able to establish a network of functional relationship between DDR, RNA export, chromatin modifications and double strand breaks (DSBs), the main goal of this study. 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 will focus our work on those factors that should give us clues about the mechanisms by which not only R loops are formed and stabilized, but most importantly, by which they cause genome instability. We selected representative factors of different biological categories (mRNA biogenesis and chromatin remodeling) to carry out a genome-wide analysis of R loops by DRIPc-seq (Immunoprecipitation of R loops followed by sequencing of the RNA fraction) in K562 human cell lines depleted of these factors to get an integrated view of R loop metabolism.
To complete the study, a parallel analysis of cis elements contributing to R loop formation was undertaken to define putative hotspots regions for R loop accumulation. We are developing 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 with enhanced deaminase activity under the GAL inducible promoter. The plasmid is being used now in yeast wild-type and hpr1Î” cells, the latter as a control for R loop accumulating strain, to set up the proper conditions of analyses, which included the genome-wide sequencing of independent isolates to establish the levels and distribution of AID-mediated hypermutation as a measure of RNA-DNA hybrids.
Finally, even a bit earlier as planned, to explore the role of the new factors in a multicellular eukaryote we are initiating the characterization of putative members of the C. elegans TREX2/THSC complex, involved in mRNA biogenesis at the nuclear periphery and genome instability (the thp-1 gene homolog to yeast THP1 and human PCID2, and the dss-1 homolog to yeast SEM1 and human DSS1). We are analyzing the effect of the lack of these factors on cell viability, DNA damage accumulation (measured by increased ATR, RPA and RAD51 foci) and replication by Cy3-dUTP incorporation in the nematode germline.
As stated in the plan of the project we expect to select a full representative subset of nuclear proteins involved in R loop protection covering different nuclear metabolic factors from RNA metabolism, chromatin remodeling, DNA replication and DDR, and our results are heading successfully in this direction. After exhaustive genetic and molecular characterization, as performed already with some of the candidates, and a comparative genome-wide analysis of the distribution of these proteins and their impact in R loop accumulation and DNA breakage along the eukaryotic genomes, our work should give us the clues to understand how eukaryotic cell prevents in a coordinated manner R loop accumulation and associated genome instability and whether these effects apply globally or to specific genomic regions, that could define a new feature for potential fragile sites.