Abstract
The study of the so called 4D genome has clarified that RNA molecules play a key role in organizing the DNA into High-Order Chromatin Structures (HOCS). In particular, transient and highly frequent DNA:RNA hybrids can stabilize somewhere in the genome by adopting a triple-stranded structures called R-loop. R-loops were identified as the result of abortive transcripts accumulation, but more recently it has been demonstrated that they actively regulate transcription by serving both as platforms for the recruitment of transcription factors and terminators. Moreover R-loops can also form in trans independently from the transcription process and affect genome stability and DNA damage response (DDR). In this proposal we aim to identify new cis and trans regulators of R-loops and evaluate the direct impact of R-loops accumulation on genome stability and DSB repair. In Task 1 we aim to describe a new epigenetic mechanism that controls R-loops metabolism. We have demonstrated that a newly identified HDAC complex, named HRDAC, supervises the acetylation of H2B at lysine 120 thus controlling R-loops formation, in particular in correspondence to genomic loci subjected to double-strand breaks (DSBs). Here by inhibiting or by knocking-down key HRDAC subunits we aim to correlate RNAP2 processivity to R-loops by means of DRIP-seq, DRIVE-seq and PRO-seq. Colorectal cancer (CRC) cell lines and Patients’ derived organoids (PDOs) will be engineered to express mega-endonucleases and DDR-reporters and quantify the impact of R-loops on the repair of well-defined DSBs. In Task 2 the topological stress and the replication/transcription (R/T) conflicts will be investigated as cis and trans sources of R-loops. Topoisomerase I inhibition will be used as an exogenous inducer of topological and replication stress. DRIP-seq and TRIPn-Seq will be adopted to correlate R-loops formation to R/T conflicts. The genomic instability induced by R-loops accumulation will be quantified both at single cell level and by omics approaches. In Task 3, ATM/Tel1 signaling will be correlated to R-loops formation. Mutant yeast strains will be adopted to increase or decrease R-loops, while DRIP-seq and ChIP-seq will be used for the co-mapping of Tel1 and DNA:RNA hybrids. The levels of DNA damage after CPT treatment in cells lacking Tel1 and R-loop resolving factors will be evaluated and compared to wild-type strain. Finally, synthetic lethality will be exploited in Task 1 by combining FOLFOX therapy and epigenetic drugs on CRC PDOs. On the opposite, in Task 3 a yeast genetic screening will be adopted to identify compensatory pathways overcoming ATM/Tel1, Sen1 and RNaseH deficiency thus allowing survival in the presence of CPT and hydroxyurea. In conclusion, our project will identify new R-loops regulators; moreover, by quantifying the impact of R-loops on genome stability, we also aim to explore new approaches of personalized medicine for the treatment of genetic diseases and cancer. Results Achieved The project, carried out independently by the three research units, successfully achieved all of its intended objectives. In particular, a detailed summary of the activities performed and the research objectives achieved by each operational unit, with a focus on the timeline of their implementation, is provided below. Unit 1 – PI: Jessica Marinello. Unit 1 was involved in Task 2, focusing on the relationship between R-loop formation, DNA supercoiling, transcription–replication conflicts and genomic instability. Initial phase (Months 1–6): the unit developed the necessary know-how for genome-wide DNA damage mapping by END-Seq protocol and implemented a methodology for detecting micronuclei in cells at various stages of the cell cycle by immunofluorescence (recently published in STAR Protocols, Pepe et al. 2025). Intermediate phase (Months 7–18): genome-wide mapping of DNA breaks was carried out, together with the mapping of R-loops and their correlation to transcription–replication conflicts. The data obtained demonstrated that, following inhibition of topoisomerase 1 catalytic activity and the consequent accumulation of Top1 cleavage complexes (Top1cc), RNA Pol II is induced to undergo backtracking, leading to the formation of R-loops ahead of the enzyme. These R-loops are responsible for DNA damage arising from transcription–replication conflicts in highly transcribed genes and during a specific phase of cell cycle (Duardo et al., Science Advances, 2024). Final phase (Months 19–24): data were consolidated and finalized, leading to publications and the preparation of preliminary data to support potential future grant submissions. Unit 2 – PI: Eros Di Giorgio. Unit 2 was responsible for Task 1, aimed at elucidating the epigenetic and molecular mechanisms controlling R-loop metabolism and their impact on replication dynamics and therapeutic response in colorectal cancer (CRC). Initial Phase (Months 1–12): during the first 12 months, the unit focused on identifying the chromatin-modifying machinery responsible for regulating R-loop formation and stability. Through integrated molecular and genomic approaches, we identified a protein complex governing chromatin acetylation/deacetylation, functionally centered on HDAC4. We demonstrated that HDAC4 plays a pivotal role in modulating chromatin accessibility at genomic regions prone to R-loop accumulation. These regions were characterized by RPA32 hyperphosphorylation and replication fork slowing. Mechanistically, HDAC4-dependent chromatin deacetylation emerged as a key upstream regulator of R-loop homeostasis (Di Giorgio et al., Nucleic Acids Research, 2024). Intermediate Phase (Months 13–18): we investigated the molecular determinants underlying the persistence of R-loops at hyperphosphorylated RPA32 regions. We identified regulation of the RPA complex as a critical mechanistic node. Our data indicate that hyperphosphorylated RPA modulates RNaseH1 recruitment and/or activity, contributing to the inability of RNaseH1 to efficiently recognize and process G-loops at these sites. This mechanistic insight explains the stabilization of R-loops in replication-stressed chromatin domains and their correlation with replication fork slowing (manuscript in press). Final Phase (Months 19–24): we translated these mechanistic findings into a therapeutic context. We demonstrated that inhibition of HDAC4 or disruption of the RPA–RNaseH1 interaction restores R-loop processing and re-sensitizes CRC cell lines and patient-derived organoids (PDOs) to FOLFOX therapy (Tolotto et al., Molecular Oncology, 2025). During the last six months, datasets were consolidated and three scientific manuscripts finalized: two have been published in peer-reviewed journals, while a third is in press. Final reports were prepared, and the generated results provide a strong basis for future competitive grant applications. Unit 3 – PI Diego Bonetti. Unit 3 was responsible for Task 3, aimed at elucidating the cellular sensing of abnormal R-loops and the subsequent response in yeast, with a particular focus on the conserved Tel1/ATM kinase. During the first phase (months 1–12), the unit investigated whether Tel1 directly regulates R-loop levels, its genome-wide localization, and the underlying molecular mechanisms. We demonstrated that Tel1/ATM does not act upstream by modulating R-loop formation or stability, but rather participates in the response to DNA damage caused by their abnormal accumulation, particularly during collisions between R-loops and replication forks. In cells lacking Sen1 helicase or RNaseH enzymes, Tel1 loss increased DDR activation (Rad53 phosphorylation) and Rad52 foci formation, indicating elevated DSBs. Consistently, ChIP-seq analyses revealed Tel1 enrichment at R-loop-prone regions of the yeast genome, including promoters, centromeres, tRNA genes and Ty elements, especially in Sen1- or RNaseH-deficient cells and after CPT treatment. During the second phase (months 13–24), the unit focused on events occurring at stalled replication forks in the absence of Tel1. Experiments addressed: 1) ssDNA and RPA accumulation at replication forks; 2) recombination events using genetic assays; and 3) genome-wide mapping of Tel1, R-loops and DSBs through the implementation of new methodologies (BLESS/END-seq). In parallel, genetic screens identified two major candidates interacting with Tel1: Pif1 helicase and Sir4, a component of a conserved histone deacetylation complex. These findings fit well with the project goals and complement results from Unit 2. We further characterized Pif1 and obtained preliminary insights into Sir4 function. Notably, loss of either gene suppressed the toxicity associated with R-loop accumulation. Preliminary data suggest that Sir4-dependent chromatin regulation may limit R-loop formation. In contrast, Pif1 helicase activity appears detrimental under excessive R-loop conditions. We found that Pif1 does not affect R-loop levels directly but acts downstream, similarly to Tel1, by modulating damage generated by R-loop accumulation. Importantly, we uncovered a previously uncharacterized role for Pif1 in DSB and replication fork processing (5′–3′ resection), now included in a manuscript ready for submission. Further studies are ongoing to clarify how defects in end processing suppress R-loop-associated toxicity and how this pathway is connected to Tel1/ATM. This structured approach ensured timely completion of the tasks and the generation of high-quality datasets, supporting both scientific output and further research development.
Dettagli del progetto
Responsabile scientifico: Jessica Marinello
Strutture Unibo coinvolte:
Dipartimento di Farmacia e Biotecnologie
Coordinatore:
ALMA MATER STUDIORUM - Università di Bologna(Italy)
Contributo totale di progetto: Euro (EUR) 229.750,00
Contributo totale Unibo: Euro (EUR) 80.000,00
Durata del progetto in mesi: 24
Data di inizio
28/09/2023
Data di fine:
28/02/2026
