Periodic Reporting for period 4 - ConflictResolution (Transcription-replication conflicts in disease and development)
Periodo di rendicontazione: 2024-08-01 al 2025-11-30
Conflicts are a normal part of our social life. After all, it can’t be expected that two people agree on everything at any time. Thus, managing conflicts and finding ways how to avoid and resolve them is an important skill that we need to acquire early-on and continue practicing on an every-day basis in our life. Unexpectedly, conflicts are not only a social phenomenon among human individuals, but they can also arise inside of our body within our cells. More precisely at the molecular level, many molecular machines progress using the same DNA template. ConflictResolution investigates how two of these machineries, namely transcription to read the information of genes and DNA replication to duplicate the genome are regulated and coordinated to avoid so-called transcription-replication conflicts (TRCs). In our social environment, mismanagement of conflicts can lead to great harm, and this holds also true for cellular conflicts. TRCs are a potent endogenous source for genetic and epigenetic instability, and can therefore contribute to cancers, aging, developmental and neurological diseases. But how and where do TRCs provoke these instabilities? How frequently do the machineries meet and which fraction of encounters are harmful or get easily resolved? Unfortunately, these questions are largely unanswered owing to the lack of suitable cellular systems. ConflictResolution deciphers the contribution of collisions to induce pathological transformation of cancer cells as well as physiological changes in embryonic cells.
To address where these molecular accidents occur, the project utilized a sophisticated episomal reporter system to characterize the genetic and epigenetic consequences of collisions. Using ChIP-qPCR on the mAIRN reporter gene, we observed strong promoter-proximal pausing of the transcription machinery (RNAPII) coinciding with an accumulation of replication factors. This indicates that the preferred location of collisions is close to the promoter, regardless of the orientation of the DNA strands. This suggests that RNAPII promoter escape is a fundamental mechanism for preventing harmful TRCs. By mapping chromatin accessibility and histone marks, we discovered a "chromatin scar" at these sites, characterized by increased accessibility and specific enrichment of H3K4me3 and H3K79me2/3. We found that the mark H3K79me2/3, deposited by the methyltransferase DOT1L, serves as a functional bookmark; inhibiting DOT1L led to reduced transcriptional output and exacerbated DNA damage. This proves that specific epigenetic modifications are required for effective transcription recovery. Furthermore, screens identified that functional homologous recombination, specifically involving BRCA2 and RAD51B, is essential for resolving R-loop-dependent conflicts.
Beyond model systems, the project investigated these dynamics within human cancer cells using a cutting-edge APEX2 proximity labeling system. Established in MCF7 breast cancer cells, this system allowed us to identify the "proxisome"—the specific proteins that gather at the interface of colliding machineries. From a pool of 88 candidate proteins, we identified CGGBP1 (CGG-trinucleotide repeat binding factor 1) as a vital mitigator of TRCs at short CGG-repeats. Our research showed that CGGBP1 binds to human gene promoters prone to R-loop formation and recruits DEAD-box helicases DDX41 and DHX15 to unwind these structures. In cells lacking CGGBP1, we observed massive RNAPII stalling and transcription elongation defects. This demonstrates that cells rely on specialized DNA-binding factors to police repetitive elements that would otherwise be highly unstable.
The project also uncovered a critical role for transcription-replication interference during early mammalian development. By studying mouse embryos and embryonic stem cells (mESCs), we discovered a surprising developmental shift. While early embryos maintain high coordination, pluripotent stem cells naturally exhibit poor coordination and high levels of TRCs. We identified the RNA:DNA helicase Aquarius (AQR) as a primary protector of cell identity. When AQR is depleted in stem cells, the existing poor coordination is exacerbated, leading to increased cell fate heterogeneity. These findings reveal that maintaining transcription–replication coordination is not just a housekeeping task for DNA repair, but is essential for maintaining the stability and identity of stem cells during development.
ConflictResolution has significantly advanced our understanding of how life manages its most fundamental internal conflicts. By identifying the H3K79me2/3 mark as a signature of resolution and uncovering the role of factors like CGGBP1 and Aquarius, we have provided a new framework for understanding genomic health. These findings offer potential new avenues for cancer therapy by targeting the pathways cells use to survive replication stress and provide a new lens through which to view developmental diseases and the aging process. Through this work, we have moved from simply knowing that these molecular "traffic jams" exist to understanding the sophisticated mechanical and epigenetic systems that keep our genetic information flowing smoothly.
To address where these molecular accidents occur, the project utilized a sophisticated episomal reporter system to characterize the genetic and epigenetic consequences of collisions. Using ChIP-qPCR on the mAIRN reporter gene, we observed strong promoter-proximal pausing of the transcription machinery (RNAPII) coinciding with an accumulation of replication factors. This indicates that the preferred location of collisions is close to the promoter, regardless of the orientation of the DNA strands. This suggests that RNAPII promoter escape is a fundamental mechanism for preventing harmful TRCs. By mapping chromatin accessibility and histone marks, we discovered a "chromatin scar" at these sites, characterized by increased accessibility and specific enrichment of H3K4me3 and H3K79me2/3. We found that the mark H3K79me2/3, deposited by the methyltransferase DOT1L, serves as a functional bookmark; inhibiting DOT1L led to reduced transcriptional output and exacerbated DNA damage. This proves that specific epigenetic modifications are required for effective transcription recovery. Furthermore, screens identified that functional homologous recombination, specifically involving BRCA2 and RAD51B, is essential for resolving R-loop-dependent conflicts.
Beyond model systems, the project investigated these dynamics within human cancer cells using a cutting-edge APEX2 proximity labeling system. Established in MCF7 breast cancer cells, this system allowed us to identify the "proxisome"—the specific proteins that gather at the interface of colliding machineries. From a pool of 88 candidate proteins, we identified CGGBP1 (CGG-trinucleotide repeat binding factor 1) as a vital mitigator of TRCs at short CGG-repeats. Our research showed that CGGBP1 binds to human gene promoters prone to R-loop formation and recruits DEAD-box helicases DDX41 and DHX15 to unwind these structures. In cells lacking CGGBP1, we observed massive RNAPII stalling and transcription elongation defects. This demonstrates that cells rely on specialized DNA-binding factors to police repetitive elements that would otherwise be highly unstable.
The project also uncovered a critical role for transcription-replication interference during early mammalian development. By studying mouse embryos and embryonic stem cells (mESCs), we discovered a surprising developmental shift. While early embryos maintain high coordination, pluripotent stem cells naturally exhibit poor coordination and high levels of TRCs. We identified the RNA:DNA helicase Aquarius (AQR) as a primary protector of cell identity. When AQR is depleted in stem cells, the existing poor coordination is exacerbated, leading to increased cell fate heterogeneity. These findings reveal that maintaining transcription–replication coordination is not just a housekeeping task for DNA repair, but is essential for maintaining the stability and identity of stem cells during development.
ConflictResolution has significantly advanced our understanding of how life manages its most fundamental internal conflicts. By identifying the H3K79me2/3 mark as a signature of resolution and uncovering the role of factors like CGGBP1 and Aquarius, we have provided a new framework for understanding genomic health. These findings offer potential new avenues for cancer therapy by targeting the pathways cells use to survive replication stress and provide a new lens through which to view developmental diseases and the aging process. Through this work, we have moved from simply knowing that these molecular "traffic jams" exist to understanding the sophisticated mechanical and epigenetic systems that keep our genetic information flowing smoothly.
Aim 1 characterized the genetic and epigenetic consequences of collisions using an mAIRN episomal model. ChIP-qPCR revealed that RNAPII and replication factors accumulate at promoter-proximal sites regardless of construct orientation, suggesting that RNAPII promoter escape is a primary mechanism for preventing transcription-replication conflicts (TRCs). MNase digestion confirmed increased chromatin accessibility at these sites, supporting the “chromatin scar” hypothesis. Epigenetic mapping identified specific enrichment of H3K4me3 and H3K79me2/3 during TRCs. Specifically, inhibiting the H3K79 methyltransferase DOT1L reduced transcriptional output and increased DNA damage, indicating that this mark is required for transcription recovery and collision resolution. Finally, a siRNA screen identified BRCA2, RAD51B, and the helicase Aquarius (AQR) as essential factors for resolving R-loop-dependent conflicts.
Aim 2 established a Split-APEX2 proximity labeling system in MCF7 cells to map the TRC "proxisome." Mass spectrometry identified 88 candidate proteins, most notably CGGBP1. We found that CGGBP1 binds promoter-proximal CGG-repeats to mitigate R-loop formation and TRCs. Loss of CGGBP1 led to deregulated transcription and RNAPII stalling at both episomal and endogenous loci. Mechanistically, CGGBP1 recruits DEAD-box helicases DDX41 and DHX15 to unwind R-loops. This work identifies short trinucleotide repeats as significant sources of genomic instability and demonstrates that specific DNA-binding factors are required to maintain coordination at these repetitive sequences.
Aim 3 explored transcription-replication interference during mouse embryonic development using RNAPII-PCNA Proximity Ligation Assays (TRC-PLA). We discovered that pluripotent mouse embryonic stem cells (mESCs) exhibit higher TRC levels and poorer coordination compared to early-stage embryos. siRNA screens identified INO80, RECQL5, and AQR as key mediators of this coordination. Further characterization revealed that AQR is a crucial protector of cell identity; its depletion in mESCs exacerbated coordination defects and increased cell fate heterogeneity. These findings establish that preventing R-loop-driven instability is essential for maintaining stem cell identity and proper zygotic genome activation.
Aim 2 established a Split-APEX2 proximity labeling system in MCF7 cells to map the TRC "proxisome." Mass spectrometry identified 88 candidate proteins, most notably CGGBP1. We found that CGGBP1 binds promoter-proximal CGG-repeats to mitigate R-loop formation and TRCs. Loss of CGGBP1 led to deregulated transcription and RNAPII stalling at both episomal and endogenous loci. Mechanistically, CGGBP1 recruits DEAD-box helicases DDX41 and DHX15 to unwind R-loops. This work identifies short trinucleotide repeats as significant sources of genomic instability and demonstrates that specific DNA-binding factors are required to maintain coordination at these repetitive sequences.
Aim 3 explored transcription-replication interference during mouse embryonic development using RNAPII-PCNA Proximity Ligation Assays (TRC-PLA). We discovered that pluripotent mouse embryonic stem cells (mESCs) exhibit higher TRC levels and poorer coordination compared to early-stage embryos. siRNA screens identified INO80, RECQL5, and AQR as key mediators of this coordination. Further characterization revealed that AQR is a crucial protector of cell identity; its depletion in mESCs exacerbated coordination defects and increased cell fate heterogeneity. These findings establish that preventing R-loop-driven instability is essential for maintaining stem cell identity and proper zygotic genome activation.
We developed CATCH-IT, a new technology designed to identify the molecular players involved in TRC resolution. While we pioneered Proximity Ligation Assays (PLA) to detect TRC levels in situ, PLA lacks genomic precision, and standard proximity labeling requires time-consuming genetic engineering. CATCH-IT combines the best of both worlds as a versatile tool applicable to any cell type or tissue sample—including patient material—without requiring genetic engineering. It allows researchers to “catch in the act” functionally active protein-protein interactions (PPIs) at sites of genomic conflict, providing an unbiased approach to understanding the proxisome of stressed replication forks. Proteins function through stable or transient complexes essential for cell growth; yet, while 130,000 human PPIs are known, only a few hundred are currently therapeutic targets. A major challenge in drug discovery is that critical PPIs often only appear when a biological process is initiated. CATCH-IT overcomes this by identifying clinically relevant PPIs as they happen. By shortcutting the laborious screening process for cancer drug targets, this technology offers immense potential for the global oncology market. The ConflictResolution project has not only mapped the mechanics of how cells manage internal traffic but has also provided the tools to translate these insights into therapeutic strategies for cancers characterized by high replication stress.