Transcription-Replication Conflicts and R-loops disrupt the local chromatin landscape in human model cell lines

Werner, Marcel (2025): Transcription-Replication Conflicts and R-loops disrupt the local chromatin landscape in human model cell lines. Dissertation, LMU München: Fakultät für Biologie

Abstract

Transcription and DNA replication are fundamental nuclear processes that control the expression of genes and ensure the correct duplication of the genome, respectively. Tight regulation of these molecular machines is of paramount importance as both dysregulated transcription and replication are associated with numerous diseases including cancer. Importantly, both processes require extensive remodeling of the underlying chromatin template to perform their function. Transcription-replication conflicts (TRCs) are nuclear events during which transcription and replication collide on the same DNA template during the S-phase of the cell cycle. Despite their rare occurrence, TRCs are potent inducers of DNA damage, mutations, and genomic instability. Given that transcription and replication extensively remodel chromatin during their progression on the genome, one hypothesis suggests that TRC sites might be particularly vulnerable to chromatin and epigenome alterations that could provoke genome instability. In this work, I engineered an inducible TRC reporter system by genomically integrating an R-loop-prone sequence previously used for conflict induction and characterized the dynamic changes of the local chromatin structure inflicted by TRCs. Importantly, the new reporter system was not only able to induce chromosomal TRCs with high efficiency but also induced drastic local replication impairments, which coincided with an activation of the DNA damage response in a local but also global manner. TRC-inducing cells were also particularly sensitive to inhibition of the Ataxia Telangiectasia and Rad3 related (ATR), a crucial kinase of the replication stress response. Analyzing the impact of TRCs on chromatin organization, I found a striking loss of nucleosome occupancy at the activated TRC reporter that was highly dependent on the stability of the associated R-loops. Furthermore, I investigated numerous histone modifications for an association with TRCs and found an increase in H3K79 methylation specifically at the R-loop forming TRC site. Through inhibition of H3K79 methylation writer enzyme disruptor of telomeric silencing-1-like (DOT1L), I showed that H3K79 methylation is actively deposited at TRC sites. Further, lack of DOT1L activity resulted in reduced transcriptional output and exacerbated DNA damage response, suggesting that deposition of this modification is required for effective transcription recovery and resolution of TRCs. Beyond the work on the reporter system, I characterized the potential of several oncogene overexpression cell lines to induce TRCs and identified CDC25 and CDC6 overexpression as potent disruptors of transcription-replication coordination. Ultimately, my work establishes a powerful new reporter system and cancer model cell lines to study TRC biology, while also uncovering novel chromatin dynamics at TRC sites and revealing a specific epigenetic modifier bookmarking TRCs, that are relevant to cancer and other diseases.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 5. August 2025
• 1. Bericht­erstatter:in: Torres-Padilla, Maria-Elena