Chromatin re-organization in response to replication stress

Project Information

ChOReS

Grant agreement ID: 101078750

DOI

10.3030/101078750

EC signature date 2 February 2023

Start date 1 March 2023

End date 29 February 2028

Funded under

• European Research Council (ERC)

Total cost

€ 1 500 000,00

EU contribution

€ 1 500 000,00

Coordinated by

ERASMUS UNIVERSITAIR MEDISCH CENTRUM ROTTERDAM
Netherlands

Periodic Reporting for period 1 - ChOReS (Chromatin re-organization in response to replication stress)

Reporting period: 2023-03-01 to 2025-08-31

Summary of the context and overall objectives of the project

Cells must copy their entire DNA during every cell division, a process known as DNA replication. However, this process is frequently disrupted by environmental stress, genetic mutations, or cancer therapies, leading to a condition called replication stress. Unresolved replication stress threatens the stability of the genome and can fuel the development and progression of cancer. Understanding how cells normally protect their DNA during replication stress, and why some fail to do so, can reveal new strategies to improve cancer diagnosis and treatment.
This project has uncovered a new form of genome protection, in which chromatin, the structure that packages DNA, is temporarily reorganized into a silenced, compact form known as heterochromatin, specifically at DNA replication sites. This process is driven by the chromatin modifier enzyme G9a, which deposits a key chemical tag (H3K9me2/3) to protect fragile DNA at stalled replication forks. Our findings, published in Nature Cell Biology (2023), demonstrate that this de novo heterochromatin assembly, and its timely removal, are essential to ensure accurate DNA replication and to avoid DNA breaks.
To understand how DNA and chromatin are affected during replication stress, the project developed a suite of advanced technologies capable of probing 3D genome architecture and DNA integrity at high resolution. These tools have revealed how changes in chromatin structure at replication forks contribute to chemotherapy resistance in cancer cells. The technologies and discoveries generated in this project are being disseminated through collaborations.
In summary, this project is identifying chromatin remodeling at replication forks as a key genome-protective mechanism, linking chromatin architecture to fork degradation, and developing innovative tools for studying replication stress responses in both normal and cancer cells.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The project was structured around three major objectives:
1. Discovering new protective mechanisms at replication forks
Using cellular and molecular approaches, we identified a dynamic form of heterochromatin that forms specifically at stalled replication forks. This protective layer is formed by the enzyme G9a and the deposition of H3K9 methylation marks (H3K9me2/3). We showed that this layer stabilizes fragile DNA, prevents breakage, and must be removed once replication resumes to avoid genome instability.

2. Developing next-generation technologies to study replication stress
We created a new set of tools that allow researchers to directly visualize how chromatin and DNA behave during replication stress:
• Rep-ChIC captures chromatin specifically from replicating regions, enabling high-resolution mapping of histone modifications at stressed replication forks.
• Rep-HiC enriches for DNA undergoing replication, revealing changes in 3D chromatin architecture, key structural features of genome organization, that standard Hi-C methods miss.
• ForkDeg-seq maps genome-wide regions of DNA degradation, identifying areas where newly replicated DNA is lost under stress, particularly in cells lacking the BRCA2 gene, which plays a key role in DNA repair.

3. Linking chromatin structure to chemotherapy resistance
By combining these tools, we are investigating and have demonstrated to some extent that chromatin architecture protects newly synthesized DNA from degradation. In cancer cells, leading to uncontrolled DNA damage and resistance to chemotherapy. This provides a molecular explanation for why some cancers do not respond to treatment.

Outcomes of the Action
• A new model of genome protection via de novo heterochromatin formation at stalled replication forks
• High-resolution tools (Rep-ChIC, Rep-HiC, ForkDeg-seq) to map chromatin structure and fork stability
• Mechanistic link between chromatin architecture and chemotherapy resistance
• A clinical proof-of-concept study in ovarian cancer patients to evaluate H3K9me3 as a biomarker of treatment response
• Development of ChromStretch, a single-molecule assay to measure chromatin flexibility and predict treatment outcomes

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

This project goes beyond existing knowledge by revealing how 3D chromatin architecture actively protects the genome under stress. Previous studies focused primarily on DNA repair pathways. In contrast, this project highlights how the physical organization of chromatin itself serves as a first line of defense during replication stress.
The development of Rep-ChIC, Rep-HiC, and ForkDeg-seq represents a leap forward in the tools available for genome research. These technologies are now enabling the community to study replication stress responses with unprecedented resolution, particularly in cancer models.
Our discovery that chromatin architecture is essential for protecting DNA has major implications for cancer therapy. We are gaining new insights into how 3D genome organization differs between chemoresistant and chemosensitive BRCA1/2-mutated tumors, particularly in relation to their ability to establish protective epigenetic marks. These findings may open up new avenues for therapeutic intervention.
To support clinical translation, we are also launching a proof-of-concept study in ovarian cancer patients. This study aims to determine whether high levels of H3K9me3, a marker of heterochromatin formed at replication forks,can predict poor response to chemotherapy. If successful, this could lead to earlier and more personalized treatment decisions for patients.

Overview of Results
• Novel genome-protective mechanism discovered (heterochromatin at replication forks)
• Three state-of-the-art technologies developed for replication stress profiling
• Fundamental insights into chemotherapy resistance mechanisms in BRCA-deficient cancers
• Ongoing efforts toward clinical biomarker validation and single-molecule diagnostics
• Broad potential impact in cancer research, diagnostics, and therapy personalization