Advances in single-cell omics to study genome organization, regulation, and repair

Abstract

This dissertation explores intercellular variation in genome organization, regulation, and repair. My research advances two areas in particular: (1) technological innovation of single-cell sequencing tools for chromatin profiling and analysis of DNA-binding proteins, and (2) investigation of heterogeneity in DNA repair and its interplay with genome topology. The work focuses on technology- and discovery-driven insights: it addresses fundamental regulatory principles using molecular biology and engineering. While highly relevant for the development of therapeutic strategies and companion diagnostics, it does not aim to provide direct clinical applications. Additionally, although I incorporate microscopy-based approaches throughout the thesis, a workflow for fully integrated single-cell imaging and sequencing remains a future goal. Chapter 2 presents a detailed experimental protocol to perform single-cell DamID sequencing. It focuses on studying nuclear organization through genome-wide mapping of nuclear lamina-associated domains. The protocol implements multiplexing to improve throughput and reduce technical variability, making large-scale single-cell chromatin mapping more efficient and cost-effective. Chapter 3 describes EpiDamID, a novel approach for detecting post-translational histone modifications using genetically engineered chromatin binders. In combination with scDam&T-seq, this enables joint profiling of chromatin state and transcriptional output in single cells, and remains the only antibody-free technique to do so. Chapter 4 details the development of a dual readout system combining DamID and ChIC, with two distinct manifestations: 1) As a multi-factorial method, two different DNA-binding proteins can be simultaneously profiled. 2) As a multi-modal method, the same protein can be measured with two different temporal resolutions, namely the cumulative signal of DamID versus the snapshot signal of ChIC, contributing a unique view on binding dynamics. Chapter 5 presents the first genome-wide single-cell maps of DNA repair protein localization at double-strand breaks. By applying the techniques described in the previous chapters to this novel context, I present a single-cell genomics toolbox to study heterogeneity in DNA repair, from epigenetic chromatin context to transcriptional changes in response to damage. Chapter 6 builds upon the data generated in Chapter 5 and investigates single-cell coordination of repair proteins at sites of damage. I present evidence that damaged loci cluster in space, forming higher-order contacts that are coordinately bound by repair protein, indicating cooperative behavior. Chapter 7 provides a comprehensive overview of recent progress in single-cell and single-molecule chromatin profiling, positioning the methodologies developed in this dissertation within the broader landscape of genome regulation research. Chapter 8 contextualizes above findings, first addressing the technical features of current single-cell techniques. In relation to Chapter 3, I briefly discuss epigenetic regulation with a focus on Polycomb repressive complexes in development and disease. Then I consider my work on DNA repair profiling and genome organization, highlighting recent insights from other work and proposing future directions. Lastly, I reflect on the technological advancements presented in this dissertation, outlining potential short-term applications and long-term innovations. In sum, by advancing the scope of single-cell sequencing methodologies, this thesis provides new tools and insights into genome organization and DNA repair, laying the foundation for future studies into the chromatin-based regulation of genome stability.