Cellular response to defects in chromatin assembly: How nascent chromatin formation is linked to cell cycle progression and epigenome maintenance

Abstract

Chromatin assembly during DNA replication is a fundamental process that ensures the faithful inheritance of both genetic and epigenetic information during cell division. Disruptions to this tightly regulated mechanism can trigger profound cellular responses that impact genome stability, transcriptional fidelity, and ultimately influence cell fate decisions. Central to this process is the histone chaperone Chromatin Assembly Factor 1 (CAF-1), which deposits newly synthesized histones onto nascent DNA behind replication forks. While its canonical function in nucleosome assembly during S-phase is well established, the broader consequences of its loss, as well as its potential roles outside of chromatin replication, remain incompletely understood. In this thesis, we systematically investigated the acute and long-term consequences of defective chromatin assembly using CAF-1 as a central model system. Through the generation of specialized human cell lines and the application of advanced (single-cell) genomic, proteomic, and imaging technologies, we examined how cells respond to chromatin assembly defects at multiple levels. In addition, we studied the interactome of CAF-1 during different phases of the cell cycle to investigate potential functions outside of canonical chromatin replication. Our studies do not only reveal how CAF-1 safeguards chromatin integrity during DNA replication, but also provides insight into its interactions and possible contributions to epigenome regulation and heterochromatin stability throughout the cell cycle. This integrative approach allowed us to connect molecular mechanisms to broader cellular outcomes, expanding our understanding of the complex, fundamental and multi-faceted roles of the CAF-1 complex. Our work establishes CAF-1 as a central node in the maintenance of chromatin integrity, linking replication, transcriptional control, and cellular stress responses. It highlights that chromatin assembly is not only essential for genome duplication but is intricately connected to cellular identity, fate, and metabolic adaptation. Our work highlights that proper functioning of CAF-1 is essential for chromatin structure, cell cycle progression and genome stability, due to and beyond its canonical role in nucleosome assembly in S-phase. This thesis thus provides a framework for understanding how cells respond to chromatin disruption and sets the stage for future research into the multi-faceted roles of the CAF-1 complex.