The Pulse of Nascent Chromatin: The Interplay Between Chromatin Dynamics and DNA replication

Abstract

Faithful duplication of chromatin during DNA replication is essential for preserving genome stability, maintaining epigenetic information, and ensuring proper cell identity. Central to this process is Chromatin Assembly Factor-1 (CAF-1), a conserved histone chaperone that deposits newly synthesized histones onto nascent DNA. Although CAF-1 has long been recognized as a key component of replication-coupled nucleosome assembly, the molecular principles governing its recruitment, regulation, and functional impact on genome-wide chromatin dynamics remain incompletely understood. This thesis investigates how CAF-1 works at the interface between DNA synthesis and chromatin formation, integrating biochemical and genome-wide approaches to define its mechanistic roles in nascent chromatin assembly. To clarify how CAF-1 engages the replication machinery, we looked at its interaction with proliferating cell nuclear antigen (PCNA), a core replisome factor that coordinates protein recruitment during DNA synthesis. Biochemical analyses demonstrated that mono-ubiquitination of PCNA, a modification associated with DNA damage tolerance and lagging-strand replication, does not impair CAF-1 binding or nucleosome assembly activity. This indicates that CAF-1 can function efficiently even when PCNA is modified, supporting a model in which histone deposition proceeds under diverse replication contexts. Genome-wide strand-specific analyses further revealed that CAF-1 deposits histones equally on leading and lagging strands, establishing that its activity during physiological replication lacks detectable strand bias and defining a baseline modality for de novo nucleosome assembly. To directly assess the consequences of impaired chromatin assembly, we implemented rapid protein-depletion strategies combined with genome-wide profiling of replication dynamics and nascent chromatin structure. These experiments revealed that loss of CAF-1 causes an immediate slowdown in replication fork progression, accompanied by a striking genome-wide increase in the accessibility of newly replicated DNA. These findings demonstrate that nucleosome assembly is tightly coupled to replication speed and chromatin maturation. Notably, these acute defects arise without activation of the canonical intra-S-phase checkpoint pathways, suggesting the existence of alternative mechanisms by which the replication machinery senses chromatin assembly status. Genome-wide analyses further showed that impaired chromatin assembly differentially affects chromatin domains, with facultative heterochromatin regions displaying reduced ability to restore compact structure compared with constitutive heterochromatin. This asymmetry suggests that chromatin assembly efficiency can influence epigenetic stability and transcriptional plasticity, providing a potential mechanism through which cells modulate gene expression programs during developmental transitions or stress responses. Finally, functional dissection of CAF-1 interaction domains demonstrated that its PCNA-binding module is essential for sustaining normal replication dynamics, whereas domains implicated in heterochromatin interactions are dispensable for replication progression. These findings establish a functional separation between CAF-1’s replication-coupled and chromatin-organizational activities and refine current models of its mechanism of action. Together, this work defines CAF-1 as a central integrator of genome duplication and epigenome maintenance. By revealing how nucleosome assembly influences replication behaviour, chromatin architecture, and cellular responses, this thesis provides a mechanistic framework for understanding how defects in chromatin assembly can reshape epigenetic landscapes and impact cell fate, with potential implications for development, reprogramming, and disease.