Molecular Mechanisms of Replication-Coupled Chromatin Assembly and Maturation

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2023

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Abstract

In eukaryotic cells, The local chromatin structures play a critical role in regulating all DNA-mediated events. However, the process of DNA replication is highly disruptive to the chromatin structure. Specifically, the parental chromatin needs to be disassembled before the passing of the replication fork and re-assembled afterward. Additionally, a maturation process is required for newly assembled chromatin to completely recover to the pre-replicative state. Rapid and faithful assembly and maturation of chromatin are essential for preserving the epigenome and maintaining genome integrity. Elegant in vitro biochemistry studies have allowed the characterization of numerous factors in the process of replication-coupled chromatin assembly. Recent advancements in genomic technologies further expanded our understanding of the process, providing genome-wide views of the spatial-temporal dynamics of chromatin maturation. However, there is still a lack of mechanistic details on many aspects of chromatin assembly, and the broader phenotypic significance of this process also remains unstudied.

One such example is Chromatin Assembly Factor 1 (CAF-1). It is one of the first histone chaperones identified that deposit histones onto the newly replicated DNA, yet there remain missing puzzles of CAF-1 ranging from the mechanisms by which CAF-1 interacts with DNA and the replisomes to its impact on chromatin maturation and how that relates to genome stability. Using nascent chromatin occupancy profiling (NCOP), I tracked the chromatin maturation kinetics in WT and CAF-1 mutant cells with high spatiotemporal resolution. I found that loss of CAF-1 results in a heterogeneous rate of nucleosome assembly, with individual nucleosomes exhibiting either rapid or slow maturation kinetics, ultimately leading to a global delay in chromatin maturation. The slow-to-mature nucleosomes are enriched with poly(dA:dT) sequences, suggesting CAF-1 facilitates (H3-H4)2 tetramer deposition and nucleosome maturation on sequences resistant to nucleosome formation. The assembly defect caused by loss of CAF-1 leads to the accumulation of nucleosome intermediates whose position is also influenced by underlying DNA sequences. The finding offers a new perspective on the pathway for nucleosome assembly in vivo. In addition, there exists a long-standing paradox that CAF-1 mutant cells exhibit loss of gene silencing and increased cryptic transcription, yet their steady-state chromatin landscape is nearly indistinguishable from that of WT cells. Our work illustrates that the dysregulation of transcription is temporary and occurs specifically in S phase, presumably as a result of transient defects in chromatin maturation. These results demonstrate how the DNA replication program can directly shape the chromatin landscape and regulate gene expression through the process of chromatin maturation.

During replication, the leading and lagging strands are replicated via distinct mechanisms. Numerous studies showed that different histone chaperones are employed for nucleosome assembly on the two strands; however, it remains unclear if or how the kinetics of chromatin maturation differ between the two strands. The current NCOP method does not distinguish actions on the leading and lagging strands. To address this, I developed strand-specific NCOP that captures the kinetics of chromatin maturation on the two daughter strands separately. Strand-specific NCOP experiments on CAF-1 and Mcm2 mutant cells provide evidence for the coordinated nucleosome assembly by Mcm2 and CAF-1 to ensure symmetric histone segregation and assembly on the daughter strands. Together, this work provides mechanistic insights into the role of multiple histone chaperones in chromatin assembly and maturation and opens up new avenues for understanding how disruption to these processes might contribute to defects in gene expression programs commonly found in disease states.

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Chen, Boning (2023). Molecular Mechanisms of Replication-Coupled Chromatin Assembly and Maturation. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/30294.

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