Genome-Wide Dynamics of Chromatin Maturation Following DNA Replication

MacAlpine, David M

Gutierrez, Monica P

2019-04-02T16:27:33Z

2019-07-09T08:17:07Z

2018

Genetics and Genomics

All DNA-templated events, including replication and gene transcription, occur in the context of the local chromatin environment. The passage of the replication machinery results in disassembly of chromatin, which must be re-assembled behind the replication fork to re-establish the epigenetic state of the cell. Many of the factors and mechanisms regulating DNA replication and chromatin assembly have been identified from elegant in vitro biochemical experiments, work in model systems like Saccharomyces cerevisiae, or novel proteomic approaches. In spite of current advances in the field, it is still not clear how the chromatin landscape is organized and re-assembled during this process.

Current methods, while informative, lack the genome-wide base-pair resolution required to assess the dynamics of chromatin assembly and maturation in a spatial-temporal manner. To overcome the limitations of these studies, I have taken advantage of an epigenome mapping technique based on micrococcal nuclease (MNase) digestion followed by paired-end sequencing. This approach facilitates the analysis of chromatin structure by capturing not only nucleosomes, but also smaller DNA binding protein footprints in a factor-agnostic manner. I have developed a technique based on this approach that generates Nascent Chromatin Occupancy Profiles (NCOPs) to study the dynamics of chromatin assembly following passage of the DNA replication fork at a genome-wide level and at single base-pair resolution in S. cerevisiae. It employs a nucleoside analog to specifically enrich for nascent chromatin, which can be captured following a chase over different periods of time. Thus, NCOPs resolve the structure of nascent and mature chromatin, facilitating the analysis of chromatin maturation across the entire genome.

Using NCOPs, I provide a comprehensive description of the maturation process across different genomic regions and the dynamics of small DNA binding factor association with nascent and mature chromatin states. Our results support previous work characterizing the structure of nascent chromatin as being more disorganized and having poorly positioned nucleosomes. Importantly, using positioning and occupancy scores, I provide new details on the structure of nascent and mature chromatin at intergenic regions, including replication origins, and at highly transcribed and poorly transcribed genes. I uncovered that local epigenetic footprints have the potential to shape the dynamics of chromatin assembly, generating a chromatin maturation landscape that is dependent on the parental chromatin. Finally, I resolved patterns of transcription factor occupancy with nascent and mature chromatin, and observed transient factor association in the nascent state.

In all, this work provides insight into the dynamics of chromatin assembly, and allows for genome-wide and base-pair resolution investigation of chromatin maturation. The genomic and bioinformatic approaches developed here open the door for further investigation of the dynamics of epigenetic inheritance and the role of known and unknown players in re-establishing the eukaryotic epigenome following passage of the DNA replication fork.

https://hdl.handle.net/10161/18263

Genetics

Chromatin

Chromatin assembly

DNA replication

Epigenetics

Nucleosome

Genome-Wide Dynamics of Chromatin Maturation Following DNA Replication

Dissertation

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