Defining Replication-Coupled Chromatin Assembly Pathways and their Effect on Silencing and Responses to DNA Damage in Saccharomyces cerevisiae
Date of Award
Doctor of Philosophy (PhD)
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Committee Member 2
Committee Member 3
The genetic information of a cell is organized in a DNA and protein structure referred to as chromatin. Chromatin consists of repeating units called nucleosomes, which consists of 147 base pairs of DNA wrapped around an octamer of histone proteins. These histone proteins are post-translationally modified at specific residues that are important gene regulation and genomic stability. Altered histone modification patterns are strongly associated with multiple cancers, underlying the importance of better understanding the pathways that regulate these modifications. During DNA replication, chromatin must be disassembled, and then reassembled behind the replication fork. Defects in this process can cause alterations in these histone modifications. Failure to properly maintain these histone modifications throughout the cell cycle can lead to defects epigenetically heritable gene silencing and in responses to DNA damage. Therefore, we aim to better define these replication-coupled chromatin assembly pathways and how they influence histone modifications that impact gene silencing and responses to DNA damage. One such histone modification is acetylation of H4 K16. Presence of this residue has a preventative effect on the formation of silent chromatin and hypoacetylation of this residue is associated with multiple cancers. Here we better define the replication-coupled chromatin assembly pathways that affect H4 K16ac levels in chromatin and provide insight into how these pathways may be regulated. During DNA replication, the deposition of newly synthesized H3-H4 histones to assemble nucleosomes onto newly synthesized DNA is coordinated by H3-H4 histone chaperones Asf1p, Rtt106p, and the CAF-1 complex, consisting of Cac1p, Cac2p and Cac3p. Currently, strong evidence suggests that Asf1p initially binds H3-H4 dimers and transfers these histones to Rtt106p and CAF-1, which are then able to promote H3-H4 tetramer formation onto the newly synthesized DNA. CAF-1 is targeted to the replication fork via its interactions with PCNA, which is important for proper histone deposition. Synthetic interactions between Rtt106p and CAF-1 indicate they are partially redundant in function. Here, we demonstrate that H4 K16ac deposition occurs independently of RTT106 and is mediated through a CAF-1 pathway, indicating that Rtt106p and CAF-1 have separate functions in regards to H4 K16ac. Additionally, we demonstrate that the cell cycle-dependent kinase Cdc7p is able to regulate silencing through influencing H4 K16ac likely via cell cycle-dependent interactions with CAF-1. Overall, this study provides insight into how H4 K16ac, a critical histone modification in regulating the formation of silent chromatin that is altered in human cancer, is regulated during replication-coupled chromatin assembly and how cell cycle-dependent kinases may influence histone modifications.
Young, Tiffany J., "Defining Replication-Coupled Chromatin Assembly Pathways and their Effect on Silencing and Responses to DNA Damage in Saccharomyces cerevisiae" (2017). Open Access Dissertations. 1669.