Novel Insights into Nuclear Localization of Proteins Crucial for Chromatin Assembly Metabolism in Saccharomyces cerevisiae

Candidate: Nora Danna,

Supervisor: Dr. Jeffrey Fillingham

Abstract:

Proper chromatin assembly is critical for maintaining genetic integrity, accurate DNA repair as well as gene expression. These processes are fundamental in eukaryotic cells, and studying them in the powerful budding yeast genetic model allows us to uncover novel mechanisms in the regulation of chromatin assembly and dynamics. Chromatin is assembled by replication-independent or replication-coupled pathways, where histone chaperones and modifying enzymes (such as HATs) are key factors in regulating these mechanisms. Yet, the underlying mechanisms are highly complex and incompletely understood. Many studies provided insights into the important role of histone chaperones and HATs in maintaining epigenetic information and chromatin stability. Alterations in these factors have been implicated in cancer and multiple human diseases. Therefore, the goal is to investigate the involvement of histone chaperones and HATs in the regulation of chromatin assembly.

One interesting aspect of my research is the evolutionarily conserved histone H3-H4- specific chaperone Hif1. Hif1, as well as its human homolog NASP, is involved in a wide array of chromatin-related processes, including histone H3-H4 transport, chromatin assembly and DNA repair. This research elucidated several functional aspects of Hif1. I revealed that an acidic region interpreting the second TPR domain of Hif1 is essential for Hif1 physical interactions with Hat2 (HAT-B complex), H4 (Histone H3-H4), as well as the histone H3-H4 chaperone III Asf1. Moreover, this study was the first to demonstrate that the nuclear localization of Hif1 is regulated by a classical nuclear localization sequence (cNLS) located at its C-terminus. Another aspect of my research is to investigate the role of putative cNLSs in the complex interplay between Rtt109, Vps75 and Asf1 in H3K56ac in S cerevisiae. Proteins holding cNLSs are designated to function in the nucleus, and their entry is critical to this compartment. Dysregulation of a nuclear protein can be deleterious and results in several human diseases, including cancer. In yeast, H3K56ac plays a key role in DNA replication and genome stability, and the basic-patch at the C-terminus of Rtt109 is also important for H3K56ac. Therefore, I began to investigate the fungus-specific HAT Rtt109 that is required for the Asf1-dependant acetylation of K56 on histone H3. Here, I showed that this basic-patch of Rtt109 is indeed a functional cNLS. I further provided evidence that Rtt109 is imported to the nucleus through its cNLS in a redundant manner with the histone chaperon Vps75. Furthermore, I identified a cNLS within the acidic C- terminus of Asf1 in S. cerevisiae. I confirm that this cNLS is critical for Asf1 nuclear localization. My work also demonstrates that this cNLS of Asf1 is required for full H3K56 acetylation.

Additionally, this research expounds on how the cNLSs of Hif1, Rtt109 and Asf1 can mediate their nuclear import pathway. I specifically showed that Kap123 is partially involved in Rtt109 nuclear localization and import, conversely, Hif1 and Asf1 might use Kap123 as a secondary transporter. Finally, I provide evidence that Kap95 might be one major importin that plays an important role in Hif1, Asf1 and Rtt109 nuclear import pathway. To my knowledge, this study is the first to reveal the presence of functional cNLS motifs within Hif1, Asf1 and Rtt109. This novel finding may provide insights into learning about analogous pathways in human cells.