|dc.description.abstract||The Anaphase-Promoting Complex (APC) plays an important role in cell cycle progression. This evolutionarily conserved multi-subunit ubiquitin ligase is responsible for targeting proteins that hinder passage through mitosis and G1 progression for ubiquitination and proteasome-dependent degradation. Our laboratory has previously linked the APC with mitotic chromatin metabolism, as APC mutants were shown to exhibit impaired chromatin assembly. Chromatin assembly occurs when appropriately acetylated histones are deposited onto DNA. To date the only chromatin assembly factor linked to the cell cycle is the evolutionarily conserved CAF-I, a three-subunit complex of Cac1, Cac2 and Msi1. CAF-I associates with the histone chaperone Asf1, which first presents histones H3 and H4 to the histone acetyltransferases (HATs), Gcn5 and Rtt109, for acetylation. Following acetylation the histones are then passed on to CAF-I, which facilitates chromatin formation. Defective chromatin assembly has been linked to mitotic defects, leading to chromosomal rearrangements and aneuploidy. In addition to chromatin assembly, histone modifications have been linked to transcriptional activity and mitotic progression. The molecular mechanisms employed by the APC to govern chromatin biogenesis are unknown. In this thesis project, a modified genetic screen was performed to identify HAT and histone deacetylase (HDAC) mutants that interacted with APC mutants in the budding yeast Saccharomyces cerevisiae. This thesis focuses on the genetic and biochemical interactions observed between the APC and the HATs, Elp3 and Gcn5. As the majority of the proteins involved in chromatin assembly and histone modification are evolutionarily conserved, the insights obtained from the studies presented here utilizing the budding yeast S. cerevisiae should be directly applicable to research in human cells.
Via Western assays, this thesis demonstrates that yeast cells harboring mutations to the APC exhibit altered histone acetylation levels as well as altered total histone levels. Our genetic screen found that the temperature sensitive apc5CA (chromatin assembly) mutant genetically interacted with a number of HATs and HDACs. Combining the apc5CA allele with deletion of the genes ELP3, GCN5, HDA1 or SAS3 worsened the growth of the apc5CA mutant, whereas deletion of HOS1, HOS2, HOS3 or SAS2 improved the growth of the apc5CA mutant. Consistent with the genetic interaction results, increased expression of genes encoding the HATs Elp3, Gcn5 and Rtt109 (binds to Asf1) rescued the apc5CA temperature sensitive phenotype. The temperature sensitive phenotype of the apc5CA mutant was also rescued by increased expression of the genes encoding the CAFs Asf1 and Msi1 (a CAF-I subunit), as well as those encoding histones H3 and H4. These results suggest that increased deposition of acetylated histones is beneficial to APC function. Further analysis demonstrated that the APC and the HATs Elp3 and Gcn5 interact in the same pathway: cells lacking ELP3 or GCN5 accumulated in mitosis, whereas cells lacking both accumulated in G1 regardless of whether the APC was mutated or not. Additionally, increased APC5 expression partially rescued the severely slow growing elp3∆ gcn5∆ double mutant. Elp3 and Gcn5 do not activate the APC, as the APC target Clb2 remained unstable in elp3∆ gcn5∆ cells. Our analysis suggests that Elp3, Gcn5 and the APC work together to promote mitotic progression. However, as increased expression of ELP3 or GCN5 causes cells to arrest in G1 this may reflect a need to degrade Gcn5 and/or Elp3 to exit G1. This is consistent with previous findings that show Gcn5 is required to transcribe genes necessary for mitotic exit. Using protein degradation assays we determined that Gcn5 is unstable during G1 in an APC dependent manner. Furthermore, wild-type Elp3 modification patterns are dependent on various APC subunits, the E2 Ubc1 and the proteasomal ubiquitin receptor Rpn10.
This thesis presents a model where the activities of Elp3 and Gcn5, along with the APC, promote mitotic exit and G1 progression, but that Gcn5, and possibly Elp3, must be degraded to allow progression into S-phase. The APC is further linked to chromatin assembly in that the APC physically interacted with the CAFs Asf1 and Cac2 (a CAF-I subunit). This interaction with Cac2 still occurred in the absence of Asf1. The literature has genetically linked Cac2 with Gcn5 and here my findings demonstrate that Cac2 and Gcn5 physically associate. Taken together, the data presented in this thesis suggest that the APC may bring the proteins involved in chromatin assembly and histone modification into close proximity in order to facilitate and possibly optimize chromatin assembly and subsequently genomic stability.||en_US