Description: Regulating levels of centromeric histone H3 (CenH3) variant is crucial for genome stability. Interaction of Psh1 , an E3 ligase, with the C terminus of Cse4 has been shown to contribute to its proteolysis. Here, we demonstrate a role for ubiquitination of the N terminus of Cse4 in regulating Cse4 proteolysis for faithful chromosome segregation and a role for Doa1 in ubiquitination of Cse4 .

Description: Centromeric localization of the evolutionarily conserved centromere-specific histone H3 variant CENP-A ( Cse4 in yeast) is essential for faithful chromosome segregation. Overexpression and mislocalization of CENP-A lead to chromosome segregation defects in yeast, flies, and human cells. Overexpression of CENP-A has been observed in human cancers; however, the molecular mechanisms preventing CENP-A mislocalization are not fully understood. Here, we used a genome-wide synthetic genetic array (SGA) to identify gene deletions that exhibit synthetic dosage lethality (SDL) when Cse4 is overexpressed. Deletion for genes encoding the replication-independent histone chaperone HIR complex ( HIR1 , HIR2 , HIR3 , HPC2 ) and a Cse4 -specific E3 ubiquitin ligase, PSH1 , showed highest SDL. We defined a role for Hir2 in proteolysis of Cse4 that prevents mislocalization of Cse4 to noncentromeric regions for genome stability. Hir2 interacts with Cse4 in vivo , and hir2 strains exhibit defects in Cse4 proteolysis and stabilization of chromatin-bound Cse4 . Mislocalization of Cse4 to noncentromeric regions with a preferential enrichment at promoter regions was observed in hir2 strains. We determined that Hir2 facilitates the interaction of Cse4 with Psh1 , and that defects in Psh1 -mediated proteolysis contribute to increased Cse4 stability and mislocalization of Cse4 in the hir2 strain. In summary, our genome-wide screen provides insights into pathways that regulate proteolysis of Cse4 and defines a novel role for the HIR complex in preventing mislocalization of Cse4 by facilitating proteolysis of Cse4 , thereby promoting genome stability.

Description: The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAPII) is required to regulate transcription and to integrate it with other essential cellular processes. In the budding yeast Saccharomyces cerevisiae , the CTD of Rpb1p consists of 26 conserved heptad repeats that are post-translationally modified to orchestrate protein factor binding at different stages of the transcription cycle. A long-standing question in the study of the CTD is if there are any functional differences between the 26 repeats. In this study, we present evidence that repeats of identical sequence have different functions based on their position within the CTD. We assembled plasmids expressing Rpb1p with serine to alanine substitutions in three defined regions of the CTD and measured a range of phenotypes for yeast expressing these constructs. Mutations in the beginning and middle regions of the CTD had drastic, and region-specific effects, while mutating the distal region had no observable phenotype. Further mutational analysis determined that Ser5 within the first region of repeats was solely responsible for the observed growth differences and sequencing fast-growing suppressors allowed us to further define the functional regions of the CTD. This mutational analysis is consistent with current structural models for how the RNAPII holoenzyme and the CTD specifically would reside in complex with Mediator and establishes a foundation for studying regioselective binding along the repetitive RNAPII CTD.

Description: Stringent regulation of cellular levels of evolutionarily conserved centromeric histone H3 variant (CENP-A in humans, CID in flies, Cse4 in yeast) prevents its mislocalization to non-centromeric chromatin. Overexpression and mislocalization of CENP-A has been observed in cancers and leads to aneuploidy in yeast, flies, and human cells. Ubiquitin-mediated proteolysis of Cse4 by E3 ligases such as Psh1 and Sumo-Targeted Ubiquitin Ligase (STUbL) Slx5 prevent mislocalization of Cse4. Previously, we identified Siz1 and Siz2 as the major E3 ligases for sumoylation of Cse4. In this study, we have identified lysine 65 (K65) in Cse4 as a site that regulates sumoylation and ubiquitin-mediated proteolysis of Cse4 by Slx5. Strains expressing cse4 K65R exhibit reduced levels of sumoylated and ubiquitinated Cse4 in vivo . Furthermore, co-immunoprecipitation experiments reveal reduced interaction of cse4 K65R with Slx5, leading to increased stability and mislocalization of cse4 K65R under normal physiological conditions. Based on the increased stability of cse4 K65R in psh1 strains but not in slx5 strains, we conclude that Slx5 targets sumoylated Cse4 K65 for ubiquitination-mediated proteolysis independent of Psh1. In summary, we have identified and characterized the physiological role of Cse4 K65 in sumoylation, ubiquitin-mediated proteolysis, and localization of Cse4 for genome stability.

Description: Predicting phenotypes based on genotypes and understanding the effects of complex multi-locus traits on plant performance requires a description of the underlying developmental processes, growth trajectories, and their genomic architecture. Using data from Brassica rapa genotypes grown in multiple density settings and seasons, we applied a hierarchical Bayesian Function-Valued Trait (FVT) approach to fit logistic growth curves to leaf phenotypic data (length and width) and characterize leaf development. We found evidence of genetic variation in phenotypic plasticity of rate and duration of leaf growth to growing season. In contrast, the magnitude of the plastic response for maximum leaf size was relatively small, suggesting that growth dynamics vs. final leaf sizes have distinct patterns of environmental sensitivity. Consistent with patterns of phenotypic plasticity, several QTL-by-year interactions were significant for parameters describing leaf growth rates and durations but not leaf size. In comparison to frequentist approaches for estimating leaf FVT, Bayesian trait estimation resulted in more mapped QTL that tended to have greater average LOD scores and to explain a greater proportion of trait variance. We then constructed QTL-based predictive models for leaf growth rate and final size using data from one treatment (uncrowded plants in one growing season). Models successfully predicted non-linear developmental phenotypes for genotypes not used in model construction and, due to a lack of QTL-by-treatment interactions, predicted phenotypes across sites differing in plant density.