Antagonistic Gcn5-Hda1 interactions revealed by mutations to the Anaphase Promoting Complex in yeast

Abstract

BackgroundHistone post-translational modifications are critical for gene expression and cell viability. A broad spectrum of histone lysine residues have been identified in yeast that are targeted by a variety of modifying enzymes. However, the regulation and interaction of these enzymes remains relatively uncharacterized. Previously we demonstrated that deletion of either the histone acetyltransferase (HAT) GCN5 or the histone deacetylase (HDAC) HDA1 exacerbated the temperature sensitive (ts) mutant phenotype of the Anaphase Promoting Complex (APC) apc5CA allele. Here, the apc5CA mutant background is used to study a previously uncharacterized functional antagonistic genetic interaction between Gcn5 and Hda1 that is not detected in APC5 cells.ResultsUsing Northerns, Westerns, reverse transcriptase PCR (rtPCR), chromatin immunoprecipitation (ChIP), and mutant phenotype suppression analysis, we observed that Hda1 and Gcn5 appear to compete for recruitment to promoters. We observed that the presence of Hda1 can partially occlude the binding of Gcn5 to the same promoter. Occlusion of Gcn5 recruitment to these promoters involved Hda1 and Tup1. Using sequential ChIP we show that Hda1 and Tup1 likely form complexes at these promoters, and that complex formation can be increased by deleting GCN5.ConclusionsOur data suggests large Gcn5 and Hda1 containing complexes may compete for space on promoters that utilize the Ssn6/Tup1 repressor complex. We predict that in apc5CA cells the accumulation of an APC target may compensate for the loss of both GCN5 and HDA1.

The Anaphase Promoting Complex Regulates Yeast Lifespan and rDNA Stability by Targeting Fob1 for Degradation

“…In the absence of Rpn10, we previously demonstrated that potential APC substrates accumulate within cells (Islam et al 2011). Consistent with Fob1 modification, and perhaps ubiquitination, we observed an accumulation of higher molecular weight bands when we expressed Fob1-HA from the GAL-inducible promoter in asynchronous rpn10Δ cells ( Figure 6B).…”

“…The short lived apc5 CA mutant has a broad range of genomic instability issues including chromatin assembly and histone metabolism defects (Harkness et al 2002;2005;Arnason et al 2005;Turner et al 2010;Islam et al 2011), increased rDNA recombination ( Figure 3D), and increased plasmid loss ( Figure 3E; Harkness et al 2002), which is an approximation of chromosome loss.…”

“…These functions include maintenance of genomic stability and control of chromatin metabolism. Several reports demonstrate that APC mutants result in loss of chromosomes and plasmids at accelerated rates (Hartwell and Smith 1985;Palmer et al 1990;Harkness et al 2002) and lack the ability to effectively assemble chromatin in vitro, as well as properly regulate histone post-translational modifications (Harkness et al 2002Arnason et al 2005;Harkness 2005;Turner et al 2010;Islam et al 2011). The compilation of defects observed in APC mutants manifest as reduced lifespan in yeast and mice (Baker et al 2004;Harkness et al 2004).…”

Sumoylation controls the timing of Tup1-mediated transcriptional deactivation

“…To this end, we examined whether other proteins known to interact physically or genetically with Tup1 are also SUMO targets, and whether their sumoylation is induced by SM treatment. These include histone H3 42 , Cti6, which relieves Tup1 transcriptional repression 25 , Gcn5 43 , Gal11 24 , 26 and Cyc8 20 , 44 , 45 . We generated strains expressing HA-tagged versions of each of these proteins from their chromosomal loci, and subjected them to SM treatment for 20 min.…”

Mitotic degradation of yeast Fkh1 by the Anaphase Promoting Complex is required for normal longevity, genomic stability and stress resistance

“…Cdc20 controls APC function through mitosis, while Cdh1 regulates APC-dependent processes through G1 passage. We have described biological roles affected by the APC that go beyond lifespan, including critical functions in stress response, mitotic chromatin assembly, and mitotic-associated histone modifications [ 4 , 26 - 29 ]. We observed that deletion of both FKH genes was necessary to further impair mutant APC phenotypes, such as sensitivity to temperature and oxidative stress, and reduced lifespan, indicating how important this combination of genes is to cell health and adaptive survival.…”