Hos1 Is a Lysine Deacetylase for the Smc3 Subunit of Cohesin

Bo Xiong, Shuai Lu, Jennifer L. Gerton
2010 Current Biology  
Chromosome cohesion is a cell-cycle-regulated process in which sister chromatids are held together from the time of replication until the time of separation at the metaphaseto-anaphase transition, ensuring accurate chromosome segregation [1] [2] [3] [4] [5] [6] [7] [8] [9] . Chromosome cohesion is established during S phase, and this process requires the four subunits of the cohesin complex (Smc1, Smc3, Mcd1/Scc1, and Irr1/ Scc3) and the acetyltransferase Eco1 [10] [11] [12] [13] . Acetylation
more » ... f Smc3 by Eco1 at two evolutionarily conserved lysine residues promotes cohesion establishment during S phase in budding yeast and humans [14] [15] [16] . Here we report that Hos1, a member of the evolutionarily conserved class I histone deacetylase family, acts as a deacetylase for Smc3 in S. cerevisiae. We examine the Smc3 acetylation level in nine histone deacetylase deletion strains and find that the acetylation level is increased specifically in a hos1D strain post-S phase. Coimmunoprecipitation experiments show that Hos1 interacts with Smc3 and that the interaction is most pronounced as cells reach anaphase. We provide direct evidence that Hos1 can deacetylate Smc3 and retains a soluble pool of deacetylated Smc3. Overexpression of Hos1 results in less acetylation of Smc3 and cohesion defects in both WT and eco1 mutant strains; mutation of the Hos1 active site abolishes the defects. Hos1 may help to maintain a pool of unacetylated Smc3 that can be used for new chromosome cohesion. Results and Discussion Identification of a Deacetylase for Smc3 Reversible acetylation of histone and non-histone proteins by HATs (histone acetyltransferases) and HDACs (histone deacetylases) has been increasingly reported to regulate many cellular processes, including gene transcription; DNA replication, repair, and recombination; metabolism; cytoskeletal dynamics; apoptosis; protein folding; and cellular signaling [17] [18] [19] [20] [21] . Because the establishment of chromosome cohesion depends on Smc3 acetylation by the acetyltransferase Eco1, it is reasonable to speculate that a deacetylase for Smc3 might exist. In budding yeast, ten known histone deacetylases have been identified and divided into three main classes (I, II, and III) on the basis of sequence homology [22, 23] . Class I and II deacetylases use zinc-dependent catalysis, whereas class III deacetylases are NAD+ dependent. To examine whether one of the histone deacetylases is responsible for Smc3 deacetylation, we immunoprecipitated the cohesin complex from extracts of deacetylase deletion strains and then examined the Smc3 acetylation level by using anti-acetyl-lysine antibody. As shown in Figure 1A , the Smc3 acetylation level in a hos1D strain was significantly increased over that in wild-type and other deacetylase deletion strains, suggesting that Hos1 is a potential candidate for an Smc3 deacetylase. Because Smc3 acetylation by Eco1 occurs during S phase, we asked in which phase of the cell cycle Hos1 exerts its function. The cohesin complex was immunoprecipitated from a culture arrested in G1 and at various times after release for both wild-type and hos1D strains, and Smc3 acetylation was analyzed. In G1 cells, Smc3 acetylation was not detectable in either wild-type or a hos1D strain; no Smc3 was pulled down because of the instability of Mcd1 at this time. Once cells entered S phase, Smc3 acetylation could be observed. At time points following DNA replication (45, 60, and 80 min after release), the level of Smc3 acetylation was clearly higher in a hos1D strain than in the wild-type, suggesting Hos1 might normally function after S phase ( Figure 1B) . We further investigated whether acetylation was affected in the chromatinbound fraction (the cohesive fraction) of Smc3 or the soluble fraction. Although acetylated Smc3 clearly increases in the hos1D strain in the chromatin-bound fraction, what is more striking is the presence of acetylated Smc3 in the soluble fraction ( Figure 1C ). These results suggest that acetylation of Smc3, and particularly soluble Smc3, is regulated by Hos1 after S phase. Hos1 Interacts with Smc3 in a Cell-Cycle-Dependent Manner Given that Hos1 regulates acetylation of Smc3, we tested for an interaction between Hos1 and Smc3. An interaction between Hos1 and Smc3 can be detected in an asynchronous culture ( Figure 2A ). Because Smc3 deacetylation by Hos1 is cell-cycle regulated, we examined the interaction over the cell cycle. Coimmunoprecipitation (co-IP) was carried out from whole-cell extracts in a G1 release experiment similar to that in Figure 1 in the epitope-tagged strains, and immunoblotting was then performed. The interaction between Hos1 and Smc3 was most strongly detected in cells with 2N DNA content ( Figure 2B) , consistent with the idea that Hos1 might act on Smc3 after S phase. We stained the nuclei from the 60 and 80 min time point with DAPI to determine whether the cells contained one or two DAPI masses, indicative of metaphase or anaphase, respectively. At the 60 min time point, 17% of cells had two DAPI masses, and 44% had two DAPI masses at the 80 min time point. These results suggest that the interaction between Smc3 and Hos1 is increasing as the cells progress to anaphase.
doi:10.1016/j.cub.2010.08.019 pmid:20797861 fatcat:4k2lkikrijay7dbip2nw5sak6m