proteins and has been proposed to recognize unmodified histone tails [20]. Lastly, the LisH domain has been shown to be important in binding to hypoacetylated histone H4 tails [21]. hif2D, set3D and snt1D mutants display cytokinesis defects upon perturbation of the cell division machinery If Hif2p, Set3p, and Snt1p act as part of a protein complex with a common function, then one would expect the respective loss of function mutants to exhibit similar phenotypes. To determine if this were the case, hif2D, set3D, and snt1D strains, as well as the respective double and triple mutant strains, were grown to mid-log phase in liquid YES at 30uC and 10-fold serial dilutions were plated onto YES plates containing LatA or DMSO (solvent control). Interestingly, all mutants displayed a reduced capacity for growth in the presence of LatA in comparison to wild type (Figure 2A). Next, the cells were examined at the microscopic level to determine if the observed sensitivity to LatA was related to defects in cytokinesis. The deletion mutants were treated with LatA (0.5 mM) or DMSO (solvent control) for 5 hours in liquid YES growth medium, and subsequently fixed and stained with DAPI and aniline blue to visualize the nucleus and cell wall/septum, respectively [22]. The cells were classified into four different phenotypic categories: i) uni-nucleate cells, ii) bi-nucleate cells with a morphologically normal septum (i.e. the septum completely bisects the cell), iii) bi-nucleate cells with a fragmented septum (i.e. the septum is non-functional and does not completely bisect the cell), and iv) tetra-nucleate cells. As shown in Figure 2B hif2D, snt1D, set3D and wild-type strains did not display any obvious growth defects when cultured in the presence of DMSO. In contrast, large proportions of hif2D, snt1D, and set3D mutant cells (49–75%) displayed a tetra-nucleate phenotype when treated with 0.5 mM LatA, whereas only 11% of wild type were tetra-nucleate (Figure 2B,C). Importantly, in support of the hypothesis that the three gene-products work within the same pathway, no significant synthetic effects were observed when the respective double and triple gene deletion mutants were assayed using only 0.1 mM LatA (Figure 2C). Moreover, genetic analysis with strains bearing mutations in genes encoding various cytokinesis regulators (myo2-E1, cdc12-112, cdc15-140) revealed specific synthetic interactions between the set3, snt1, and hif2 gene deletions and the temperature sensitive cdc15-140 mutation. The presence of either the hif2D, snt1D, or set3D gene deletions reduced the restrictive temperature of cdc15-140 strains to a similar extent (but did not affect the restrictive temperatures of myo2-E1 or cdc12- 112 strains) (Figure S2). Taken together, the similarities in phenotype observed in hif2D, snt1D, and set3D gene deletion mutants are consistent with a model in which the three proteins modulate a common biological process that influences the successful completion of cytokinesis in S. pombe. Set3p works in parallel to other regulators of the cytokinesis monitoring system The cytokinesis failure observed in hif2D, snt1D, and set3D mutants was reminiscent of that seen in the previously character- ized cytokinesis regulators, clp1D and lsk1D [8,13]. It was thus of interest to determine if these proteins functioned within the same pathway or in parallel to Clp1p (a Cdc14 family phosphatase) or Lsk1p (a Ser-2 CTD kinase), respectively. If Set3p functioned in a linear pathway with Clp1p or Lsk1p, one would expect set3D clp1D and set3D lsk1D double mutants to exhibit similar hyper-sensitivity to LatA relative to the respective single mutants. Conversely, if Set3p functioned in parallel, one would expect set3D clp1D and SET Domain Protein Regulates S. pombe Cytokinesis set3D lsk1D double mutants to exhibit a more severe sensitivity to LatA than either of the respective single mutants. To differentiate between these two possibilities, a wild-type strain, as well as the respective single and double deletion mutants, were treated with LatA (0.1 mM) or DMSO for 5 hours in liquid YES growth medium. This was followed by fixation and staining with DAPI and aniline blue to visualize the nucleus and cell/wall septum, respectively. Interestingly, both set3D clp1D and set3D lsk1D double mutants showed a significant increase in sensitivity to LatA (Figure 3). At a concentration of 0.1 mM both double mutants displayed a large proportion of tetra-nucleate cells with fragment- ed septa. In contrast, the phenotypes of single mutants at this dose were far less severe. These results are consistent with a model in which Set3p functions in parallel to both Clp1p and Lsk1p. Thus, set3 defines a novel regulatory pathway governing the successful completion of cytokinesis in fission yeast. Set3p, Hif2p and Snt1p form a nuclear-localized complex Bioinformatics suggested that the hif2, set3, and snt2 gene products exist as part of a conserved histone deacetylase complex. In order to function in chromatin remodeling, one would expect Hif2p, Set3p, and Snt2p to localize to the nucleus. To test this prediction, strains expressing GFP-tagged fusion proteins – integrated at their normal genomic loci and under the control of their native promoters – were constructed. Consistent with a role in chromatin modification, all three proteins localized to the nucleus (Figure 4A). The localization of the fusion proteins was not altered as a function of cell cycle position or as a function of LatA concentration in the growth medium (data not shown). Next, to determine if Hif2p, Set3p, and Snt1p physically interacted in vivo, co-immunoprecipitation experiments using Myc- or HA-epitope tagged alleles were performed. Interestingly, Set3-HA and Hif2-HA fusion proteins could be detected in anti- Myc immuno-precipitates of Snt1-Myc Set3-HA and Snt1-Myc Hif2-HA extracts (Figure 4B, top and middle panels). Likewise, Hif2-HA proteins could be detected in anti-Myc immuno- precipitates of Set3-Myc Hif2-HA extracts (Figure 4B, bottom panel). Taken together, these data demonstrate that Hif2p, Set3p, and Snt1p exist as part of a nuclear-localized protein complex in S. pombe. Protein levels of Set3p, Hif2p and Snt1p increase upon LatA treatment Since hif2D, set3D, and snt2D mutants display cytokinesis defects upon LatA treatment, we next asked if the expression levels of these proteins might be responsive to the presence of LatA in the growth medium. To this end, protein extracts were made from LatA (0.5 mM) or DMSO treated strains expressing Set3-HA, Snt1-HA and Hif2-HA fusion proteins. The levels of Set3-HA, Snt1-HA and Hif2-HA were then quantified by western blotting. Remarkably, while no significant changes were noted in DMSO treated cells, the protein levels of Set3p, Snt1p, and Hif2p increased 2–3 fold upon LatA treatment (Figure 5A–C). Increasing the dose of LatA to 1 mM did not increase the level of induction (compare Figure 5A, left and right panels). Thus, the magnitude of the increase is not dosage dependent in this range of LatA concentrations. To ensure that changes in protein level were not due to genotype specific delays at a particular cell cycle stage (since wild- type cells engage the cytokinesis monitoring system, whereas the respective gene deletion mutants have this system abrogated) we examined the levels of the respective epitope tagged proteins upon release from a G2/M block produced through the use of the temperature sensitive cdc25-22 mutation (Figure S3A). The PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e31224