Subcellular Localization of Class II HDAs Figure 2. Subcellular localization of Class II HDAs. Protoplast transient expression using HDA-YFP fusion constructs were used to determine the subcellular localization of Class II HDAs. HDA5, HDA8, and HDA14 exhibited cytoplasmic localization while HDA15 concentrates inside the nucleus. VirD2NLS fused with mCherry was used as a nuclear marker. Scale bars were calibrated to 10 mm. doi:10.1371/journal.pone.0030846.g002 retention, and Class IIb, which relies on its own NLS and NES for nuclear import and export. Although all the Class II HDAs in Arabidopsis contain three conserved Ser & Thr residues which are potential binding sites for 14-3-3, only HDA15 and HDA18 both contain nuclear localization (NLS) and nuclear export signals (NES) indicating their potential to be classified as Class IIb HDAs. Based on the neighbor joining phylogenetic tree generated from Class II HDAs in humans, Drosophila, C. elegans, and yeast, HDA5 and HDA18 are more closely related to HsHDA504, HsHDA505, HsHDA509, and HsHDA507, which have been well established as Class IIa HDACs. On the other hand, HDA15 is in the middle between Class IIa and Class IIb (HsHDA506 and HsHDA510) showing both potential to be classified as Class IIa and Class IIb [4]. To determine if these histone deacetylases can be subclassified as Class IIa or Class IIb, biflourescence complementation (BiFC) assay was undertaken to assess if HDA5 and HDA15 can interact with 14-3-3 k and e. 14-3-3 proteins are generally known to have high sequence homology with very low specificity to its target proteins. As shown in Figure 6, HDA5 exhibited positive interactions with both 14-3-3 k and e in the cytoplasm. Whether HDA5 was fused with the YFP amino end with 14-3-3 k and 14-3- 3 e fused with the YFP carboxyl end or vice versa, consistent cytoplasmic interactions were observed. This interaction was further validated using coimmunoprecipitation where HDA-GFP was co-transfected with myc-tagged 14-3-3 k or 14-3-3 e (Figure 6C). Unfortunately, this association failed to interact in yeast-two- hybrid (data not shown) indicating the need for a kinase, which may be absent in yeast, to catalyze this interaction. Similar findings were also found in Class IIa human HDAC5, which positively binds with 14-3-3 in vivo but fails to associate in yeast suggesting its interaction to be largely dependent on Ca2+/ calmodulin-dependent kinases [61]. These results suggest that HDA5 relies on 14-3-3 proteins for its nuclear export and cytoplasmic retention considering that it does not contain any NES. This classifies HDA5 as a Class IIa histone deacetylase. On PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e30846