Groucho Central Domains Instruct Development Figure 9. Subcellular localization of Gro central domain deletion variants. (A–E) Third instar imaginal wing discs were stained with Myc antibodies (red) to detect Myc-tagged Gro and DAPI (blue) to stain DNA. GroWT (A) and GroDSP (D) localized exclusively to the nucleus. GroDGP (B) localized exclusively to the cytoplasm. GroDCcN (C) localized primarily to the nucleus, but we also detected a low level of cytoplasmic localization and GroDCR (E) localized to the nucleus and cytoplasm. Wing disc images were obtained using a 1006 objective. Scale bars = 10 mm. (F–J) Drosophila S2 cells were stained with FLAG antibodies (red) to detect FLAG-tagged Gro and DAPI (blue) to stain DNA. GroWT (F) and GroDSP (I) localized exclusively to the nucleus. GroDGP (G) and GroDCR (J) localized exclusively to the cytoplasm. GroDCcN (H) localized primarily to the nucleus. S2 cell images were obtained using a 1006 objective. Scale bars = 5 mm. doi:10.1371/journal.pone.0030610.g009 nonspecifically through interactions with histones [22,34]. This could lead to promiscuous repression. Indeed, we find that GroDSP represses normal Gro targets as well as genes not normally targeted by Gro such as cin and Rpt3, and therefore a normal function of the SP domain may be to safeguard against promiscuous repression. It could do so, for example, by limiting the ability of Gro to spread along chromatin. If phosphorylation of the SP domain further limits spreading, this would explain how this modification could attenuate repression [28,30]. Regardless of the mechanism by which the SP domain modulates specificity, our results suggest that co-regulators are likely to be broadly important in target gene selection and cannot be ignored in attempts to predict the network of transcription factor/cis-regulatory module interactions that control development. Positive roles for the GP and CcN domains in Gro- mediated repression Both gain and loss-of-function experiments demonstrate that the GP and CcN domains are essential for Gro function in vivo. Previous studies have suggested that the GP domain is required for the binding of Gro to the histone deacetylase Rpd3 and that Rpd3 function is required for Gro-mediated repression [23,25,35]. We thus considered this interaction to be a prime candidate for the essential function of the GP domain. We were surprised to discover, however, that the GP domain has an essential function in nuclear localization. Since the GP domain does not have homology to any known NLS, this suggests the existence of a novel mechanism for Gro nuclear import or that this region interacts with an unknown protein containing a more conventional nuclear localization signal. Because the deletion mutant lacking the GP domain does not enter the nucleus, we are, at this point unable to address the question of whether the GP domain/Rpd3 interaction is required for function in vivo. Our findings show that although the CcN domain contains a sequence with similarity to a canonical NLS, this domain is not required for nuclear import. This is consistent with previous studies in S2 cells showing that deletion of the putative nuclear localization signal in the CcN domain did not lead to relocaliza- tion of the bulk of the Gro to the cytoplasm, whereas deletion of the entire central region did [52]. While the CcN domain is not required for nuclear import, it nonetheless has a critical, but currently unidentified, function in repression. PLoS ONE | www.plosone.org 11 February 2012 | Volume 7 | Issue 2 | e30610