Figure 10. Central region contains redundant determinants of binding to the histone H3 and H4 tails. (A) GST-tagged histone H3 and H4 tails were expressed in bacteria and purified on glutathione beads. 750 ng of GST-H3 (lane 1), GST-H4 (lane 2), and GST (lane 3) were subjected to 10% SDS-PAGE followed by staining with Coomassie Blue. Lane 4 shows size markers with molecular mass indicated in kDa. (B) GroWT (lane 1), GroDGP (lane 2), GroDCcN (lane 3), GroDSP (lane 4), and GroDCR (lane 5) were translated in vitro in the presence of [35S] methionine. 10% of the translation products were subjected to 8% SDS- PAGE and the gel imaged by autoradiography. (C) The translation products from (B) were incubated with glutathione bead bound GST-H3 (Lanes 1–5), GST-H4 (Lanes 6–10), or GST (Lane 11). After extensive washing, bound proteins were eluted with SDS gel sample buffer and subjected to 8% SDS-PAGE. The gel was imaged by autoradiography. The percentage of input protein that bound the immobilized GST fusion proteins is indicated at the bottom of each lane. doi:10.1371/journal.pone.0030610.g010 Intrinsic disorder in the central domains may enable them to serve diverse regulatory functions Previous studies identifying lethal alleles of Gro failed to turn up mutations that mapped to the central domains [16]. Despite this fact, the findings presented here demonstrate that the central region plays critical roles in Gro function and in development. It is perhaps not surprising that it is difficult or impossible to identify missense mutations in the Gro central region given the lack of sequence conservation in this region between Drosophila Gro and its vertebrate orthologs. This lack of conservation suggests that these domains might not be highly ordered and therefore might be resistant to inactivation by single amino acid changes, a suggestion that is supported by our analysis of the Gro amino acid sequence. A large fraction of proteins, especially in higher eukaryotes, are thought to contain disordered domains subject to rapid evolution. Many proteins with profound significance to human disease, such as the tumor suppressor p53, contain such domains [39]. Contrary to what is sometimes assumed, this is not a sign that these domains are without required function. Rather the disorder in these domains may play a useful role by allowing them to adopt multiple structural states, which could each mediate interactions with different partners, allowing the domains to function as hubs of large regulatory networks [39]. Thus, by facilitating a large Groucho Central Domains Instruct Development number of regulatory interactions, the disordered central domains in Gro could account for its ability to repress multiple targets via diverse mechanisms. Furthermore, disordered domains may serve as regulatory targets because they can bind their partners with both high specificity and low affinity, a type of binding that can be easily reversed by posttranslational modification [39]. This is consistent with the previous findings showing that the central domains often serve as posttranslational regulatory targets [26,27,28,30,53]. While the central region in Gro family proteins undergo rapid sequence evolution, their functions are often conserved even when the sequence conservation is hard to recognize. For example, the GP domain appears to be responsible for Rpd3/HDAC1 binding both in Drosophila Gro and its mammalian orthologs [23,24]. Similarly, the SP domain seems to be a target for regulation by some of the same protein kinases in vertebrates and invertebrates alike [28,30,31,53,54]. Thus, it seems that disordered domains in related proteins may be conserved at the level of function long after evolutionary drift has erased easily recognizable sequence conservation. Evidence for target gene-specific mechanisms of Gro- mediated repression Previous studies suggest that Gro may repress transcription by multiple mechanisms including histone deacetylase-dependent and histone deacetylase-independent mechanisms (see introduction). In accord with this idea, some of the findings presented here suggest that the mechanism of Gro-mediated repression may be target gene specific. For example, we show that hkb and tll are more sensitive to increases in GroWT levels than ftz. In contrast, the effect of deleting the SP domain is much more dramatic for ftz than it is for hkb and tll. This may reflect gene specific differences in the mechanism of repression that are intrinsic to the structure of the regulatory region of the gene. In conclusion, it seems that the Gro central domains, although not well conserved, have essential roles in Gro-mediated repression and in the regulation of development. A full understanding of the mechanisms of repression by Gro will require the identification and characterization of the many proteins that are likely to interact with the Gro central region and a determination of the roles of these partner proteins in repression. At least some of these partners may well be target gene specific. Materials and Methods Plasmid Construction The plasmids encoding the Gro central domain deletion variants were generated using pET17b-Gro as a template for PCR [17]. The PCR primers used to generate the DGP, DCcN, DSP, and DCR deletions are listed in Table S1. The resulting PCR products contained the entire pET17b vector and sequences encoding all of Gro with the exception of the domain being deleted, and contained an AscI restriction site at the site of the deletion. To generate transgenic UAS constructs that can be expressed in somatic and germ line cells, PCR fragments encoding GroWT, GroDGP, GroDCcN, GroDSP, and GroDCR and that also contained 59 NotI and 39BamHI restriction sites were inserted into the pUASP transformation vector [55]. PCR products encoding the above Gro variants that contained 59 XhoI and 39 StuI restriction sites were inserted into the p131 pUAST transformation vector so that the Gro coding region was in frame with the amino-terminal 6XMyc epitope tag [56]. PLoS ONE | www.plosone.org 12 February 2012 | Volume 7 | Issue 2 | e30610