Less is known about the poorly conserved central region. This region, along with the Q domain, mediates binding of Gro to hypoacetylated histone N-terminal tails [22]. The GP domain binds the histone deacetylase Rpd3/HDAC1, which is required for optimal Gro function [23,24,25]. The CcN domain contains a putative nuclear localization signal (NLS), as well as phosphory- lation sites for the cyclin-dependent kinase family member Cdc2 and casein kinase II (CKII) [14]. Association of Gro with chromatin is negatively regulated by Cdc2 phosphorylation and positively regulated by CKII phosphorylation of the CcN domain [26,27]. The SP domain contains phosphoacceptor sites for mitogen activated protein kinase (MAPK) [28,29] and homeodo- main-interacting protein kinase (HIPK) [30,31]. Phosphorylation of Gro by both these kinases leads to decreased transcriptional repression, although the mechanism behind this phenomenon is unclear. A model for Gro-mediated repression that incorporates some of the above-described biochemical functions of its domains is as follows: Once Gro is recruited via interactions with repressors, its ability to self-associate and to bind histone tails allows it to polymerize along the template establishing a transcriptionally silent domain [32]. The template bound Gro may then recruit histone deacetylase Rpd3 leading to histone deacetylation and possibly to an increase in nucleosome density [25,33]. In support of this model, Gro family proteins are able to condense chromatin arrays in vitro thereby preventing the transcriptional machinery from having access to the template [34]. Any or all of these steps could be regulated by phosphorylation of the CcN and SP domains. While the above pathway may account for many examples of Gro-mediated repression, additional findings suggest that Gro may repress by other mechanisms as well, including histone deacetylase independent mechanisms [25,34,35,36]. Further evidence for mechanistic diversity comes from studies showing that Gro may function in both long- and short-range repression [37,38]. It appears that mechanisms of Gro-mediated repression may vary depending on the target gene and developmental context. In this study, we examined the roles of the unconserved Gro central domains in Gro-mediated repression and pattern forma- tion by analyzing multiple Gro deletion variants lacking one or more of the central domains. Our findings indicate that the SP region has a negative role in repression and helps to determine target gene specificity, while the GP and CcN domains are required for repression and viability, thus challenging the notion that essential function and evolutionary conservation go hand in hand. Our studies also indicate that development requires the correct balance between positively and negatively acting Gro domains as excess Gro activity results in the inappropriate resetting of repressor concentration thresholds sufficient to mediate repression. This implies that ubiquitous co-regulators such as Gro may actively cooperate with graded DNA-binding transcription factors in setting the boundaries of developmental domains. Our findings further suggest that such factors cannot be neglected in efforts to predict the targets of DNA binding transcription factors and to predict how such targets will respond to developmental cues. Results The Gro central domains are likely to be intrinsically disordered The lack of conservation in the Gro central region suggests that this region might not be highly ordered. To explore this idea, we analyzed the Gro amino acid sequence using well-established Groucho Central Domains Instruct Development algorithms for predicting protein disorder [39,40]. This analysis shows that the central domains display all the hallmarks of intrinsically disordered protein domains – in particular they contain a high proportion of charged amino acid residues and a low proportion of hydrophobic amino acid residues [40,41] (Figure 1). Numerous predictors of disorder indicate that, while the Q and WD repeat domains are ordered, the GP, CcN, and SP domains are all very likely to be disordered (Figure 1). Gro central domains are not required for repressor binding or self-association To test the idea that the lack of lethal point mutations in the central domains reflects disorder (and therefore an ability to tolerate single amino acid changes) rather than a lack of function, we set out to examine both the recessive and dominant phenotypes associated with transgenes encoding Gro deletion variants lacking one or more of the central domains (Figure 2A). Since the aim of these experiments was to identify essential functions for the Gro central domains, it was necessary to show that the functions of the conserved Q domain in self-association and the conserved WD- repeat domain in repressor binding were not adversely impacted by the internal deletions. Accordingly, before examining the effects of these deletions in vivo, we expressed the deletion variants in vitro and assessed their ability to self-associate and to bind a repressor protein (Brk). In the self-association assays (Figure 2B), full-length Gro or internal deletion variants of Gro were cotranslated with an N- terminal 194 amino acid residue long His-tagged Gro fragment containing the intact Q domain (His-GroNWT). The His-tagged protein was immobilized on Ni-NTA beads and co-immobilization of the 35S-labeled untagged internal deletion variants was assessed by SDS-PAGE followed by autoradiography. Immobilization of the untagged variants was completely dependent on the presence of His-GroNWT (compare lanes 1 and 2), while full length Gro and all the internal deletion variants bound His-GroNWT with comparable efficiency (lanes 2–6). The specificity of the assay was demonstrated by the results obtained with His-GroN40/89D and His-GroN38/87D. Both of these double mutants contain amino acid substitutions in the Q domain coiled-coil motifs required for self-association. In accord with previous findings, the mutations in His-GroN40/89D do not inhibit self-association (lane 7), while the mutations in His-GroN38/87D do (lane 8) [18]. In the GST-Brk pull down assays (Figure 2C), in vitro translated full-length Gro or internal deletion variants of Gro were incubated with a GST-Brk fusion protein immobilized on glutathione beads. Full-length Gro and all internal deletion variants bound GST-Brk with comparable efficiency (lanes 1 and 3–11). The specificity of the assay is demonstrated by the failure of full-length Gro to bind to GST alone (lane 2). Thus, we conclude that deletion of the central domains does not interfere with the essential functions of the conserved Q and WD- repeat domains in self-association and repressor binding. There- fore, any effects of these deletions on repression most likely reflect roles of the internal domains in other biochemical interactions essential for repression. The Gro central domains are required for viability To determine if the central domains are required for viability, we generated genomic constructs encoding full-length Gro (GroWT), or Gro deletion variants lacking the GP, CcN, or SP domains (GroDGP, GroDCcN, and GroDSP, respectively) in a vector containing the bacterial attachment site (attB) for bacteriophage phiC31. These were then introduced into the fly germ line by site- specific integration into a second chromosome phiC31 attachment PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e30610