The Unconserved Groucho Central Region Is Essential for Viability and Modulates Target Gene Specificity Wiam Turki-Judeh, Albert J. Courey* Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California Los Angeles, Los Angeles, California, United States of America Abstract Groucho (Gro) is a Drosophila corepressor required by numerous DNA-binding repressors, many of which are distributed in gradients and provide positional information during development. Gro contains well-conserved domains at its N- and C- termini, and a poorly conserved central region that includes the GP, CcN, and SP domains. All lethal point mutations in gro map to the conserved regions, leading to speculation that the unconserved central domains are dispensable. However, our sequence analysis suggests that the central domains are disordered leading us to suspect that the lack of lethal mutations in this region reflects a lack of order rather than an absence of essential functions. In support of this conclusion, genomic rescue experiments with Gro deletion variants demonstrate that the GP and CcN domains are required for viability. Misexpression assays using these same deletion variants show that the SP domain prevents unrestrained and promiscuous repression by Gro, while the GP and CcN domains are indispensable for repression. Deletion of the GP domain leads to loss of nuclear import, while deletion of the CcN domain leads to complete loss of repression. Changes in Gro activity levels reset the threshold concentrations at which graded repressors silence target gene expression. We conclude that co-regulators such as Gro are not simply permissive components of the repression machinery, but cooperate with graded DNA-binding factors in setting borders of gene expression. We suspect that disorder in the Gro central domains may provide the flexibility that allows this region to mediate multiple interactions required for repression. Citation: Turki-Judeh W, Courey AJ (2012) The Unconserved Groucho Central Region Is Essential for Viability and Modulates Target Gene Specificity. PLoS ONE 7(2): e30610. doi:10.1371/journal.pone.0030610 Editor: Pedro Fernandez-Funez, University of Florida, United States of America Received November 14, 2011; Accepted December 26, 2011; Published February 3, 2012 Copyright: 2012 Turki-Judeh, Courey. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by NIH grant GM44522 to AJC and by NIH National Research Service Award GM86122 to WTJ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: courey@chem.ucla.edu Introduction As corepressors, Drosophila Groucho (Gro) and its orthologs in other metazoans lack DNA-binding domains and are recruited to the template by numerous DNA-binding repressor proteins including HES family repressors, Engrailed, Dorsal, Capicua (Cic), and Brinker (Brk) [1,2,3,4,5]. Once recruited to the template, Gro regulates a variety of developmental processes including neurogenesis, sex determination, and patterning of the embryo and imaginal discs [6]. In addition, Gro regulates signaling through multiple signal transduction pathways, including the Ras, Notch, Wingless, and Decapentaplegic (Dpp) pathways [7]. Mammalian Gro orthologs exhibit similarly widespread roles in signaling and development and have been implicated in tumorigenesis [8,9]. Many of the DNA binding repressors through which Gro acts, including Dorsal, Cic, and Brk, are distributed in concentration gradients that provide positional information along developmental axes. A graded factor is able to subdivide fields of developing cells because different target promoters respond to different threshold concentrations of the factor and are therefore expressed in domains with differing borders. Previous efforts to understand how target promoters can respond to different concentrations of DNA binding transcription factors have usually focused on the role of the DNA binding factors themselves, with particular attention to such parameters as binding site affinity, cooperative binding to DNA, synergy and antagonism between DNA bound factors, etc. [10,11,12,13]. Broadly distributed co-regulators, such as Gro, have generally been viewed as required components of the regulatory system that are needed for activation or repression by graded DNA binding transcription factors, but that do not have active roles in target gene selection or in determining borders of target gene expression. Gro/TLE family members are characterized by a conserved N- terminal domain (the Q domain), a variable middle region that can be subdivided into GP, CcN, and SP domains, and a conserved C-terminal WD-repeat domain [14]. Sequencing of lethal Gro point mutant alleles has revealed mutations that map to the WD repeat and Q domains, demonstrating the functional importance of these two domains [15,16]. In contrast, none of the known point mutations map to the GP, CcN, or SP domains, suggesting that these regions may not be required for viability [16]. However, an alternative possibility, which is suggested by the poor conservation in these regions, is that they are not well ordered and are therefore resistant to inactivation by point mutagenesis. The roles of the conserved Q and WD-repeat domains are well studied. The Q domain is required for Gro homo-oligomerization, and point mutations within this domain that disrupt self- association also interfere with Gro-mediated repression [17,18]. This region also binds to several repressors including Tcf/Lef and Myc [19,20]. The WD-repeat domain forms a b-propeller that contacts peptide motifs found in many Gro-binding corepressors, and thus is critical for the recruitment of Gro to many of its target genes [2,4,15,21]. PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e30610