Groucho Central Domains Instruct Development Figure 7. Central domains regulate Gro-mediated repression in the wing disc. Clones of Gro variant overexpressing cells were generated in third instar wing discs contained the vgQ-lacZ reporter. Discs were stained with antibodies against CD2 (green) and b-galactosidase (red). Overexpression clones are marked by the absence of CD2. Arrows mark clones that overlap the vg expression domain. GroWT clones (B) and GroDSP clones (E), exhibited ectopic repression of vgQ-lacZ, while control clones (A), GroDGP clones (C), and GroDCcN clones (D) exhibited no repression of vgQ- lacZ. GroDCR clones (F) exhibited slight expansion of vgQ-lacZ into regions in which the reporter is not normally expressed. Clones containing a mixture of overexpressed GroWT and GroDCR (G) show significantly reduced ectopic repression of vgQ-lacZ, relative to clones overexpressing GroWT alone. Wing disc images were obtained using a 206 objective. Scale bars = 50 mm. doi:10.1371/journal.pone.0030610.g007 GroWT and GroDSP are exclusively nuclear in both wing discs and S2 cells (Figure 9A, D, F, I). Surprisingly, GroDGP was completely cytoplasmic, while GroDCcN was primarily nuclear, implying that the GP rather than the CcN domain is primarily responsible for nuclear localization (Figure 9B, C, G, H). Therefore, the lack of an overexpression phenotype observed with the GroDGP variant likely reflects, in large part, a failure to localize to the nucleus. These observations also explain why GroDGP does not function as a dominant negative, while GroDCcN does. Interestingly enough, the GroDCR variant localized to the nucleus and cytoplasm in third instar wing discs (Figure 9E). This is consistent with its strong dominant negative function, but seems to be at odds with the notion that the GP domain is required for nuclear import since GroDCR lacks the GP domain. A plausible interpretation of this finding is that the central region contains a nuclear import signal or interacts with a protein that aids in Gro import in the GP domain and a nuclear export signal somewhere outside this region and that the subcellular localization of Gro depends on the interplay between these two signals. In S2 cells, GroDCR is cytoplasmic suggesting that the relative importance of the import and export signals may be cell type specific (Figure 9J). Promiscuous repression by GroDSP could reflect binding to histones The studies presented above suggest that in the absence of the SP domain, Gro can act promiscuously to repress genes that it normally does not repress. This raises the question of how Gro is recruited to such genes. Coimmobilization studies have shown that Gro binds directly to the N-terminal tails of histones H3 and H4 with a preference for the hypoacetylated forms of these histone tails [22,51]. This histone binding suggests a mechanism for promiscuous recruitment. To explore this possibility further, we examined the ability of the internal deletion variants to bind the H3 and H4 tails. These experiments involved the use of GST-H3 tail and GST-H4 tail fusions (Figure 10A) together with in vitro transcribed and translated Gro deletion variants (Figure 10B). GST-pulldown assays show that deletion of any single domain was insufficient to inhibit H3 or H4 binding, while deletion of the entire central region reduced H3 and H4 tail binding by at least 4- fold (Figure 10C). Thus, there appear to be multiple redundant determinants of histone binding in the central region, and the observation that GroDSP maintains an ability to bind to histone tails could provide a partial explanation for its promiscuous function. Discussion The findings presented in this paper suggest that the poorly conserved central domains of Gro may play essential roles in the developmental regulation of transcription. Specifically, we have shown that the GP, CcN, and SP domains have important roles in repression that are either essential for viability or greatly increase viability. In these studies, we employed deletion variants that remove entire domains as opposed to point mutations because a previous study suggested that the central region was resistant to point mutagenesis [16]. As discussed further below, we believe that this resistance to point mutagenesis is a reflection of the intrinsic disorder of these domains. Now that we know that the internal domains are essential for function, it will be worthwhile to carry out studies to further dissect the functions of these domains by, for example, generating finer deletions or clustered substitution mutations to map regions responsible for specific protein:protein interactions. While the use of deletion variants is subject to the caveat that deletions alter spacing and juxtapose parts of proteins that are not normally juxtaposed, we have demonstrated that our internal Gro deletions do not alter the functions of the adjacent conserved domains, i.e., the self-association function mediated by the Q domain or the repressor binding function mediated by the WD- PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e30610