Groucho Central Domains Instruct Development Figure 2. Central domain deletions impair neither Q nor WD-repeat domain function. (A) Structure of the Gro deletion variants, GroDGP, GroDCcN, GroDSP, and GroDCR. (B) Gro self-association assays. Untagged wild-type full-length Gro (lanes 1, 2, 7 and 8) or internal deletion variants of Gro (lanes 3–6) were translated alone (lane 1); or cotranslated with the wild-type His-tagged Gro N-terminal region (His-GroNWT, contains the first 194 amino acids of Gro including the intact Q domain) (lanes 2–6), with the 40D/89D double point mutant form of the His-tagged Gro N-terminal region (His-GroN40D/89D) (Lane 7), or the 38D/87D double point mutant form of the His-tagged Gro N-terminal region (His-GroN38D/87D) (Lane 8). Samples were incubated with Ni-NTA beads, and after extensive washing, Ni-bound proteins were eluted and analyzed by 10% SDS-PAGE and autoradiography. 10% of input is shown on the left, while the Ni-bound proteins are shown on the right. The percentage of input that was immobilized on the beads is indicated at the bottom of each lane. (C) GST-Brk pulldown assays using Gro deletion variants. [35S]-methionine-labeled GroWT (lanes 1–3), GroDGP (lanes 4–5), GroDCcN (lanes 6–7), GroDSP (lanes 8–9), or GroDCR (lanes 10–11) were incubated with glutathione-agarose beads bearing immobilized GST (lane 2) or a GST-Brk fusion protein (lanes 3, 5, 7, 9 and 11). After extensive washing, bound proteins were eluted and analyzed by 8% SDS-PAGE and autoradiography. The percentage of input protein that bound the glutathione-agarose beads is indicated at the bottom of the GST and GST-Brk lanes. doi:10.1371/journal.pone.0030610.g002 and GroDCR may function as dominant negatives, presumably through the formation of mixed oligomers with endogenous wild- type Gro. Overexpression of the GroDSP variant resulted in significantly higher levels of repression than overexpression of GroWT, indicating that the SP domain has a negative regulatory function, and explaining the incomplete rescue by GroDSP. The relative importance of the SP domain appears to be target dependent. For example, deletion of the SP domain increases repression of Cic targets hkb and tll by a factor of ,2, while deletion of the SP domain increases repression of the Hairy target ftz by a factor of more than 6. kni functions as a control to show that repression due to Gro overexpression is specific for Gro targets. kni is not a direct Gro target, but rather it is a target for repression by Tll. Therefore, overexpression of Gro indirectly activates kni by directly repressing tll. The effects of the other deletion variants on kni expression are consistent with the effects of these variants on tll expression with the exception of GroDSP, which unexpectedly acts to repress both tll and kni. One possible explanation for this paradox is that SP domain deletion leads to a loss of Gro specificity, turning Gro into a promiscuous repressor of genes that it does not normally repress. To explore this possibility further, we looked at two additional genes not expected to be Gro targets. Genome-wide chromatin PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e30610