Two-hybrid interactions between Runt and the Gro and H corepressors. (A) Schematic diagram of Gro showing the location of different protein motifs. The conserved N-terminal glutamine-rich (Q) domain and C-terminal WD-repeat domains are separated by nonconserved glycine/proline (GP)- and serine/proline (SP)-rich regions and a loosely conserved CCN motif that contains putative phosphorylation sites for Cdc2 and Casein kinase II as well as a nuclear localization signal. The segments of Gro contained within the two clones recovered in the two-hybrid screen translate into protein fragments (blue lines) initiating at either amino acid Leu-181 or Pro-338 and extending to the C terminus. The longer clone overlaps the CCN, SP, and WD-repeat domains, whereas the shorter fragment contains only a portion of the SP motif and the WD-repeats. (B) One clone from the H gene that extends from Ser-672 to the C terminus was identified. This fragment overlaps a significant portion of the Groucho-interacting domain (ID) of Hairless (though not the eh1 domain) and the entire CtBP-ID. It does not contain the Su(H)-ID near the N terminus of H protein. (C) Schematic of Runt with boxes indicating each of the eight conserved regions. The largest conserved region contains the entire Runt Domain (RD), which mediates interaction with DNA and the Bro protein, as well as N- and C-terminal extensions of 14 and 54 amino acids, respectively. The segments removed in the Runt deletion constructs used in yeast two-hybrid experiments are indicated in the schematics below (red stippled regions), with amino acid coordinates provided for each deletion. Note that Runt[Δ3] removes the highly conserved 54–amino acid C-terminal extension (Ser-232 to Lys-285, inclusive), and not the conserved N-terminal extension of the Runt Domain. The strength of the yeast two-hybrid interaction for each deletion derivative with Gro and H is provided to the right of each schematic. ++, a strong two-hybrid signal (clear lacZ signal in <2 h); +. a positive but weak signal (lacZ activity detected between 2 and 6 h of incubation); - indicates the absence of any detectable interaction.

Distinct roles for Gro and H in the repression of eve and hairy. In situ hybridization showing the mRNA expression of eve and hairy in late-blastoderm-stage embryos. In wild-type embryos, both eve (A) and hairy (B) are expressed in seven stripes. The stripes that are subject to Runt-dependent repression are indicated by numbers. Note, hairy also is expressed in a dorsal domain of cells at the anterior end of the embryo. (C) Embryo showing partial repression of eve stripe 2 in response to ectopic Runt. This embryo is from a cross between NGT40; NGTA heterozygous females and UAS-runt[15];UAS-opa[14] homozygous males. At this level of ectopic Runt, ∼50% of late blastoderm stage embryos show stronger, full repression of eve stripe 2. (D) Late blastoderm embryo from this same cross showing repression of hairy stripes 1 and 6. At this level of NGT-driven Runt, all mid- to late-cellular blastoderm-stage embryos show repression of these two hairy stripes. (E, F) expression of eve and hairy in embryos from a similar cross using NGT40; NGTA heterozygous females that are also heterozygous for Gro[BX22]. The reduction of maternal Gro dosage relieves the Runt-dependent, stripe-specific repression of these two pair-rule genes. In contrast, in parallel experiments with NGT40;NGTA, females that are also heterozygous for H[1], NGT-driven Runt and Opa produces stripe-specific repression of both eve (G) and hairy (H) with penetrance similar to control crosses. Similar results were also obtained with H[P8] and H[E31].

Hairless associates with Runt and slp1 regulatory elements in vivo. (A) Screen shot of an ∼25-kb interval spanning the slp1 locus from UCSC genome browser showing the location of the CG3407, slp1, and slp2 transcription units. The results of ChIP/chip assays for Runt and Pol II with chromatin from blastoderm-stage embryos obtained by the Berkeley Drosophila Transcription Network Project are provided below this map (MacArthur et al., 2009). The two most prominent regions of Runt association correspond well to the minimal Runt-responsive early stripe elements identified from 8.1–7.2 kb and from 3.1–2.5 kb upstream of the slp1 start site. Asterisks below this map indicate the location of PCR products generated with primer pairs located at 10 kb, 7.5 kb (DESE), 2.8 kb (PESE) upstream, and flanking the slp1 promoter. (B) Results of ChIP assays with anti-Hairless and control goat serum using chromatin from 3–4 h y w[67c23]) embryos. All four intervals give a ChIP signal for H greater than obtained with the control goat serum, with the strongest association detected with the Runt-associated DESE and PESE intervals. The lower signals observed at −10 kb and at the promoter may be due to generally higher background with the anti-H serum, or reflect some level of H association across the entire locus. (C) Results of similar ChIP assays using chromatin from similarly staged embryos from a cross between females homozygous for both the NGT40 and NGTA GAL4 drivers and males homozygous for UAS-runt[15] and UAS-ftz[263]. All gastrula-stage embryos in this cross show uniformly strong repression of slp1 in all cells (Wang et al., 2007). Increased H association is observed with the DESE interval, and potentially also at the promoter in these slp1-repressed embryos. (D) Results of coimmunoprecipitation assays using an anti-Hairless antibody and Drosophila embryo extracts. The left panel shows the Coomassie stained gel, and the right panel shows the results of a Western blot using a cocktail of anti-Runt monoclonal antibodies. The gel lanes are as labeled (1) whole cell (WC) lysate from 0–6 h embryos, (2) nuclei-enriched (P10) fraction, (3) nuclei-depleted (S10) fraction, (4) last wash (w) from the P10 anti-Hairless immunoprecipitation, (5) P10 immunoprecipitation with preimmune (PI) serum, (6) P10 anti-Hairless (α-H) immunoprecipitation, (7) S10 immunoprecipitation with preimmune (PI) serum, and (8) S10 anti-Hairless (α-H) immunoprecipitation. The anticipated migration of Runt at ∼53 kDa is indicated by the arrow on the right. The bands detected between 20 and 25 kDa are presumed to be degradation products; the more slowly migrating bands detected near 100 kDa may reflect covalent modifications that alter mobility.

H participates in the maintenance of Runt-dependent en repression. In situ hybridization shows en expression in gastrula stage (left column) and germband extension stage (right column) embryos. Embryos in this and other figures are shown anterior toward the left, dorsal side upwards. (A, B) Wild-type (C, D) expression in progeny of females heterozygous for both the NGT40 and NGTA GAL4-drivers crossed to males homozygous for the UAS-runt[232] transgene. This level of NGT-driven Runt blocks expression of the odd-numbered en stripes in all embryos at both stages. (E, F) Embryos from a cross between NGT40; NGTA heterozygous females that are also heterozygous for Gro[BX22] with UAS-runt[232] males. Although the odd-numbered en stripes are fully repressed early, expression is restored to intermediate levels similar to that shown in >50% of germband extension stage embryos in such crosses, with ∼10% showing expression that is indistinguishable from the wild-type pattern. (G, H) Embryos from a parallel cross but using NGT40; NGTA heterozygous females that are heterozygous for H[E31]. The fully penetrant repression observed in gastrula-stage embryos is partially relieved in more than half of the germband extension stage embryos. For H[E31], the full derepression as shown is found in 6% (n = 747) of the progeny in this cross. A similar proportion, (7% of 905 germband extended embryos) show full derepression in experiments with the H[P81] allele. (I, J) A parallel cross with females heterozygous for NGT40, NGTA, and the CtBP[03463] mutation yields a similar proportion of germband extension stage embryos with full derepression of en.

H, not Gro, contributes to slp1 repression. In situ hybridization showing slp1 mRNA expression in gastrula-stage embryos. For these experiments, slp1 expression was scored in embryos that show evidence of mesodermal invagination but that have not yet undergone significant germband extension. This narrow developmental window of <15 min allows for a careful comparison of expression phenotypes that can change dynamically during development. (A) In wild-type embryos at this stage, slp1 is expressed in 14 two-cell wide stripes, corresponding to the two posterior-most cells in parasegments 1–13 as well as a stripe 0 that is anterior to parasegment 1. There is also expression in a band of dorsal cells anterior to the presegmental region. The level of ectopic Runt and Ftz obtained in embryos from a cross between females heterozygous for both the NGT40 and NGTA drivers and males homozygous for both UAS-runt[15] and UAS-ftz[263] is sufficient to fully repress slp1 expression in 20% of the embryos. Most of the remaining embryos show weak expression in the head region with very weak (B) to weak (C) expression of a subset of stripes in the presegmental region. None of the embryos show evidence of all 14 slp1 stripes in response to this level of NGT-driven coexpression of Runt and Ftz. The level of NGT-driven Runt in these embryos is approximately threefold greater than obtained in the cross-es with UAS-runt[232] that were used to examine en repression (Li and Gergen, 1999). Note that the embryo in (B) is from an experimental cross with NGT40; NGTA heterozygous females that are otherwise wild-type, whereas the embryo in (C) is from females that are also heterozygous for ttk[1e11]. These two maternal genotypes produce an identical range of slp1 expression phenotypes, indicating that, unlike the Runt-dependent repression of en, Ttk does not make an important dose-dependent contribution to slp1 repression. (D) Representative embryo from a cross with NGT40; NGTA heterozygous females that are also heterozygous for Gro[BX22] showing slp1 repression in response to Runt and Ftz. Approximately half (35 of 67 = 52%) of the gastrula and early germband extension stage embryos in this cross show slp1 repression at this level if not stronger, with 14 of 67 embryos (21%) showing some evidence of all 14 stripes. Similar results were obtained in crosses with Gro[E48], with 54 of 97 (56%) embryos showing weak to no slp1 expression and 22 of 97 embryos (23%) with evidence of all 14 stripes. (E) Representative embryo from a cross with NGT40, NGTA females that are also heterozygous for H[E31]. In this cross, only 14 of 119 (12%) gastrula and early germband extension stage embryos showed strong slp1 repression, whereas 82 embryos (69%) showed evidence of all 14 stripes similar to that shown here. Similar results were obtained with H[P81]. (F) Representative embryo from an experimental cross using females heterozygous for NGT40; NGTA and CtBP[03463]. In this experiment, 11 (28%) of gastrula-stage embryos scored showed an even more severe, fully repressed phenotype. (G) Representative embryo from a cross with NGT40; NGTA heterozygous females that are also heterozygous for Rpd3[04556]. In this case, the most common phenotype at the gastrula stage (49%, n = 39) is faint expression of most of the stripes; full repression was still obtained in 20% of the embryos.

Gro and H have common effects on other Runt targets. In situ hybridization reveals expression of of slp1 (A–F) and ftz (G–L) mRNAs in gastrula and cellular blastoderm stage embryos, respectively. (A) Wild-type gastrula-stage embryo shows 14 two-cell-wide stripes of slp1. Expression in odd-numbered parasegments appears later than in the even parasegments and is weaker at this stage. (B) Embryo from a cross between NGT40; NGTA heterozygous females that are otherwise wild-type and males that are homozygous for UAS-Runt[15] and UAS-Opa[14] showing slp1 activation in the anterior head region with expanded expression domains in cells from odd-numbered parasegments that would normally express the homeodomain protein Eve and not Ftz. The exception is parasegment 11, where slp1 fails to be expressed due to the expanded expression of Ftz in these cells (see below). (C, D) Anterior slp1 activation in response to NGT-driven Runt and Opa is obtained in similar crosses using NGT40; NGTA heterozygous females that are also heterozygous for the Gro[BX22] and H[E31] mutations, respectively. Expression in parasegment 11 reappears in embryos from both of these crosses with the other stripes also showing a more mottled appearance. (E, F) Embryos from crosses involving NGT40; NGTA heterozygous females that are also heterozygous for the CtBP[03463] and Rpd3[04566] mutations, respectively, show expression similar to that found in embryos from NGT40; NGTA females that are otherwise wild-type, including loss of stripe 11. (G) Wild-type ftz expression in a late cellular blastoderm stage embryo consists of seven approximately four-cell-wide stripes. (H) The ftz stripes expand in embryos from a cross between NGT40; NGTA females and homozygous UAS-Runt[15] UAS-Opa[14] males, with nearly complete fusion of stripes 5 and 6 (representing expression in parasegments 10 and 12). (I, J) The activation of ftz in response to Runt and Opa is reduced in embryos from similar crosses with females that are also heterozygous for Gro[BX22] or H[E31], respectively, with the loss of fusion of stripes 5 and 6. (K, L) In contrast, embryos from similar crosses with females that are heterozygous for CtBP[03463] or Rpd3[04566] have broadened ftz stripes with fusion of stripes 5 and 6.