H interacts directly with Gro and with dCtBP in vitro. (A) Domain diagram of the H protein. The Su(H)-interaction domain is shown in yellow; motifs that resemble Gro-binding and CtBP-binding domains are shown in red. The C-terminal region of H deleted in the H²² mutation is indicated by the purple bracket; the H fragments used in panels C and E are represented by the black bars; the region of H included in the H^C peptide is represented by the blue bar. (B) The proposed Gro-interaction and dCtBP-interaction domains of H are conserved in dipteran evolution. Protein sequence identity between D. melanogaster H and its apparent orthologs in D. hydei and in the mosquito Anopheles gambiae is shown in 20-residue blocks. Below, the sequences of the proposed Gro-interaction and dCtBP-interaction motifs are aligned with the corresponding sequences in D. hydei and A. gambiae, along with similar motifs from other Gro- and CtBP-binding repressor proteins. (C) In vitro-translated ³⁵S-Gro binds to purified GST-H (lane 7), but not to GST alone (lane 5), or appreciably to GST-Su(H) (lane 13). GST-H^618–723, which includes the YSIHSLLG motif, binds directly to Gro (lane 9); GST-H^618–723m, a mutated fusion protein in which this motif has been changed to AAAHSAAG, does not bind Gro (lane 11). Gro-GST-Hairy interaction (lane 3) serves as a positive control for Gro binding, while in vitro-translated ³⁵S-Emc (even-numbered lanes) serves as a negative control. Approximate amounts of purified protein used: GST-Hairy, 0.2 μg; GST, 31 μg; GST-H, 0.8 μg; GST-H^618–723 and GST-H^618–723m, 15 μg; GST-Su(H), 3 μg. (D) Interaction between a biotinylated peptide (H^C) representing the C-terminal 24 aa of H, including the PLNLS motif, and GST-dCtBP beads. Interaction was assayed by retention of ³⁵S-streptavidin (SA; see Materials and Methods). Amounts of H^C peptide used: lane 16, 100 μg; lane 17, 1 μg; lane 18, 100 ng; lane 19, 10 ng; lane 20, 1 ng; lane 21, no peptide. (E) Purified GST-dCtBP binds to an in vitro-translated, 6xHis-tagged C-terminal H fragment (H^1036–1077; lane 26), but not to a fragment in which the PLNLSKH motif has been deleted (H^1036–1070; lane 27).

Reduction of H, gro, or dCtBP function suppresses N loss-of-function phenotypes affecting the socket/shaft and pIIA/pIIB cell fate decisions in the bristle lineage. (A–E) Abdomens of adult male flies of the following genotypes: wild type (A), N^ts1/Y (B), and N^ts1/Y flies lacking one dose of H (C), gro (D), or dCtBP (E), shifted to a restrictive temperature (31°C) at 22 h after puparium formation. (F) Quantification of N^ts1 effects on the socket-shaft cell fate decision. The x-axis represents the average number of socket cell fate defects on the dorsal abdomen of at least eight flies of each genotype. Asterisks denote the statistical significance of differences between N^ts1/Y flies and each of the other genotypes, by Student's t-test: *, P < 0.05; **, P < 0.005. Error bars indicate standard error.

Replacement of putative repression domains of H with a transcriptional activation domain creates dominant-negative effects in vivo. (A) Diagram of transgenic GAL4-inducible H expression constructs. Protein interaction domains of H are indicated. UAS-H^wt contains wild-type H driven by five UAS sites. UAS-H^ΔC encodes a H protein lacking the C-terminal-most seven aa (PLNLSKH), deleting the identified dCtBP interaction domain (see Fig. 1). UAS-H^ΔC-VP16 encodes a H protein containing the transcriptional activation domain (TAD) of the VP16 protein in place of the PLNLSKH residues of H. UAS-H^N-VP16 encodes a H protein lacking both putative repression domains, containing instead the VP16 TAD fused to an N-terminal fragment of H. (B–E) Dorsal abdomens of adult (C–E) or pharate adult (B; note reduced pigmentation in posterior tergites and in bristle shafts) flies carrying both sca-GAL4 and one of the UAS-H transgenes described above. (F,G) The effects of H misexpression on the socket cell fate are cell-autonomous. Shown are SEMs of head macrochaetes of a wild-type fly (F) and a UAS-H^wt ASE5-GAL4 fly, in which H is misexpressed specifically in socket cells (G).

Genetic interactions among gro, dCtBP, H, and Su(H) during adult mechanosensory bristle development. (A,B) Percentage of bristles affected at 20 macrochaete positions on the adult head and thorax. The lower (black) part of each bar represents bristle loss, or balding, events; the upper part represents shaft-to-socket transformation phenotypes. “TOTAL” bars combine data from all 20 positions; error bars indicate standard error. At least 52 bristles were scored for each genotype at each position. (A) Genetic interactions between null alleles of dCtBP, gro, and H. (B) Effects of heterozygous gro and/or dCtBP mutations in flies homozygous for H²², an allele that deletes the dCtBP interaction domain of the H protein (see Fig. 1). The y-axis represents the number of normal shaft structures observed at 42 macrochaete bristle positions (the 20 pairs shown in A, plus the posterior sternopleurals) per fly. Twenty-five flies of each genotype were scored. Error bars indicate standard error. (C) Interactions between dCtBP or gro and a derepressed Su(H) transgene that produces a gain-of-function Su(H) phenotype (Barolo et al. 2000). Data are presented in the same manner as described for A.

Su(H)-H protein complexes bind efficiently to DNA in vitro. Gel retardation analysis of a ³²P-labeled oligonucleotide probe containing a high-affinity Su(H) binding site (5′-CGT GTGAA-3′) from the Su(H) autoregulatory enhancer. Mutation of a single base pair of this sequence (to CGTGTCAA) abolishes binding of Su(H) to the probe, indicating that Su(H) binding is sequence-specific (Barolo et al. 2000). His-tagged Su(H) and H proteins were expressed in bacteria and affinity-purified. H alone does not bind to the probe (lane 2). Su(H) alone binds to a large percentage of the probe (lane 3, white arrowhead). The addition of both proteins produces a band of substantially lower mobility, or supershift (lanes 4–6, black arrowhead). Approximate amounts of protein used: His-Su(H), 180 ng (lanes 3–6); His-H, 10 ng (lane 4), 25 ng (lane 5), 50 ng (lanes 2,6). fp: free probe.

Proposed transcriptional roles for Su(H), H, N, Gro, and dCtBP during socket/shaft cell fate determination. (A) In the absence of N signaling, Su(H) binds H, which in turn binds the corepressors Gro and dCtBP, repressing N target genes and promoting the shaft cell fate (“default repression”). A similar H repression complex, including H and Gro but perhaps not dCtBP, promotes the SOP cell fate during lateral inhibition. (B) In the presence of N signaling, a N^IC activation complex, probably including Mastermind (Mam), displaces the H repression complex and activates N target genes, promoting the socket cell fate. “Local activators” are transcription factors, bound to N target promoters, that cooperate with Su(H)/N^IC to activate transcription during N signaling, and which must be counteracted by Su(H)/H repression complexes in the absence of signaling (Barolo et al. 2000). (C–F) Proposed mechanisms underlying phenotypes caused by experimental changes in levels of H and/or Su(H). (C) In H mutant flies, N/Su(H) target genes are derepressed, leading to N gain-of-function phenotypes. (D) Upon H overexpression, H repression complexes displace N^IC activation complexes, repressing N target genes and causing N loss-of-function phenotypes. (E) Su(H) overexpression causes titration of H repression complexes, thereby derepressing N target genes. (F) Su(H) overexpression strongly enhances the H overexpression phenotype, due to an increased number of Su(H)/H repression complexes.