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. 2001 Feb 1;20(3):510-9.
doi: 10.1093/emboj/20.3.510.

FTZ-Factor1 and Fushi tarazu interact via conserved nuclear receptor and coactivator motifs

C J Schwartz  1 , H M Sampson, D Hlousek, A Percival-Smith, J W Copeland, A J Simmonds, H M Krause
Affiliations

FTZ-Factor1 and Fushi tarazu interact via conserved nuclear receptor and coactivator motifs

C J Schwartz et al. EMBO J. .

Abstract

To activate transcription, most nuclear receptor proteins require coactivators that bind to their ligand-binding domains (LBDs). The Drosophila FTZ-Factor1 (FTZ-F1) protein is a conserved member of the nuclear receptor superfamily, but was previously thought to lack an AF2 motif, a motif that is required for ligand and coactivator binding. Here we show that FTZ-F1 does have an AF2 motif and that it is required to bind a coactivator, the homeodomain-containing protein Fushi tarazu (FTZ). We also show that FTZ contains an AF2-interacting nuclear receptor box, the first to be found in a homeodomain protein. Both interaction motifs are shown to be necessary for physical interactions in vitro and for functional interactions in developing embryos. These unexpected findings have important implications for the conserved homologs of the two proteins.

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Figures

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Fig. 1. Domains in FTZ required to bind FTZ-F1. (A) Line drawings of the FTZ deletion constructs that affect binding to FTZ-F1. The N- and C-terminal regions of FTZ are represented by open boxes and the homeodomain by a hatched box. FTZΔ112–118 is missing conserved amino acids that comprise a consensus nuclear receptor box. FTZΔ257–265 is missing the first nine residues of the homeodomain. FTZΔΔ contains both deletions. (B) A far western blot used to test binding of a 35S-labeled FTZ-F1 probe to the immobilized FTZ polypeptides depicted in (A). The FTZ protein present in each lane is indicated above. (C) A far western blot in which radiolabeled Paired protein (PRD) was used to probe the same polypeptides as in (B). (D) A Coomassie Blue stained gel of the same proteins loaded in (B) and (C).
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Fig. 2. The FTZ nuclear receptor box is required for FTZ-F1-dependent FTZ activities. Full-length FTZ and FTZΔ112–118 proteins were tested for rescuing activity in transgenic embryos. (A and B) FTZ expression; (C and D) EN expression; (E and F) WG expression; (G and H) cuticular phenotypes. (A)–(F) are also double-stained for β-galactosidase expression. (A), (C), (E) and (G) are internal ftz+ controls from ftzΔ112–118 rescue crosses. These contain a wild-type ftz chromosome marked with hb-lacZ. The embryos in (B), (D), (F) and (H) are homozygous mutant for the endogenous ftz gene and express only the ftzΔ112–118 transgene.
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Fig. 3. Mapping domains in FTZ-F1 required for binding to FTZ. (A) Schematic drawings of the full-length and deleted FTZ-F1 constructs initially used to map FTZ-interacting regions. The light and dark hatched regions at the N-terminal ends of the proteins represent the alternative N-termini of the α and β FTZ-F1 isoforms. The black region is the zinc-finger DNA-binding domain. The vertically striped region immediately behind is the A-box and the diagonally hatched region at the C-terminus is the LBD. (B) A far western blot in which the FTZ-F1 deletion constructs shown in (A) were radioactively labeled and used to probe full-length FTZ immobilized on a membrane after SDS–PAGE. Lanes 1–4 and lane 5 are from separate blots.
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Fig. 4. Drosophila melanogaster and B.mori FTZ-F1 sequences encode conserved AF2 motifs. (A) The corrected DNA and protein sequences for the final 34 amino acids of the Drosophila FTZ-F1 coding region. The boxed sequence above position 3006 indicates the repeat that is present in the previously published sequence but not found in ours. The numbering above the DNA sequence is for the α transcript. The corrected protein sequence is provided below the DNA sequence. (B) The corrected DNA and protein sequences for the final 34 amino acids of the B.mori FTZ-F1 coding region. The boxed adenosine at position 1660 represents a missing nucleotide in the previously published sequence. The corrected protein sequence is provided below the DNA sequence. (C) A comparison of the AF2 helices of FTZ-F1 homologs and the most closely related Drosophila receptor DHR39. Dark shading indicates identical residues and light shading similar residues. DmFTZ-F1 is the Drosophila protein, Bm FTZ-F1 the B.mori protein and XFTZ-F1 the Xenopus laevis protein. FTF is human fetoprotein transcription factor, SF-1 is steroidogenic factor 1 and DHR39 is the Drosophila hormone receptor at genomic position 39.
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Fig. 5. Binding to FTZ requires the AF2 helix of FTZ-F1. (A) A far western blot in which various FTZ-F1 derivative proteins are probed with 35S-labeled FTZ. Each of the FTZ-F1 polypeptides begins at the A-box and contains all (A+LBD) or most of the LBD. A+LBDΔAF2 has the AF2 helix deleted. A+LBD/FTF contains the FTF AF2 in place of the FTZ-F1 AF2. Similarly, A+LBD/SF-1 and A+LBD/DHR39 contain the SF-1 and DHR39 AF2 helices in place of the FTZ-F1 AF2. (B) A Coomassie Blue-stained gel loaded with the same amounts of proteins loaded in (A). (C) A far western blot in which different full-length and deleted nuclear receptor proteins were 35S-labeled and used to probe blots in which equivalent amounts of FTZ were loaded. The probe used is indicated above each lane. FTFΔAF2 is FTF with its AF2 helix removed.
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Fig. 6. Phenotypes of rescued Ftz-F1209 embryos. FTZ-F1 constructs were used to rescue embryos lacking maternally provided αFTZ-F1. The embryos shown are representative of the cuticular phenotypes obtained with each of the listed genetic backgrounds. (A) A wild-type cuticle. (B) A Ftz-F1209 cuticle. (C) A Ftz-F1209 embryo rescued with a full-length FTZ-F1 transgene. Embryos in (D)–(F) were rescued with a FTZ-F1ΔAF2 transgene (D), a FTZ-F1/DHR39 transgene (E) or a FTZ-F1/SF-1 transgene (F). (G) A typical example of an embryo in which full-length FTZ-F1 was expressed in a wild-type background.
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