Mutations in two regions of Adf-1 decrease DNA-binding affinity. (A) Wild-type and amino-terminal mutant forms of Adf-1 were tested for their abilities to bind an Adf-1 binding site in mobility shift experiments. Lane 1, background DNA binding due to endogenous Adf-1 in extracts from cells transfected with a negative-control expression vector; lanes 2 to 12, mobility-shifts for cell extracts transfected with the indicated expression vectors. (B) Wild-type and carboxy-terminal mutant forms of Adf-1 were tested for their abilities to bind an Adf-1 binding site-containing probe. Lanes 1 to 5, mobility shifts obtained with extracts from cells transfected with wild-type and mutant Adf-1 as described for panel A; lanes 6 to 11, mobility shifts obtained with 5 (lanes 6, 8, and 10) and 50 ng (lanes 7, 9, and 11) of purified recombinant wild-type and mutant Adf-1 proteins, respectively; lanes 12 and 13, mobility shifts obtained with 0.5 and 1 μl, respectively, of partially purified recombinant 5A228 protein. The positions of aberrant mobility shifts are indicated by arrowheads. (C) Chemical cross-linking of wild-type and mutant forms of Adf-1. Wild-type and mutant forms of Adf-1 were incubated in the presence or absence of 0.0025% glutaraldehyde and then subjected to SDS-PAGE followed by Western blotting and probing with anti-Adf-1 antibodies. The positions of prestained molecular weight standards and the presumed oligomeric states of the Adf-1 species are indicated. The lower band in the N228 samples (lanes 7 and 8) indicates a degradation product. kD, kilodaltons. (D) Schematic of Adf-1 in which the regions that affect DNA binding and the region of Myb homology are indicated. The amino-terminal region is the bona fide DBD, while the carboxy-terminal region is required for dimerization.

Activation by Adf-1 requires the presence of TAFs, and Adf-1 is able to interact directly with the TFIID complex. (A) The TFIID complex is required by Adf-1 to activate transcription. The transcription system used for Fig. 3B was depleted of TFIID activity. Purified TFIID (lanes 1 and 2) or purified recombinant TBP (lanes 3 and 4) was added back either in the absence of added activator (lanes 1 and 3) or with 10 ng of purified wild-type Adf-1 (lanes 2 and 4). (B) Adf-1 can interact directly with purified TFIID, while some mutants cannot. Purified TFIID was immunoprecipitated with beads containing anti-TAF_II250 antibodies. These beads (lanes 4 to 6) or negative-control GST beads (lanes 7 to 9) were incubated with wild-type or mutant forms of Adf-1, washed extensively, and subjected to SDS-PAGE followed by Western blotting and probing with anti-Adf-1 (α-Adf-1) antibodies. Results for 5% of the input wild-type and mutant Adf-1 are also shown (lanes 1 to 3). The upper bands seen in lanes 1 and 4 are believed to result from an isomeric form of Adf-1 (see Materials and Methods). The lower band seen in lane 2 results from a degradation product.

The carboxy terminus of Adf-1 is homologous to ADA2 and to Myb DBDs. (A) The carboxy terminus of Adf-1 is homologous to a region of the S. cerevisiae and human ADA2 proteins. An alignment between amino acids 182 and 247 of Adf-1 (Adf C), amino acids 65 and 110 of yeast ADA2 (yADA2), and amino acids 75 and 120 of human ADA2 (hADA2) is shown. The position of a 15-residue insertion in Adf-1 is indicated. All residues that are highly similar or identical to those of yADA2 are indicated by shading. The percent similarities between all sequences and that of yADA2 are indicated. Similar amino acids are PG, ST, DE, NQ, RHK, AVLIM, and FTW. (B) The carboxy terminus of Adf-1 may be a Myb-like domain. AdfC and yADA2 are aligned to two DNA-binding Myb domains, the amino terminus of Adf-1 (AdfN; amino acids 6 to 69) and M. musculus Myb repeat 2 (Myb2; amino acids 95 to 141). Amino acid positions in AdfC are indicated, as are the positions of sequence insertions in AdfC and AdfN. Residues that are identical or highly similar to those of Myb2 are indicated as in panel A. Percent similarities between all sequences and that of Myb2 are indicated. The positions of helices 1, 2, and 3 in Myb2 are indicated by brackets. Seven residues whose side chains project from the exposed face of mMyb helix 3 are indicated by asterisks.

The mutagenesis of Adf-1 allowed the mapping of DNA-binding and transactivation activities by using Schneider cell transfections. Twenty-six mutant forms of Adf-1 were constructed and assayed following Schneider cell transfections. The amino and carboxy halves of Adf-1 are shown with nonnative amino acids highlighted. Internally deleted amino acids are indicated with dashes. Data obtained from quantitating mobility shift experiments using extracts from Schneider cells transfected with wild-type and mutant expression vectors and Adf-1 binding site probes are summarized in the “DNA binding” column. Data obtained from quantitating CAT assays using extracts of cells cotransfected with Adf-1 expression vectors and an Adf-1-responsive CAT vector are summarized in the “Transactivation” column. Symbols (both columns): +++, activity at least 70% of that of the wild type; ++, activity between 30 and 69% of that of the wild type; +, activity between 10 and 29% of that of the wild type; −, activity less than 10% of that of the wild type. Background activity was standardized to 0% of that of the wild type.

Regions of Adf-1 that are required for transcriptional activation. (A) Extracts from transfected cells were used for CAT assays to measure the transcriptional activities of wild-type and mutant forms of Adf-1. Lane 1, background CAT activity from extracts of cells transfected with a negative-control expression vector; lanes 2 to 6, results of CAT assays using extracts of cells transfected with Adf-1 expression vectors. Converted and unconverted forms of [¹⁴C]chloramphenicol are indicated. (B) Purified wild-type and mutant forms of Adf-1 behave similarly in in vitro and in vivo transcription. Lane 1, in vitro transcription signal from a reaction mixture containing a partially purified Drosophila embryo extract; the Adh distal position −86 template contains Adf-1 binding sites, while the Adh distal position −46 template does not; lanes 2 to 6, the same transcription system as in lane 1 with 15 ng of purified wild-type or mutant Adf-1 added. (C) A schematic of Adf-1 in which the regions that affect activation are indicated. The DBD is partially filled since it is not possible to discriminate between direct and indirect effects on activation by mutations in this region.

Regions of TAF_II110 and TAF_II250 bind the carboxy terminus of Adf-1. (A) The carboxy terminus of Adf-1 binds TAF_II110 and TAF_II250. TAF_II110 and TAF_II250 (ΔN660) were incubated with glutathione beads bound to GST alone (lanes 2 and 8), GST–Adf-1 amino acids 162 to 253 (C92) (lanes 3 and 9), GST–Adf-1 amino acids 92 to 253 (C162) (lane 4), or GST–Adf-1 full length (wt) (lane 5). Bound TAFs were visualized by autoradiography of SDS-PAGE gels after extensive washing. in (lanes 1 and 7), 10% lysate input; −, binding to negative-control beads. The positions of the TAF products are indicated by open circles. (B) One region of TAF_II110 and multiple regions of TAF_II250 are responsible for Adf-1 binding. Full-length and truncated forms of TAF_II110, TAF_II250, and hTAF_II250 were incubated with glutathione beads bound to GST–Adf-1 C92 or to GST alone and visualized by autoradiography after extensive washing. +, binding to GST–Adf-1 C92; in and − are as defined for panel A. The positions of TAF products are indicated by open circles. (C) Adf-1 binds the same regions of TAF_II110 and TAF_II250 in vivo as it does in vitro. A C92 fragment of Adf-1 fused to the GAL4 AAD and portions of TAF_II110 and TAF_II250 fused to the GAL4 DBD or the DBD alone (as indicated) were coexpressed in S. cerevisiae containing a GAL4-responsive LacZ gene. Schematics of TAF_II110 and hTAF_II250 are shown with shading indicating the relative amounts of binding to Adf-1 in vitro, as shown in panel B. Below these are bars representing the portions of TAF_II110 and hTAF_II250 fused to the DBD. The amounts of LacZ activity seen are summarized in the “Lac-Z” column; the symbols represent the relative level of LacZ signal observed. (D) Some of the Adf-1 mutants are defective for TAF binding. Purified Adf-1 proteins were incubated with beads bound to hTAF_II250 (lanes 4 to 6), TAF_II110 (lanes 7 to 9), NTAF_II110 (amino acids 75 to 208) (lanes 10 to 12), NTAF_II250 (amino acids 1 to 414) (lanes 15 to 18), and GST (lanes 19 to 22). The beads were washed extensively and subjected to SDS-PAGE, followed by Western blotting and probing with anti-Adf-1 antibodies. Lanes 1 to 3 and 11 to 14 show results for 10% input wild-type and mutant Adf-1 proteins. The upper bands seen in some of the lanes represent isomeric forms of Adf-1 (see Materials and Methods), and the lower bands in the N228 samples (lanes 2 and 12) represent degradation products. (E) Schematic of Adf-1, with the region required for binding to TAF_II110 and TAF_II250 indicated.

A synthesis of structural and functional information about Adf-1. (A) Secondary structure prediction of Adf-1 correlates with mutagenesis data and protein homologies. α-Helices predicted by the nearest-neighbor secondary-structure prediction algorithm are indicated by shading. The positions of helices 1, 2, and 3 predicted by an analysis of the two Myb domain homologies (Fig. 6B) are indicated below the protein. Regions required for DNA binding, activation, TAF binding, and dimerization are indicated above the protein. (B) A model for the structure of Adf-1. The data summarized in panel A are interpreted as a possible three-dimensional structure showing the activities of Adf-1. A dimer of two Adf-1 molecules is depicted bound to DNA. A monomer of Adf-1 contains a DNA-binding helix-turn-helix domain at its amino terminus. The recognition helix that is expected to lie in the major groove of the DNA is shown as an extended helix. The relationship between this helix and DNA is unknown, and the helical kink depicted is arbitrary. The positions and orientations on DNA of the two monomers with respect to each other are also arbitrary. The activities and structures in the central portion of Adf-1 are not well understood, although the region is predicted to contain several α-helices. The carboxy terminus of Adf-1 also contains a Myb-like helix-turn-helix domain; this domain is able to dimerize as well as bind TAF_II110 and TAF_II250. Potential interactions are indicated by arrows.