Nipped-A, the Tra1/TRRAP Subunit of the Drosophila SAGA and Tip60 Complexes, Has Multiple Roles in Notch Signaling during Wing Development

The Tra1/TRRAP gene and locations of Nipped-A mutations. Shown is a diagram of the Tra1/TRRAP gene, starting with the most 5′ RACE product and ending with the polyadenylylation site. The numbering is in kilobase pairs. The exons present in the major Tra1/TRRAP transcript are shown and numbered consecutively on the line, and the exons in the previously reported (29) rare transcript are shown below, numbered consecutively (1a, 2a, etc.). While most of the exons that overlap in the major and minor transcripts are identical, the positions of the splice acceptor or donor sites differ in three, as indicated by asterisks. Locations of the Nipped-A mutations listed in Table 1 are indicated by vertical lines.

Tra1/TRRAP proteins encoded by Nipped-A mutants. The diagrams show the proteins encoded by the major Tra1/TRRAP transcript in the indicated Nipped-A mutants (Fig. 2; Table 1). The locations of the PI3 kinase motif, the FAT and FATC motifs associated with phosphatidylinositol-3 kinases, and two HEAT repeats are indicated by boxes. The Nipped-A ^NC105 and Nipped-A ^NC106 mutations cause frame shifts that add nonnative residues at the ends of the truncated proteins.

Effects of heterozygous Nipped-A mutations on Notch and mastermind mutant phenotypes. The left panel shows that the heterozygous mam ^g2 null mastermind mutation (45) causes rare wing margin nicks and some Nipped-A mutations increase the frequency of these nicks, indicating reduced mastermind function. The wild-type Nipped-A allele is the parental allele in the cn bw stock used to generate the EMS-induced Nipped-A mutations (NC). The error bars indicate the 95% confidence intervals. The middle panel shows box plots of the distribution of wing widths of N ^nd-1 hypomorphic Notch mutant flies relative to the wild type (wt) in the presence of the indicated Nipped-A alleles. Wing widths were measured from the anterior to the posterior margin between the anterior and posterior crossveins, as shown at the bottom. Some Nipped-A mutations decrease the width, indicating reduced Notch function. The median width is indicated by the black line within each box, the ends of each box indicate the 25th and 75th percentiles, and the ends of the “whiskers” indicate the 10th and 90th percentiles. Circles show the lowest and highest values. The right panel shows the relative lengths of the L4 vein in N ^Ax-E2 hypermorphic Notch mutant flies in the presence of the Nipped-A alleles. As shown below, the veins were measured from the posterior crossvein toward the tip of the wing. Some Nipped-A mutations increase the length of the vein, indicating reduced Notch activity. The error bars show the 95% confidence intervals.

Western blot assay of Nipped-A mutants. Each lane contained 10 or 20 μl of extract of 0- to 12-h-old embryos from mothers of the indicated genotypes: +/+, wild type; 105/+, Nipped-A ^NC105/+; 194/+, Nipped-A ^NC194/+. The amounts loaded are the equivalent of 115 and 230 whole embryos. The blot was probed with affinity-purified NipA12 antibody. Densitometry indicates that the mutants contain 35 to 53% of the full-length Nipped-A protein present in the wild type.

Fluorescent immunostaining of larval tissues for Nipped-A. The examples shown were performed with affinity-purified NipA14 antibody, and essentially identical results were obtained with NipA12 antibody. The genotypes are indicated on the right: +/+, wild type; 106/Df, Nipped-A ^NC106/Nipped-E ⁴³; 105/Df, Nipped-A ^NC105/Nipped-E ⁴³; 194/Df, Nipped-A ^NC194/Nipped-E ⁴³; +/Df, +/Nipped-E ⁴³; 96/Df, Nipped-A ^NC96/Nipped-E ⁴³. The micrographs were taken with a 60× objective. All second-instar micrographs were taken with identical exposure times, and both third-instar micrographs were identical exposures.

Effects of Ada2b mutations on Notch and mastermind mutant phenotypes. The effects of the indicated Ada2b mutations and the parental P insertion [EP(3)3412] used to generate them on the mam ^g2 wing nicking phenotype, the N ^nd-1 wing width phenotype, and the N ^Ax-E2 vein-shortening phenotype were determined and diagrammed as described in the legend to Fig. 1. Homozygous Ada2b ^HD-5 mutations occasionally survive to eclosion, indicating that they are hypomorphic, while the Ada2b ¹ and Ada2b ² alleles both delete significant portions of the transcribed regions and die during pupal development (44).

Effects of domino mutations on Notch and mastermind mutant phenotypes. Dominant effects of the recessive lethal dom ¹ P insertion allele of domino (44) and a viable revertant (dom ^R12) generated for this work on the mam ^g2, N ^nd-1, and N ^Ax-E2 phenotypes were measured as described in the legend to Fig. 1, except that the wing widths and vein lengths are presented in micrometers instead of relative values.

Nipped-A and mastermind bind to the same sites on salivary gland chromosomes. The top panels show double immunostaining with the two Nipped-A antibodies, NipA12 and NipA14. The staining patterns are virtually identical, providing evidence additional to the Western blot data (Fig. 5) and the tissue immunostaining (Fig. 6) that the antibodies are specific for Nipped-A. The bottom panels show double immunostaining for Nipped-A and mastermind. As seen in the merge, virtually all sites that stain for mastermind also stain for Nipped-A, and there are several minor Nipped-A staining sites that that do not stain for mastermind. Arrowheads indicate puffs that stain for both Nipped-A and mastermind.