Transcriptional adaptor ADA3 of Drosophila melanogaster is required for histone modification, position effect variegation, and transcription

Abstract

The Drosophila melanogaster gene diskette (also known as dik or dAda3) encodes a protein 29% identical to human ADA3, a subunit of GCN5-containing histone acetyltransferase (HAT) complexes. The fly dADA3 is a major contributor to oogenesis, and it is also required for somatic cell viability. dADA3 localizes to chromosomes, and it is significantly reduced in dGcn5 and dAda2a, but not in dAda2b, mutant backgrounds. In dAda3 mutants, acetylation at histone H3 K9 and K14, but not K18, and at histone H4 K12, but not K5, K8, and K16, is significantly reduced. Also, phosphorylation at H3 S10 is reduced in dAda3 and dGcn5 mutants. Variegation for white (w(m4)) and scute (Hw(v)) genes, caused by rearrangements of X chromosome heterochromatin, is modified in a dAda3(+) gene-dosage-dependent manner. The effect is not observed with rearrangements involving Y heterochromatin (bw(D)), euchromatin (Scutoid), or transvection effects on chromosomal pairing (white and zeste interaction). Activity of scute gene enhancers, targets for Iroquoi transcription factors, is abolished in dAda3 mutants. Also, Iroquoi-associated phenotypes are sensitive to dAda3(+) gene dosage. We conclude that dADA3 plays a role in HAT complexes which acetylate H3 and H4 at specific residues. In turn, this acetylation results in chromatin structure effects of certain rearrangements and transcription of specific genes.

FIG. 1.

dAda3 gene expression. (A) Northern blots showing expression from unfertilized eggs (lane UE); 4- to 10-h embryos (lane E); first-, second-, and third-instar larvae (lanes LI-II and LIII); pupae (lane P); and adults (lane A). Two mRNAs of 2.8 and 2.6 kb are detected. Note the continuous expression throughout development and the intensity of the maternal component of the unfertilized eggs. (B) In situ hybridization of an antisense RNA probe to a sagittal section of a third-instar larva showing the brain (br) and ventral ganglion (vg). Note the hybridization signal (blue). Bar = 35 μm. D, dorsal; A, anterior. (C) Equivalent hybridization to a horizontal section of an adult female abdomen showing nurse cells (n) in cysts (cys) of various stages. This ovarian signal represents the maternal component to the unfertilized eggs. Bar = 80 μm.

FIG. 2.

Specificity of the anti-dADA3 antibody. (A) Western blot from nuclear extracts of wild-type adults stained with the affinity-purified serum (ap), the preimmune serum (pi), and the peptide-preabsorbed serum (pap). For a loading control, the Western blot was hybridized with an anti-actin antibody. (B) Western blot from bacterial (bact.) extracts expressing a GFP-tagged dADA3 construct coupled to glutathione S-transferase. The tagged product justifies the higher molecular mass (in kDa, listed to the left) of dADA3. Note that the anti-GFP and anti-dADA3 antibodies recognize the same protein. The Coomassie-stained gel is included below as a loading control. (C) The dADA3 immunosignal localizes on the polytene chromosomes, counterstained with DAPI to identify DNA, and provides the in situ evidence for the specificity of the antibody. (D) Polytene chromosomes hybridized with anti-dADA3 (red) and anti-RNA Pol II (green) antibodies and DAPI (blue) to mark DNA. The X chromosome is indicated. Note the colocalization of dADA3 and RNA Pol II at some sites (arrowheads) but not others (arrows) (see also Fig. S3 in the supplemental material).

FIG. 3.

Mutant dAda3 phenotypes. (A) Normal wing disk from a third-instar male larva (genotype y w dAda3¹; E4RX/+). (B) Equivalent disk from a sibling y w dAda3¹ larva. Bar = 150 μm. Note the reduced size of the disk. The images in panels A and B were taken at the same magnification. Phase-contrast images of polytene chromosome from male wild-type (C) and dAda3² mutant (D) third-instar larvae. Note the aberrant banding pattern, particularly the X chromosome (arrowheads), in the mutant (see the text). Bar = 50 μm.

FIG. 4.

dADA3 is depleted in some HAT mutants. Polytene chromosomes stained with RNA Pol II (green) and dADA3 (red) antibodies in wild-type (A) and dAda2a (B), dAda2b (C), and Gcn5 (D) mutant backgrounds. Note the severe reduction of anti-dADA3 signal in Gcn5 and dAda2a mutants but not in dAda2b mutants. (E and F) Western blots from nuclear extracts of whole larvae from the same mutants stained with the anti-dADA3 antibody.

FIG. 5.

Histone acetylation phenotypes in dAda3 mutants. Polytene chromosomes immunostained with antibodies specific for individual acetylated lysine residues of H3 and H4 (red) as indicated on the left, and a RNA Pol II-specific antibody (green). Genotypes are indicated at the top. Each experiment is represented by four panels, including panels showing RNA Pol II staining as an internal control. The images from the wild type (+) and the dAda3² mutant were obtained with identical datum-recording settings. Note the severe reduction of H3 K9 acetylation (A and D) and the lack of effect on H4 K5 acetylation (B). (C and D) Also note the reduction of H4 K12 acetylation. (D) Western blots further demonstrate the histone acetylation reduction in dAda3 mutants.

FIG. 6.

H3 S10 phosphorylation in HAT mutant components. +, wild type. (A) Polytene chromosomes immunostained with antibodies specific for the phosphorylated form of H3 S10 (H3 P-S10; red) and the DNA marker DAPI (blue). The immunosignal is clearly reduced in dAda3 and dGcn5 mutants. (B) Western blot of dAda3 mutants confirming the previously described observation. All images were taken under the same laser and filter settings.

FIG. 7.

dAda3 mutant effects on PEV and gene expression. (A) Adult eye of a y w/In(1)w^m4h female showing the characteristic white variegation. (B) Enhanced variegation in y w dAda3¹/In(1)w^m4h females. (C) Reduced variegation in y w dAda3¹/In(1)w^m4h; E4RX/E4RX females. The genomic E4RX fragment carries a copy of dAda3⁺. The same effect was detected with other alleles and dAda3⁺-containing duplications [i.e., Dp(1;3)JC153]. (D) Dorsal view of an adult thorax of the genotype Df(3L)iro-2/+ showing the normal pattern of bristles. Note the scutum (scu) and scutellum (sc) regions of the thorax. The anterior is at the top of the panel. (E) Similar view of a sibling dAda3²/+; Df(3L)iro-2/+ adult. Note the absence of scutellum and several bristles from the remaining scutum in the thorax. (F) Normal wing disk from male third-instar larvae showing the expression of the LacZ reporter under the control of enhancer 1.1 from the AS-C gene complex. (G) Equivalent disk from a dAda3² AS-C1.1-LacZ male larva. Note the reduced disk size and the absence of LacZ expression. The same effect was detected with AS-C enhancers 1.2 and 3.8.