Drosophila Ada2b is required for viability and normal histone H3 acetylation

Abstract

Regulation of chromatin through histone acetylation is an important step in gene expression. The Gcn5 histone acetyltransferase is part of protein complexes, e.g., the SAGA complex, that interact with transcriptional activators, targeting the enzyme to specific promoters and assisting in recruitment of the basal RNA polymerase transcription machinery. The Ada2 protein directly binds to Gcn5 and stimulates its catalytic activity. Drosophila contains two Ada2 proteins, Drosophila Ada2a (dAda2a) and dAda2b. We have generated flies that lack dAda2b, which is part of a Drosophila SAGA-like complex. dAda2b is required for viability in Drosophila, and its deletion causes a reduction in histone H3 acetylation. A global hypoacetylation of chromatin was detected on polytene chromosomes in dAda2b mutants. This indicates that the dGcn5-dAda2b complex could have functions in addition to assisting in transcriptional activation through gene-specific acetylation. Although the Drosophila p53 protein was previously shown to interact with the SAGA-like complex in vitro, we find that p53 induction of reaper gene expression occurs normally in dAda2b mutants. Moreover, dAda2b mutant animals show excessive p53-dependent apoptosis in response to gamma radiation. Based on this result, we speculate that dAda2b may be necessary for efficient DNA repair or generation of a DNA damage signal. This could be an evolutionarily conserved function, since a yeast ada2 mutant is also sensitive to a genotoxic agent.

FIG. 1.

The Drosophila Ada2b locus. (A) The dAda2b locus is drawn to scale. It spans 3.4 kb and contains four exons (open boxes). The alternative splicing that occurs in exon 3 is indicated, together with the stop codons in exon 4 that terminate the alternative reading frames. Stippled boxes indicate the last exons of the flanking genes CG9636 and DNA pol iota. The location of the P-element EP(3)3412 is indicated by a triangle. Two of the deletions generated by imprecise excision of this P element are shown. The gray box indicates the genomic sequence present in the transgene that rescues the dAda2b alleles. (B) Comparison of the two protein isoforms generated by alternative splicing. Isoform a is present in the EST clone LD 24527 and is the same protein sequence as described in reference . The b isoform was described in reference . The conserved ZZ and SANT domains are boxed, and Ada boxes 1 to 3 are shaded gray. (C) RT-PCR results with RNA prepared from wild-type (wt) or dAda2b¹ or dAda2b² homozygous mutant pupae are shown at the left. The upper panel shows 930- and 1,008-bp products generated with an Ada2b primer pair, and the lower panel shows the RP49 product (control). With a different primer pair, the alternative splice site usage in dAda2b exon 3 is evident (upper right panel). Isoform a generates a 262-bp product, whereas isoform b gives rise to a 184-bp product.

FIG. 2.

Drosophila Ada2b and Gcn5 interact and are expressed in similar patterns during embryogenesis. (A to I) Lateral views of embryos hybridized with digoxigenin-labeled RNA probes or with a dAda2b antibody. Anterior is to the left and dorsal is up. (A to C) Hybridization of a dGcn5 probe to wild-type embryos. Precellular (A), cellularized (B), and advanced-stage embryos (C) are shown. In early embryos the message is ubiquitously distributed due to its maternal contribution. In late-stage embryos, expression is strongest in the CNS, but is also present in the gut. (D to F) Wild-type embryos incubated with a dAda2b probe. Ubiquitous staining is observed in precellular embryos (D). Maternal dAda2b mRNA is absent from cellularized embryos (E). In advanced-stage embryos, expression is strongest in the CNS, but low levels of message are also found in the gut (F). (G and H) Stage 16 embryos stained with a dAda2b antibody. In wild-type embryos (G), strong staining is observed in the CNS. Most of the dAda2b protein has disappeared by this stage of development in dAda2b mutant embryos (H). (I) In dAda2b mutant embryos, no zygotic dAda2b mRNA can be detected. (J) Interaction between dAda2b and dGcn5 proteins in vitro. The dAda2b isoform a cDNA was in vitro translated and incubated with GST (control) or a GST-dGcn5 fusion protein. Ten percent of the input was run on a gel together with eluates of GST and GST-dGcn5 resins. The dAda2b-a protein is efficiently retained by GST-dGcn5.

FIG. 3.

Reduced histone H3 acetylation in dAda2b mutant embryos. Antibodies that recognize different acetylated forms of histone H3 and histone H4 were used to stain Drosophila embryos. All four antibodies show strongest signals in the CNS and epidermis. Ventral views of stage 16 embryos are shown, with anterior to the left. Homozygous dAda2b mutant embryos were identified by the absence of Ubx-lacZ expression, which is present in embryos containing a balancer chromosome. (A and B) Wild-type (wt) (A) and dAda2b mutant (B) stage 16.1 embryos were stained with an antibody recognizing histone H3 acetylated at lysine 14. A substantial reduction in staining intensity was found in dAda2b mutant embryos. (C and D) wt (C) and dAda2b mutant (D) stage 16.1 embryos incubated with an antibody directed against acetylated histone H3 lysine 9. The staining intensity is diminished in dAda2b mutant embryos compared to wt embryos. (E and F) wt (E) and mutant (F) stage 16.2 embryos stained with an acetylated histone H4 lysine 8 antibody. The staining pattern and intensity are indistinguishable between wt and dAda2b mutant embryos. (G and H) Stage 16.2 wt (G) and dAda2b mutant (H) embryos incubated with a tetra-acetylated histone H4 antibody. Comparable staining intensities were observed in wt and mutant embryos.

FIG. 4.

Histone H3 acetylation on salivary gland polytene chromosomes is reduced in dAda2b mutants. Polytene chromosomes were prepared from wild-type (wt) and dAda2b homozygous mutant third-instar larval salivary glands, stained with antibodies directed against different acetylated forms of histone H3 or H4, and counterstained with DAPI. For wt (A and A′) and dAda2b mutant (B and B′) chromosomes incubated with an anti-acetylated histone H3 lysine 14 antibody, the staining intensity was greatly diminished on dAda2b mutant chromosomes compared to wt. A reduction in staining intensity was also found on dAda2b mutant chromosomes incubated with anti-acetylated H3 K9 (D and D′) compared to the wt (C and C′). By contrast, we observed similar staining intensities for wt and dAda2b mutant chromosomes incubated with anti-acetylated H4 K8 (E to F′) or a tetra-acetylated H4 antibody (G to H′).

FIG. 5.

Ada2b mutants are hypersensitive to radiation-induced apoptosis and can induce expression of the p53 target gene reaper. (A to D) Wild-type (wt) and dAda2b mutant third-instar larvae were mock treated or irradiated with 800 rads. Wing imaginal disks were dissected after 4 h and incubated in the vital dye acridine orange, which preferentially stains apoptotic cells. Very few apoptotic cells are observed in mock-treated wt (A) and dAda2b mutant (C) wing disks. Following irradiation, extensive apoptosis is observed in the wt wing disk (B). More apoptotic cells are found in dAda2b mutant disks than in the wt after irradiation (D). (E to H) Expression of a p53-dependent radiation-responsive enhancer from the reaper gene does not require dAda2b. Embryos were incubated with a β-galactosidase antibody that recognizes reaper-driven lacZ expression and identifies dAda2b homozygous mutant embryos by distinguishing Ubx-lacZ expression from the balancer chromosome. Stage 16.2 embryos are shown from a ventral view, with anterior to the left. In the absence of irradiation, lacZ expression is strong in the epidermis but largely absent from the gut in both wt (E) and dAda2b mutant (G) embryos. Expression in the gut is strongly induced in both wt (F) and dAda2b mutant (H) embryos following irradiation (arrows).

FIG. 6.

dAda2b acts upstream of reaper in p53-dependent apoptosis. (A and B) Wild-type (wt) and dAda2b mutant third-instar larvae containing an hs-reaper transgene (hs-rpr) were heat shocked, and wing imaginal disks were incubated in acridine orange after a 1-h recovery period. Similar numbers of apoptotic cells are observed in wt (A) (1,092 ± 125 stained cells) and dAda2b mutant (B) (1,056 ± 202 stained cells) wing disks containing the hs-reaper transgene. (C to J) Radiation-induced apoptosis in dAda2b mutants is p53 dependent. wt (C and G), dAda2b mutant (D and H), p53 mutant (E and I), and dAda2b p53 double mutant (F and J) third-instar larvae were mock treated or irradiated with 4,000 rads. After 4 h, wing imaginal disks were dissected and incubated in acridine orange. Very few apoptotic cells are observed in mock-treated wt (C), dAda2b mutant (D), and p53 mutant (E) wing disks. Slightly more apoptotic cells are found in mock-treated dAda2b p53 double mutant wing disks (F). In response to 4,000 rads of irradiation, massive apoptosis is observed in wt wing disks (G), and even more is observed in dAda2b mutant wing disks (H). As expected, no apoptosis is found in p53 mutant wing disks (I). In dAda2b p53 double mutant wing disks (J), there is no increase in the amount of apoptosis compared to mock-treated disks (F).

FIG. 7.

A yeast ada2 mutant strain is sensitive to DNA damage. (A) Tenfold serial dilutions of isogenic wild-type (wt) and ada2 mutant yeast strains were plated on rich medium without MMS and on medium containing 0.02% MMS. (B) Growth of wt yeast on 0.02% MMS was compared to that of gcn5 mutant and ada3 mutant yeast. The strain lacking Ada3p is highly sensitive to MMS, whereas the gcn5 mutant grows a little more slowly than the wt.