The histone H3 acetylase dGcn5 is a key player in Drosophila melanogaster metamorphosis

Abstract

Although it has been well established that histone acetyltransferases (HATs) are involved in the modulation of chromatin structure and gene transcription, there is only little information on their developmental role in higher organisms. Gcn5 was the first transcription factor with HAT activity identified in eukaryotes. Here we report the isolation and characterization of Drosophila melanogaster dGcn5 mutants. Null dGcn5 alleles block the onset of both oogenesis and metamorphosis, while hypomorphic dGcn5 alleles impair the formation of adult appendages and cuticle. Strikingly, the dramatic loss of acetylation of the K9 and K14 lysine residues of histone H3 in dGcn5 mutants has no noticeable effect on larval tissues. In contrast, strong cell proliferation defects in imaginal tissues are observed. In vivo complementation experiments revealed that dGcn5 integrates specific functions in addition to chromosome binding and acetylation. Surprisingly, a dGcn5 variant protein with a deletion of the bromodomain, which has been shown to recognize acetylated histones, appears to be fully functional. Our results establish dGcn5 as a major histone H3 acetylase in Drosophila which plays a key role in the control of specific morphogenetic cascades during developmental transitions.

FIG. 1.

dGcn5 is required for Drosophila metamorphosis. (A) Genetic map of the dGcn5 region. The Drosophila dGcn5 gene with its two introns (white boxes) is depicted in black. The deletion sex204 was generated by imprecise excision of the 1479/10 P element (black triangle) inserted in the CG14121 gene. dGcn5 alleles are indicated, as well as the position of the genomic fragment included in the PL transgenic construct. (B) Impaired metamorphosis in Gcn5^E333st mutants. (Left side) Homozygous Gcn5^E333st animals failed to formal normal puparium compared to Gcn5^E333st/TM3 control animals. Salivary glands from control or Gcn5^E333st late-third-instar larvae (top) were immunostained with a dGcn5 antibody. (Right side) Squashed salivary glands from wild-type (control) and homozygous Gcn5^E333st (E333st) late-third-instar larvae. Brackets indicate chromosomal regions corresponding to 2B (top) and 74EF-75B (bottom) early puffs, respectively. (C) Gcn5^E333st/Gcn5^C137T animals mostly died as pharate adults with abnormally elongated metathoracic legs (black arrowhead), strong defects in abdominal cuticle deposition (open arrowhead), and rough eyes. Note that the eye pigmentation was stronger in Gcn5^E333st/Gcn5^C137T animals than in Gcn5^C137T/TM3 control animals because of the presence of a FRT79D white⁺ marker on the mutagenized chromosomes. A metathoracic twisted and crooked leg (black arrow) from a Gcn5^E333st/Gcn5^C137T adult escaper is shown to the right. The same defects and lethality were observed for the Gcn5^E333st/Gcn5^ΔT280-F285 heteroallelic combination (not shown). (D) Structures of the wild-type and variant dGcn5 proteins expressed from pUAST-derived transgenic constructs. The N-terminal domain conserved in vertebrate Pcaf, the catalytic HAT domain, the Ada domain, and the bromodomain are indicated as shaded boxes.

FIG. 2.

dGcn5 is required for cell proliferation in imaginal tissues. (A) Reduced and misshapen imaginal wing disks from homozygous Gcn5^E333st mutants compared to a wing disk from a Gcn5^E333st/TM3 control third-instar larva. (B) Wings from control animals (+/+) and en-GAL4/UAS-IR[Gcn5] adult escapers with vein and cross-vein defects (black arrows) in the smaller posterior compartment. (C) Wings from vg-GAL4/UAS-IR[Gcn5] adult escapers. (D) Complete absence of abdominal adult cuticle in esg-GAL4/UAS-IR[Gcn5] pharate adults compared to a control animal (+/+).

FIG. 3.

dGcn5 loss of function induces cell cycle defects and apoptosis. The results of a confocal analysis of GFP (green) and dGcn5 or CBP (red) expression in wing disks from en-GAL4 UAS-GFP/UAS-IR[Gcn5] (A and B) or en-GAL4/UAS-GFP (A′) late-third-instar larvae are shown. BrdU incorporation experiments (C) and anti-phospho(S10)-histone H3 immunostaining (D) revealed a greater proportion of cells in S phase and at mitosis, respectively, in the dGcn5 silenced compartment from en-GAL4 UAS-IR[Gcn5] wing disks. The results of a TUNEL analysis of Gcn5^E333st/Gcn5^E333st (E), Gcn5^E333st/TM6 Tb (F), en-GAL4/UAS-IR[Gcn5] (G), and ptc-GAL4/UAS-IR[Gcn5] (H) wing disks are also shown. UAS-IR[GFP] transgenes did not induce apoptosis in control experiments (not shown).

FIG. 4.

dGcn5 is required for acetylation of histone H3 K9 and H3 K14 residues in polytene chromosomes. Polytene chromosomes from a Gcn5^E333st/sex204 mutant and a lio-GAL4/UAS-H2b-YFP wild-type animal were squashed together and costained with DAPI (blue), anti-GFP (green), and an antibody (red) against acetylated H3 K14 (A), acetylated H3 K9 (C), acetylated H4 K8 (D), phosphorylated H3 S10 (E), dimethylated H3 K4 (F), dimethylated H3 K9 (G), or HP1 (H). Panels A and B show the same field through the red channel (acetylated H3 K14) and the green channel (H2b-YFP), respectively. The H2b-YFP signal in the green channel is not shown for immunostaining of the other histone modifications.

FIG. 5.

dGcn5 RNA interference depletes imaginal disks of acetylated H3 K9 and H3 K14 residues. The results of a confocal analysis of GFP (green) and acetylated H3 K9 (A), acetylated H3 K14 (B), acetylated H4 K8 (C), acetylated H4 K16 (D) dimethylated H3 K4 (E), or dimethylated H3 K9 (F) in wing disks from en-GAL4 UAS-GFP/UAS-IR[Gcn5] late-third-instar larvae are shown. (G) Western blot of da-GAL4/UAS-IR[Gcn5] (IR[Gcn5]) and +/UAS-IR[Gcn5] (WT) late-third-instar larvae probed with antibodies against dGcn5, H3-AcK9, H3-AcK14, and H4-AcK8, as indicated.

FIG. 6.

Complementation of histone acetylation and dGcn5 chromosome binding in dGcn5 mutants. Polytene chromosomes from Gcn5^E333st/sex204 da-GAL4 third-instar mutant larvae heterozygous for the indicated transgenes were stained with an antibody directed against acetylated H3 K9 and H3 K14 residues (A, C, E, G, and I) or an antibody against the bromodomain of the dGcn5 protein (B, D, F, H, and J). A Western blot of da-GAL4 late-third-instar larvae heterozygous for the indicated dGcn5 variant transgenes (K) was probed with an antibody raised against the dGcn5 bromodomain. The asterisk indicates a degradation product of the Gcn5ΔHAT protein.

FIG. 7.

Gcn5ΔPcaf variant restores the viability of dGcn5 mutants. (A) Gcn5^E333st/sex204 da-GAL4 adult fly heterozygous for the UAS-Gcn5 transgene. Rescued animals are indistinguishable from wild-type animals as well as from fully rescued UAS-Gcn5ΔBromo/+ Gcn5^E333st/sex204 da-GAL4 animals (not shown). (B) Gcn5^E333st/sex204 da-GAL4 adult fly heterozygous for the UAS-Gcn5ΔPcaf transgene. Rescued adults displayed held-out, notched wings (white arrow), rough eyes, and crooked legs (black arrow).