MOLECULAR AND CELLULAR BIOLOGY, Sept. 2005, p. 8228–8238
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 25, No. 18
The Histone H3 Acetylase dGcn5 Is a Key Player in
Drosophila melanogaster Metamorphosis
Cle ´ment Carre ´,† Dimitri Szymczak,†‡ Josette Pidoux, and Christophe Antoniewski*
Laboratory of Drosophila Genetics and Epigenetics, Department of Developmental Biology, CNRS URA 2578,
Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France
Received 20 April 2005/Returned for modification 20 May 2005/Accepted 22 June 2005
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.
Gene expression in eukaryotes has to accommodate the
presence of nucleosomes and the packaging of DNA into high-
er-order chromatin structures. Nucleosomes are composed of
octamers of histone proteins H2a, H2b, H3, and H4, whose
N-terminal tails project outward from the nucleosomal core
and are subjected to covalent modifications such as acetylation,
methylation, phosphorylation, and ubiquitination. The variety
of these modifications and their association with distinct states
of gene transcription suggested that they may act as a combi-
natorial code to specify downstream events such as the recruit-
ment of transcription factors or modifications of the chromatin
structure (23, 58).
The acetylation of lysine residues is one of the most studied
histone modifications and has long been linked to gene acti-
vation. For instance, a twofold up-regulation of transcription
from the male X chromosome in Drosophila melanogaster is
correlated with histone hyperacetylation (60), while gene si-
lencing in heterochromatin or X chromosome inactivation in
mammals are correlated with histone hypoacetylation (27).
Numerous sequence-specific activators, such as the nuclear
receptors MyoD and CREB, have been shown to recruit co-
activator complexes with histone acetyltransferase (HAT) ac-
tivity, while transcriptional repressors have been found associ-
ated with corepressor complexes with histone deacetylase
activity (13). HAT activity is also associated with more general
transcription factors, such as TATA-binding protein-associ-
ated factor 1 (TAF1) and yeast elongation factor 3 (44, 63).
Collectively, these data point to a causal role of histone acet-
ylation in transcriptional activation. In support of this hypoth-
esis, the acetylation of lysine 8 in histone H4 (H4-AcK8) and
lysine 14 in histone H3 (H3-AcK14) has been implicated in the
sequential recruitment of transcription factors leading to the
activation of the human beta interferon gene in vitro (1), and
distinct patterns of histone acetylation have been associated
with groups of coexpressed genes in genome-wide studies (36,
The yeast adaptor Gcn5 was the first transcription factor
identified as a bona fide HAT (14, 35). It defines a family of
evolutionarily conserved Gcn5-related N-acetyltransferases
(GNATs) whose members were purified as essential subunits
of ADA and SAGA complexes in yeast and of PCAF, STAGA,
and TFTC complexes in mammals. The GNAT complexes
contain Ada transcriptional adapters, Spt proteins, and a set of
TATA-binding protein-associated factors, and they are struc-
turally related, suggesting that they perform similar functions
in transcription (13, 65). In vitro, Gcn5 acetylates lysine 14 of
free, but not nucleosomal, histone H3. In contrast, Gcn5 acety-
lates an expanded set of lysines of nucleosomal histone H3
when copurified with native ADA or SAGA complexes (26),
suggesting that one function of these complexes in vivo is to
modulate the activity and specificity of Gcn5.
Two distinct genes, hGCN5 and hPCAF, encode Gcn5 ho-
mologues in humans. Both hGCN5 isoforms, GCN5S and
GCN5L, and hPCAF (P300/CBP-associated factor) share a
close similarity with the complete yeast Gcn5 sequence, includ-
ing more matches within the HAT catalytic domain as well as
the bromodomain, which has been shown to bind acetylated
histone lysines (21, 30–32, 46, 47). PCAF and GCN5L share an
additional N-terminal domain (Pcaf homology domain) of
about 350 amino acids involved in binding to CBP/p300 and to
several nuclear receptors. While the GCN5 gene knockout
results in early embryonic lethality, the PCAF gene knockout
* Corresponding author. Mailing address: Laboratory of Drosophila
Genetics and Epigenetics, Department of Developmental Biology,
CNRS URA 2578, Institut Pasteur, 25 rue du Docteur Roux, 75724
Paris Cedex 15, France. Phone: 33 1 44 38 93 35. Fax: 33 1 40 61 39 18.
† These authors contributed equally to this work.
‡ Present address: Laboratory of Polarity and Morphogenesis, Insti-
tut Jacques Monod, 75251 Paris Cedex 05, France.
has no detectable consequences on mouse development (66).
However, GCN5-PCAF double mutants die earlier than single
GCN5 mutants, indicating that PCAF and GCN5 functions are
not completely redundant.
Although the Gcn5 HAT has been clearly involved in the
control of Arabidopsis growth, development, and homeostasis
(8, 61), its contribution to the control of specific morphoge-
netic events during animal development remains poorly under-
stood. There is only one Gcn5 homologue in Drosophila (56),
which thus provides a simplified model for the study of the
function of a GNAT in the context of a whole organism. Dro-
sophila Gcn5 (dGcn5) has been isolated in at least two GNAT
complexes that contain distinct Ada2 variants (37, 45). A 1.8-
MDa SAGA-like complex includes the Ada2b variant, the
Ada3 and Spt3 homologues, and several TAFs. An Ada2b
loss-of-function mutation is lethal and suppresses the histone
H3 acetylation of polytene chromosomes (49), indicating that
the SAGA-like complex plays an essential role in gene expres-
sion in Drosophila. In addition, dGcn5 associates with the
Ada2a variant and with Ada3 in a 440-kDa non-SAGA-like
complex. Interestingly, Ada2a and Ada2b mainly localize to
different bands on polytene chromosomes, suggesting that
GNAT complexes may play distinct functions depending on
In order to characterize the function of dGcn5 and GNAT
complexes during Drosophila development, we have under-
taken two complementary approaches. We isolated dGcn5
loss-of-function mutants from a genetic screen, and we per-
formed in vivo targeting of dGcn5-specific RNA interference
using inverted repeat transgenes. Here we show that dGcn5 is
the major HAT for the lysine residues K9 and K14 of histone
H3, while the acetylation of histone H4 involves other HATs.
Our data indicate that dGcn5 is required for larva-to-adult
metamorphosis and suggest an essential function of dGcn5 in
the control of the cell cycle in imaginal tissues. An analysis of
dGcn5 variant proteins revealed that the Pcaf homology do-
main, the domain of interaction with the Ada proteins, and the
catalytic domain are all required for the function of the dGcn5
protein. In contrast, the bromodomain appears to be dispens-
MATERIALS AND METHODS
DNA constructs. An EcoRV-KpnI fragment encompassing the dGcn5 and
CG14121 genes was subcloned from the bacterial artificial chromosome
BACR48G06 into the pBluescript vector. Clones containing this fragment were
selected by colony hybridization with dGcn5 cDNA excised from the LD17356
expressed-sequence-tag clone (BDGP). The PL genomic rescue construct was
then generated by subcloning the CG14121-dGcn5 region as a 4.7-kb KpnI-NotI
fragment into the pCaSper-4 transformation vector.
pUAS-Gcn5 and its derivatives were all made by subcloning the dGcn5 cDNA
from the LD17356 expressed-sequence-tag clone into the pUAST vector. Dele-
tions were generated by the excision of dGcn5 regions and cloning of the ap-
propriate PCR-amplified fragments. Gcn5 variant constructs contained the fol-
lowing deletions: pUAS-Gcn5?Pcaf, deletion of the first 361 amino acids of the
dGcn5 peptide; pUAS-Gcn5?HAT, deletion of amino acids M554 to K595;
pUAS-Gcn5?Ada, deletion of amino acids F635 to P699; and pUAS-
Gcn5?Bromo, deletion of amino acids S703 to T788. The structure of all Gcn5
variant constructs was confirmed by DNA sequencing. The cloning procedures
and PCR primers used are available upon request.
Generation of dGcn5 mutants. Sixteen Df(3L)iro-2 noncomplementing P ele-
ments on chromosome III (a generous gift from Peter Maroy) were mapped by
inverse PCR. A P-element insertion, 1479/10, that was localized in the CG14121
gene, 521 bp upstream of the dGCN5 transcription start site, is homozygous
lethal at puparium formation. This P element was mobilized by crossing with a
strain expressing transposase. Derived lines with both a lost white marker gene
and early embryonic lethality associated with chromosome III were analyzed
using combinations of PCR primers. The flanking regions of the sex204 defi-
ciency were PCR amplified and sequenced.
Males isogenic for an FRT79D ebony marked chromosome were treated with
35 mM ethyl methanesulfonate (EMS) and mated en masse to TM3SerGFP/
TM6b virgin females at 25°C. F1 males (FRT79D ebony/TM6b or FRT79D ebony/
TM3SerGFP) were then pair mated with females with the deficiency sex204, and
crosses were scored for the absence of the F2 progeny class that was heterozy-
gous for the mutagenized chromosome and the deficiency. If this class was
absent, stocks were established from FRT79D ebony/TM3SerGFP or TM6b flies.
We recovered 11 lethal mutations that failed to complement the sex204 defi-
ciency from 6,000 fertile pair matings scored. Each of these mutations was placed
in complementation groups by complementation tests with all other mutations
and with the P-element insertions 1479/10 (a CG14121 allele) and KG01697 (a
Citron allele from BDGP). Four dGcn5 alleles were rescued in crosses with a
PL/PL; sex204/TM3TbSb line homozygous for the PL genomic transgene (Fig. 1)
and in crosses with a UAS-Gcn5/UAS-Gcn5; sex204/da-GAL4/TM3TbSb line
which constitutively expresses the dGcn5 cDNA. Six mutations were genetically
mapped in Citron. The remaining allele was mapped in CG14121 by noncomple-
mentation with the P-element 1479/10 and was rescued with a short genomic
transgene encompassing only the CG14121 locus. We did not isolate a mutation
in CG10686 in our genetic screen because the loss of function of this gene is
likely not lethal. Six pairs of primers were used to PCR amplify the dGcn5 coding
region from flies heterozygous for dGcn5 mutations and a balancer chromosome.
We identified the dGcn5 alleles by comparison of the sequence from mutant
heterozygotes to that obtained from the homozygous parental strain upon which
the mutations were induced. The primer sequences used for this study are
available upon request.
Germ line transformations and fly strains. Transgenic lines were established
as described previously (11). The GAL4-UAS system (10) was used to express
dGcn5, dGcn5 variants, and the green fluorescent protein (GFP) reporter. The
da-GAL4 (GAL4daG32) driver line has been described previously (64). The
lio-GAL4P15line (55) was provided by Jean-Maurice Dura, IGH, Montpellier,
France. The engrailed-GAL4 (en-GAL4) and UAS-GFP lines were provided by
Jean-Paul Vincent, MRC, London, United Kingdom. The vestigial-GAL4 (vg-
GAL4) driver line was provided by Fre ´deric Bernard, IJM, Paris, France. The
distal-less-GAL4 (dll-Gal4) and patch-GAL4 (ptc-GAL4) driver lines were ob-
tained from the Bloomington Stock Center. The escargot-GAL4 (esg-GAL4, or
NP5153) line was obtained from Drosophila Genetic Resources, Japan (http:
Immunohistochemistry and Western blotting. Immunostaining of imaginal
disks dissected from mid-third-instar larvae was performed as described previ-
ously (20). Rabbit anti-dGcn5 (50) and anti-CBP (a gift from A. Mazo, Kimmel
Cancer Center, Philadelphia, Pa.) were used at a 1:1,000 dilution. Antibodies
against the histone tail modifications were obtained from Upstate and were used
at the follow dilutions: anti-Ac-H3K9, 1:1,000; anti-Ac-H3K14, 1:200; anti-Ph-
H3S10, 1:1,000; anti-Ac-H4K8, 1:1,000; and anti-Ac-H3K9/K14, 1:1,000. Second-
ary antibodies were used at a 1/200 dilution (Jackson Laboratories). Preparations
were mounted in Citifluor AF1, and imaging was carried out using a Leica
TCS-SP confocal microscope.
Western blots were performed as described previously (11), using primary
antibodies at dilutions of 1:5,000 (anti-dGcn5), 1:1,000 (anti-Ac-H3K9), 1:1,000
(anti-Ac-H3K14), 1:1,000 (anti-Ac-H4K8), and 1:50,000 (anti-MBF-1; a gift of
Marek Jindra, Institute of Entomology, CAS, Ceske Budejovice, Czech Repub-
lic). A horseradish peroxidase-conjugated anti-rabbit secondary antibody (Vec-
tor) was used at a 1:5,000 dilution and detected using the SuperSignal West Pico
chemiluminescent substrate (Pierce).
Polytene chromosome staining. Glands from third-instar larvae raised at 18°C
were dissected in phosphate-buffered saline (PBS), fixed for 10 min in 50% acetic
acid–3.7% formaldehyde, and squashed on glass slides. Immunostaining of poly-
tene chromosomes was performed essentially as described previously (67). The
primary antibodies used were mouse anti-GFP (1:200) from Roche to reveal the
H2b-YFP marker protein (4) and anti-Ac-H3K9 (1:100), anti-Ac-H3K14 (1:100),
anti-Ac-H3K9/K14 (1:100), anti-Ph-H3S10 (1:100), and anti-Ac-H4K8 (1:100)
from Upstate. The anti-HP1 antibody (a gift from S. Elgin, Washington Univer-
sity, St. Louis, Mo.) was used at a 1:500 dilution. The secondary antibodies were
Cy3-conjugated anti-rabbit (1:200) and fluorescein isothiocyanate-conjugated
anti-mouse (1:200) from Jackson Laboratories. Slides were mounted in Vectash-
ield (Vector Laboratories) with 1.5 ?g/ml DAPI (4,6-diamidino-2-phenylindole),
and imaging was carried out using a Zeiss Axiovert 200 M microscope with a
Roper Scientific Coolsnap HQ camera.
BrdU incorporation and terminal deoxynucleotidyltransferase-mediated
VOL. 25, 2005dGcn5 IS REQUIRED FOR DROSOPHILA METAMORPHOSIS8229
dUTP-biotin nick end labeling (TUNEL) experiments. For the BrdU (5-bromo-
2?-bromodeoxyuridine) incorporation procedure, imaginal disks from third-in-
star larvae were dissected in Schneider medium and incubated for 15 min in 75
?g/ml BrdU at room temperature. Disks were washed twice with PBS and fixed
for 30 min at 4°C in PBS with 4% formaldehyde. Disks were then washed three
times for 10 min each time with 1? PBS–0.3% Triton X-100 (PBT). DNAs were
denatured in 3 N HCl for 30 min and washed three times for 10 min each time
with PBT. The disks were blocked (three times for 10 min each time) in 1?
PBS–0.3% Triton X-100–1% bovine serum albumin (PBTA), incubated over-
night at 4°C with an anti-BrdU monoclonal antibody (Upstate) according to the
manufacturer’s instructions, and washed with PBTA. The disks were then incu-
bated for 1 h at room temperature with a Cy3-conjugated anti-mouse secondary
antibody (Jackson ImmunoResearch) in PBTA, washed with PBT (three times
for 10 min each time), and mounted in Vectashield (Vector Laboratories).
TUNEL assays were performed using a TUNEL apoptosis detection kit (Up-
state). Wing disks from wandering third-instar larvae were dissected and fixed in
PBS with 4% formaldehyde for 20 min at 4°C. The disks were washed with PBS
for 10 min, blocked in 1? PBS–0.05% Tween 20–0.2% bovine serum albumin for
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 Gcn5E333stmutants. (Left side) Homozygous Gcn5E333stanimals failed to formal normal puparium compared to
Gcn5E333st/TM3 control animals. Salivary glands from control or Gcn5E333stlate-third-instar larvae (top) were immunostained with a dGcn5
antibody. (Right side) Squashed salivary glands from wild-type (control) and homozygous Gcn5E333st(E333st) late-third-instar larvae. Brackets
indicate chromosomal regions corresponding to 2B (top) and 74EF-75B (bottom) early puffs, respectively. (C) Gcn5E333st/Gcn5C137Tanimals 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 Gcn5E333st/Gcn5C137Tanimals than in Gcn5C137T/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 Gcn5E333st/Gcn5C137Tadult escaper is shown to the right. The same defects and lethality were observed for the Gcn5E333st/Gcn5?T280-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.
8230 CARRE´ET AL.MOL. CELL. BIOL.
10 min, and treated for the TUNEL reaction according to the manufacturer’s
instructions. The disks were washed three times with PBS and mounted in
Vectashield (Vector Laboratories).
Isolation of dGcn5 mutant alleles. We determined the in-
sertion sequences of several P elements which cytologically
mapped close to the 69C4 position of dGcn5 on polytene
chromosomes and were lethal over the Df(3L)iro-2 deficiency
(P. Maroy, personal communication). The 1479/10 P element
was found inserted 521 bp upstream of the dGcn5 transcription
start site, in the coding sequence of the CG14121 gene (Fig.
1A). We used this insertion to generate deletions via trans-
posase-mediated P-element excision and recovered the lethal
deficiency sex204 that removes a part of the CG14121 coding
sequence, the dGcn5 and CG10686 genes, and the transcrip-
tion start site of the citron gene (Fig. 1A). To obtain dGcn5-
specific alleles, we screened for EMS-induced dGcn5 mutants
by noncomplementation of the sex204 deficiency. We recov-
ered 11 mutants that failed to complement the sex204 defi-
ciency for viability. Five mutants were rescued by a PL trans-
genic construct encompassing the dGcn5 and CG14121 genes
(Fig. 1A). Four of these alleles were fully rescued by a UAS-
Gcn5 cDNA transgene (Fig. 1D) expressed under the control
of the ubiquitous da-GAL4 driver, indicating that these alleles
specifically impair dGcn5 function. The remaining allele is a
mutation of the CG14121 gene (see Materials and Methods).
We sequenced the dGcn5 mutations and found that they all lie
within the first dGcn5 exon. Two of them are localized in the
Pcaf homology domain and result in a cysteine-to-tyrosine sub-
stitution (C137T) or a small deletion from tyrosine 280 to
phenylalanine 285 (?T280-F285). The two other mutations
generate stop codons (Q186st and E333st). Gcn5E333stmutants
were outcrossed and then rescued as homozygous by the PL
transgene, indicating that they do not bear other irrelevant
EMS-induced lethal mutations.
dGcn5 is required for metamorphosis and oogenesis. To
determine the lethal phase associated with the dGcn5 loss of
function, we maintained the Gcn5E333stallele over a GFP-
expressing balancer chromosome. About 26% of eggs laid by
females from this stock did not reach the first larval instar, a
number attributable to embryonic lethality from the homozy-
gous balancer chromosome. Nonfluorescent homozygous
Gcn5E333stlarvae developed similarly to their GFP-expressing
fluorescent heterozygous siblings until the end of the third
larval instar, despite the absence of detectable dGcn5 protein
in mutant tissues at this stage (Fig. 1B and data not shown).
However, Gcn5E333stmutant larvae did not form their pupa-
rium at the normal time and continued to wander for 4 to 5
additional days. They eventually stopped moving but failed to
evert spiracles, formed abnormally long prepupae, and died
with partial separation of the posterior part of the prepupa
from the pupal case (Fig. 1B). Similar developmental arrest
and defects were observed with the heteroallelic null combi-
nation Gcn5Q186st/Gcn5E333st(not shown).
Puparium formation is triggered by a pulse of 20-hydroxy-
ecdysone at the end of the third larval instar. This involves the
coordinated induction of a small number of early puff genes,
whose products in turn regulate the expression of a larger set
of late puff genes (3). Homozygous Gcn5E333stmutants were
taken at various times during their extended wandering stage,
and polytene chromosomes from their salivary glands were
squashed (Fig. 1B, right panels). We observed a strong reduc-
tion of the size of the early puffs 2B, 74EF, and 75B in these
animals, indicating a failure of the ecdysone-controlled genetic
program in dGcn5 mutants.
Gcn5?T280-F285mutant larvae formed their puparium normally,
indicating that the Gcn5C137Tand Gcn5?T280-F285variant pro-
teins retain partial function. However, these mutants failed to
elongate their metathoracic legs correctly, had rough eyes, and
displayed a partial to complete absence of abdominal cuticle
deposition (Fig. 1C). Both heteroallelic combinations gave rise
to rare adult escapers with misshapen wings, rough eyes, and
crooked and twisted metathoracic legs (Fig. 1C and data not
shown). These escapers died a few hours after eclosion. Alto-
gether, these data point to an essential function of dGcn5 at
the onset of and during metamorphosis.
Since an important stock of dGcn5 protein is detected in
oocytes and presyncytial embryos (data not shown), this ma-
ternal contribution of dGcn5 may be sufficient to allow embry-
onic and larval development. To generate embryos lacking a
maternal contribution, we took advantage of the absence of
expression of the pUAST-derived construct UAS-Gcn5 in the
femalegerm line (10). We
Gcn5E333st/sex204 da-GAL4 rescued adults and found oogen-
esis in females to be arrested at stages 5 and 6 (data not
shown). This effect was due to the lack of UAS-Gcn5 expres-
sion in the germ line, since control females rescued by the
dGcn5 genomic construct (PL/?; Gcn5E333st/sex204) were fer-
tile. With dGcn5 being required for oogenesis, we were not
able to analyze its contribution to embryonic development.
dGcn5 is required for cell proliferation. Imaginal disks from
homozygous Gcn5E333stmutant third-instar larvae are mis-
shapen and severely reduced in size (Fig. 2A), suggesting that
a dGcn5 loss of function impairs the proliferation of imaginal
cells during larval instars. In a previous report, we made use of
the inverted-repeat transgenic construct UAS-IR[Gcn5] to tar-
get RNA interference (RNAi) against dGcn5 (50). To further
analyze the role of dGcn5 in the cell proliferation of imaginal
tissues, a UAS-IR[gcn5] transgenic line was crossed with en-
GAL4 UAS-GFP individuals expressing the GAL4 activator.
The en-GAL4 driver induced both GFP expression and specific
dGcn5 depletion in the posterior (P) compartments of imagi-
nal disks of third-larval-instar progeny (compare Fig. 3A and
A?). In a control experiment, silencing of the unrelated CBP
protein was not observed (Fig. 3B). The dGcn5-depleted P
compartments often had a reduced size and appeared flatter
than anterior compartments, suggesting that cell proliferation
is slowed down in these compartments (most visible in Fig.
3B). Although dGcn5 RNAi in the P compartments of imagi-
nal disks induced strong lethality during late pupal develop-
ment, few animals survived until the adult stage. The posterior
part of their wings was reduced in size and displayed abnormal
veins and cross veins. In the most dramatic cases, a bubble
indicative of abnormal adhesion between the dorsal and ven-
tral wing epithelial sheets was observed (Fig. 2B). The cell
density was not significantly changed in the silenced compart-
ment (data not shown), indicating that the size reduction is due
to a reduction in the cell number. A vg-GAL4 driver triggered
VOL. 25, 2005dGcn5 IS REQUIRED FOR DROSOPHILA METAMORPHOSIS8231
dGcn5 RNAi in the wing margin and induced a strong reduc-
tion of this structure in silenced adults (Fig. 2C), while the
induction of dGcn5 RNAi using a dll-GAL4 driver led to a
reduction in the size of the distal part of the tarsus and wings
(not shown). Finally, the induction of dGcn5 RNAi by an
esg-GAL4 driver, which is strongly expressed in imaginal his-
toblasts, resulted in lethality of pharate adults, with a partial to
complete absence of the abdominal cuticle (Fig. 2D). Collec-
tively, these data strongly suggested that dGcn5 RNAi limits
Surprisingly, however, a greater proportion of cells in S
phase (Fig. 3C) as well as a significantly larger number of cells
undergoing mitosis (Fig. 3D) was observed in the P compart-
ments of wing disks silenced by UAS-IR[Gcn5] upon activation
by en-GAL4. Notably, we also detected a high level of apopto-
sis in imaginal disks from third-instar dGcn5 mutant larvae
(Fig. 3E) as well as in response to dGcn5 RNAi triggered by
either en-GAL4 (Fig. 3G) or ptc-GAL4 (Fig. 3H). Together,
these data suggest that the net effect of the dGcn5 loss of
function on the compartment size results from a deregulation
of the cell cycle coupled to cell death.
Histone H3 acetylation of polytene chromosomes depends
on dGcn5. To investigate the contribution of dGcn5 to histone
acetylation, we immunostained polytene chromosomes from
larval salivary glands using antibodies specific for the acetyla-
tion of various lysine residues of histones H3 and H4. In order
to compare their acetylation levels, polytene chromosomes
from wild-type and mutant larvae were squashed and stained
together on the same slide. The expression of a histone 2B
yellow fluorescent fusion protein (H2b-YFP) in wild-type lar-
vae allowed us to distinguish their chromosomes from dGcn5
mutant chromosomes (Fig. 4B and data not shown). We de-
tected the acetylation of the H3 K14 and K9 residues in nu-
merous loci of wild-type polytene chromosomes (Fig. 4A and
C). In striking contrast, the acetylation of H3 K14 was unde-
tectable and the acetylation of H3 K9 was barely detectable in
dGcn5 mutant chromosomes. Antibodies against the acety-
lated H4 K8 (Fig. 4D) and H4 K16 residues (data not shown)
revealed unchanged acetylation of these residues in dGcn5
mutants compared to that in wild-type animals. We then ex-
amined whether the loss of H3 K9 and K14 acetylation in
dGcn5 mutants affects other modifications of histone H3 res-
idues. Like H3 K9 and H3 K14 acetylation, the phosphoryla-
tion of H3 S10 and di- and trimethylation of H3 K4 have been
associated with the transcriptional activation of target loci. We
found that global levels of these modifications are not affected
in dGcn5 mutant polytene chromosomes (Fig. 4E and F). On
the other hand, methylation of the H3 K9 residue acts as a
marker for the recruitment of the heterochromatin protein
HP1 and transcriptional silencing. The loss of H3 K9 acetyla-
tion in dGcn5 mutants could have favored ectopic H3 K9
methylation and the subsequent delocalization of HP1. How-
ever, we found that both H3 K9 methylation levels and HP1
localization in the pericentromeric regions of polytene chro-
mosomes were unchanged in dGcn5 mutants (Fig. 4G and H).
We also analyzed the contribution of dGcn5 to histone acet-
ylation in imaginal tissues by immunostaining of imaginal disks
in which dGcn5 was silenced in the P compartment by UAS-
IR[gcn5] under the control of en-GAL4. The acetylation of H3
K9 and H3 K14 residues was strongly reduced in the nuclei of
the dGcn5-depleted imaginal cells (Fig. 5A and B), while the
acetylation of H4 K8 and H4 K16 residues was not affected
(Fig. 5C and D). As seen with polytene chromosomes of dGcn5
mutants, dGcn5 depletion did not affect the level of H3 K4 or
H3 K9 methylation in the nuclei of imaginal disks (Fig. 5E and
F). When expression of the UAS-IR[Gcn5] transgene was
driven by the ubiquitous da-GAL4 driver, animals arrested
their development at the onset of metamorphosis. Western
blot analysis of such late-third-instar larvae showed a strong
depletion of histone H3 acetylated at the K9 and K14 residues,
while the level of histone H4-AcK8 remained unchanged (Fig.
5G). Hence, the effect of the dGcn5 loss of function on poly-
tene chromosomes, together with the results of our dGcn5
RNAi studies with imaginal disks and larval tissues, points to
dGcn5 as the major histone H3 K9 and H3 K14 acetyltrans-
ferase in Drosophila.
Functional analysis of dGcn5 domains. To analyze the func-
tional requirement of the evolutionarily conserved domains of
FIG. 2. dGcn5 is required for cell proliferation in imaginal tissues.
(A) Reduced and misshapen imaginal wing disks from homozygous
Gcn5E333stmutants compared to a wing disk from a Gcn5E333st/TM3
control third-instar larva. (B) Wings from control animals (?/?) and
en-GAL4/UAS-IR[Gcn5] adult escapers with vein and cross-vein de-
fects (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 (?/?).
8232 CARRE´ET AL.MOL. CELL. BIOL.
the dGcn5 protein, we established lines transgenic for dGcn5
variant genes under the control of the UAS promoter (Fig.
1D). The variant genes were designed in order to express
dGcn5 with a deletion of the Pcaf homology domain (?Pcaf),
the catalytic domain for acetylation (?HAT), the conserved
domain shown in yeast to be involved in interaction with the
Ada2 protein (?Ada), or the bromodomain (?Bromo). We
verified by Western blot analysis that the variant transgenes are
expressed in the presence of da-GAL4 at levels comparable to
that of the UAS-Gcn5 wild-type transgene (Fig. 6K) and then
tested their ability to rescue the dGcn5 function. In contrast to
the UAS-Gcn5 wild-type construct, the UAS-Gcn5?HAT con-
struct did not rescue the lethal phenotype of the Gcn5E333st/
sex204 heteroallelic combination. As expected from the disrup-
tion of the catalytic acetyltransferase domain, the acetylation
of polytene chromosomes at the H3 K9 and K14 residues was
not restored by the Gcn5?HAT variant in the mutant animals
compared to the rescue of acetylation by the wild-type dGcn5
protein (Fig. 6A and C). However, it should be noted that the
Gcn5?HAT variant still localized to the interbands of the
polytene chromosomes in a manner similar to that of the wild-
type dGcn5 protein expressed under the control of the da-
GAL4 driver (Fig. 6B and D).
It was recently shown that the dAda2b component of the
Drosophila SAGA-like complex directly interacts with dGcn5
and is required for the acetylation of histone H3. This result
suggested that the interaction with dAda2b is essential either
for the HAT activity of dGcn5 or for its targeting to chromatin
(49). To our surprise, the Gcn5?Ada protein appeared nor-
mally distributed on polytene chromosomes and restored his-
tone H3 acetylation in dGcn5 mutants (Fig. 6E and F). How-
ever, dGcn5 mutants were not rescued by Gcn5?Ada and still
arrested at puparium formation (not shown).
The Gcn5?Pcaf variant protein also appeared to be nor-
mally distributed at the interbands of polytene chromosomes
and to restore the acetylation of histone H3 (Fig. 6G and H).
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
Gcn5E333st/Gcn5E333st(E), Gcn5E333st/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).
VOL. 25, 2005dGcn5 IS REQUIRED FOR DROSOPHILA METAMORPHOSIS8233
In addition, about half of the dGcn5 mutant animals expressing
Gcn5?Pcaf formed their puparium and completed metamor-
phosis but died before eclosion as pharate adults, while the
other half gave rise to adult flies (Fig. 7). However, rescued
adults displayed held-out, misshapen wings, legs with a severe
femur kink, and rough eyes. They died a few hours after eclo-
sion, indicating that the UAS-Gcn5?PCAF construct only par-
tially rescues dGcn5 mutants.
Strikingly, the Gcn5?Bromo variant protein not only re-
stored histone H3 acetylation (Fig. 6I) but also restored com-
plete viability of the dGcn5 mutants. Adult flies were indistin-
guishable from dGcn5 mutant flies rescued by the full-length
dGcn5 protein (not shown). The only observed defect was
female sterility, a result expected from the absence of activity
of the UAS promoter in the female germ line. We were not
able to examine the chromosomal localization of the
Gcn5?Bromo protein because our dGcn5 antibody was pre-
pared against the dGcn5 bromodomain (Fig. 6J). Nevertheless,
the full phenotypic rescue by this variant strongly suggests that
its localization to chromosomes was restored.
GNAT complexes are essential actors of transcriptional
gene regulation in eukaryotes. Their structure and composition
have been conserved throughout evolution from yeast to hu-
mans, and they are thought to exert their function through the
catalytic activity of an HAT component belonging to the
GNAT family. Vertebrate Gcn5 and Pcaf proteins have been
involved in many biological processes, including the response
to steroid/retinoid hormones, cell proliferation, and cell differ-
entiation. However, how the function of these HATs is inte-
grated in higher organisms to control development has still to
In an extensive EMS-based genetic screen, we isolated and
characterized mutations of dGcn5, the unique Drosophila
member of the GNAT family. dGcn5 mutants have no discern-
ible phenotype before the onset of metamorphosis. The con-
siderable dGcn5 maternal stock (37; our unpublished data)
may be sufficient to fulfill dGcn5 functions in mutant embryos.
However, the dGcn5 protein is undetectable in dGcn5 mutant
mid-third-instar larvae, and the proliferation of imaginal disks
is already strongly impaired in such mutants at this stage. In
addition, mutant larvae keep wandering when control animals
have already formed a puparium, dramatically extending the
duration of their third larval instar by 4 to 5 days before they
die. Ada2b mutants die during pupal development (49), while
Ada2a mutants fail to form a puparium in a manner very
similar to that of dGcn5 mutant larvae (47a). Together with
these observations, our results strongly suggest that the func-
tion of the GNAT complexes is not required for Drosophila
The absence of normal induction of the three early puffs at
2B, 74EF, and 75B in dGcn5 mutants is indicative of a major
failure in the regulatory hierarchy controlled by the steroid
hormone ecdysone at the end of the third larval instar. The
extended duration of this stage in dGcn5 null mutants is also
characteristic of defects in pathways controlled by ecdysone.
For instance, it is observed with EcR-B1-specific alleles of the
ecdysone receptor gene (6, 53) and with mutant alleles of the
broad gene (5). The Pcaf homology domain has been involved
in interactions of human Pcaf with various nuclear receptors
(9, 41, 54). Two dGcn5 hypomorphic alleles isolated in this
work change amino acids in the Pcaf homology domain, result-
ing in a partial loss of function of the protein. In heteroallelic
combination with the null Gcn5E333stallele, they led to im-
paired metamorphosis, with strong defects in adult appendage
formation. dGcn5 adult mutant escapers rescued by the
Gcn5?Pcaf variant consistently display very similar defects of
leg elongation and blistered wings. These defects are also
highly suggestive of an impaired ecdysone regulatory cascade
during metamorphosis (17, 18, 19). From these observations,
FIG. 4. dGcn5 is required for acetylation of histone H3 K9 and H3
K14 residues in polytene chromosomes. Polytene chromosomes from a
Gcn5E333st/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 (acety-
lated 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.
8234CARRE´ET AL.MOL. CELL. BIOL.
we propose that dGcn5 acts as a nuclear receptor coactivator at
metamorphosis. The finding that the EcR protein is coimmu-
noprecipitated from embryonic nuclear extracts with dGcn5 in
an ecdysone-dependent manner (A. Mazo, personal commu-
nication) points to the ecdysone receptor itself as a dGcn5
target. However, we did not detect interactions between our
dGcn5 alleles and EcR mutant alleles in a trans-heterozygous
genetic test. Although these results do not rule out a functional
interaction between dGcn5 and the ecdysone receptor, they
may also indicate that dGcn5 acts in the metamorphic ecdy-
sone regulatory hierarchy through interactions with other Dro-
sophila nuclear receptors.
We have shown that the lack of dGcn5 in the female germ
line arrests oogenesis at an early stage. In addition, somatic
dGcn5 mutant clones in ovarian follicles induce the formation
of compound egg chambers indicative of an oocyte packaging
defect (J. R. Huynh, personal communication). Together,
these results indicate a strict requirement for dGcn5 during
oogenesis, in both the germ line and the somatic line. In light
of the role of dGcn5 during ecdysone-triggered metamorpho-
sis, it is noteworthy that ecdysone regulatory hierarchies have
also been shown to regulate Drosophila oogenesis (33).
dGcn5 mutant imaginal disks, as well as imaginal tissues
silenced by dGcn5 RNAi, display slower cell proliferation. The
role of CBP in the cell cycle has been documented for verte-
brates (39), and a domain of interaction of CBP with the Gcn5
homologue Pcaf is targeted by viral and cellular factors to
regulate cell cycle progression (51). The acetylation of E2F1 by
Pcaf also results in cell cycle modulation (43). In yeast, Gcn5
regulates genes required for mitotic exit (34). Together, these
data suggest that functions of Gcn5 in cell cycle regulation
have been conserved throughout evolution. Interestingly, a
mouse knock-in mutant for Trrap, a conserved component of
GNAT complexes, results in aberrant mitosis (29, 40). Simi-
larly, the higher mitotic index, together with the increased
BrdU incorporation, of dGcn5-silenced imaginal cells suggests
that they undergo an aberrant cell cycle. We have shown that
apoptosis induced in imaginal tissues by the loss of dGcn5
function may contribute to the net reduction in cell number.
The Drosophila SAGA-like complex interacts in vitro with
Dmp53 (37), and several authors reported a role of dAda2b in
DNA damage-induced Dmp53-dependent apoptosis (49, 47a).
However, this is the first time that apoptosis in a mutant with
a deletion of a component of the Drosophila SAGA-like com-
plex has been observed in the absence of X-ray-induced DNA
damage. Strikingly, Gcn5 mouse mutants also display apoptosis
in the absence of any exogenous inducers (66). Although fur-
ther analysis is required to understand the complex roles of
dGcn5 in cell proliferation, we propose that apoptosis in the
dGcn5 mutant might be a consequence of cell cycle defects.
Our data demonstrate that dGcn5 is the major acetylase for
two distinct histone residues, H3 K9 and H3 K14, in Drosoph-
ila. In contrast, its contribution to histone H4 acetylation could
not be detected in our global analysis. The loss of Ada2b
results in a partial loss of H3 K9 and H3 K14 acetylation on
polytene chromosomes, while the loss of Ada2a has no detect-
able effect on chromosome acetylation (49, 47a). In light of our
results, these data suggest that the Drosophila GNAT com-
plexes may retain partial dGcn5 HAT activity in the absence of
the Ada2 components. Alternatively, dGcn5 could exert its
HAT activity in other complexes which remain to be charac-
terized. The substrate specificities of Drosophila and yeast
Gcn5 proteins appear to be identical (26). However, it is in-
teresting that in contrast to what we found for Drosophila, both
the Gcn5 and Elp3 HATs must be invalidated in yeast to
significantly impair histone H3 K9 and K14 acetylation (62).
Recent studies have extensively documented the relationship
between histone H3 acetylation and gene transcription (7, 36,
52), but whether or not patterns of histone modification con-
stitute a true code for the control of gene activity in eukaryotes
is still a matter of debate. In either case, our results strongly
suggest that specific histone acetylation profiles may be estab-
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.
VOL. 25, 2005dGcn5 IS REQUIRED FOR DROSOPHILA METAMORPHOSIS 8235
lished in vivo through the activity of a very limited set of
substrate-specific enzymes. The observation that dGcn5 mu-
tant larvae survive for several days without detectable acetyla-
tion of H3 K9 and H3 K14 residues is striking and suggests that
transcriptional regulation in larval versus embryonic or adult
insect tissues involves distinct mechanisms.
Numerous data indicate that histone modifications can in-
fluence each other (24). The loss of H3 K9 and H3 K14 acet-
ylation in imaginal disks or salivary glands had no detectable
effect either on the levels of H3 S10 phosphorylation and H3
K4 methylation, both of which have been associated with tran-
scriptional activation, or on the level of H3 K9 methylation,
which marks HP1 recruitment and silencing. These results
suggest that histone H3 acetylation is either a terminal or
independent process in the cascade of histone H3 modifica-
Using transgenic dGcn5 variants, we investigated the func-
tions of the conserved regions in the dGcn5 protein. The de-
letion of the HAT domain completely abolished the ability of
dGcn5 to acetylate histones and to rescue dGcn5 mutant lar-
vae. This result provides the first demonstration that Gcn5 in
metazoans exerts its regulatory function during development
through its histone acetylase activity. As discussed above, de-
fects displayed by dGcn5 mutant adults rescued by the
Gcn5?Pcaf variant suggest a role of dGcn5 in the ecdysone
regulatory hierarchy during metamorphosis, possibly through
interactions with nuclear receptors. However, it is noteworthy
that Gcn5?Pcaf retains important functions. The variant pro-
tein was distributed along polytene chromosomes similarly to
the dGcn5 wild-type protein and provided apparently normal
H3 K9 and K14 acetylation. This suggests that the core func-
tions of chromatin binding and substrate acetylation may be
performed by the ancestral portion of Gcn5, which has been
conserved throughout evolution from yeast to humans, and
that the Pcaf homology domain has evolved to perform more
specific functions in metazoans. The Ada2 interaction domain
has been mapped in Gcn5 to an evolutionarily conserved re-
gion between the HAT domain and the bromodomain (15, 16,
42). A direct interaction in vitro between dGcn5 and Ada2b
FIG. 6. Complementation of histone acetylation and dGcn5 chro-
mosome binding in dGcn5 mutants. Polytene chromosomes from
Gcn5E333st/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 het-
erozygous 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 Gcn5?HAT protein.
FIG. 7. Gcn5?Pcaf variant restores the viability of dGcn5 mutants.
(A) Gcn5E333st/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-Gcn5?Bromo/? Gcn5E333st/
sex204 da-GAL4 animals (not shown). (B) Gcn5E333st/sex204 da-GAL4
adult fly heterozygous for the UAS-Gcn5?Pcaf transgene. Rescued
adults displayed held-out, notched wings (white arrow), rough eyes,
and crooked legs (black arrow).
8236CARRE´ET AL.MOL. CELL. BIOL.
was recently shown for Drosophila (49). The finding that the
Gcn5?Ada variant was completely unable to rescue the lethal-
ity of the dGcn5 mutants at the time of puparium formation
provides strong evidence that the interaction of dGcn5 with
Ada2 proteins is crucial for the function of the Drosophila
GNAT complexes. Strikingly, however, in dGcn5 mutants
Gcn5?Ada could bind to polytene chromosomes and restore
H3 K9 and H3 K14 acetylation patterns that were indistin-
guishable from those provided by the wild-type dGcn5 protein.
This result is at odds with the finding that histone H3 acetyla-
tion is strongly reduced in Ada2b null mutant polytene chro-
mosomes (49). It is possible that the loss of interaction be-
tween Ada2b and dGcn5 is deleterious for a specific function
of the multiprotein SAGA-like complex but is not sufficient to
disrupt the architecture of this complex as well as its ability to
The bromodomain is conserved in a large number of tran-
scription factors and has been shown to bind to acetylated
lysines on histone tails. The yeast Gcn5 bromodomain has been
shown to be involved in the stabilization of the SWI/SNF
remodeling complex through interactions with acetylated nu-
cleosomes at the PHO5 promoter (59). The bromodomain-
containing complexes SWI/SNF and TFIID have been shown
to be targeted to acetylated histone H3 and H4 tails on the beta
interferon promoter in vitro (1, 2). Collectively, these findings
suggest that the bromodomain is essential for targeting a large
set of transcription factors to acetylated nucleosomes. In this
context, the apparently normal chromosome acetylation by the
Gcn5?Bromo variant, and most significantly, the complete res-
cue of dGcn5 mutant viability by this protein, raises a number
of questions. We cannot exclude the possibility that the over-
expression of the Gcn5?Bromo variant may overcome the lack
of an essential dGcn5 function or that Gcn5?Bromo may be
unable to supply an essential function under particular stress
conditions. However, our observations clearly indicate that the
dGcn5 bromodomain is not as strictly required as the other
domains of the protein, since variants with truncations of these
domains were expressed under the same conditions. Interest-
ingly, others have reported a minor contribution of the bro-
modomain to the functions of various transcription factors.
Notably, in yeast the deletion of the bromodomain of dGcn5
has little consequence on its transcriptional activity (16, 57),
while deletion of the bromodomain of Swi2/Snf2 (38) or Spt7
(25) has no phenotypic effect. Similarly, the Brahma protein
with a deletion of its bromodomain binds normally to chromo-
somes in Drosophila and fully rescues brahma null mutants
(22). The apparent lack of requirement for the bromodomain
in dGcn5 may be due to a functional redundancy of various
components of dGcn5 complexes in targeting chromatin. This
role has been proposed for the Spt7 factor and the large Tra1
protein, which are both components of the SAGA complex (12,
28). The recent demonstration that a chromodomain of the
yeast SAGA component Chd1 interacts with methylated lysine
4 of histone H3 (48) also raises the interesting possibility that
different factors in GNAT complexes interact with different
In summary, the deletion of the HAT domain in dGcn5
abolished both its HAT activity and its ability to rescue dGcn5
mutants. dGcn5 variants deleted in either the Pcaf homology
domain or the Ada2 interaction domain acetylated chromo-
somes. However, Gcn5?Pcaf was able to partially rescue the
dGcn5 mutants, while Gcn5?Ada was not. Finally, the deletion
of the dGcn5 bromodomain had no noticeable consequences.
This remarkable variety of effects revealed in our complemen-
tation experiments strongly suggests that dGcn5 integrates
multiple functions during development.
We are grateful to P. Maroy for the gift of the 1479/10 line, to N.
Dos-Santos, E. Scola, A. Ikmi, D. Cohen, and J.-R. Huynh for their
technical help, and to the Institut Pasteur Dynamic Imaging Platform
for assistance and advice. We thank I. Boros, L. Tora, and A. Mazo for
sharing data before publication, T. Grange and J. A. Lepesant for
helpful discussions during the course of this work, and M. Bucking-
ham, D. Fagegaltier, H. Thomassin, and L. The ´odore for critical read-
ings of the manuscript.
This work was supported by the Pasteur Institute and the C.N.R.S.
(URA 2578) and by grants from the A.R.C. (7742/4383/3202). D.S. and
C.C. were supported by the University of Paris VI and the Ministe `re de
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