The Essential Role of Drosophila HIRA
for De Novo Assembly of Paternal Chromatin
Emilie Bonnefoy1,2[, Guillermo A. Orsi1,2[, Pierre Couble1,2, Benjamin Loppin1,2*
1 Universite ´ Lyon 1, Lyon, France, 2 CNRS, UMR5534, Centre de Ge ´ne ´tique Mole ´culaire et Cellulaire, Villeurbanne, France
In many animal species, the sperm DNA is packaged with male germ line–specific chromosomal proteins, including
protamines. At fertilization, these non-histone proteins are removed from the decondensing sperm nucleus and
replaced with maternally provided histones to form the DNA replication competent male pronucleus. By studying a
point mutant allele of the Drosophila Hira gene, we previously showed that HIRA, a conserved replication-independent
chromatin assembly factor, was essential for the assembly of paternal chromatin at fertilization. HIRA permits the
specific assembly of nucleosomes containing the histone H3.3 variant on the decondensing male pronucleus. We report
here the analysis of a new mutant allele of Drosophila Hira that was generated by homologous recombination.
Surprisingly, phenotypic analysis of this loss of function allele revealed that the only essential function of HIRA is the
assembly of paternal chromatin during male pronucleus formation. This HIRA-dependent assembly of H3.3
nucleosomes on paternal DNA does not require the histone chaperone ASF1. Moreover, analysis of this mutant
established that protamines are correctly removed at fertilization in the absence of HIRA, thus demonstrating that
protamine removal and histone deposition are two functionally distinct processes. Finally, we showed that H3.3
deposition is apparently not affected in Hira mutant embryos and adults, suggesting that different chromatin assembly
machineries could deposit this histone variant.
Citation: Bonnefoy E, Orsi GA, Couble P, Loppin B (2007) The essential role of Drosophila HIRA for de novo assembly of paternal chromatin at fertilization. PLoS Genet 3(10):
The assembly of nucleosome particles on nuclear DNA is
the initial step for the formation of chromatin. Nucleosome
assembly initiates with the formation of a H3-H4 histone
tetramer on DNA followed by the addition of two H2A-H2B
dimers to form the octameric particle [1,2]. Although this
organisation of genomic DNA is remarkably conserved in
eukaryotes, sperm cells of many species are characterized by a
very different type of chromatin architecture involving non-
histone proteins such as protamines . The replacement of
histones with protamines or other sperm nuclear basic
proteins (SNBPs) during the differentiation of post-meiotic
spermatids is generally associated with a high level of nuclear
condensation, a general shutdown of transcriptional activity,
and a state of chromatin that is incompatible with DNA
replication [3–5]. Although the precise advantages of acquir-
ing a specialized type of chromatin for the sperm cell are
poorly known, the protamine type of chromatin could
protect the paternal DNA from damaging agents or allow
the resetting of epigenetic marks carried by histones [6–8]. In
any case, once entered in the egg cytoplasm, the fertilizing
sperm nucleus must replace its SNBPs with maternally
provided histones that are stored in the egg cytoplasm. This
process, called sperm chromatin remodelling (SCR), allows
the paternal DNA to recover a nucleosomal chromatin and
thus guarantees the ability of the male pronucleus to
replicate its DNA in coordination with its female counterpart
[3–5]. SCR can be separated into two key processes. The first
process is the removal of SNBPs from the paternal DNA once
the sperm nucleus is released in the egg cytoplasm. The
second is the assembly of nucleosomes from maternal
components before the first round of DNA replication. SCR
has been almost exclusively studied in animal models that
produce large quantities of eggs, such as amphibians or sea
urchins, thereby facilitating the biochemical characterization
of factors capable of remodelling sperm nuclei in vitro .
Drosophila embryonic extracts have also been used as a source
of sperm chromatin decondensation factors [9–12], but none
of the identified molecules has been demonstrated so far to
have a function in SCR in vivo. In Drosophila, the sperm DNA
is packaged with two protamines, whereas core histones are
not detectable in male gamete nuclei [13,14]. In this sense,
Drosophila represents a good model for the functional study of
SCR in vivo. In previous publications, we characterized se ´same
(ssm), a Drosophila maternal effect mutation that specifically
prevented male pronucleus formation  and SCR . This
Editor: Asifa Akhtar, European Molecular Biology Laboratory, Germany
Received May 2, 2007; Accepted September 7, 2007; Published October 26, 2007
A previous version of this article appeared as an Early Online Release on September
10, 2007 (doi:10.1371/journal.pgen.0030182.eor).
Copyright: ? 2007 Bonnefoy et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author
and source are credited.
Abbreviations: HR1, homologous recombination 1; RC, replication coupled; RI,
replication independent; SCR, sperm chromatin remodelling; SNBP, sperm nuclear
basic protein; snky, sneaky; sra, sarah; ssm, se ´same; TC, trancription coupled
[These authors contributed equally to this work.
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mutation was subsequently shown to cause a single amino
acid substitution (R225K) in the Hira gene .
HIRA is a conserved chromatin assembly factor that allows
the replication-independent (RI) deposition of core histones
on DNA, in contrast to the CAF-1 complex whose replication-
coupled (RC) nucleosome assembly activity is strictly linked to
DNA synthesis . Accordingly, it has been established in
vitro that HIRA specifically deposits H3-H4 dimers that
contain the histone H3 variant H3.3, which is expressed
throughout the cell cycle, whereas CAF-1 deposits H3-H4
dimers that contain the replicative histone H3.1 . Our
functional analysis of the Drosophila Hira gene allowed us to
demonstrate in vivo that HIRA was indeed involved in the RI
deposition of H3.3 . In addition, we observed that
maternal HIRA localized in the decondensing sperm nucleus
where it deposited H3.3-H4 histones before the first zygotic S
phase, thus establishing the essential role of HIRA in SCR.
Recently, the Hirassmallele was found to enhance the
variegation of a white reporter transgene, indicating that
HIRA could help counteract the spread of heterochromatin
by mediating histone replacement at specific sites .
However, because of the subtle nature of the Hirassmmutation
and the absence of obvious phenotype in mutant adults, it was
not clear whether HIRA could have important functions
during development or in adult flies. In this paper, we report
the characterization of a loss of function Hira allele that we
have generated by homologous recombination. Surprisingly,
we show that paternal chromatin assembly at fertilization is
the only developmental process that absolutely requires
HIRA. We also demonstrate that protamine removal does
not depend on HIRA and is thus functionally distinct from
the paternal nucleosome assembly process. Finally, we show
that H3.3 is deposited in the chromatin of mutant embryos
and adults, suggesting that other factors are implicated in the
assembly of H3.3 nucleosomes.
Targeting the Hira Gene by Homologous Recombination
The original ssm185ballele (referred to as Hirassm) is a point
mutation that replaces an evolutionary conserved arginine
with a lysine (R225K) in the N-terminus region of HIRA .
This region is characterized in all HIRA proteins by the
presence of a well-conserved domain containing seven WD-
repeats. WD-repeats assemble into a structure called beta-
propeller . The Hirassmmutation does not affect the
normal recruitment of HIRA in the male nucleus at
fertilization . Nevertheless, it completely prevents the
deposition of histones on paternal DNA [16,17], suggesting
that the beta-propeller domain is important for the nucle-
osome assembly activity of HIRA. To gain insight into other
possible functions of Hira not evident from the subtle Hirassm
mutation, we generated a new mutant allele using ends-out
homologous recombination . The targeting construct was
designed to delete a 319 bp DNA fragment encompassing the
complete predicted 59 UTR, the first exon, the first intron,
and the 59 part of the second exon of Hira. In addition, the
recombination arms used in this construct did not overlap
any other predicted coding sequence, thus minimizing the
risk of damaging adjacent genes. Finally, in the recombined
allele, the 319 bp deletion was replaced with a 4778 bp
sequence from the pW25 vector , containing the white
marker gene flanked with stop codons in the six reading
frames (Figure 1A). We recovered 59 independent recombi-
nation events on the X chromosome that did not complement
the 100% female sterility associated with the Hirassmmutation
(Table 1). Surprisingly, all these lines produced viable and
fertile mutant males. In all the lines that were further
examined (n ¼ 7), homozygous mutant females were also
viable but produced embryos that never hatched (unpub-
lished data). One line, named HiraHR1(homologous recombi-
nation 1), was arbitrarily chosen to conduct the rest of the
analysis. The nature of the molecular lesion at the HiraHR1
locus was verified by PCR analysis and sequencing of genomic
DNA, and the expected recombination event was found, with
no other detectable alteration (Figure 1B and unpublished
data). We verified that the maternal effect phenotype
associated with HiraHR1remained unchanged in hemizygous
HiraHR1/Df(1)ct4b1 females, Df(1)ct4b1 being a large X chro-
mosome deficiency that covers the Hira region . In
addition, the HiraHR1phenotype was fully rescued by a single
copy of a wild-type Hira transgene , demonstrating that
no other important gene was affected by the HiraHR1
recombination event (unpublished data).
The HiraHR1mutation was expected to destroy the normal
transcriptional regulation of Hira. However, transcriptional
activity was detected by RT-PCR analysis at the junction
between the pW25 vector and the beginning of the Hira
sequence (unpublished data), suggesting that the HiraHR1
allele could be transcribed from the hsp70 promoter
associated with the whsmarker gene or from another
promoter in or upstream from the pW25 vector. To check
for the translation of any truncated HIRA protein from the
HiraHR1allele, we first established transgenic lines containing
a pW25-HiraHR1-Flag transgene (Figure 1A). This construct is
identical to the donor transgene used for the homologous
recombination with the exception of a 3X-Flag tag fused in
frame to the C-terminus of HIRA. RT-PCR analysis of two
independent pW25-HiraHR1-Flag lines confirmed that the
Hira sequence in these transgenes is also transcribed
(unpublished data). However, western-blot analysis of embryo
extracts from both lines did not detect any HIRA-FLAG
protein (Figure 1C).
We then directly tested the presence of HIRA in eggs from
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HIRA Function during Drosophila Development
Chromatin is composed of basic units called nucleosomes, in which
DNA wraps around a core of histone proteins. HIRA is a histone
chaperone that is specifically involved in the assembly of
nucleosomes containing H3.3, a universally conserved type of
histone 3. To understand the function of HIRA in vivo, the authors
generated mutant fruit flies with a non-functional Hira gene.
Surprisingly, mutant flies were viable, but females were completely
sterile. By analysing the female fruit flies’ eggs, the authors found
that in the absence of HIRA protein, the sperm nucleus was unable
to participate in the formation of the zygote. In Drosophila, as in
many animals, the condensed sperm chromatin contains protamines
instead of histones. The authors found that the only crucial role of
HIRA in flies was to assemble nucleosomes containing H3.3 in the
male pronucleus, after the removal of protamines. This fundamental
process, which is presumably also controlled by HIRA in vertebrates,
allows the paternal DNA to reconstitute its chromatin and
participate in the development of the embryo.
HiraHR1females using two independent HIRA polyclonal
antibodies. The first antibody was raised against a mix of two
synthetic HIRA oligopeptides  whose cognate DNA
coding sequences are intact in the HiraHR1allele. The second
antibody was raised against a recombinant protein contain-
ing residues 381–935 of HIRA (see Methods). Both sera
readily detect maternal HIRA in wild-type and Hirassmfixed
eggs, as the protein specifically accumulates in the male
pronucleus (Figure 2A, 2C, and 2D). As reported before ,
at the pronuclear apposition stage in Hirassmeggs, the male
pronucleus appeared much more condensed and smaller than
the female pronucleus and brightly stained with anti-HIRA
antibodies (Figure 2D). In HiraHR1eggs at the same stage, the
male pronucleus looked identical to that in Hirassmeggs, but
did not contain any detectable HIRA protein (Figure 2B and
Considering the fact that maternal HIRA protein is
immediately available at fertilization to assemble paternal
chromatin, we speculated that the protein must accumulate
in growing oocytes during oogenesis. Indeed, wild-type
ovaries stained with anti-HIRA antibodies revealed a specific
signal in the oocyte nucleus (also called germinal vesicle) that
was well visible from stage 10 of egg chamber formation
(Figure 3A). The same staining of the oocyte nucleus was
obtained with transgenic Hira-Flag ovaries stained with anti-
FLAG antibodies (Figure 3C). Strikingly, the germinal vesicle
staining was absent in HiraHR1ovaries and HiraHR1-Flag
ovaries stained with anti-HIRA or anti-FLAG antibodies,
respectively (Figure 3B and 3D). Altogether, these results
strongly support the hypothesis that no HIRA protein is
produced from the HiraHR1mutant allele.
The HiraHR1and HirassmPhenotypes at Fertilization Are
Previous studies of the Hirassmallele had revealed that the
male nucleus in mutant eggs was unable to undergo SCR .
Despite the fact that the mutant HIRA protein normally
accumulates in the male nucleus in Hirassmeggs ( and
Figure 2D), it is unable to assemble chromatin. Consequently,
the male nucleus does not achieve its decondensation and
does not replicate its DNA.
At the cytological level, fertilized eggs from HiraHR1females
appeared phenotypically identical to Hirassmeggs. In all cases
observed (n . 100), the male pronucleus remained abnor-
mally small and condensed after pronuclear apposition
(Figure 2E) and was unable to participate in the formation
of the zygote (see Figure 4). As a consequence of this early
defect, embryos from HiraHR1females were haploid, with only
the maternal chromosome set.
To check for any RI nucleosome assembly in HiraHR1eggs,
we used an anti-acetylated histone H4 antibody that brightly
and specifically stains the decondensing male nucleus in wild-
type eggs . As expected, the massive RI nucleosome
assembly that normally occurs during male pronucleus
Figure 1. Targeting the Hira Gene by Homologous Recombination
(A) Schematic representation of the wild-type (WT) Hira locus, the HiraHR1recombined allele, and the pW25-HiraHR1-Flag reporter transgene. The dotted
lines indicate the region that is replaced by the pW25 vector sequence in HiraHR1. The gray and white boxes indicate the Hira and white exons,
respectively, and the black box is the 3X-Flag tag at the 3’ end of the pW25-HiraHR1-Flag transgene. The dark gray hexagons represent termination
codons in the six reading frames. The positions of the primer pairs used in (B) are shown (arrows).
(B) Example of a genomic PCR with the primer pairs shown in (A). Note that the primer pair #1 does not amplify the large pW25 insertion in the HiraHR1
allele. The tested male genotypes are indicated.
(C) Anti-FLAG and anti-tubulin western blot analysis of embryo extracts from Hira-Flag and HiraHR1-Flag transgenic lines. The arrow indicates the HIRA-
FLAG protein. Other smaller bands are interpreted as HIRA-FLAG degradation products.
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HIRA Function during Drosophila Development
formation was not detected in HiraHR1eggs (Figure 4A and
4B). In contrast, RC deposition of acetylated H4 was normally
detected in maternal nuclei (Figure 4C). Thus both Hirassm
and HiraHR1mutant alleles specifically prevent assembly of
paternal chromatin and do not affect maternal nuclei.
HIRA Is Not Involved in the Removal of Protamines from
the Fertilizing Sperm Nucleus
In Drosophila, during spermiogenesis, post-meiotic sperma-
tid nuclei progressively elongate and condense to eventually
reach the typical needle-shape of mature sperm nuclei .
This complex process is also characterized by the replace-
ment of histones with SNBPs, including two closely related
protamines, ProtA and ProtB [13,14]. At fertilization, prot-
amines are removed from the paternal chromatin, and
nucleosomes are assembled in an RI process before the onset
of the first zygotic S phase. The incapacity of the male nucleus
to form in Hirassmeggs led us to hypothesize that this
phenotype could result from a defect in protamine removal
. Indeed, we would expect the persistence of protamines
on paternal DNA to prevent nucleosome assembly and male
nucleus decondensation. However, the presence of the HIRA
protein in the male nucleus in Hirassmeggs precluded drawing
any conclusion about its role in protamine removal . In
contrast, the HiraHR1allele allowed us to address this point
because in this case the protein is absent from the male
nucleus. To document the dynamics of protamine removal at
fertilization, we used transgenic males expressing ProtA-GFP
or ProtB-GFP in their germ line . These males are fertile
and their testes contain groups of spermatid nuclei that
achieve maximum fluorescence toward the end of the
condensation process (Figure 5A, left panel). To verify that
protamine-GFP can be detected in eggs, we crossed wild-type
females with ProtA-GFP males homozygous for sneaky (snky), a
paternal effect mutation that prevents sperm plasma mem-
brane breakdown at fertilization and sperm activation .
We found that fertilizing sperm nuclei from ProtA-GFP ; snky
males were brightly fluorescent in all cases observed (Figure
5B). We then looked at wild-type and HiraHR1eggs fertilized
with ProtA-GFP or ProtB-GFP sperm. Even in the earliest eggs
we observed, we never detected any trace of Prot-GFP in the
decondensing male nucleus (Figure 5C and 5D). We thus
concluded that the removal of protamines from the fertilizing
sperm nucleus is a fugacious, HIRA-independent process that
must occur immediately after sperm plasma membrane
breakdown and before the onset of the second meiotic
RI Paternal Chromatin Assembly Does Not Depend on Egg
In Drosophila, mature oocytes are arrested in metaphase of
the first meiotic division until egg ovulation and activation. In
contrast to many animals, egg activation in flies is not
dependent on fertilization. Instead, eggs are reactivated
during ovulation and immediately resume meiosis .
Drosophila females with a mutated sarah (sra) gene lay eggs
that are defective in several aspects of egg activation,
including a meiotic block in anaphase of the first division
. Interestingly, these authors observed that the male
pronucleus in fertilized sra eggs remained abnormally
condensed and did not replicate its DNA. This aspect of the
sra phenotype presents striking similarities with the Hira
mutant phenotype, raising the possibility that HIRA activity
could depend on egg activation. In their paper, Horner et al.
observed that the male nucleus and maternal chromosomes
stained, although rather diffusely, with an anti-histone H1
antibody. They concluded that paternal chromatin remodel-
ling was not impaired in sra eggs. However, it has been
previously reported that early Drosophila embryos lack histone
H1 , opening the possibility that anti-H1 antibodies could
cross-react with a non-H1 epitope. To directly analyse
paternal chromatin assembly in sra eggs, we used anti-
acetylated-H4 antibodies. In all cases, the condensed male
nucleus, but not the maternal chromosomes, brightly stained
with the anti-acetylated-H4 antibody, confirming that pater-
nal chromatin assembly is not dependent on egg activation
(Figure 6A). In addition, we verified that ProtA-GFP was not
detected from the male nucleus in sra eggs fertilized with
ProtA-GFP males (unpublished data).
In sra eggs blocked in anaphase of the first meiotic division,
the male nucleus frequently presented a rather irregular
shape (Figure 6A) and an apparent level of DNA condensa-
Table 1. Hira Ends-Out Targeting
Donor LineChromosomePhenotypeNumber of
Number of wþ
Results of the targeting experiments are given for each independent pW25-Hira transgenic donor line.
Chromosome, chromosome insertion of the donor line; Phenotype, Phenotype of the homozygous donor insertion; WT, viable and fertile; fs, female sterile; HR, homologous
aNumber of screened F1 virgin females with the pW25-Hira donor transgene and the Pf70FLPg11 Pf70I-SceIg2B, Sco Chromosome with white or mosaic eyes.
bNumber of vials of four females crossed with Pf70FLPg10 males that gave whiteþ progenies.
cNumber of independent whiteþ chromosomes that did not complement the Hirassmphenotype.
dc/a 3 100. Note that the actual rate of homologous recombination is possibly slightly underestimated because females were screened in vials of four individuals.
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HIRA Function during Drosophila Development
tion that was comparable with the highly condensed maternal
chromosomes blocked in anaphase I of the first meiotic
division. Hence, the high level of cyclin B in sra eggs that
causes the meiotic block  could also affect the male
nucleus and force it to recondense its unreplicated chroma-
tin. In comparison to sra, the male nucleus in Hirassmmutant
eggs is a uniformly round nucleus that systematically adopts
its definitive shape by the end of female meiosis II . To see
if the Hirassmmale nucleus could recondense in sra eggs, we
constructed double mutant Hirassm/Hirassm; sraA108/Df(3R)sbd45
females. In fertilized eggs from these double mutant females,
we observed that the male nucleus did not stain with anti-
acetylated-H4 antibodies and looked identical in shape and
size to the male nucleus in Hirassmeggs (Figure 6B). Thus, in
the absence of an assembled chromatin, the male nucleus is
unable to recondense in response to the meiotic block of sra
The ASF1 Histone Chaperone Is Not Involved in the RI
Assembly of Paternal Chromatin
SCR provides a unique opportunity to study de novo
nucleosome assembly in vivo at the scale of a whole nucleus
and in the absence of DNA synthesis or transcription. A
striking feature of this process is the very specific use of the
H3.3 histone variant to assemble paternal nucleosomes,
despite the presence of large quantities of canonical H3
Figure 2. HIRA Is Not Detected in HiraHR1Eggs
Confocal sections of eggs or embryos stained for DNA (red) and anti-HIRA antibodies (green).
(A) In wild-type (WT) fertilized eggs, HIRA is specifically detected in the male nucleus (arrowhead in the inset).
(B) In eggs from HiraHR1females, HIRA is not detected in the male nucleus (inset). Note that the HIRA antibody 830 non-specifically binds the sperm tail
(elongated structure visible in the green channel) .
(C) A Cycle 5 haploid embryo from a Hirassmfemale stained with antibody 830. The only stained nucleus is the condensed male nucleus (arrowheads).
(D) Apposed pronuclei in a Hirassmegg stained with HIRA antibody PG1 showing a strong signal in the male nucleus (arrowhead).
(E) A HiraHR1egg at the same stage stained with the same antibody. F: Female pronucleus. PB: Polar Bodies. Bars: 10 lm.
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HIRA Function during Drosophila Development
stored in the egg cytoplasm. ASF1 is a conserved histone
chaperone involved in the assembly of chromatin during
DNA replication (reviewed in ). Recent studies have
shown that ASF1 specifically interacts with H3-H4 dimers
[30,31] and with HIR proteins [32,33], and could play a key
role in presenting dimers containing specific H3 variants to
their corresponding chaperones, such as H3 to CAF-1 and
H3.3 to HIRA [29,31,33]. Accordingly, ASF1 proteins are
found in both H3.1 and H3.3 complexes in human cells .
To investigate this possibility in our model, we stained
fertilized eggs with an antibody against the unique Drosophila
ASF1 protein . We observed that ASF1 was systematically
detected in replicating nuclei, including the pronuclei
(Figure 7C). However, ASF1 was not found on the decondens-
ing male nucleus in wild-type eggs or in the male nucleus in
Hira mutant eggs (Figure 7A, 7B, 7D, and 7E). Thus, ASF1 does
not directly cooperate with HIRA during the RI assembly of
paternal chromatin. This is consistent with a recent report
showing that ASF1 is dispensable for direct de novo histone
deposition in Xenopus egg extracts . So far, HIRA is the
only H3-H4 chaperone involved in SCR in vivo.
H3.3 Deposition Is Not Globally Affected in HiraHR1Mutant
Embryos and Adults
The analysis of the HiraHR1allele confirmed the essential
role of maternal HIRA for the RI chromatin assembly in the
male pronucleus. In Drosophila, early development is under
maternal control and zygotic transcription essentially begins
at the blastoderm stage . In embryos, HIRA antibodies did
not produce any detectable staining, suggesting that the
protein, if it plays any role, does not accumulate at high levels
in embryo nuclei like in the male pronucleus (unpublished
data). Haploid embryos laid by HiraHR1females (named
HiraHR1embryos for simplicity) arrest their development just
before hatching. We used this situation to study H3.3
deposition in wild-type and HiraHR1early embryos. We used
a previously described transgenic line expressing H3.3-FLAG
under the regulatory sequences of the Drosophila His3.3A gene
. Maternally expressed H3.3-FLAG was then revealed
using anti-FLAG antibodies. Zygotically expressed H3.3-
FLAG becomes detectable in chromatin only at the gastrula
stage (Figure 8I and 8J) and was thus not detected in our
experiments on early embryos. As reported before , in
wild-type eggs, H3.3-FLAG is first detected in the decondens-
ing male nucleus shortly after fertilization (Figure 8A). As
expected, the male nucleus does not contain any H3.3-FLAG
in HiraHR1eggs, confirming the absence of chromatin
assembly in the male nucleus (Figure 8B). At the pronuclear
apposition stage in wild-type eggs, after the first round of
DNA replication, H3.3-FLAG is still abundant in the male
nucleus, but a faint staining is also visible in the female
Figure 3. HIRA Accumulates in the Germinal Vesicle in Wild-Type but Not in HiraHR1Oocytes
Stage 10 egg chambers stained for DNA (red) and anti-HIRA PG1 or anti-FLAG antibodies (green).
(A) In wild-type egg chambers, HIRA is specifically detected in the germinal vesicle where it occupies the whole nuclear volume. The karyosome, the
compact structure containing the maternal chromosomes, is visible in the DNA channel (arrow).
(B) In HiraHR1egg chambers, the antibody does not detect HIRA in the germinal vesicle (arrow).
(C) In transgenic Hira-Flag egg chambers, HIRA-FLAG protein is found in the germline vesicle (arrows) like the endogenous protein.
(D) No HIRA-FLAG protein is detected in the oocyte nucleus in HiraHR1-Flag transgenic egg chambers.
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HIRA Function during Drosophila Development
pronucleus (Figure 8C) and polar bodies (unpublished data).
Interestingly, this H3.3-FLAG staining in the female pronu-
cleus is also detected in HiraHR1eggs at the same stage (Figure
8D). H3.3 can be deposited on DNA through a transcription-
coupled (TC) assembly mechanism, suggesting that the
passage of the RNA polymerase complex displaces nucleo-
somes and creates a need for RI assembly . In the absence
of transcription in early Drosophila embryos, the observed
H3.3-FLAG must occur through a transcription-independent
process, presumably during DNA replication. In wild-type
embryos, we observed that the initial enrichment of H3.3-
FLAG on paternal chromosomes was still detectable during
the first 3 or 4 nuclear cycles (Figure 8E). In HiraHR1early
embryos, only a faint H3.3-FLAG staining was detected on the
sole maternally derived set of chromosomes (Figure 8F). The
paternal H3.3 mark in wild-type embryos was no longer
detectable in later embryos (unpublished data) suggesting a
rapid dilution by the massive RC deposition of H3 that occurs
at each S phase. To verify this point, we used a transgenic line
that expresses H3-FLAG with the regulatory sequences of
His3.3A . Both H3-Flag and H3.3-Flag transgenes produce
equivalent levels of tagged histones in embryos  and allow
a direct comparison of their respective deposition during
early development. During the earliest mitoses, the H3-FLAG
staining on chromosomes was much stronger than the H3.3-
FLAG staining (Figure 8K, compare with Figure 8E),
confirming that H3 is much more efficiently incorporated
in chromatin than H3.3 at this stage. The difference between
H3.3-FLAG and H3-FLAG chromosome staining was also
visible in blastoderm embryos (Figure 8G and 8L). At the
blastoderm stage, H3.3-FLAG clearly marked the chromatin
of all nuclei in both WT and HiraHR1(Figure 8G and 8H). In
conclusion, although H3 is preferentially deposited during
the early nuclear cycles, our results demonstrate that H3.3 is
also deposited at this stage, through a HIRA-independent
assembly pathway. Further work will be required to deter-
mine whether this HIRA-independent H3.3 deposition occurs
during or independently of DNA replication.
The migration of nuclei at the embryo periphery correlates
with the onset of zygotic transcription, with the notable
exception of germ line pole cells that are kept silent until
stage 9/10 of embryo development . Interestingly, we
observed that H3.3-FLAG is deposited at equivalent levels in
somatic and in pole cell nuclei in both wild-type and HiraHR1
embryos (Figure 9). Thus, TC assembly does not seem to
contribute substantially to the observed level of H3.3-FLAG
in chromatin at this stage. The activation of the zygotic
genome in blastoderm embryos correlates well with the
Figure 4. HiraHR1Eggs Are Unable to Assemble Paternal Chromatin at Fertilization
Confocal sections of eggs and embryos stained for DNA (red) and anti-acetylated histone H4 antibody (green).
(A) A wild-type egg in meiosis II with the elongated fertilizing male nucleus (M) that brightly stains for acetylated-H4 (arrow).
(B) A HiraHR1egg at the same stage with no acetylated-H4 detected in the male nucleus.
(C) A cycle 3 haploid embryo from a HiraHR1mother. The maternal nuclei, but not the male nucleus, stain for acetylated-H4. Bar: 10 lm.
PLoS Genetics | www.plosgenetics.orgOctober 2007 | Volume 3 | Issue 10 | e1821997
HIRA Function during Drosophila Development
apparition of histone post-translational modifications asso-
ciated with transcriptionally active chromatin, such as the
methylation of histone H3 at lysine 4 . Figure 9 shows that
this active mark is normally detected in HiraHR1embryos,
suggesting that HIRA is not required for the remodelling of
chromatin associated with the onset of zygotic transcription.
Accordingly, HiraHR1embryos develop without obvious
problems until late embryogenesis and eventually arrest
development with a phenotype typical of haploid embryos
produced by other mutants ([39,40] and unpublished data).
That HiraHR1flies are viable offered us the possibility to
evaluate the impact of the mutation on H3.3-FLAG distribu-
tion in adult tissues. We chose to focus on the testis, an organ
where H3.3 distribution had been characterized already .
In wild-type transgenic adult testis, we observed a strong
nuclear staining of H3.3-FLAG in all somatic and germline
nuclei with the exception of late spermatid and sperm nuclei,
similar to previous reports . In HiraHR1testis we found no
detectable alteration of the distribution of H3.3-FLAG in
both somatic and germ line nuclei (Figure 10). We then
looked at other adult tissues including ovaries, malpighian
tubules, and gut; again, we found no difference between
control and mutant (unpublished data). We conclude that,
with the sole exception of the male pronucleus, HIRA does
not seem to play any crucial role for the assembly of H3.3
nucleosomes during Drosophila development.
HIRA and SCR
The analysis of maternal effect mutations in the Drosophila
Hira gene has revealed that SCR at fertilization involves at
least two functionally distinct steps. The first step is a HIRA-
independent process that allows the rapid removal of
protamines from the activated sperm nucleus. The second
step is the RI nucleosome assembly on paternal DNA and
requires maternal HIRA. That the male pronucleus seems to
be the only nucleus where H3.3 deposition is critically
dependent on HIRA (see below) indicates a peculiar case of
RI assembly. This could reflect specific features of the sperm
nucleus itself or constraints inherent to the tightly time-
controlled, whole paternal genome assembly at fertilization.
At least we know that this specific requirement of HIRA for
SCR is not directly linked to the removal of protamines.
Figure 5. HIRA Is Not Required for Protamine Removal from the Decondensing Sperm Nucleus
(A) Left panel: in a fixed ProtA-GFP transgenic testis, the GFP fluorescence is very strong in the most condensed spermatid nuclei (asterisk), whereas less
condensed nuclei are much less bright (arrow). Middle panel: the same testis stained with an anti-GFP antibody considerably enhances the GFP
detection in less condensed nuclei (arrow, compare with left panel), whereas highly condensed nuclei are comparatively less stained. Right panel: the
same testis stained with the DNA dye TO-PRO3.
(B) In wild-type (WT) eggs fertilized with sperm from snky1; ProtA-GFP males, the sperm nucleus is not activated (arrow), remains at the egg periphery,
and its protamines are not removed.
(C) In wild-type eggs fertilized with ProtA-GFP sperm and fixed before the end of meiosis II (MII), ProtA-GFP is never detected in the decondensing male
(D) The same result is obtained for HiraHR1eggs. Eggs in (B–D) were stained with an anti-GFP antibody revealed with a green secondary antibody to
cumulate the GFP and secondary antibody respective fluorescence in the green channel of the confocal microscope. Identical results were obtained
with ProtB-GFP transgenic males (unpublished data). PB: Polar Body. Bar: 10 lm.
PLoS Genetics | www.plosgenetics.org October 2007 | Volume 3 | Issue 10 | e1821998
HIRA Function during Drosophila Development
Our finding that SNBP removal activity is functionally
uncoupled to nucleosome assembly in Drosophila does not
apply to all known cases of SCR in animals. In fact, in the
classical example of SCR in Xenopus laevis, it was demon-
strated through in vitro experiments that a unique histone
chaperone, nucleoplasmin, was necessary and sufficient to
perform both SNBP removal and histone deposition [42,43].
Nucleoplasmin is a small, acidic protein that is highly
abundant in amphibian oocytes and forms pentameric
complexes that associate with core histones [2,44,45]. It is
important to consider, however, that the protein composition
of Xenopus sperm chromatin is rather peculiar since it
essentially retains H3-H4 tetramers on paternal DNA, where-
as H2A and H2B are replaced with protamine-like proteins
named SPs [43,46]. In vitro, nucleoplasmin allows the
replacement of SPs with H2A and H2B and reconstitute
nucleosomes [43,44]. There is apparently no need for a H3-
H4 assembly factor such as HIRA for Xenopus SCR. A
nucleoplasmin-like protein exists in Drosophila, but studies
of its ability to decondense demembranated Xenopus sperm
nuclei in vitro have led to contradictory results [11,12]. The
actual function of Drosophila nucleoplasmin remains to be
determined. In addition, other Drosophila embryonic nuclear
factors are known to decondense Xenopus sperm in vitro, such
as DF31  and NAP-1 , but their protamine removal
activity has not been confirmed in vivo. In mouse, as in
Drosophila, sperm chromatin is essentially packaged with
protamines . Interestingly, the knock-out of NPM2, the
mouse ortholog of Xenopus nucleoplasmin, does not affect
SCR . In contrast, HIRA is very likely involved in the
assembly of paternal chromatin in the mouse zygote. Indeed,
in this species, HIRA is detected in the decondensing male
nucleus  and H3.3 is specifically deposited on paternal
DNA in an RI manner [49,50]. We thus expect HIRA to be
generally involved in the assembly of paternal chromatin in
animal species in which histones H3 and H4 are totally or
partially replaced with SNBPs in the mature sperm. As an
H3.3-H4 deposition factor, HIRA itself is not expected to
mediate the deposition of H2A-H2B required for the
completion of nucleosome assembly on paternal DNA. It will
be interesting to identify this H2A-H2B chaperone and see if
it is dedicated to RI assembly or involved in both RI and RC
In Hira mutant eggs, the male nucleus is a small, round
nucleus that appears homogeneously condensed when
stained with a DNA dye. How the paternal DNA is organised
in this nucleus is not known. That it is surrounded by a de
novo assembled nuclear lamina  probably participates in
the maintenance of its round shape. Also, it is established that
the four centromeric regions are the only regions that are
organized with histones, most likely because centromeric
chromatin is not replaced with protamines in the sperm
nucleus . In this paper, we have demonstrated that the
male nucleus in Hira mutant eggs is also devoid of prot-
amines, strongly suggesting that most paternal DNA is free of
chromosomal proteins. A similar situation was reported in
decondensation assays using sperm from Bufo japonicus, a toad
species whose sperm chromatin only contains protamines
. In the presence of nucleoplasmin, protamines are
efficiently removed but nucleosomes are not assembled.
Consequently, B. japonicus sperm nuclei decondensed with
egg extracts containing the protamine removal activity
possess neither protamines nor core histones, and are very
fragile . Similarly, in Hira mutant eggs, the removal of
protamines from the male nucleus permits its partial
decondensation as the sperm nuclear volume increases when
the nucleus loses its specific needle shape and becomes
round. However, in the absence of a nucleosomal organisa-
tion, the male nucleus cannot achieve its decondensation and
does not replicate its DNA. This unique, inert state of the
male nucleus in Hira mutant eggs is also well illustrated by its
incapacity to recondense in blocked sra mutant eggs.
Figure 6. The Male Nucleus Does Not Recondense in Hira ; sra Double Mutant Eggs
(A) In sraA108/Df(3R)sbd45 mutant eggs, the female meiosis arrests in anaphase of the first meiotic division (MI). The male nucleus (arrowhead and
bottom panels) appears condensed but irregular in shape and stains with anti-acetylated histone H4 antibodies (bottom right panel). Note that the DNA
positive dots that are visible in this egg are Wolbachia bacteria that naturally infect the stock.
(B) In Hirassm; sraA108/Df(3R)sbd45 Double Mutant Eggs, the Male Nucleus Is Round and Does Not Stain with Anti-Histone Antibodies. Bars: 2 lm.
PLoS Genetics | www.plosgenetics.org October 2007 | Volume 3 | Issue 10 | e1821999
HIRA Function during Drosophila Development
The Function of HIRA during Drosophila Development
A surprising aspect of this study is the viability of HiraHR1
homozygous flies. This was unexpected, because in mouse the
Hira knock-out is embryonically lethal . From a genetic
point of view, both Hirassmand HiraHR1alleles behave as null
alleles with respect to the Df(1)ct4b1 deficiency. In addition,
several lines of evidence indicate that no HIRA protein is
translated in HiraHR1flies, including the absence of detection
of HIRA in the germinal vesicle and the male pronucleus, and
the absence of HIRA-FLAG protein expressed from the
Figure 7. ASF1 Is Not Directly Involved in the RI Paternal Chromatin Assembly
Confocal sections of eggs stained for DNA (red) and anti-ASF1 antibody (green).
(A) In wild-type fertilized eggs, ASF1 is not detected in the male nucleus or in maternal nuclei during the decondensation phase.
(B) ASF1 is not detected in the male nucleus during pronuclear migration.
(C) ASF1 stains both pronuclei in a wild-type egg during the first S phase.
(D) ASF1 is not detected in the male nucleus in Hirassmeggs.
(D) The same result was obtained with the HiraHR1allele. F: Female pronucleus, M: Male pronucleus, PB: Polar Bodies. Bar: 10 lm.
PLoS Genetics | www.plosgenetics.orgOctober 2007 | Volume 3 | Issue 10 | e1822000
HIRA Function during Drosophila Development
pW25-HiraHR1-Flag reporter transgene. In the alternative
possibility that some truncated HIRA protein would be
translated from this allele and escaped our detection, the first
possible translation initiation codon downstream from the
deleted region in HiraHR1is at position 61, after the second
WD repeat. Such a truncated HIRA would thus be expected to
have, at best, a destabilized beta-propeller domain, which
represents the most evolutionarily conserved part of HIRA
proteins [53,54]. The fact that both Hirassmand HiraHR1alleles
display identical mutant phenotypes also highlights the very
important role of the arginine 225 mutated in Hirassm, and by
extension, the important role of the beta-propeller domain
for the assembly of paternal chromatin. A recent study
implicated Drosophila HIRA and the GAGA factor–FACT
complex in a histone replacement mechanism that prevents
the spreading of heterochromatin into a white reporter
transgene inserted near centromeric heterochromatin .
Nakayama et al. observed that silencing of this variegating
transgene was enhanced in Hirassmmales, and concluded that
the mutation affected H3.3 replacement at a site near the
white gene. Their work suggests that Drosophila HIRA could
indeed function in RI assembly in other situations and is
consistent with the fact that Hira is expressed throughout
development, in addition to its strong maternal expression
[17,53,54]. Nevertheless, the fact that HiraHR1mutant adults
are viable indicates that this function is dispensable.
H3.3 Deposition without HIRA
Another important aspect of this study lies in the fact that
the HiraHR1mutation does not have detectable effect on the
deposition of H3.3-FLAG in embryos or adult cells. First, it
clearly establishes that H3.3 nucleosomes can be efficiently
assembled in the absence of functional HIRA in vivo. So far
HIRA is the only chaperone known to deposit the H3.3
variant. This study demonstrates the existence of at least one
alternative assembly pathway for H3.3 nucleosomes, although
the nature of the histone chaperone(s) involved is unknown.
A simple hypothesis is the deposition of H3.3 by the CAF-1
complex. In fact, we have shown that in early embryos, the
bulk of H3.3 is deposited independently of transcription,
Figure 8. Dynamics of H3.3 Deposition in Wild-Type and HiraHR1Early Embryos
Confocal sections of eggs/embryos stained with propidium iodide (red) and anti-FLAG antibody (green).
(A) In wild-type (WT) eggs, RI deposition of maternal H3.3-FLAG is observed in the decondensing male nucleus (M) before the first zygotic S phase.
(B) H3.3-FLAG is not detected in the male nucleus in HiraHR1eggs.
(C) At pronuclear apposition, during the first S phase, limited RC deposition of H3.3-FLAG is detected in the female pronucleus (arrow) in WT eggs.
(D). The same, faint H3.3-FLAG staining of the female pronucleus is observed in HiraHR1eggs (arrow).
(E) A WT embryo in anaphase of the third nuclear division. At this early stage, the stronger H3.3-FLAG staining of the paternally derived chromosomes
(arrowheads) is still detectable (note that paternal and maternal chromosomes tend to remain separated during the early syncytial mitoses).
(F) A HiraHR1haploid embryo in its fourth mitosis showing a weak H3.3-FLAG staining on maternally derived chromosomes (arrows).
(G) A wild-type, diploid blastoderm embryo in metaphase showing a strong H3.3-FLAG chromosomal staining on all nuclei.
(H) H3.3-FLAG is also detected on the chromosomes of HiraHR1haploid blastoderm embryos.
(I) Embryos from wild-type mothers crossed with H3.3-Flag/CyO males showing no detection of zygotic H3.3-FLAG at this stage.
(J) Zygotic H3.3-FLAG appears in the chromatin of gastrula embryos.
(K) A wild-type, cycle 3 embryo in anaphase showing a strong H3-FLAG staining on all chromosomes.
(L) A blastoderm embryo with a strong maternal H3-FLAG staining. Gray panels or insets show the H3.3-FLAG or H3-FLAG staining for a representative
group of nuclei. Bar: 10 lm.
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HIRA Function during Drosophila Development
presumably at each S phase of the early nuclear cycles.
Indeed, these cycles consist on a very rapid succession of S
and M phases and lack gap phases . The S phase
deposition of H3.3 is consistent with a previous report
showing that overexpressed H3.3-GFP was deposited during
DNA replication in Drosophila Kc cells . In human cells,
only the small subunit of CAF-1 was found in the H3.3
complex, whereas all three subunits of the complex were
copurified with the replicative histone H3.1 . In early
cycles, H3 is preferentially deposited compared with H3.3.
However, a peculiarity of Drosophila embryos is the storage of
large maternal pools of both H3 and H3.3, a situation that
could favour a competition of these histones for their
interaction with CAF-1. In contrast, in differentiated cells,
the massive expression of S phase histones at the onset of
DNA replication could strongly reduce the use of H3.3-H4
dimers by the CAF-1 complex. The early Drosophila embryo
should represent a good model to address this point.
A study of Hira ?/? mouse ES showed that these cells
undergo early differentiation, suggesting that core histone
deposition during this process could use HIRA-independent
pathways . Although it is well established that H3.3
deposition correlates with active chromatin in many instan-
ces, there is yet no link between HIRA and transcription in
higher eukaryotes . In budding yeast, nucleosome reas-
sembly at the PHO5 promoter absolutely requires the histone
H3-H4 chaperone Spt6 , whereas Hir1 is not absolutely
required [59,60]. In Drosophila, Spt6 is clearly involved in
transcription elongation [61,62] and thus represents an
interesting candidate for TC deposition of H3.3 . The
biochemical analysis of H3.3 complex in HiraHR1mutant
could help identify alternative H3.3 chaperone(s).
Our results support the hypothesis that multiple and
possibly redundant pathways are involved in the assembly
of H3.3 nucleosomes in multicellular organisms. Besides, it is
now established that H3.3 nucleosomes can be assembled
independently of RC and TC assembly pathways. For
example, nucleosome replacement mechanisms at cis-regu-
latory elements implicating the deposition of H3.3 have been
recently reported in Drosophila [20,63]. The ability of cells to
assemble chromatin independently of DNA replication is
apparently common to all eukaryotes. In fact, some organisms
such as yeasts have only one type of histone H3, which is
related to H3.3 and is deposited throughout the cell cycle
. The coexistence of RC and RI histone H3s in most other
eukaryotes indicates that these distinct modes of chromatin
assembly fulfil important complementary functions. Interest-
ingly and surprisingly, the deletion of all RI H3 histone genes
in the protist Tetrahymena thermophila does not compromise
survival and, in particular, does not affect nucleosome density
at highly transcribed regions . However, RI H3 genes in T.
thermophila appear to be critical for the production of viable
sexual progeny and for the function of germline micronuclei
, suggesting that sexual reproduction and/or developmen-
tal processes could have played an important role in the
evolution of the RI mode of nucleosome assembly. RI H3.3
replacement at fertilization is apparently a conserved
mechanism in nematodes, insects, vertebrates, and plants
[17,49,50,66,67]. That the paternal chromatin assembly is the
only essential function of Drosophila HIRA suggests that this
factor acquired new important roles during the evolution of
vertebrates. So far, in mammals, the implication of the HIRA/
H3.3 complex has been shown or at least suspected in various
remodelling processes, including heterochromatin repair
Figure 9. H3.3-FLAG Is Deposited in the Germ Line Chromatin in Blastoderm Embryos
Confocal sections of blastoderm embryos stained for DNA (blue), H3.3-FLAG (green), and H3K4me3 (red).
(A) H3.3-FLAG (left inset) is deposited at equivalent levels in somatic (arrows) and germ line (arrowheads) nuclei in wild-type embryos. H3K4me3 is
enriched in somatic nuclei (right inset).
(B) An identical situation is observed in HiraHR1embryos. Bar: 10 lm.
PLoS Genetics | www.plosgenetics.org October 2007 | Volume 3 | Issue 10 | e1822002
HIRA Function during Drosophila Development
, mammalian meiotic sex chromosome inactivation ,
fertilization [49,50], and possibly, formation of senescence-
associated heterochromatin foci  and histone exchange
during spermiogenesis . More functional studies should
reveal if all these processes strictly rely on HIRA, in the
context of the developing organism.
Note: After the preparation of our manuscript, a paper by
A. Konev et al.  was published that reported the
implication of the motor protein CHD1 in the deposition
of histone H3.3 in Drosophila. This finding supports our own
conclusions about the existence of Hira-independent H3.3
Figure 10. HiraHR1Does Not Affect the Distribution of H3.3-FLAG in Adult Testis
Testis and accessory glands from H3.3-Flag/CyO transgenic adult males with a wild-type (A–C) or HiraHR1(D–F) X chromosome, stained with anti-FLAG
antibody and propidium iodide.
(A) Apical tip of a wild-type testis.
(B) A group of elongating spermatids in a wild-type testis showing a bright H3.3-FLAG nuclear staining that disappears in late condensing spermatid
(C) Nuclei from a wild-type accessory gland.
(D) Apical tip of a HiraHR1testis.
(E) Spermatid nuclei in a HiraHR1testis. H3.3-FLAG is not detected in late spermatid nuclei (arrows).
(F) Nuclei from a HiraHR1accessory gland. Bars: 10 lm.
PLoS Genetics | www.plosgenetics.orgOctober 2007 | Volume 3 | Issue 10 | e1822003
HIRA Function during Drosophila Development
Materials and Methods
Flies. The w1118ssm185b/FM7c stock was described before . The
ProtamineA/B-GFP stocks  are a gift from S. Jayaramaiah Raja and
R. Renkawitz-Pohl. The sraA108allele  is a gift from V. Horner and
M. Wolfner. Df(3R)sbd45 is a deficiency that covers the sra locus. The
H3.3-Flag, H3-Flag, and Hira-Flag stocks have been described before
. The y w67cand w1118stocks were used as wild-type controls. All
the other stocks or chromosomes used in this paper were obtained
from the Bloomington Drosophila stock center.
Hira targeting by homologous recombination. The Hira gene was
targeted by ends-out homologous recombination as described in
[22,23]. Two DNA fragments from the Hira locus were PCR-amplified
from the cosmid genomic DNA clone 107B5 (European Drosophila
Genome Project) using the following primers: 59-ATGAAAT-
GAGTGCCAGCAGC-39 and 59-GGTACCTATCGGTAACGATGCC-
CATC-39 for the Hira upstream arm (4209 bp) and 59-
GGCGCGCCGTGGTCATCTGGAATCTGCT-39 and 59-CGTACGA-
TATTGGTTCCCGGTACCAG-39 for the Hira downstream arm
(3530 bp). These fragments were ligated in the pW25 vector 
using the following restriction sites: Sac II and Acc65I for the
upstream arm and AscI and BsiWI for the downstream arm. The final
construct, named pW25-Hira, was verified by PCR and restriction
analysis (unpublished data).
Six independent autosomal pW25-Hira transgenic lines were
established in a y w67cbackground. Batches of 15–20 virgin y w;
Pf70FLPg11 Pf70I-SceIg2B, Sco/CyO females were crossed with
approximately 10 males from a given donor line in plastic vials.
Vials containing 24-h egg collections from these crosses were heat
shocked for 90 min at 37 8C in a water bath on days 3, 4, and 5 after
egg laying. pW25-Hira /Pf70FLPg11 Pf70I-SceIg2B, Sco virgin F1
females with white or mosaic eyes were collected and crossed with
w ; Pf70FLPg10 males. Non-mosaic, coloured-eyed progenies were
then crossed again with w ; Pf70FLPg10 to establish individual lines.
Each line with a whiteþchromosome resistant to constitutive Flipase
activity was tested for its complementation with the w Hirassm
chromosome. Chromosomes that did not complement the maternal
effect embryonic lethality associated with Hirassmwere selected,
outcrossed with w1118for five generations, and balanced with the
pW25-HiraHR1-Flag transgenes. The pW25-HiraHR1-Flag transgene
was constructed by replacing an AgeI-BsiWI restriction fragment in
the 3’ Hira arm from the pW25-Hira vector with a 729 bp fragment
excised from the pW8-Hira-Flag transgene  to introduce the 3X-
Flag tag at the 3’ end of Hira. The final construct was verified by
sequencing, and transgenic lines were established.
RT-PCR. Total RNA was extracted by the Trizol method
(Invitrogen) and first-strand cDNAs were synthesized with the
Superscript II reverse transcriptase (Invitrogen) and oligo-dT
primers. The primer sequences used for PCR amplification of the
cDNAs or genomic DNA are available on request.
Antibodies for immunofluorescence. Anti-Flag M2 mouse mono-
clonal antibody (F-3165, Sigma Aldrich) was used at 1:2000, rabbit
anti-acetylated histone H4 polyclonal antibody (06–598, Upstate) at
1:500, rabbit anti-H3K4me3 polyclonal antibody (ab8580, Abcam) at
1:250, and mouse monoclonal anti-GFP antibody (Roche 1814460,
clones 7.1 and 13.1) at 1:500 (IF). The anti-Drosophila ASF1 antibody
 is a gift from F. Karch and was used at a 1:1000 dilution. The
HIRA 830 anti-peptide antibody was described before and used at
For the production of the PG1 anti-HIRA polyclonal antibody, a
plasmid PW8-Hira-Flag  was used as a template to amplify a 1943-
pb fragment from 1241 to 3183 (amino acids 381–935) by PCR using
primers 59-ACATATGGTGAACGGTCTGGGAAAGTC-39 and
5’TGGATCCGTACCCGTTGTCACAGCCAT-39. The fragment was
cloned into the NdeI and BamHI of pET15b vector (Novagen) in
frame with the His?Tagt at the N-terminus end of the recombinant
protein. The recombinant plasmid was transformed into Escherichia
coli BL21-CodonPlust (DE3)-RIL competent cells (Stratagene) and
expression of the recombinant protein was induced by IPTG
(isopropyl b-D-thiogalactoside) as described by the manufacturer
and analysed by SDS-PAGE. Two rabbits were immunized with the
purified HIRA-HIS-TAG protein purified on a Nickel column. Crude
sera were purified on a Proteine-G column (Proteogenix) and were
used at 1:1000.
Immunofluorescence. Eggs and embryos were collected, fixed in
methanol, and immunostained as described . For each experi-
ment, we observed a minimum of 25 eggs/embryos at the desired
stage. Testes and ovaries were dissected in PBS-Triton 0.1%, fixed in
4% paraformaldehyde for 20 min (testis) or 30 min (ovaries) at room
temperature, rinsed in TBST (0.1% Triton), and stained as for
embryos. DNA was stained either with propidium iodide as described
 or with TO-PRO-3 (Molecular Probes) used at a 1:10,000 dilution.
Preparations were observed under a Zeiss LSM Meta confocal
microscope. Images were processed with the LSM and Photoshop
Western blot. WT and transgenic O/N embryos were collected,
washed, dechorionated, and homogenized in Laemmli 2X sample
buffer (125 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 100mM
DTE, 1% bromophenol blue) with an Eppendorf fitting pestle-
homogenizer using the bio-vortexerTMmixer (Roth). Protein samples
were centrifuged 5 min at 5000 rpm, boiled for 10 min at 95 8C, and
subjected to electrophoresis on an 10% SDS-PAGE gel. Immunoblot-
ting was performed using a tank transfer system (Mini Trans-Blot
Cell, Bio-Rad) and Hybond-C Extra nitrocellulose membranes
(Amersham Biosciences) in transfer buffer (25 mM Tris, 20 mM
glycine, 20% ethanol, 0.05% SDS). Antibodies incubation was in
TBST (20 mM Tris-HCl [pH 7.5], 130 mM NaCl, 0.1% Tween 20)
supplemented with 1% (w/v) nonfat dry milk as blocking agent.
Detection was performed using the ECL western blotting detection
system (Amersham Pharmacia). Anti-FLAG M2 (F-3165, Sigma
Aldrich) and anti-a-tubulin Dm1A (T-9026, Sigma Aldrich) mouse
monoclonal antibodies were used at a 1:20,000 dilution. Goat anti-
mouse horseradish peroxidase–conjugated antibody (170-5047, Bio-
Rad) was used at 1:15,000 dilution. Note that our HIRA antisera did
not work on western blots using the extraction and detection
procedures that worked very well with the HIRA-FLAG recombinant
protein detected with the anti-FLAG antibody.
We thank R. Renkawitz-Pohl, S. Jayaramaiah Raja,V. Horner, M.
Wolfner, Y. Rong, K. Golic, F. Karch, the Bloomington Drosophila
Stock Center, and the Drosophila Genome Resource Center for flies,
antibodies, and plasmids. We also thank E. Cortier, J. Schmitt, and
the CTl microscopy center for technical assistance. We are grateful
to F. Berger and B. Horard for helpful comments on the
manuscript. This paper is dedicated to the memory of our colleague
Author contributions. All authors conceived and designed the
experiments and analysed the data. EB, GAO, and BL performed the
experiments. BL wrote the paper.
Funding. This work was supported by the Centre National de la
Recherche Scientifique (CNRS), the A. N. R. Young Researcher
Fellowship, and the French Ministry of Research.
Competing interests. The authors have declared that no competing
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