Download Protection of Drosophila chromosome ends through minimal

© 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 1969-1981 doi:10.1242/jcs.167825
RESEARCH ARTICLE
Protection of Drosophila chromosome ends through minimal
telomere capping
ABSTRACT
In Drosophila, telomere-capping proteins have the remarkable capacity
to recognize chromosome ends in a sequence-independent manner.
This epigenetic protection is essential to prevent catastrophic ligations
of chromosome extremities. Interestingly, capping proteins occupy a
large telomere chromatin domain of several kilobases; however, the
functional relevance of this to end protection is unknown. Here, we
investigate the role of the large capping domain by manipulating HOAP
(encoded by caravaggio) capping-protein expression in the male
germ cells, where telomere protection can be challenged without
compromising viability. We show that the exhaustion of HOAP results
in a dramatic reduction of other capping proteins at telomeres,
including K81 [encoded by ms(3)K81], which is essential for male
fertility. Strikingly however, we demonstrate that, although capping
complexes are barely detected in HOAP-depleted male germ cells,
telomere protection and male fertility are not dramatically affected. Our
study thus demonstrates that efficient protection of Drosophila
telomeres can be achieved with surprisingly low amounts of capping
complexes. We propose that these complexes prevent fusions by
acting at the very extremity of chromosomes, reminiscent of the
protection conferred by extremely short telomeric arrays in yeast or
mammalian systems.
KEY WORDS: Drosophila melanogaster, Telomere, Capping protein,
Male germline, Spermatogenesis
INTRODUCTION
Telomeres are specialized structures that organize the extremities of
linear chromosomes in a way that prevents their recognition as DNA
double-strand breaks (DSBs) by the DNA repair machinery.
Another important function of telomeres is to counteract the
gradual erosion of chromosomes that naturally occurs over cell
divisions and results from the incomplete replication of DNA ends
(reviewed in Fulcher et al., 2014). In the vast majority of eukaryotes,
including human and yeasts, telomeres comprise arrays of variable
lengths (from several hundred base pairs in yeast to 5–60 kb in
mammals) of small G-rich DNA repeats that are added by the
enzymatic complex telomerase (reviewed in Jain and Cooper,
2010). These DNA repeats are specifically recognized and bound
by capping-protein complexes that inhibit the processing of
chromosome extremities through the non-homologous end-joining
(NHEJ) repair pathway (reviewed in Oganesian and Karlseder,
2009). In mammals, for instance, TRF1 and TRF2 (also known as
TERF1 and TERF2, respectively) recognize the TTAGGG repeats
and interact with other capping proteins of the shelterin complex
Centre de Gé né tique et de Physiologie Molé culaire et Cellulaire, CNRS UMR 5534,
Université Claude Bernard Lyon 1, Université de Lyon, 69100 Villeurbanne, France.
*Author for correspondence ([email protected])
Received 17 December 2014; Accepted 18 March 2015
(reviewed in Palm and de Lange, 2008; Stewart et al., 2012; Lu
et al., 2013).
Dipterans represent an exception in Metazoa as they lack
telomerase (Mason et al., 2008; Villasante et al., 2008). Telomere
elongation thus uses different mechanisms, such as homologous
recombination or transposition of telomere-specific retrotransposons.
In Drosophila melanogaster, terminal sequences contain an array –
called the HTT array – of complete and incomplete copies of
three non-long-terminal-repeat (non-LTR) retrotransposons (Het-A,
TAHRE and TART) (reviewed in Pardue and DeBaryshe, 2011) that
has been estimated to vary in length from approximately 20 to 150 kb
(Capkova Frydrychova et al., 2008). Drosophila chromosome ends
are also bound by a protective protein complex that constitutes a
functional analog of the mammalian shelterin complex (Raffa et al.,
2011; Raffa et al., 2013). This capping complex includes the fast
evolving, non-conserved proteins HP1/ORC-associated protein
(HOAP; encoded by caravaggio), HipHop, Verrocchio (Ver) and
Modigliani (Moi), which are called terminin proteins, and the
conserved Heterochromatin Protein 1a [HP1a; encoded by
Su(var)205] (Raffa et al., 2011; Raffa et al., 2013). HP1a localizes
not only at telomeres but also throughout heterochromatin and at
many euchromatic loci, whereas terminin proteins are specifically
enriched at telomeres throughout the cell cycle and are involved in
chromosome-end protection. Capping proteins are essential for
viability, and loss-of-function mutants usually die during larval
stages, displaying chromosome end-to-end fusions at high frequency
in rapidly dividing cells (Fanti et al., 1998; Cenci et al., 2003; Raffa
et al., 2009; Raffa et al., 2010; Gao et al., 2010). In addition – and
despite the fact that HOAP, HipHop, Ver and Moi are not conserved
outside Drosophilidae and closely related Diptera – these proteins
share some properties with their mammalian or yeast counterparts
(Cenci et al., 2005). For instance, Ver and Moi localize exclusively
at the tip of the chromosome end, where Ver would bind to the
single-stranded DNA 3′-overhang through its oligonucleotide/
oligosaccharide-binding (OB)-fold domain, in a manner similar to
that of POT1 in mammals (Raffa et al., 2010; Raffa et al., 2013). By
contrast, HOAP, HipHop and HP1a form a complex that localizes not
only in the direct vicinity of the chromosome extremities, where
HOAP interacts directly with Moi (Raffa et al., 2009), but also
occupies a relatively large terminal region of at least 10 kb (Gao et al.,
2010). Importantly, unlike their mammalian counterparts, Drosophila
capping proteins all share the remarkable property of recognizing
chromosome ends independently of the underlying DNA sequence
(Perrini et al., 2004; Cenci et al., 2005; Pimpinelli, 2006; Rong, 2008;
Raffa et al., 2013). Indeed, chromosomes with a terminal deletion that
completely removes the HTT array can be generated and maintained
through generations (Levis, 1989; Ahmad and Golic, 1998; Gao et al.,
2010). Remarkably, such chromosomes accumulate an apparently
normal amount of capping proteins at their extremities (Gao et al.,
2010). Similarly, no change in the level of capping is observed on
chromosomes that have naturally longer HTT arrays (Gao et al.,
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Journal of Cell Science
Raphaë lle Dubruille and Benjamin Loppin*
Journal of Cell Science (2015) 128, 1969-1981 doi:10.1242/jcs.167825
Fig. 1. K81 and HipHop show a complementary localization at telomeres in the male germline. (A) Schematic representation of Drosophila
spermatogenesis. Spermatogenesis is organized in differentiating units called cysts that progress along the axis of the testis (Fuller, 1993). At the apical end of
the testis, a group of somatic cells called the hub forms the niche of germline stem cells (GSCs) and somatic cyst stem cells (not represented on the scheme). The
asymmetric division of a GSC produces another GSC and a spermatogonial cell. Spermatogonia undertake four rounds of mitosis without complete cytokinesis
leading to a cyst of 16 interconnected spermatocytes. After a growth step, spermatocytes undergo meiotic divisions in synchrony giving rise to 64 haploid
spermatids. The maturation of spermatids is notably characterized by the elimination of most histones and their replacement by sperm-specific proteins, such as
Mst77F and the protamine-like proteins Mst35Ba/b (not shown on the scheme). At the end of spermatogenesis, sperm cells individualize and are transferred to the
seminal vesicle. (B–K) Confocal images of whole-mount testes from 5′hiphop-GFP::hiphop hiphop1/5′hiphop-GFP::hiphop hiphopEY7584 males (B,B′,D–G) and
5′K81-GFP::K81/5′K81-GFP::K81; K812/K812 males (C,C′,H–K) to locate HipHop and K81, respectively. (B–C′) View of the apical tip of testes stained with
an antibody against Vasa (blue) to identify germ cells, an antibody against GFP (green) and for DNA (red). GFP–HipHop foci are visible in the nuclei of GSCs
and early spermatogonia (B,B′, magnification of the ‘boxed’ islet is shown in D), whereas GFP–K81 is barely detected in these cells (C,C′, magnification of the
‘boxed’ islet is shown in H). In spermatocyte nuclei, only a few GFP–HipHop foci (from 0 to 2) are still visible (E, arrows), whereas GFP–K81 decorates all
telomeres (I, arrows). During meiosis (F, two anaphase-II nuclei) and in spermatid nuclei (G), GFP–HipHop is not detected. By contrast, bright GFP–K81 foci are
visible throughout meiosis (J, two metaphase-I nuclei) and in spermatid nuclei (K). The dashed white lines in E and I delineate the nucleus. Scale bars: 5 µm.
2010). Hence, although the size of the capping domain in organisms
that express telomerase is directly linked to the length of terminal
DNA repeats, the establishment of a functional cap in the absence of
telomerase remains poorly understood. Several studies have
nevertheless established that some effectors of the DNA damage
response (DDR), including the conserved Mre11–Rad50–Nbs
complex and the ATM and ATR checkpoint kinases, are required
for the prevention of telomere fusions and for the initial recruitment of
HOAP onto chromosome ends (Ciapponi et al., 2004; Bi et al., 2005;
Oikemus et al., 2006; Ciapponi et al., 2006). It has been proposed that
1970
the spreading of capping complexes toward the centromere follows
this initial recruitment of a protective cap at the extremity (Pimpinelli,
2006; Gao et al., 2010), although the mechanism that regulates the
size of the capping domain is currently unknown. Moreover, the
relative contribution of this large domain to telomere protection is
unclear.
Among the functionally characterized capping proteins, HOAP
and HipHop play a pivotal role in establishing a functional telomere.
They are interdependent for their stability (Gao et al., 2010), and
HOAP is required for the localization of Ver, Moi and HP1a at
Journal of Cell Science
RESEARCH ARTICLE
RESEARCH ARTICLE
telomeres. However, the fact that these proteins are essential for
viability is a serious limitation when analyzing their function during
cell proliferation and differentiation. Spermatogenesis represents a
powerful system for functional analysis of capping genes in vivo.
Their invalidation in the male germline does not compromise
viability, and post-meiotic spermatids are an abundant source
of synchronized, differentiated nuclei that are particularly tractable
for imaging of telomeres. Furthermore, the existence of a HipHop
paralog that is specific to the male germline, K81 [encoded by
ms(3)K81], provides a unique opportunity to manipulate cappingcomplex assembly throughout spermatogenesis (Dubruille et al.,
2010; Gao et al., 2011; Dubruille and Loppin, 2011).
By specifically reducing the expression of HOAP in the male
germline, we show that this protein plays a central role in
determining the size of the capping domain. Surprisingly, we
demonstrate that the amount of capping proteins at telomeres can be
dramatically decreased without severely disrupting chromosomeend protection and male fertility.
RESULTS
HipHop and K81 successively contribute to telomere
protection in male germ cells
We have recently discovered that, in the male germline, HipHop is
replaced by K81 in spermatid nuclei (Dubruille et al., 2010). At
fertilization, K81 is transmitted to the zygote, where it is required to
protect paternal telomeres during the first division. Interestingly,
although K81 is absolutely necessary for telomere protection at
fertilization, it does not seem to be required during spermatogenesis
(Dubruille et al., 2010; Gao et al., 2011). Indeed, no chromosomal
defects have been observed in the male germline of K81-mutant
males (Fuyama, 1984; Yasuda et al., 1995), suggesting that other
types of capping complexes, probably involving HipHop, are
required in these cells.
To gain insight into the respective contributions of HipHop and K81
in the protection of telomeres during spermatogenesis, we first
analyzed their expression dynamics in the male germline. We used
previously characterized transgenic flies expressing green fluorescent
protein (GFP)-fusion proteins under the regulation of their
own promoter (Dubruille et al., 2010). The 5′hiphop-GFP::hiphop
transgene was introduced into an otherwise lethal trans-heterozygous
hiphop-mutant background to avoid competition with the endogenous
Journal of Cell Science (2015) 128, 1969-1981 doi:10.1242/jcs.167825
protein. In these rescued animals, GFP–HipHop protein was detected
as discrete foci in the nuclei of germline stem cells (GSCs) and
their progeny, spermatogonial cells (see Fig. 1A for a schematic
description of Drosophila spermatogenesis; Fig. 1B,B′,D). Then,
the intensity of GFP–HipHop foci decreased progressively in dividing
spermatogonia. In primary spermatocytes, a weak staining often
persisted as two foci (Fig. 1E). Then, GFP–HipHop became
undetectable throughout meiosis and in spermatid nuclei (Fig. 1F,G).
For GFP–K81, very weak signals were occasionally detected in
GSCs and the more apical spermatogonia (Fig. 1C,C′,H). Then, the
signals increased in late spermatogonia and in primary spermatocytes,
forming several GFP foci in each nucleus (Fig. 1I). Finally, GFP–K81
brightly decorated telomeres during meiotic divisions and in
spermatid nuclei, as previously reported (Fig. 1J,K) (Dubruille et al.,
2010; Dubruille and Loppin, 2011). Thus, these paralogs displayed
complementary expression patterns, suggesting that, in contrast to the
post-meiotic function of K81, HipHop is required for telomere
protection in early male germ cells. To address this question, we chose
to knock down HipHop in the male germline by using RNA
interference through the UAS-GAL4 system. We generated transgenic
flies expressing a small hairpin (sh)RNA targeting hiphop that were
then crossed to flies bearing a nos-Gal4 transgene, which drives
expression of GAL4 in GSCs and early spermatogonia (WhiteCooper, 2012). The hiphop-knockdown males were totally sterile
(Table 1). They had severely atrophied testes that were almost
systematically devoid of germ cells (97.6%, n=84; Fig. 2A–D). This
phenotype, which is characteristic of testes in which GSCs have been
genetically ablated (Castrillon et al., 1993; Gönczy and DiNardo,
1996; Kauffman et al., 2003), was also obtained when an shRNA
targeting the GFP coding sequence was expressed in the germline of
GFP::hiphop-rescued males (Table 1 and data not shown). Our results
thus demonstrate that HipHop is necessary for the survival of GSCs.
Interestingly, a single copy of a 5′hiphop-GFP::K81 transgene
[which allows expression of GFP–K81 under the control of the
hiphop promoter (Dubruille et al., 2010) and is not targeted by
hiphop shRNAs] partially rescued the fertility of the hiphopknockdown flies (Table 1). Accordingly, we observed that 33% of
the 5′hiphop-GFP::K81, nos-Gal4/UAS-sh-hiphop flies had testes
that appeared normal in shape and size (n=60) and contained germ
cells (Fig. 2E). These results demonstrate that K81 can partially
replace HipHop during the division of early male germ cells.
Table 1. Fertility tests
Number of eggs
Number of larvae
Hatching rate
w/Y
w/Y; nos-Gal4/+
w/Y; nos-Gal4/UAS-sh-hiphop
w/Y; 5′hiphop-GFP::K81 nos-Gal4/UAS-sh-hiphop
w/Y; 5′hiphop-GFP::hiphop, UAS-sh-GFP, hiphop1/Df(3L)BSC776, nos-Gal4
w/Y; 5′K81-GFP::K81/+; K812/K812
5′hiphop-GFP::cav 2A w/Y; cav1/+
w/Y; 5′hiphop-GFP::cav 62E cav1/Df(3R)Exel6198
5′hiphop-GFP::cav 2A w/Y ; cav1/Df(3R)Exel6198
5′hiphop-GFP::cav 2A w/Y; 5′K81-GFP::K81/+; cav1 K812/K812
5′hiphop-GFP::cav 2A w/Y; 5′K81-GFP::K81/+; cav1 K812/Df(3R)Exel6198 K812
5′hiphop-GFP::cav 2A w/Y; cav1 K812/Df(3R)Exel6198
w/Y; hiphop1 Df(3R)Exel6198/+
5′hiphop-GFP::cav 2A w/Y; cav1/hiphop1 Df(3R)Exel6198
5′hiphop-GFP::cav 2A w/Y; cav1 K812/hiphop1 Df(3R)Exel6198
w/Y; ver2 Df(3R)Exel6198/+
5′hiphop-GFP::cav 2A w/Y; cav1/ver2 Df(3R)Exel6198
w/Y; moi1 Df(3R)Exel6198/+
5′hiphop-GFP::cav 2A w/Y; cav1/moi1 Df(3R)Exel6198
217
511
877
1629
891
518
106
284
763
356
335
777
553
374
603
1122
238
222
236
193
437
1
1062
0
477
93
219
591
280
31
315
475
228
330
1058
140
205
208
88.9%
86.5%
0%
65.2%
0%
92.1%
87.7%
77.1%
77.5%
78.7%
9.3%
40.6%
85.9%
61.0%
54.7%
94%
58.8%
92.3%
88.1%
Journal of Cell Science
Genotype of males
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RESEARCH ARTICLE
Journal of Cell Science (2015) 128, 1969-1981 doi:10.1242/jcs.167825
Low levels of capping proteins are sufficient to protect
telomeres during spermatogenesis
The observed functional redundancy between HipHop and K81
suggests that K81 ensures telomere capping in spermatocytes when
HipHop is no longer detected. However, as mentioned above,
K81-null mutants do not show any defects during spermatogenesis
(Fuyama, 1984; Yasuda et al., 1995), opening the possibility that a
low, residual amount of HipHop can protect telomeres during
meiotic divisions. To more generally test the adaptability of
telomeres to a limiting amount of capping proteins, we turned to
HOAP, an interacting protein of HipHop encoded by the caravaggio
(cav) gene. In contrast to hiphop, cav has a much stronger expression
in testis (Flybase) and does not have a paralog that is specific to the
male germline in D. melanogaster (Dubruille et al., 2012). Using an
antibody against HOAP, we indeed observed bright HOAP foci in all
germ cell nuclei during pre-meiotic and meiotic stages (Fig. 3C,D).
We also observed HOAP foci in spermatid nuclei (Fig. 3E), as
1972
previously described (Dubruille et al., 2010), suggesting that this
capping protein is required to protect telomeres at all stages of
spermatogenesis. Because cav is essential for viability, we designed
a partial rescue strategy to stably and specifically reduce its
expression in the male germline. We generated flies expressing a
GFP–HOAP fusion protein under the control of the hiphop promoter
(5′hiphop-GFP::cav; Fig. 3A). We reasoned that this transgene,
introduced into a cav-null-mutant background, would restore HOAP
expression in somatic cells but limit cav expression to GSCs and
proliferative spermatogonia in testes.
As expected, the 5′hiphop-GFP::cav transgene fully rescued
the lethality of cav mutants. We obtained 39.6% of the rescued
5′hiphop-GFP::cav; cav1/Df(3R)Exel6198 genotype (designated as
Rescav for simplicity; see Materials and Methods and Fig. 3A) over
the 33% expected Mendelian ratio (n=416). GFP–HOAP was
normally observed at somatic telomeres of 5′hiphop-GFP::cav
animals, such as in salivary gland polytene chromosomes (Fig. 3F).
Journal of Cell Science
Fig. 2. hiphop knockdown in the male germline dramatically impairs early spermatogenesis. (A,B) Confocal images showing a pair of testes from a control
male (A) and a hiphop knockdown male (B, nos-Gal4/UAS-sh-hiphop), stained with a pan-histone antibody. The scale is the same for both images.
Note the drastic reduction in size of the hiphop knockdown testes. AT, apical tip; AD, anterior ejaculatory duct; SV, seminal vesicle. Almost all of the hiphop
knockdown testes were vestigial as shown in B (97.6%, n=84). We observed a few testes with a normal shape but with a reduced size (2.4%, n=84).
(C–E) Confocal sections of whole-mount testes stained for the hub [Fas III (green)], stem cells [Vasa (blue)] and DNA (red). Asterisks indicate the apical tip
of the testes. The scale is the same for all images. Approximately 5% of the testis is visible on the panels C and E, whereas in D, more than half of the testis is
visible. In the testes from hiphop knockdown males (D), the staining of Vasa appears to be severely reduced compared with control testis (C), showing that germ
cells are depleted. As previously reported, hub cells expand in agametic testes, as revealed by using the Fas III marker (Gö nczy and DiNardo, 1996). This
phenotype is partially rescued with a 5′hiphop-GFP::K81 transgene, as about 33% of the males (5′hiphop-GFP::K81 nos-Gal4/UAS-sh-hiphop) have testes of
wild-type size, in which the hub and germ cells are clearly detected (E). Scale bars: 250 µm (A,B); 10 µm (C–E).
RESEARCH ARTICLE
Journal of Cell Science (2015) 128, 1969-1981 doi:10.1242/jcs.167825
In addition, adult Rescav males had testes of normal size, indicating
that telomere protection was not compromised, at least in early male
germ cells. However, semi-quantitative reverse-transcription (RT)PCR analysis confirmed the severe reduction of cav transcripts in
Rescav testes compared with that of the wild-type control (Fig. 3B).
Accordingly, in testes of adult Rescav males, GFP–HOAP could not
be detected above background levels in spermatocytes or spermatid
nuclei using antibodies against either GFP or HOAP (Fig. 3G–J).
Surprisingly, however, the fertility of Rescav males was only
slightly affected by the observed reduction of HOAP levels at
telomeres in the male germline (Table 1). In somatic cells, HOAP is
crucial for the recruitment or the stabilization of other capping
proteins at telomeres (Raffa et al., 2009; Gao et al., 2010; Raffa
et al., 2010). We thus wondered whether the accumulation of the
capping proteins HP1a and K81, which are present in spermatocytes
and early spermatids (Dubruille et al., 2010), are affected in germ
cells of Rescav males. We thus systematically analyzed the level of
these capping proteins in Rescav spermatid nuclei by quantifying
immunofluorescence signals. To exclude any impact of nuclear
condensation on antibody accessibility, we only considered
spermatid nuclei at the canoe stage and distinguished in the
analyses those that were negative or positive for Mst77F, a spermspecific nuclear protein that is incorporated at the histone-toprotamine transition (Rathke et al., 2010). Using an antibody
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Journal of Cell Science
Fig. 3. cav is downregulated in male germ
cells of Rescav flies. (A) A scheme
representing the genotype of Rescav males
[5′hiphop-GFP::cav/Y; cav1/Df(3R)
Exel6198]. The organization of the
5′hiphop-GFP:cav transgene is represented
with filled and empty boxes corresponding
to, respectively, coding exons and UTRs;
and with lines for introns and intergenic
regions. The position relative to the ATG of
the corresponding gene is indicated. This
transgene was inserted into the 2A platform
on the X chromosome (chr) and combined
with a cav-null allele (cav1) and a deficiency
that uncovers cav [Df(3R)Exel6198]. bp,
base pair. (B) RT-PCR analyses were
performed on total RNAs from the testes of
control (w1118) or Rescav flies. Rp49 was
used as a control gene. +, with reverse
transcriptase; −, without reverse
transcriptase. The expression level of cav
mRNA is reduced in Rescav testes. The
downregulation of cav does not affect the
expression of the capping genes K81
and Su(var)205 (encoding HP1a).
(C–E) Confocal images of squashed testes
from w1118 males stained with an antibody
against HOAP (green) and for DNA (red).
(C) A nucleus of a primary spermatocyte
(the white dashed line delineates the
nucleus), (D) metaphase of meiosis II, (E) a
cyst of spermatid nuclei. (F) A confocal
image of squashed salivary glands of a 5′
hiphop-GFP::cav third instar larvae stained
with an antibody against GFP (green) and
for DNA (red). The fusion protein GFP–
HOAP localizes at telomeres of polytene
chromosomes. (G–J) Confocal images of
squashed testes from Rescav males
stained for HOAP or GFP (green) and for
DNA (red). (G) A nucleus of a mature
primary spermatocyte (white dashed line),
(H) prophase of meiosis, (I) anaphase of
male meiosis II and (J) nuclei of a cyst of 64
spermatids. In Rescav males, HOAP is
undetectable at telomeres (arrows in I)
during meiosis and in spermatid nuclei.
Scale bars: 10 µm.
specific to HOAP, we first confirmed the severe reduction of HOAP
at spermatid telomeres in Rescav males. HOAP foci were readily
detected in control spermatid nuclei before and after the histone-toprotamine transition, whereas HOAP was barely detectable in
Rescav spermatids at both stages (Fig. 4). We then analyzed HP1a
and K81 accumulation using, respectively, an antibody against
HP1a and the 5′K81-GFP::K81 transgene that was introduced in the
Rescav background. Strikingly, both the size and intensity of HP1a
and GFP–K81 foci were also drastically reduced in spermatids of
Rescav males (Fig. 4).
Thus, the reduction of HOAP levels in Rescav males has a general
effect on the accumulation of the capping proteins HP1a and K81 at
spermatid telomeres. However, we know that at least the capping
function of K81 is absolutely required for male fertility, by
preventing fusion of paternal telomeres at fertilization (Loppin et al.,
2005; Dubruille et al., 2010; Gao et al., 2011). Our analysis thus
demonstrates that the very limited quantity of K81 present at
telomeres in Rescav post-meiotic male germ cells is sufficient to
ensure efficient protection of paternal chromosomes during zygote
formation.
Sensitized telomere protection in Rescav males unveils a
protective role of K81 in meiosis
Although the reduction of capping-protein recruitment at telomeres
in Rescav testes had no significant impact on spermatogenesis and
fertility, we nevertheless occasionally noticed the presence of
spermatid nuclei connected by a chromatin thread. This indicated
that telomeres occasionally fuse before or during meiotic divisions,
thus suggesting that telomere capping is sensitized in the Rescav
male germline. To test this hypothesis, we challenged telomere
capping by knocking down other capping genes in the Rescav
background. Strikingly, in Rescav males that were homozygous for
a null allele of K81 (K812), spermatogenesis appeared severely
disorganized, with the majority of spermatid nuclei failing to
differentiate normally (Fig. 5B). They were notably connected
through gigantic chromatin threads of several tens of micrometres
(Fig. 5G–J). Moreover, in telophase of meiosis, we could frequently
observe chromatin bridges (Fig. 5E), which are likely to result from
the fusion of uncapped telomeres. The chromatin threads
connecting spermatid nuclei probably result from the stretch of
unbroken chromatin bridges that form during male meiosis II, as
similarly reported by Titen and colleagues (Titen et al., 2014).
We also observed chromatin bridges, albeit at a lower frequency,
when Rescav males had a single copy of K81. Quantification of this
phenotype confirmed that Rescav; K812/+ testes contained
significantly more abnormal nuclei than Rescav testes (Fig. 5L).
Accordingly, Rescav; K812/+ males had reduced fertility compared
to control flies (Rescav; Table 1). Taken together, these results
demonstrate that, in the Rescav background, K81 is required for
telomere protection during male meiosis. As we hypothesized
above, the fact that no defects are observed in meiosis in K81mutant males suggests that HipHop efficiently protects telomeres
until completion of male meiosis. Interestingly, when we combined
Rescav with the hiphop1 null allele, we also observed a significant
increase of abnormal spermatid nuclei compared with that Rescav
testes (Fig. 5L). This confirms that HipHop is also involved in
telomere protection during male meiosis in the Rescav background.
These genetic interactions between Rescav and K81 or hiphop
show that a further decrease of capping-protein expression in the
Rescav background destabilizes capping complexes and impairs
chromosome protection during meiosis. We thus tested, in this
context, a partial loss of other capping genes, such as ver and
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Journal of Cell Science (2015) 128, 1969-1981 doi:10.1242/jcs.167825
moi, that are essential to prevent telomere fusions in somatic cells
and for viability (Raffa et al., 2009; Raffa et al., 2010). We
introduced the ver2 mutant allele into the Rescav background and
examined whole-mount testes of these flies. Interestingly, we again
observed the presence of many spermatids with giant chromatin
threads. The quantification of abnormal spermatid nuclei in these
flies revealed a phenotype similar to that of Rescav, K812/+ and
Rescav, hiphop1/+ males (Fig. 5L). This implies that Ver, like
HipHop, is involved in telomere protection in the male germline, at
least until completion of meiosis. By contrast, however, combining
the moi1 null allele with Rescav did not aggravate the Rescav
phenotype (Fig. 5L). This result suggests that either Moi is not
required for telomere protection in the male germline or that Moi is
not limiting in this context. The fact that, in larval salivary glands,
Ver and Moi are interdependent for their localization at telomeres
supports the latter hypothesis (Raffa et al., 2010).
Taken together, our results show that during male meiosis,
telomere protection relies on capping complexes that contain
HipHop or K81, and at least the proteins HOAP and Ver.
Forcing hiphop expression in spermatids increases the
recruitment of capping complexes at telomeres
Our analysis of Rescav males showed that reducing HOAP expression
level dramatically decreases the level of capping proteins present at
telomeres. On the opposing side, we wondered whether it would be
possible to increase the amount of capping proteins recruited at
telomeres. Interestingly, over the course of our experiments, we
noticed in a particular genotype that HOAP foci in young spermatid
nuclei appeared brighter and larger than those in control flies
(Fig. 6A). These flies bear two copies of a 5′K81-GFP::hiphop
transgene, which drives ectopic expression of a GFP–HipHop fusion
protein in spermatids (Dubruille et al., 2010). To confirm our
observation, we quantified HOAP signals in spermatid nuclei. We and
others have previously observed that telomeres cluster in spermatid
nuclei and in various somatic cell types (Dubruille et al., 2010;
Wesolowska et al., 2013). We thus first evaluated the clustering in
spermatid nuclei of 5′K81-GFP::hiphop flies by counting the
number of foci in individual nuclei. We did not observe any
statistically significant changes of the clustering between 5′K81GFP::hiphop flies and control flies (Fig. 6B). To nevertheless exclude
any clustering effect, we quantified the total area and the mean
intensity of all HOAP foci detected in individual nuclei (Fig. 6C, top
panels). This analysis confirmed that both the area and intensity of
HOAP foci were higher in spermatids of 5′K81-GFP::hiphop males
compared with those of control males. We then asked whether the
increase of HOAP levels affected the recruitment of other capping
proteins. Therefore, we quantified HP1a and GFP–HipHop signals,
which appeared, indeed, to be significantly higher than in control flies
(GFP–HipHop signals were compared to GFP–K81 expressed with
the same promoter; Fig. 6C, middle and bottom panels). These results
thus demonstrate that in flies expressing high levels of GFP–HipHop
in post-meiotic stages, capping complexes are more abundant at
telomeres.
DISCUSSION
A capping complex switch during spermatogenesis
We have previously shown that capping protein K81, which is
specific to the male germline, localizes at telomeres in spermatids
and is essential for the protection of sperm telomeres at fertilization
(Dubruille et al., 2010). However, despite the essential role of K81
for male fertility through a paternal effect, spermatogenesis itself,
including meiotic divisions, occurs normally in K81-null mutant
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Fig. 4. The reduction of HOAP in spermatid nuclei affects the localization of K81 and HP1a at telomeres. (A) Representative confocal images of squashed
testes stained for HOAP, HP1a or GFP (green), Mst77F (blue) and DNA (red). For HOAP and HP1a staining, flies were w1118 (Control) or 5′hiphop-GFP::
cav/Y; cav1/Df(3R)Exel6198 (Rescav). For GFP–K81, control flies were 5′hiphop-GFP::cav/Y; 5′K81-GFP::K81/+; K812 cav1/K812 and Rescav flies were
5′hiphop-GFP::cav/Y; 5′K81-GFP::K81/+; K812 cav1/K812 Df(3R)Exel6198. For each staining, spermatid nuclei before and after the histone-to-protamine
transition are visualized through, respectively, the presence or the absence of the Mst77F staining (blue). (B) Quantification of the mean intensity of fluorescence
(graphs on the left) and area (graphs on the right) of particles detected in A are represented as box plots in which whiskers show minimum and maximum values,
boxes show the middle 50% of the values, and horizontal lines show the median. The dashed red lines show the threshold under which fluorescence signals were
considered as background. Each graph corresponds to the quantification of a single experiment that was repeated two or three times. The number of nuclei
(Nb. nuclei) and detected particles (Nb. particles) analyzed are shown under the graph. In all experiments, the mean intensity of fluorescence and the area of
HOAP, HP1a and GFP–K81 particles were significantly reduced in the Rescav background compared to in control flies. Mann–Whitney test; ****P<0.0001.
a.u., arbitrary unit. Scale bar: 10 µm.
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Fig. 5. Loss of K81 in the Rescav background results in chromatin bridges at high frequency. (A,B) Representative images of a testis from a control
fly bearing a protB::GFP transgene in a wild-type (A) or in a Rescav; K812/K812 (B) background stained with a pan-histone antibody (red) and DNA
(blue). In Rescav; K812/K812, the cysts of spermatids are disorganized (B). (C,D) Confocal images of nuclei in testes of wild-type flies stained with an
antibody against histone. (C) Anaphase of male meiosis II. (D) A group of young spermatids. (E–K) Examples of chromatin bridges observed in the
Rescav; K812/K812 flies that have been stained with an antibody against histone (E–I) or expressing a protB::GFP transgene (J,K). (E) Telophase of
meiosis. Arrows indicate chromosome bridges between haploid nuclei. (F–H) Young spermatid nuclei presenting chromatin bridges (arrows).
Some nuclei present very long chromatin threads (>50 µm; arrows in G). (I) A larger view of a testis with disorganized spermatid nuclei that harbor long
chromatin threads. (J,K) Chromatin threads observed in spermatid nuclei that have incorporated protamines (arrows). Scale bars: 10 µm.
(L) Quantification of abnormal spermatid nuclei in the indicated genotypes are represented as a scatter plot in which each point shows the number of
nuclei with a chromatin thread counted in a single testis. Horizontal lines represent means. The loss of a single copy of K81 or hiphop in the Rescav
background results in chromatin bridges at high frequency. Mann–Whitney test. n.s., non significant; ***P<0.001; ****P<0.0001. Note that abnormal
spermatid nuclei in Rescav; K812/K812 testes were too numerous for reliable quantification (>300).
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Fig. 6. The expression of a GFP–HipHop fusion protein in spermatids increases the recruitment of capping proteins at spermatid telomeres.
(A) Representative confocal images of spermatid nuclei in squashed testes stained with an antibody against HOAP (green) or Mst77F (blue) and for DNA (red).
Flies were 5′K81-GFP::K81/5′K81-GFP::K81; K812/K812 (top panel) and 5′K81-GFP::hiphop K812/5′K81-GFP::hiphop K812 (bottom panel). We show here
Mst77F-negative nuclei because GFP–HipHop is lost after the histone-to-protamine transition (Dubruille et al., 2010). The settings for the acquisition of both
images were the same. In Mst77F-negative spermatid nuclei, telomere foci appear brighter and larger in 5′K81-GFP::hiphop, K812 flies. Scale bar: 5 µm.
(B) A representative experiment to evaluate the clustering in spermatid nuclei. Telomere foci were revealed using an antibody against HP1a. The bar histogram
represents the percentage of nuclei observed for each category in control flies (w1118; n=52) or 5′K81-GFP::hiphop K812/5′K81-GFP::hiphop K812 flies
(GFP::hiphop; n=67). In all experiments, telomere clustering in spermatid nuclei was not significantly different (Kolmogorov–Smirnov test). (C) Quantification of
the total mean intensity of fluorescence (graphs on the left) or total area (graphs on the right) of telomere foci measured in each analyzed nucleus are represented
as box plots in which whiskers show the minimum and maximum values, boxes show the middle 50% of the values, and horizontal lines show the median.
The dashed red line shows the threshold under which fluorescence signals were considered as background. For HOAP and HP1a, flies were w1118 (control) or
5′K81-GFP::hiphop, K812/5′K81-GFP::hiphop, K812 (GFP::hiphop). 5′K81-GFP::K81/5′K81-GFP::K81; K812/K812 flies (GFP::K81) were used as control for
GFP. Each set of graphs (mean intensity and area) corresponds to the quantification of a single experiment that was repeated two or three times. For each
staining, the number of individual nuclei analyzed for quantification is written in the box. In all experiments, the mean intensity of fluorescence and total area
of telomere foci in 5′K81-GFP::hiphop flies were significantly higher than in control flies. Mann–Whitney test; ***P=0.0002; ****P<0.0001. a.u., arbitrary unit.
males (Fuyama, 1984; Yasuda et al., 1995). Here, we show that
telomere protection in male germ cells relies on two types of
capping complexes, each involving one of the HipHop and K81
paralogs. The onset of spermatogenesis requires HipHop, which is
expressed in GSCs and is crucial for their survival, demonstrating
that this ubiquitous capping protein is essential in order to protect
telomeres in these cells. The requirement of HipHop in GSCs is thus
similar to its role in somatic cells. In proliferative spermatogonia and
in spermatocytes, our data show that a gradual switch between
somatic-like and male-germline-specific complexes occurs, leading
to the complete replacement of HipHop by K81 in post-meiotic
cells. Although K81 is specialized in the capping of sperm
chromosomes, it has been previously reported that K81 is
efficiently targeted to somatic telomeres (Dubruille et al., 2010)
where it can achieve at least some level of protection (Gao et al.,
2011). Our observation that K81 expression in GSCs partially
restores testicular development and fertility of nos-Gal4/UAS-shhiphop flies further supports the notion that K81 is able to form
functional capping complexes outside of the sperm chromatin
environment.
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The fact that the HipHop-K81 switch occurs gradually in
dividing cells suggests that HipHop is progressively diluted as
K81 is loaded onto telomeres during each cell cycle. Although the
precise dynamics of capping-protein loading onto telomeres is
currently unknown, it is tempting to speculate that the recruitment of
capping-protein complexes is coupled with DNA replication as new
chromosome extremities are produced. Supporting this assumption,
we have previously shown that, in fertilized eggs, HipHop is not
detected in the decondensing male pronucleus but is subsequently
present at paternal telomeres when the apposed pronuclei initiate the
first zygotic replication (Dubruille et al., 2010). A coupling of DNA
replication with the recruitment of capping proteins would explain
why K81, the function of which is essential in post-meiotic nuclei,
is loaded during pre-meiotic stages.
Telomere protection with a minimal amount of capping
The most surprising result of our study is the finding that the amount
of capping proteins present at telomeres can be severely reduced
without dramatically impairing chromosome-end protection.
Indeed, K81, HOAP and HP1a are barely detected in spermatid
nuclei of Rescav males, which are nevertheless fertile. This implies
that the residual amount of K81 in Rescav spermatid nuclei is
sufficient to protect paternal chromosomes at fertilization.
Moreover, the relatively low number of spermatid nuclei that are
connected by a chromatin thread in Rescav testes demonstrates that
telomeres are efficiently protected during male meiosis, despite the
fact that HOAP is barely detected at this stage. Finally, we also
propose that, in absence of K81, residual levels of HipHop can
ensure telomere protection in meiosis. Using chromosomes with
terminal deletions that removed the HTT array allowing ChIP
analyses, Gao and colleagues (Gao et al., 2010) have previously
shown that HipHop–HOAP–HP1a capping complexes bind to a
large region (≥10 kb) of terminal chromatin. The current model
suggests that the recruitment of terminin complexes onto chromosome
extremities is mediated by some effectors of the DDR, such as the
MRN complex and the ATM and ATR–ATRIP kinases (Raffa et al.,
2011). Following this initial recruitment, other HipHop–HOAP–
HP1a capping complexes spread over a broader terminal region
(Gao et al., 2010). The fact that, in Rescav males, telomere protection
is globally not disrupted implies that the presence of a small amount
of terminin complexes is sufficient to efficiently inhibit their
recognition as DNA DSBs. Although we could not investigate the
chromatin occupancy of the HOAP, K81 and HP1a proteins in
Rescav males, we hypothesize that the few capping complexes
concentrate within the last base pairs of the DNA molecule.
Interestingly, several studies have reported that very short
telomeres in human cells and yeast are efficiently protected.
Telomeres with extremely short tracts called ‘t-stumps’ have been
characterized in transformed and cancer cells (Xu and Blackburn,
2007). Surprisingly however, these ‘t-stumps’ do not compromise
cell proliferation. In the yeast Saccharomyces cerevisiae, the
characterization of telomere fusion events revealed that a majority
of chromosome end-to-end fusions engage with short telomeric
tracts of less than 100 bp (Chan and Blackburn, 2003). Similarly, in
cultured human cells, telomeres become fusogenic when they
contain fewer than ca. 13 repeats, suggesting that 78 bp would be
sufficient to recruit shelterin complexes and efficiently protect
chromosome ends (Capper et al., 2007). Moreover, in vitro studies
have demonstrated that seven TTAGGG repeats are bound by at
least one molecule of the human shelterin proteins TRF1 or TRF2
(Xu and Blackburn, 2007) and that a minimum of 12 TTAGGG
repeats is required to inhibit the fusion of DNA ends in the presence
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Journal of Cell Science (2015) 128, 1969-1981 doi:10.1242/jcs.167825
of TRF2 and RAP1 (also known as TERF2IP in mammals) shelterin
proteins (Bae and Baumann, 2007). Thus, in species that rely on
telomerase, an extremely small number of DNA repeats is actually
sufficient to prevent end-to-end fusions. Remarkably, our study
supports that Drosophila telomeres, like their yeast and vertebrate
counterparts, can be protected from telomere fusions with a small
amount of capping proteins.
What could be the functions of a large telomeric capping
domain?
The fact that telomere protection can be achieved with low amounts
of terminin complexes raises questions about the role of the
relatively large chromatin domain that is occupied by capping
proteins at Drosophila telomeres. In a variety of organisms, neotelomeres can be formed through a mechanism called chromosome
healing, which comprises the stabilization of chromosome breaks
through the recruitment of capping proteins. In Drosophila,
chromosomes with new telomeres have been recovered in the
progeny of wild-type males in which chromosome breaks have been
induced in the germline or from irradiated females mutant for the
mutator-2 gene (Mason et al., 1984; Levis, 1989; Ahmad and Golic,
1998; Beaucher et al., 2012; Titen et al., 2014). De novo telomere
formation has also been described in S. cerevisiae and in
mammalian cells (Kramer and Haber, 1993; Sprung et al., 1999;
Diede and Gottschling, 1999; Lo et al., 2002; Gao et al., 2008;
Kulkarni et al., 2010). Interestingly, in all these systems, including
Drosophila, it has been observed that chromosome healing is
facilitated when the break occurs relatively near to the natural ends
of the chromosomes (Levis, 1989; Tower et al., 1993; Sprung et al.,
1999; Diede and Gottschling, 1999; Lo et al., 2002; Capkova
Frydrychova et al., 2008; Gao et al., 2008; Kulkarni et al., 2010).
Kulkarni and colleagues have demonstrated that de novo telomere
formation in human tumor cells is tenfold less frequent when the
break occurs 100 kb from the extremity compared with 3 kb
(Kulkarni et al., 2010). Importantly, they rule out the possibility that
this is due to the loss of genetic material essential for cell viability.
It has been alternatively proposed that de novo telomere formation is
facilitated by the availability of capping proteins (Mason et al.,
2008; Beaucher et al., 2012). Hence, a large capping domain might
constitute a reserve of capping proteins that could be rapidly
mobilized to stabilize a DNA break occurring in the vicinity of the
natural telomere. In addition, this ‘reserve’ of capping proteins
might also be involved in the stabilization of new chromosomal
extremities that are generated when chromosomes replicate or when
a telomeric retrotransposon is added to a chromosome end during
telomere elongation.
Capping proteins could also be implicated in the formation and
the stabilization of DNA structures that further protect telomeres.
Indeed, in vertebrates, although very short telomeres are still
protected from DNA repair machinery, they are too short to fold into
a t-loop (Bae and Baumann, 2007), which is thought to participate in
the protection of the chromosome extremity (Griffith et al., 1999;
de Lange, 2004; Giraud-Panis et al., 2013). T-loops are formed
through the folding and the sequestration of the 3′ single-strand
DNA overhang in the double-strand extremity, and their formation
is thought to hide the DNA extremity from the repair machinery
(Griffith et al., 1999; Doksani et al., 2013). It has been shown that
the human shelterin protein TRF2 can induce the formation of
t-loops both in vitro and in vivo (Griffith et al., 1999; Stansel et al.,
2001; Amiard et al., 2007; Doksani et al., 2013). The ability of
Drosophila telomeres to adopt a t-loop structure in vivo remains to
be established (Pimpinelli, 2006). However, among the terminin
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proteins, Verrocchio contains an OB-fold domain known to bind to
single-strand DNA, which suggests that Drosophila telomeres
terminate with a 3′ single-strand overhang (Raffa et al., 2010).
Moreover, the sequence of the telomeric retrotransposon Het-A
is prone to form a DNA structure called G-quadruplex, thought
to appear in t-loops (Abad and Villasante, 1999). It would thus
be interesting to determine whether HOAP or HipHop have
biochemical properties similar to those of TRF2.
The 5′hiphop-GFP::cav and UAS-sh-hiphop transgenic lines were
obtained using the phiC31-mediated integration system (Bischof et al.,
2007). The pValium22-sh-hiphop construct (see below) was inserted into
the P{y[+t7.7]=CaryP}attP2 platform (68A4) and the pW8-attB-5′hiphopGFP::cav construct (see below) was inserted into the M{vas-int.Dm}ZH2A (2A3) and the PBAc{y[+]-attP-3B}VK00031 (62E1) platforms. The
5′hiphop-GFP::cav 2A transgene was combined with the cav1 null allele
and Rescav males were obtained by crossing virgin 5′hiphop-GFP::cav/
5′hiphop-GFP::cav; cav1/TM6 females with w; Df(3R)Exel6198/TM6 males.
The telomeric domain occupied by capping complexes is
remarkably plastic
Fertility test
Although limiting the expression of HOAP severely reduced the
level of HP1a and K81 capping proteins recruited at telomeres,
forcing the expression of HipHop in post-meiotic stages increased
the abundance of HipHop, HOAP and HP1a at chromosome ends.
This observation strengthens the view that HOAP and HipHop and
K81 are interdependent for their localization at telomeres.
Furthermore, although we cannot exclude that the density of
capping complexes is higher within the capping domain of the
5′K81-GFP::hiphop spermatid telomeres, we favor the hypothesis
that capping proteins bind to a larger region of terminal chromatin.
Indeed, it has been proposed that in the absence of sequence
recognition, capping proteins are recruited through a spreading
mechanism, from the end towards the centromere (Gao et al., 2010).
In this model, the relative availability of key components of the
complex, such as HOAP and HipHop, can directly influence the
length of the capping domain. This is also supported by the fact that
HOAP is limiting for the assembly of capping complexes on
telomeres, as shown using Rescav males.
In mammals, very short tracts of telomeric repeats are sufficient
to achieve efficient chromosome-end protection through the
recruitment of the shelterin proteins TRF2 and RAP1 (Bae and
Baumann, 2007; Price, 2007). Because Drosophila telomeres are
epigenetically determined structures, the sizes of the HTT array and
the capping domain are naturally uncoupled (Gao et al., 2010; Raffa
et al., 2013). Therefore, a shorter HTT array has no impact on the
amount of capping proteins that are recruited onto chromosome
termini. Remarkably, our work shows that limiting the expression of
the capping protein HOAP leads to a dramatic decrease of the
capping complexes at telomeres without impairing their protection.
This study supports that, despite their divergence, Drosophila and
telomerase-dependent telomeres share the ability to efficiently
inhibit chromosome-end fusions when their capping domain is
severely reduced. Further studies should aim to understand how the
size of the fly capping domain is regulated.
MATERIALS AND METHODS
Male fertility tests were performed as described previously (Dubruille et al.,
2010).
Plasmid constructions
To obtain the pW8-attB-5′hiphop-GFP::cav construct, the hiphop coding
sequence in the previously described pW8-attB-5′hiphop-GFP::hiphop
construct (Dubruille et al., 2010) was removed and replaced by a fragment
encoding cav that was amplified, by using PCR, from genomic DNA using
the 5′-TAGCGGCCGCCATGTCGGGGACGCAAATGTC-3′ and 5′-GAGGATCCAGAACTAGACAGTGGC-3′ primers.
The pValium22-sh-hiphop construction was obtained by cloning the
two annealed primers 5′-CTAGCAGTTAAGTTGTGACTAGAGAACAATAGTTATATTCAAGCATATTGTTCTCTAGTCACAACTTAGCG-3′
and 5′-AATTCGCTAAGTTGTGACTAGAGAACAATATGCTTGAATATAACTATTGTTCTCTAGTCACAACTTAACTG-3′ into a pValium22
vector following instructions of the Transgenic RNAi Project at Harvard
Medical School (http://www.flyrnai.org/TRiP-HOME.html) (Ni et al.,
2011). The cloned DNA fragment encoded a small hairpin that targets a
21-nucleotide sequence in the 3′UTR of hiphop.
Reverse-transcription and semi-quantitative PCR
Total RNAs were extracted from 50 pairs of testes using the Trizol method
(Invitrogen, Thermo Fisher Scientific, Waltham, MA). They were then
treated with DNase1 (Ambion, Thermo Fisher Scientific) and reverse
transcribed with Superscript II (Invitrogen) and polyA primers. A control
reaction was set up without Superscript. cDNAs were PCR amplified
using the following primers – 5′-ATGTCGGATTCGCCCCATG-3′ and
5′-TAGTGGTTGATTCTTGCTCCTC-3′ for K81, 5′-AAGATCGTGAAGAAGCGCAC-3′ and 5′-ACTCGTTCTCTTGAGAACGC-3′ for Rp49,
5′-CAAGCGAAAGTCCGAAGAA-3′ and 5′-ACCATTTCTGCTTGGTCCAC-3′ for Su(var)205 and 5′-ATCTGATCTTAGCGGCATCG-3′ and
5′-AGTCCGGATCAGACTCCGTA-3′ for cav.
Immunostaining
Polytene chromosomes
Polytene chromosomes were prepared, as described previously (Dubruille
et al., 2010), and stained with a mouse monoclonal antibody against GFP,
clones 7.1 and 13.1 (Roche, Basel, Switzerland) at a 1:250 dilution and an
AlexaFluor-conjugated goat anti-mouse secondary antibody (Molecular
Probes, Thermo Fisher Scientific) at a 1:300 dilution.
Drosophila genetics
Squashed and whole-mount testes
Squashed and whole-mount testes were stained as described previously
(Dubruille et al., 2010) with the following antibodies – mouse IgG1 anti-GFP
(1:100; Roche), mouse anti-HP1a, clone C1A9 [1:25; Developmental Studies
Hybridoma Bank (DSHB), University of Iowa], mouse IgG2a anti-FasIII,
clone 7G10 (1:50; DSHB), guinea pig anti-HOAP (1:200) (Gao et al., 2010)
and rabbit anti-Mst77F (1:1000) (Rathke et al., 2010), rat anti-VASA (1:50;
DHSB), mouse monoclonal anti-pan-histone, clone F152 (1:1000; Millipore).
Secondary antibodies include AlexaFluor goat anti-mouse, anti-mouse IgG1,
anti-mouse IgG2a and anti-rat, anti-rabbit (Molecular Probes or Jackson
ImmunoResearch, West Grove, PA) and DyLight donkey anti-guinea pig
(Jackson ImmunoResearch). They were used at a 1:300 dilution for squashed
or 1:1000 for whole-mount testes. The immunostaining of Mst77F allowed us
to distinguish spermatid nuclei that undergo the histone-to-protamine
transition. All tissues were directly mounted in mounting medium (Dako,
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Journal of Cell Science
w1118 flies were used as a control. The K812, hiphop1 and hiphopEY7584
alleles and the 5′K81-GFP::K81, 5′hiphop-GFP::hiphop, 5′hiphop-GFP::
K81 and 5′K81-GFP::hiphop transgenic stocks have been described
previously (Dubruille et al., 2010).
The cav1 allele (Cenci et al., 2003) and moi1 and ver2 (l(3)S147910)
mutant stocks (Raffa et al., 2009; Raffa et al., 2010) were generous
gifts from, respectively, Stéphane Ronsseray (Université Pierre et Marie
Curie, Paris, France), Maurizio Gatti and Grazia Raffa (Università di
Roma, Roma, Italy). The ProtB-GFP transgenic strain was kindly provided
by Renate Renkawitz-Pohl (Philipps-Universität Marburg, Marburg,
Germany) (Jayaramaiah-Raja and Renkawitz-Pohl, 2005). Df(3L)BSC776
and Df(3R)Exel6198 deficiencies, UAS-sh-GFP strain (y[1] sc[*] v[1]; P{y
[+t7.7] v[+t1.8]=VALIUM20-EGFP}attP2), nos-GAL4 (w[1118]; P{w
[+mC]=GAL4::VP16-nos.UTR}CG6325[MVD1]) were obtained at the
Bloomington Drosophila Stock Center.
Glostrup, Denmark) containing 5 µg/ml propidium iodide (Sigma-Aldrich,
St Louis, MO) or 1 µM DRAQ 5 (BioStatus, Leicestershire, UK).
Imaging and quantification of fluorescent signals in spermatid
nuclei
Single-stack images were acquired on an LSM510 confocal microscope
(Zeiss, Oberkochen, Germany) in an 8-bit (for analyses in Fig. 4) or 12-bit
mode (for analyses in Fig. 6). For quantification, optimal settings (laser
power, amplifier offset, etc.) were set using the ‘palette’ command of the
LSM software to avoid saturation of fluorescence signals. Then, all images
were acquired using the same settings for samples that were compared to each
other. Quantification of results was performed using the ImageJ64 software.
First, areas corresponding to nuclei were selected in the red channel using the
‘threshold’ and the ‘analyze particle’ commands and the region of interest
(ROI) manager. Then, the particles corresponding to HOAP, HP1a or GFP
foci in the nucleus areas were selected in the green channel using a threshold
above the background level, and particles were analyzed using the ‘analyze
particle’ command. Results were plotted and statistical analyses were
performed using the Prism software (GraphPad, La Jolla, CA).
Quantification of chromatin bridges in fly testes
Whole-mount testes stained with an antibody against histone (Millipore,
Billerica, MA) were observed on a fluorescence microscope (Zeiss). All
spermatid nuclei that exhibited a chromatin thread were counted. Note that
protamine-positive nuclei were not taken into account. We used the Prism
software to plot and analyze data.
Acknowledgements
We thank Renate Renkawitz-Pohl, Christina Rathke (Philipps-Universitä t
Marburg, Marburg, Germany), Yikang Rong (National Cancer Institute NIH,
Bethesda, MD, USA), Sté phane Ronsseray (Université Pierre et Marie Curie,
Paris, France), Grazia Raffa and Maurizio Gatti (Università di Roma, Roma, Italy)
for antibodies and/or fly stocks. We also thank the Transgenic RNAi project
(TRiP) at Harvard Medical School [grant NIH/NIGMS R01-GM084947] and the
Bloomington Stock Center for providing fly stocks and plasmid vectors used in
this study. We are grateful to Julien Falk for his help in using ImageJ for
quantification analyses and Laure Sapey-Triomphe for her technical assistance.
We also thank all members of B. Loppin’s lab for helpful discussions and
feedback on this work. Confocal microscopy was performed at the Centre
Technologique des Microstructures of the University Claude Bernard Lyon1.
Competing interests
The authors declare no competing or financial interests.
Author contributions
R.D. and B.L. designed the experiments, R.D. performed the experiments, R.D. and
B.L. analyzed the data, R.D. and B.L. wrote the paper.
Funding
This work was supported by the Centre National de Recherche Scientique and a
grant from the Fondation ARC pour la Recherche sur le Cancer to B.L. [grant
SFI20121205765]. R.D. was supported by a post-doctoral fellowship from the
Fondation ARC pour la Recherche sur le Cancer.
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