Ph.D.Thesis

Studies on the structure and the roles ofDrosophila G9a histone H3 methyltransferase

Yuta Ushijima
2013
Kyoto Institute of Technology

Contents
Chapter1
General Introduction

3

Chapter2
Identification of nuclear localization signals of Drosophila G9ahistone H3 methyltransferase
INTRODUCTION

11

MATERIALS AND METHODS14

RESULTS AND DISCUSSION

18

FIGURES

24

REFERENCES

31

Chapter3
Roles of histone H3K9 methyltransferases during Drosophila
spermatogenesis
INTRODUCTION

36

MATERIALS AND METHODS40

RESULTS42

DISCUSSION

48

FIGURES52

REFERENCES

61

2

Chapter1

General Introduction

3

Epigenetic regulation by histone modification

In eukaryotic cells, the epigenetic regulation and control of gene
expression by the covalent modification of histones are important for
chromatin structure and various biological processes. Post-translational
modification of histones have acetylation, methylation, phosphorylation,ubiquitination and sumoylation of N-terminal and/or C-terminal regions ofhistones (Fig.1) (Bhaumik et al., 2007). Especially, methylation of a lysine
residue(s)onhistoneH3

caninducetranscriptional

regulation,

recombination, repair, heterochromatin formation and X-chromosome
inactivation (Tachibana et al., 2007). Such roles of post-translational
modifications of histone, especially histone lysine methylation, has been arapidly growing research subject in the last decade.

Fig. 1. Covalent modifications of histones

4Drosophila G9a, a histone H3-specific methyltransferase
G9a is one of the histone H3 lysine 9 (K9)-specific histone
methyltransferases.

In

general,themethylated

H3K9

recruits

Heterochromatin protein 1 (HP1) to repress transcription (Fig.2). Drosophila
has three histone methyltransferases (HMTases) specific to histone H3K9;
SU(VAR)3-9, DmSETDB1 and dG9a. It is reported that SU(VAR)3-9
catalyzes H3K9 dimethylation at the chromocenter and trimethylation at thecore of the chromocenter, but is not functional for monomethylation, either
at the chromocenter or along chromosome arms, nor for dimethylation at
chromosome arms and telomeres (Seum et al., 2007). DmSETDB1 is reported
to catalyze H3K9 di- and trimethylation on chromosome 4 (Yoon et al.,2008) ,while dG9a acts for H3K9 mono- and dimethylation in euchromatic
regions (Kato et al., 2008, Lee et al., 2010). In Drosophila, dG9a is a single
orthologue of mammalian G9a and GLP. Mammalian G9a contains several
distinct domains, such as GHD (G9a homology domain), ANK, preSET, SET
and PostSET. These domains are highly conserved between mammals andDrosophila. Epigenetic processes in which H3K9-specific HMTases are
involved are associated with germ line development. For example, mouse
male germ cells lacking SUV39H (a mouse homolog of SU(VAR)3-9) exhibit
chromosomal instability and severe loss of viability (Peters et al., 2001). G9a(a mouse homolog of dG9a) knockout mice are embryonic lethal at past E8.5,
and therefore this enzyme appears to play an important role during early5mouse development (Tachibana et al., 2002).

Fig. 2. Heterochromatin protein HP1 binds to methylated H3-K9
and induces chromatin condensation to repress transcription.
In Drosophila, loss-of-function mutation of dG9a exerts no effect on
viability or fertility. In contrast, in Drosophila ovaries the dG9a mutant isassociated

with

about

40%

misseddevelopment,

in

which

the

fusome/spectrosome tubular connection between cystocytes is absent or
randomly oriented (Lee et al., 2010). However, contribution of dG9a in malegerm lines remain unknown. I therefore studied subcellular localization of
dG9a

and

histoneH3K9

methylation

patterns

duringDrosophila

spermatogenesis in wild type and dG9a mutant. In the Chapter 3 of thisthesis I will describe these studies.
G9a localizes in nuclei to catalyze H3K9 mono- and dimethylation ineuchromatic regions. These subcellular localization studies have been
mainly performed with mammalian cultured cells. However, our studies
with Drosophila early embryos indicate that dG9a dynamically changes its
subcellular localization; some time in ooplasm (cytoplasm) and some time innuclei (data not shown). We are interested in this shuttling mechanism of6

dG9a between cytoplasm and nucleus. As a first step toward this, we firstly
characterized subcellular localization of dG9a in cultured Drosophila cells
and mapped the nuclear localization signals (NLS) on dG9a protein. I will
describe these studies in the Chapter 2. Once we determine the nuclear
localization signals, we could genetically engineer the recombinant dG9awith and without NLS to see the behavior in embryos after establishing thetransgenic flies carrying these genetically engineered genes.

7

REFERECES
Bhaumik S, Smith E, Shilatifard A (2007) Covalent modifications of histones
during development and disease pathogenesis. Nat Struct Mol Biol
14:1008-1016
Kato Y, Kato M, Tachibana M, Shinkai Y, Yamaguchi M (2008)
Characterization of Drosophila G9a in vivo and identification of genetic
interactants. Genes Cells 13:703-722
Lee K-S, Yoon J, Park JS, Kang Y-K (2010) Drosophila G9a is implicated ingerm cell development. Insect Mol Biol 19: 131-139
Peters A, Carroll DO, Scherthan H, Mechtler K, Sauer S, Schofer C,
Weipoltshammer K, Pagani M, Lachner M, Kohlmaier A, Opravil S,
Doyle M, Sibilia M, Jenuwein T (2001) Loss of the Suv39h histone
methyltransferases impairs mammalian heterochromatin and genome
stability. Cell 107: 323-337
Seum C, Reo E, Peng H, Rauscher FJ, Spierer P, Bontron S (2007)
Drosophila SETDB1 is required for chromosome 4 silencing. PLoS Genet
3(5): e76
Tachibana M, Nozaki M, Takeda N, Shinkai Y (2007) Functional dynamics of
H3K9 methylation during meiotic prophase progression. EMBO J 26:
3346-3359
Yoon J, Lee K-S, Park JS, Yu K, Paik S-G, Kang Y-K (2008) dSETDB1 andSU(VAR)3-9

sequentiallyfunction

8duringgermline-

stem

cell

differentiation in Drosophila melanogaster. PLoS One 3: e2234

9

Chapter 2

Identification of nuclear localization signals of

Drosophila G9a histone H3 methyltransferase

10Introduction
In eukaryotic cells, covalent histone modification is critical for chromatin
structure and defines epigenetic regulation for transcription, mitosis andheterochromatin. The binding of HP1 to histone H3 lysine 9 (H3-K9)
following H3-K9 methylation is a key for constitutive heterochromatin(Martin and Zhang, 2005).
In Drosophila, H3-K9 mono-, di- and trimethylation is enriched inpericentromeric heterochromatin including the fourth chromosome in thepolytene chromosome (Schotta et al. 2002; Ebert et al. 2004). SU(VAR)3-9
was first identified H3-K9-specific hintone methyltransferase (HMTase)(Czermin et al. 2001). In Su(var)3-9 null mutants, H3-K9 di- andtrimethylation disappear from the chromocenter of the salivary glandchromosome but not from the heterochromatic fourth chromosome (Ebert etal. 2004). Furthermore, other two HMTases in Drosophila have been
identified. One of them, dSETDB1 is required for gene silencing of the fourthchromosome (Seum et al. 2007; Tzeng et al. 2007; Brower-Toland et al. 2009)and is critical during development (Seum et al. 2007; Tzeng et al. 2007).
Another HMTase, Drosophila G9a (dG9a) appears to be a functional
homologue of human G9a and GLP/Eu-HMTase1 (Mis et al. 2006; Stabell et

al. 2006). dG9a appears to have H3-K9 methyltransferase activity both invitro and in vivo, and functions as a dominant suppressor of position effect ofvariegation (Mis et al. 2006). Although a reduced reproductivity of dG9a11mutant flies suggests the involvement of dG9a in germ cell development (Miset al. 2006; Lee et al, 2010), the other study indicates that dG9a is notessential for fly viability and reproduction (Seum et al, 2007). Furthermore,
dG9a associates with Drosophila Atrophin which is a nuclear receptor
co-repressor and is thought to be recruited to target gene promoters to
repress their transcription (Mang et al. 2008). dG9a is important for the
recruitment of HP1 in the euchromatic region to repress transcription (Katoet al. 2008). Over-expression of dG9a in eye imaginal discs inhibited thedifferentiation of pupal ommatidial cells and resulted in abnormal eyemorphology (rough eye phenotype) in the adults, although a methylase
defective mutant did not demonstrate such effects, suggesting that HMTase
activity of dG9a affects transcription of genes involved in pupal eye
formation (Kato et al. 2008). In addition, genetic interaction between dG9aand some Polycomb group genes as well as little imaginal discs wereobserved (Kato et al. 2008).
In mammals, G9a is one of the well-characterized HMTases. G9aregulates H3K9 mono- and dimethylation at euchromatic region andconsequently plays important roles in euchromatic gene regulation. A varietyof putative G9a-target genes have been identified and interestingly morethan 70 % of these genes are late-replicating (Yokochi et al. 2009). In
mammals,

G9ainteracts

withclosely

relatedhomologof

G9a,

GLP/Eu-HMTase1 during gene repression (Tachibana et al.2005) and more12

recently a functional cooperation between the four H3K9 HMTases such as
G9a, GLP, SETDB1 and Suv39h1 has been also reported (Fritsch et al. 2010).
In Drosophila, dG9a is only an orthologue of mammalian G9a and GLP.
Mammalian G9a contains several distinct domains, such as GHD (G9ahomology domain), ANK, preSET, SET and PostSET. These domains arehighly conserved between mammals and Drosophila. Although mammalian
G9a has nuclear localization signal (NLS) in its N-terminal region (Estève etal. 2005), the amino acid sequences of this region are not conserved in
Drosophila.
Here we have used a series of truncated forms of dG9a and identified
the functional domains for the nuclear localization. The identified nuclear
localization signals of dG9a are distinct from those of mammalian G9a in its
position and amino acid sequence.

13

Materials and methods

Plasmid construction
FLAG-tagged various deleted dG9a mutants were constructed with thepUAST-FLAG vector. The pUAST-FLAG-dG9a (Kato et al. 2008) was used
as a template for PCR. To construct a series of plasmids expressing HA-dG9a,various deletion cDNA fragments were amplified by PCR and inserted into
pActSV-HA vector. For detailed analysis of the nuclear localization signal,oligonucleotides

weresynthesized

topUAST-EGFP-dG9aNLS1,

constructtheplasmidpUAST-EGFP-dG9aNLS2,

pUAST-EGFP-dG9aNLS3, pUAST-EGFP-dG9aNLS4. After annealing thefollowing

pairs

of

synthetic

oligonucleotids,

thedoublestrandedologonucleotides were cloned into the pUAST-EGFP vector (Sugano andYamaguchi, 2008).
Theoligonucleotide

EGFP-dG9aNLS1,5’-GATCCCTGCTCCGAAGCGAGGAAGGGGTCGAGCCAGGAAATAGC-3’,
and 5’-TCGAGCTATTTCCTGGCTCGACCCCTTCCTCGCTTCGGAGCAGG.
The

oligonucleotide

EGFP-dG9aNLS2,5’-GATCGGGGAGAAGAACCAGGACGCAAAAGGAAACTCCCGGATTAGC3’,

and5’-TCGAGCTAATCCGGGAGTTTCCTTTTGCGTCCTGGTTTCTTCTCCCC-3’.

The

oligonucleotide

14

EGFP-dG9aNLS3,

5’-GATCGTTAAGAGAATGCGACGTAGCGTTCGTTTAGGCAATCGATAGC3’,

and5’-TCGAGCTATCGATTGCCTAAACGAACGCTACGTCGCATTCTCTTAAC.
The

oligonucleotode

EGFP-dG9aNLS4,5’-GATCAAGACCCCACCCAAAAAGCGAGGGCGGAAGGCAAAATAGC-3’,
and5’-TCGAGCTATTTTGCCTTCCGCCCTCGCTTTTTGGGTGGGGTCTT-3’.
To construct plasmids expressing HA-dG9a carrying internal deletions,pAct-HA-dG9a 334-472, pAct-HA-dG9a 834-926 and pAct-HA-dG9a 334-472 834-926
Δ

Δ

Δ

/

were created by chimera PCR. The following primers were synthesized.
5’-dG9a(481),

5'-GCAACGACGTCGACAGAGAGAT;

3’-dG9a(999),

5’-TAATAGAGGACTCCTTTTGAGCCGATGCGGGGA;

5’-dG9a(1417),

5'-CTCGGCTACGCCCTGTGAATCTGAAACATCCT;

3'-dG9a(2499),

5’-CGACAGGAGTCTCCAAGTGTTAGATGCCTGGA;

5'-dG9a(2779),

5'-CAATCTACGGACCTCCACGGTGAC;

3'-dG9a(3164),5’-GTCTCAAATGGGGCGCCCTATA. To obtain chimera PCR products, tworounds of PCR were performed. First PCR was performed with primers
5’-dG9a(481) and 3’-dG9a(999), primers 5’-dG9a(1417) and 3'-dG9a(4914),
5’-dG9a(481) and 3’-dG9a(2499), primers 5’-dG9a(2779) and 3'-dG9a(3164),
5’-dG9a(481) and 3’-dG9a(2499), respectively. Second PCR was performed withprimers 5’-dG9a(481) and 3'-dG9a(3164). To create pAct-HA-dG9a 334-472 834-926,
Δ/

pAct-HA-dG9a 334-472 was used as a template for PCR. The PCR fragmentsΔ15

were digested with SalI/SacII, and then inserted into SalI/SacII sites of
pActSV-HA-dG9a plasmid.
Fluorescent microscope analysis
2 x 105 Kc cells grown on 12-well dishes were deposited on polylysine treatedcoverslips. The cells were fixed with 4% paraformaldehyde in PBS for 30 min, andpermeabilized with 0.2% Triton X-100 in PBS for 5 min. After washing with PBS, thecells were treated with 1 mg/ml RNaseA for 30 min at 37°C followed by washingwith PBS. The samples were blocked with 1% skim milk in PBS for 10 min, thenwere incubated for 1 h with mouse monoclonal α-HA (InvivoGen, San Diego, CA,
USA) at 1:500 dilution, mouse monoclonal α-FLAG (M2, Sigma, St Louis, MO,
USA) at 5µg/ml, or rabbit polyclonal α-dG9a (Kato et al. 2008) following by
washing a further three times with 0.1% NP40 in PBS. Cells were blocked again with
1% skim milk in PBS for 10 min, then incubated for 30 min with Alexa Fluor
488-conjugated secondary antibody (Molecular Probes [Invitrogen], Carlsbad, CA,
USA) at 1:400 dilution. After washing three times with 0.1% NP-40 in PBS, thesamples were stained with 100 ⎧g/ml propidium iodide for 5 min. Coverslips were
mounted in Fluoroguard Antifade Reagent (Bio-Rad, Hercules, CA, USA), andexamined under a confocal laser scanning microscope (LSM510, Carl Zeis, Jena,
Germany).

Cell culture and transfection
Kc cells derived Drosophila melanogaster embryos were grown at 25°C in M3

16

medium (Sigma, St Louis, MO, USA) supplemented with 2% fetal calf serum inthe presence of 5% CO2 atmosphere. Cells were transfected with
pAct5C-GAL4 as the effector plasmid and pUAST constructs or pActSV-HA
constructs using CELLFECTIN reagent (Invitrogen, Carlsbad, CA, USA).

17

Results and discussion
Subcellular localization of full-length dG9a and its deletionmutants in Kc cells

Drosophila G9a localizes in nuclei in salivary glands (Kato et al. 2008) andcultured Drosophila Kc cells (Figure 1B). The observed rather weaker dG9asignal in propidium iodide (PI)-dense region may represent the localizationof dG9a in euchromatic region rather than heterochromatic region in the
nucleus as reported previously (Kato et al. 2008). Mammalian G9a has
nuclear localization signals (NLS) in its N-terminal region (Estève et al.
2005). Although some domains of G9a such as GHD, ANK, preSET, and SET
are conserved between Drosophila and mammals, N-terminal regions
including NLS is not highly conserved (Figure 1A). Amino acid sequences of
dG9a in the region from amino acids (aa) 592 to aa651 corresponding to the
NLS of mammalian G9a shows only a little conservation (Figure 1A). The
full-length FLAG-tagged dG9a (aa2-aa1637) exclusively localized in nuclei
as in the case of endogenous dG9a (Figure 1B). However the truncated
FLAG-tagged dG9a (aa547-aa809) including the aa592-aa651 region (Figure1C) was mainly localized in the cytoplasm (Figure 1D). The data indicatethat the region corresponding to the NLS of mammalian G9a is not
functional as NLS in cultured Drosophila Kc cells.
To determine the region of dG9a required for the nuclear localization,
we constructed a set of truncated forms of dG9a mutants (Figure 1C). Before

18immunocytochemical analysis, all the truncated mutant constructs weretransfected into Drosophila Kc cells, then we prepared the cell extracts andconfirmed the expression of mutant proteins as the expected size by Western
blotting analysis (data not shown). In the transfected Kc cells, both thefull-length dG9a (aa2-aa1637) and the truncated forms of dG9a containing
the region aa274-aa546 localized exclusively in nuclei (Figure 1D). In
contrast, the dG9a mutants containing ANK region (aa1085-aa1336) or
C-terminal region (aa1337-aa1637) including PreSET and SET domains
were exclusively localized in the cytoplasm. In addition, the dG9a containing
the aa2-aa273 mainly localized in the cytoplasm (Figure 1D). These results
suggest that the amino acid region aa274-aa546 is responsible for the
nuclear localization of dG9a. In this region, a putative NLS (aa506-aa512,
PKRSKRR) was found by scanning the dG9a protein sequence for NLS motif(KxxKRR) (PredictNLS; http://cubic.bioc.columbia.edu/predictNLS/) (Stabellet al. 2007). Surprisingly, the amino acid region (aa810-aa1084) containing
GHD (aa834-aa926) was also located in nuclei (Figure 1D, 810-1084). As thisdomain of mammalian G9a is responsible for the protein interaction (Gyory

et al. 2004; Nishida et al. 2007), it may be possible that dG9a interacts withother proteins via this GHD domain to be transported into nuclei.
Next, to determine whether the putative NLS (aa506-aa512,
PKRSKRR) is responsible for the nuclear localization of dG9a, weconstructed an additional set of truncated constructs (Figure 2A). However,19unexpectedly the mutant proteins lacking the putative NLS (aa2-aa375,
aa2-aa410, aa2-aa506) were all detected in the nucleus, whereas mutant
containing the aa2-aa336 region of dG9a was detected exclusively in thecytoplasm and that containing the aa2-aa340 region was detected diffusively
in both nucleus and cytoplasm (Figure 2B). These results indicate that thepredicted NLS (aa506-aa512) is not required for the nuclear localization of
dG9a and the amino acid region aa336-aa375 may be rather responsible forthe nuclear localization in cultured Kc cells.
As the truncated dG9a (aa810-aa1084) containing GHD (aa834-aa926)
localized in nucleus (Figure 1D), we also examined whether the C-terminal
region of dG9a is required for nuclear localization (Figure 3). Althoughmutant proteins containing aa927-aa1637 and aa1085-aa1637 were detecteddiffusely in both the cytoplasm and the nucleus, the protein aa835-aa1637
containing GHD did not localize in the nucleus at all (Figure 3B). Therefore
the GHD that is responsible for the protein-protein interaction may not playa role for the nuclear localization of dG9a. The data also suggest that thesequence from aa810 to aa835 might play a role for the nuclear localization,although the sequence contains neither predictable NLS sequence nor basic
lysine and arginine-rich region.
In addition to the above mentioned predictable but not functional
NLS-like sequence (aa506-aa512, PKRSKRR), the amino acid region
aa274-aa546 of dG9a contains four short stretches of amino acids20

(aa334-aa345, aa366-aa378, aa407-aa419 and aa461-aa472) that are rich inthe basic lysine and arginine residues that may function as NLS motifs (Nigg
1997; Jans et al. 2000). We therefore examined subcellular localization oftruncated forms of dG9a spanning these regions (Figure 4A). Although the
mutant proteins containing aa341-aa546 or aa377-aa546 exclusively
localized in the nucleus, that containing aa416-aa546 localized diffusely inboth the cytoplasm and the nucleus and that containing aa471-aa546 region
exclusively localized in the cytoplasm (Figure 4B). Taking account of theobservation that the mutant protein aa2-aa336 exclusively localizes in thecytoplasm (Figure 2B), the results suggest that the amino acid sequences
responsible for the NLS exist in the region aa337-aa470.

Identification of NLS motifs in dG9a
Although no potential NLS motif (WoLF PSORT; http://wolfpsort.org/)
was found in the region aa337-aa470, we found four short stretches of
positively-charged

basic

amino

acids

(NLS1,

aa334-aa345;

NLS2,

aa366-aa378; NLS3, aa407-aa419; NLS4, aa461-aa472) (Figure 5A). In order
to examine the role of the each of these potential NLS motifs within aminoacids 337-470, we introduced expression plasmids in which each of theseputative NLS was fused with the enhanced green fluorescent protein (EGFP)
into Kc cells. Because of the lack of NLS, the expressed EGFP alone
exclusively localized in cytoplasm (Figure 5B). In contrast the EGFP fused21with the NLS3 (aa407-aa419) exclusively localized in nuclei and no EGFP
signal was detectable in cytoplasm (Figure 5B). Both EGFPs fused witheither the NLS1 (aa334-aa345) or the NLS4 (aa461-aa472) mainly localized
in the nucleus with much weaker signals in the cytoplasm. Although the
EGFP fused with the NLS2 (aa366-aa378) localized in the nucleus,
significant level of the signal was also detected in the cytoplasm. These
results indicate that each of the NLS 1, 3 and 4 is sufficient for the nuclear
localization with the strongest effect with the NLS3.
To further evaluate roles of the regions aa334-aa472 including NLS1 to
4 and aa834-aa926 including GHD, we have constructed the expression
plasmids for dG9a carrying internal deletions in either one or both of theseregions (Figure 6A). dG9a carrying an internal deletion in either one of theseregions localized in nuclei (Figure 6B). However, dG9a carrying internaldeletions in both of these regions localized in the cytoplasm (Figure 6B). The
data indicate that the region aa810-aa833 is not sufficient for the nuclear
localization. The data taken together with the other data suggest that theregion aa810-aa926 in addition to the region aa334-aa472 may be necessary
for the nuclear localization.
NLS of many proteins are conserved between mammals and Drosophila.
However in the case of G9a, NLS is not conserved between mammals andDrosophila in its position and the amino acid sequence. The mammalian G9a
exclusively localize in nuclei in various cells and tissues. Although22

Drosophila G9a localize in nuclei in salivary gland cells (Kato et al. 2008)
and cultured cells as shown in this study, our preliminary data indicate that
dG9a also localize in the cytoplasm at some stages of early embryogenesis(data not shown), suggesting some shuttling activity of dG9a during
embryogenesis. The NLS of ancestor form of G9a may be therefore diverged
during evolution to adopt each regulatory mechanism for subcellular
localization of mammalian and Drosophila G9a.

23

FIGURES

Fig.

1.

Subcellular

localizationofendogenous

dG9a

and

transfected full length and deletion mutants.
(A) Sequence comparison of NLS region among hG9a, hGLP and dG9a
using

ClustalW(http://www.ebi.ac.uk/Tools/clustalw2/index.html).

Conserved residues are marked by arrowheads. (B) Subceller localization ofendogenous dG9a and FLAG-tagged dG9a in Kc cells. Kc cells were
immunostained with α-dG9a antibody (, upper panel, green). Kc cells
transfected with FLAG-tagged dG9a and immunostained with α-Flag
24

antibody (lower panel, green). DNA was stained with PI (red). Scale bar, 5µm.
(C) A schematic diagram representing proteins of dG9a wild type anddeletion mutants used to determine the subcellular localization of dG9a.
GHD, ANK repeat, pre SET, and SET domains are indicated. NLS is aputative NLS searched with NLS database. (D) Confocal laser scanningmicroscopy of Kc cells transfected with FLAG-tagged proteins (green). DNA
was stained with propidium iodide (PI, red). Scale bar, 5µm.

25

Fig. 2. Examination of the role of the N-terminal region of dG9a in
the nuclear localization.
(A) A schematic diagram of dG9a wild-type and its N-terminal deletion
derivatives used to determine the subcellular localization of dG9a. (B)Confocal laser scanning microscopy of Kc cells transfected with HA-tagged
proteins (green). DNA was stained with PI (red). Scale bar, 5µm.

26

Fig. 3. Examination of the role of the C-terminal region of dG9a in
the nuclear localization.
(A) A schematic diagram of dG9a wild-type and its C-terminal deletion
derivatives used to determine the subcellular localozation of dG9a. (B)Confocal laser scanning microscopy of Kc cells transfected with HA-tagged
proteins (green). DNA was stained with PI (red). Scale bar, 5µm.

27

Fig. 4. Dissection of N-terminal region of dG9a required for the
nuclear localization.
(A) A schematic diagram of dG9a wild-type and its mutants used todetermine the subcellular localozation of dG9a. (B) Immunostaining of Kc
cells with α-HA antibody. Kc cells transfected with HA-tagged proteins(green). DNA was stained with PI (red). Scale bar, 5µm.

28

Fig. 5. Detailed mapping of the NLS in dG9a.
(A) Candidate NLS sites within amino acids sequence 337-472 in dG9a. (B)Subcellular localization of EGFP and a set of EGFP-tagged putative NLS
sequences in Kc cells. Kc cells transfected with EGFP alone or EGFP-tagged
proteins (green). DNA was stained with PI (red). Scale bar, 5µm.

29

Fig.

6.

Subcellular

localization

of

dG9a

carrying

internal

deletions.
(A) A schematic diagram of dG9a internal deletion derivatives used for thesubcellular localization analysis. (B) Confocal laser scanning microscopy of
Kc cells transfected with HA-tagged proteins (green). DNA was stained withPI (red).

30

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Martin, C., and Zhang, Y. 2005. The diverse functions of histone lysine
methylation. Nature Review Molecular Cell 6: 838-849.
Mis, J., Ner, SS. and Grigliatti, TA. 2006. Identification of three histone
methyltrasnferases in Drosophila: dG9a is a suppressor of PEV and isrequired for gene silencing. Mol Genet Genomics 275: 513-526.
Nigg, EA. 1997. Nucleocytoplasmic transport: signals, mechanisms andregulation. Nature 386: 779-787.
Nishida, M., Kato, M., Kato, Y., Ueda, J., Tachibana, M., Shinkai, Y. andYamaguchi M. 2007. Identification of ZNF200 as a novel binding partnerof histone H3 methyltransferase G9a. Genes Cells 12: 877-888.

32

Seum, C., Reo, E., Peng, H., Rauscher, F.J.3rd, Spierer, P. and Bontron, S.
2007. Drosophila SETDB1 is required for chromosome 4 silencing. PLoS
Genet. 3: e76.
Schotta, G, Ebert, A., Krauss, V., Fischer, A., Hoffmann, J., Rea, S.,
Jenuwein, T., Dorn, R. and Reuter, G. 2002. Central role of Drosophila
SU(VAR)3-9 in histone H3-K9 methylation and heterochromatic genesilencing. EMBO J. 21: 1121-1131.
Stabell, M., Eskeland, R., Bjørkmo, M., Larsson, J., Aalen ,RB., Imhof, A.
andLambertsson A. 2006. The Drosophila G9a gene encodes amultiple-catalytic

histonemethyltransferase

requiredfor

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Sugano, W. and Yamaguchi, M. 2008. Identification of novel localization
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163-169.
Tachibana, M., Ueda J., Fukuda, M., Takeda, N. Ohta, T., Iwanari, H.,
Sakihama, T., Kodama, T., Hanakubo, T. and Shinkai, Y. 2005. Histone
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815-826.
Tzeng, T.Y., Lee, C.H., Chan, L.W. and Shen, C.K. 2007. Epigenetic
regulation of the Drosophila chromosom 4 by the histone H3K9
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dSETDB1.

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12691-12696.
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106: 19363-19368.

34

Chapter3

Roles of histone H3K9 methyltransferases during

Drosophila spermatogenesis

35Introduction
Post-translational modification of histones, such as acetylation, methylation,
phosphorylation, ubiquitination and sumoylation, is important for various
biological processes (Bhaumik et al., 2007). For example, a lysine residue(s)onhistone

H3

can

be

methylatedfortranscriptional

regulation,

recombination, repair, heterochromatin formation and X-chromosome
inactivation (Tachibana et al., 2007). Drosophila has three histone
methyltransferases (HMTases) specific to histone H3 lysine 9 (H3K9);
SU(VAR)3-9,

DmSETDB1

and

dG9a.

SU(VAR)3-9

catalyzes

H3K9

dimethylation at the chromocenter and trimethylation at the core of thechromocenter, but is not functional for monomethylation, either at thechromocenter or along chromosome arms, nor for dimethylation at
chromosome arms and telomeres (Schotta et al., 2002, Ebert A et al., 2004,
Seum et al., 2007b). DmSETDB1 catalyzes H3K9 di- and trimethylation on
chromosome 4 (Seum et al., 2007b) while dG9a acts for H3K9 mono- anddimethylation in euchromatic regions (Kato et al., 2008, Lee et al., 2010).
Epigenetic processes in which H3K9-specific HMTases are involved areassociated with germ line development. For example, mouse male germ cells
lacking SUV39H (a mouse homolog of SU(VAR)3-9) exhibit chromosomal
instability and severe loss of viability (Peters et al., 2001). G9a (a mouse
homolog of dG9a) knockout mice are embryonic lethal past E8.5, andtherefore this enzyme appears to play an important role during early mouse36

development (Tachibana et al., 2002).
In Drosophila, however, loss-of-function mutation of Su(var)3-9 exerts noeffect on viability or fertility (Tschiersch et al., 1994). Similarly a dG9amutation leading to loss of the whole dG9a open reading frame exerts noeffect.

Moreover

in

both

dG9a

loss-of-function

mutantanddG9a-overexpression flies, the methylation pattern of histone H3K9 on
salivary gland polytene chromosomes does not change (Seum et al., 2007a).
On the other hand, in Drosophila ovaries the dG9a mutant is associated with
about 40% missed development, in which the fusome/spectrosome tubularconnection between cystocytes is absent or randomly oriented (Lee et al.,2010). Furthermore, in Drosophila female germ lines, lack of DmSETDB1
causes loss of the egg chamber because of a defect in transcriptional
repression in the oocyte and/or meiotic cell cycle control (Clough et al., 2007).
Although roles of HMTases specific to H3K9 have thus been reported in

Drosophila female germ line (Yoon et al., 2008), contributions in male germlines remain unknown. In the present study, we therefore studied
subcellular localization of dG9a and histone H3K9 methylation patterns
during Drosophila spermatogenesis.
During this process, germ stem cells divide asymmetrically, producing
a differentiating daughter cell (spermatogonium) and a self-renewing stem
cell (Cheng et al., 2008). Each spermatogonium undergoes mitotic division
four times and generates sixteen spermatocytes (Fuller, 1993). During

37

division, spermatogonia are connected by ring channels (Fuller, 1993).
Sixteen spermatocytes grow to undergo meiotic divisions two times togenerate 64 spermatids in what is designated as the growth stage (Fuller,
1993). Then the nuclei condense and the mitochondria form the Nebenkern
in the onion stage (Fuller, 1993). Then, the flagella axonema elongates andthe cystic bulge is pushed backward for sweeping the cytoplasm andorganelles out of the sperm flagella in the so-called elongation stage (Fuller,
1993). Then the heads of spermatids containing nuclei, elongate and further
condensebyreplacinghistones

withprotaminestocomplete

spermatogenesis in the canoe stage ( Rathke et al., 2007, Dorogova et al.,2008).
Our present immunostaining data indicate that dG9a is localized in thecytoplasm from the growth to the elongation stages but in the early canoestage strong dG9a signals were detected exclusively in nuclei, suggestingthat a role in condensation of chromatin. While H3K9 signals were notdecreased in the dG9a null mutant at any stage, mono- and trimethylated
H3K9 signals were decreased in a heterozygous DmSetdb1 mutant during

Drosophila spermatogenesis and similar results were obtained formonomethylated H3K9 signals in a homozygous Su(var)3-9 mutant.
Therefore DmSETDB1 is likely to be responsible for mono- andtrimethylation of H3K9 and SU(VAR)3-9 mainly for monomethylation during
spermatogenesis,

althoughthisreduced38

methylationinpremeiotic

spermatocytes does not appear to influence X-Y chromosome disjunction inmale meiosis.

39Materials and methods

Fly stocks
Fly stocks were cultured at 25

on standard food. w; P{Ubi-tub56 D-GFP}( ) (Inoue et al., 2004) was used as the wild-type strain. w, dG9aRG5 was
kindly provided by Dr P. Spierer. w; DmSetdb110,1a/ CyO was kindly provided
by Dr S. Bontron. wm4; Su(var)3-91/TM3 and wm4; Su(var)3-92/TM3 and wm4;

Su(var)3-917 were kindly donated by Dr T. Hazelrigg.

Western immunoblot analysis
Testes from 30 males of wild type or dG9aRG5 mutant were homogenized in
SDS sample buffer, heated at 95

for 5min and insoluble substances wereprecipitated by centrifugation at 10,000 × g for 15 min at 4 . Protein
samples were separated by 7.5% SDS-PAGE, transferred to polyvinylidene
difluoride (PVDF) membranes (Bio-Rad) blocked with 5% dry milk, andincubated with antibodies specific for dG9a (0.691µg/ml) (Kato et al., 2008)
and a-tubulin (1/5,000) (Sigma) at 4for 16 h. HRP-conjugated anti-rabbit

and anti-mouse IgGs (GE healthcare) were used as secondary antibodies.
Visualization was carried out using ECL-plus (GE healthcare).

Immunostaining of testis
Testes were fixed according to the method of Inoue et al. (Inoue et al., 2004).

40

After dissection in TB (180 mM KCl, 47 mM NaCl, 1m M EDTA, 10 mM
Tris-HCl, pH6.8), samples were placed in liquid nitrogen, then in methanol
for 5 min at -20

and in acetone for 1 min at -20

and then rinsed threetimes in PBST (0.1% TritonX-100 in PBS), three times in PBS and once in
H2O. Following incubation for 16 h at 4with antibodies specific for mono-,

di- or trimethyl-H3-K9 (Kimura et al., 2008) or dG9a (Kato et al. 2008),
sections were incubated for 2 h at 25with Alexa Fluor 488 goat anti-mouse

IgG or Alexa Fluor 594 goat anti-rabbit IgG (Molecular probes). DNA wasstained with 4’, 6-diamidino-2-phenylindole (DAPI). Preparations were
examined using a fluorescence microscope (Olympus BX-FLA) equipped with
CCD camera (Hamamatsu Photo). The immunofluorescent signals with eachantibody in the testis obtained from several independent experiments were
quantified using Image J software and statistically analyzed by Welch’s

t-test (**P<0.01, ***P<0.001).

X-Y chromosome disjunction in male meiosis
Males carrying Dp(1;Y)BS on a genetic background hemizygous for dG9aRG5,
homozygous for Su(var)3-917 or heterozygous for DmSetdb110,1a weregenerated by genetic crosses and grown at 25
females.

41

for mating with Canton S

Results
Subcellular localization of dG9a during spermatogenesis andspermiogenesis
To examine subcellular localization of dG9a during spermatogenesis andspermiogenesis, we immunostained wild type male testes with anti-dG9a
antibody, focusing on spermatocyte growth and subsequent differentiation
stages.

Specificity

of

the

anti-dG9a

antibody

wasconfirmed

byimmunostaining with male whole testes extracts of wild type and dG9aRG5mutant flies (Seum et al., 2007a). The anti-dG9a antibody reacted with asingle dG9a band of 180kDa in extracts from the Drosophila wild type testes(Fig. 1, lane 1) as observed with extracts from Drosophila S2 cells in aprevious study (Kato et al., 2008). However, the 180 kDa dG9a band wasundetectable with extracts from the dG9aRG5 mutant (Fig. 1, lane 2),
confirming the lack of dG9a expression in dG9aRG5 testes.
The immunostaining data revealed that dG9a signals were exclusively
observed in the cytoplasm in both the growth stage (Fig. 2A-2A”) and thefollowing onion stage (Fig. 2B-2B”). In the subsequent elongation stage,
dG9a signals were mainly found in the cystic bulge into which proteins
unnecessary for spermatogenesis are normally discarded (Fig. 2C-2C”).
These results suggest that dG9a plays no significant role during these stages
and therefore rather may have roles before the spermatocyte growth stage
such as in spermatogonial stem cells and/or their mitoses. In contrast, strong42

dG9a signals were exclusively detected in the nuclei in the early canoe stage(Fig. 2D-2D”), possibly playing a role in condensing nuclei or replacement ofhistones with protamines. In the likely late canoe stage in which histones are
replaced with protamines, dG9a signals were no longer detectable (Fig.
2D-2D”). In dG9aRG5 testes, no dG9a signals were detectable throughout (Fig.
2, panels E’-H’ and I), although there was no apparent defect inspermatogenesis.

dG9a plays no apparent role for histone H3K9 methylation during
spermatogenesis and spermiogenesis
To

detectmono-,

di-andtrimethylated

histoneH3K9

duringspermatogenesis and spermiogenesis, we immunostained wild type maletestes with monoclonal antibodies to mono-, di- and trimethylated histone
H3K9 (Figs. 3-6). Specificity of each of these monoclonal antibodies has beencharacterized in vitro and in vivo using cultured mammalian cells (Kimura
et al., 2008). In the wild type fly testes, mono-, di- and trimethylated histone
H3K9 signals were all detected exclusively in nuclei in the spermatocyte
growth (Fig. 3, panels A’-C’), onion (Fig. 4, panels A’-C’), elongation (Fig. 5,
panels A’-C’) and early canoe (Fig. 6, panels A’-C’) stages. Similarly in the

dG9aRG5 mutant fly testes, mono-, di- and trimethylated histone H3K9
signals were all detected exclusively in nuclei during the same stages (Figs.
3-6, panels D’-F’). We collected data for wild type and dG9aRG5 mutant under43

the same conditions to allow direct comparison of the signal strength, but nolarge differences were apparent at these stages (Fig. 7), despite confirmed
loss of dG9a signals by immunostaining with anti-dG9a antibodies, as
described above (Fig. 2). These results, taken together with exclusive
cytoplasmic localization of dG9a in the wild type fly suggest that dG9a plays
no significant role in H3K9 methylation during these stages and that other
HMTases are involved. Since a slight increase of trimethylated histone
H3K9 signal at both growth and onion stages and slight decrease oftrimethylated histone H3K9 signal at canoe stage were observed (Fig. 7),
dG9a might play a minor role for trimethylation of H3K9 at these stage.

DmSETDB1 plays a critical role in mono- and trimethylation ofhistone H3K9 during spermatogenesis and spermiogenesis
To examine roles of HMTases other than dG9a in histone H3K9 methylation,
we firstly immunostained testes of flies heterozygous for the DmSetdb110,1amutation with monoclonal antibodies to mono-, di- and trimethylated histone
H3K9

and

comparedthesignals

with

those

of

wild

type

flies.

Monomethylated H3K9 signals were not detectable in nuclei in the
spermatocyte growth stage (Fig. 3, panels G-G”), onion stage (Fig. 4, panels
G-G”), elongation stage (Fig. 5, panels G-G”) and canoe stage (Fig. 6, panels
G-G”) even under half-dose reduction of the DmSetdb1 gene dose (Fig. 7).
Similarly extensive decrease of the trimethylated H3K9 signals in nuclei was44

observed in the growth stage (Fig. 3, panels I-I”), onion stage (Fig. 4, panelI-I”) and elongation stage (Fig. 5, panels I-I”), and signals were no more
detectable in canoe stage (Fig. 6, panels I-I”). In contrast, only a slightdecrease of dimethylated H3K9 signals in nuclei was observed at canoe stageand even an increase of dimethylated H3K9 signals were observed atelongation stage (Figs. 3-6, panels H-H’’ and Fig. 7). These results indicatethat DmSETDB1 plays a critical role in both mono- and trimethylation ofhistone H3K9 during spermatogenesis and spermiogenesis, and DmSETDB1
might play some minor roles for dimethylation of H3K9 at these stages.

SU(VAR)3-9 plays a role mainly for monomethylation of histone
H3K9 during spermatogenesis and spermiogenesis
To examine potential roles of SU(VAR)3-9, we immunostained testes ofhomozygous Su(var)3-917 mutants with monoclonal antibodies to mono-, diand trimethylated histone H3K9 (Figs. 3-6, panels J’-L’). Comparing signals
in Su(var)3-917 mutant with those of the wild type revealed thatmonomethylated histone H3K9 signals were extensively decreased from thegrowth stage to the canoe stage (Fig. 3-6, panels J-J” and Fig. 7).
Dimethylated H3K9 signals were slightly decreased at growth and onion
stages and rather enhanced signals were observed at elongation stage (Figs.
3-6, panels K-K’’ and Fig. 7). Trimethylated H3K9 signals were also
detectable throughout these stages with slightly decreased levels in onion

45

and elongation stages in compared to those in wild type flies (Figs. 3-6,
panels L-L’’ and Fig. 7). These results indicate that SU(VAR)3-9 plays a role
mainly in monomethylation of histone H3K9 during spermatogenesis andspermiogenesis, and SU(VAR)3-9 might play some minor roles for both diand trimethylation of H3K9 at these stages..

Reduced methylation of histone H3K9 in spermatocytes from

dG9a RG5 ,Su(var)3-9 17

mutantsor

males

heterozygous

for

DmSetdb1 10,1a does not influence meiotic X-Y disjunction
As a considerably reduced methylation of histone H3K9 was observed inspermatocytes from Su(var)3-917 mutants or heterozygotes for DmSetdb110,1a,
we next examined whether the hypo-methylation would influence meiotic
chromosome segregation. To examine inheritance of X and Y chromosomes inmale meiosis, we generated males carrying a dominant BS marker on Y
chromosomes and hemizygous for dG9aRG5, homozygous for Su(var)3-917 orheterozygous for DmSetdb110,1a. Then males with each genotype werecrossed with wild-type females and examined whether X-Y chromosome
disjunction in meiosis of these males occurred properly by scoring theirprogeny (Table 1). In every case, XO male progeny derived from nullo-X
sperm and Bs female progeny from sperm carrying both the marked Y and X
chromosomes scored at a very low frequency, indistinguishable from that inwild-type controls. Neither double mutant males carrying dG9aRG5 and46

Su(var)3-917/+, nor examples carrying dG9aRG5 and DmSetdb110,1a/+, orSu(var)3-917/+ and DmSetdb110,1a/+ males showed significant enhancementof X-Y nondisjunction as compared to males with each single mutation.
Therefore, we conclude that reduced methylation of histone H3K9 inpremeiotic spermatocytes from these mutants does not influence with X-Y
chromosome disjunction in male meiosis.

47

Discussion
Epigenetic regulation systems are responsible for germline cell development.
In mammals, lack of SETDB1 causes lethality because of failure of theprimitive ectoderm to develop properly (Dodge et al., 2004). Furthermore, it
is reported that G9a-knockout mice are embryonic lethal (Tachibana et al.,2002) and both male and female germ line cells of G9a mutant mice cannot
undergo meiosis, indicating an essential role of G9a during germ cell
development (Tachibana et al., 2007). Moreover, mice deficient in SUV39H
show severely impaired development and sterility affecting both males andfemales (Peters et al., 2001). Therefore these three H3K9-specific HMTases
play critical roles in germ line cell development in mammals.
In Drosophila, it is reported that Su(var)3-9 mutants are viable andfertile (Tschiersch et al., 1994). However, lack of DmSETDB1 causes very
early arrest of oogenesis (Clough et al., 2007). Although it is reported that
dG9a has no effect on viability and fertility in either males or females (Seumet al., 2007b), another study demonstrated that in about half of dG9a mutant
flies oogenesis exhibits spectrosome/fusome dysfunction (Lee et al., 2010). In
addition, in Drosophila female germ line cells, it is reported that
DmSETDB1 mainly works for trimethylation of histone H3K9 (Clough et al.,2007), dG9a for mono- and dimethylation of histone H3K9 (Lee et al., 2010)
and SU(VAR)3-9 for di- and trimethylation of histone H3K9 (Seum et al.,2007b). However, in Drosophila roles of the three H3K9-specifc HMTases in48

germ line cells have been mainly characterized in females (Yoon et al., 2008).
In the present study, we therefore analyzed roles of HMTases specific tohistone H3K9 during Drosophila spermatogenesis. In the wild type

Drosophila testes, the exclusive detection of mono-, di- and trimethylated
histoneH3K9 signals in nuclei throughout spermatogenesis is consistent
with a previous report with other antibodies specific to mono-, di-, or
trimethylated histone H3K9 (Rathke et al., 2007). However, there were noapparent large differences in nuclear signals of mono-, di- and trimethylated
histone H3K9 in hemizygous dG9aRG5 male testes (Table 2). From thespermatocyte growth stage to the elongation stage, dG9a is exclusively
localized in the cytoplasm. Given the lack of any apparent defect in malefertility or X-Y chromosome disjunction in male meiosis of the dG9aRG5mutant, dG9a may not have a critical role during spermatogenesis. However
we cannot exclude the possibilities that a small amount of dG9a localizes andfunctions in the nucleus or cytoplasm-localized dG9a plays some roles tomodify proteins other than histones. Both SU(VAR)3-9 and DmSETDB1 aremoderately

expressed

in

adult

males

(http://flybase.org/reports/FBgn0003600.html

andhttp://flybase.org/reports/FBgn0086908.html).

Althoughtheexpression

levels of both enzymes and their sub-cellular localization in testis have notbeenreported

yet,

it

is

likely

that

they

are

expressed

duringspermatogenesis, since their mutations clearly affected the methylation49

status of histone H3K9 (Table 2). Further analyses are necessary to clarify
these points.
In the homozygous Su(var)3-917 mutant, strong decrease of signals formonomethylated histone H3K9 was observed (Table 2),

suggesting that

SU(VAR)3-9 is mainly responsible for monomethylation of histone H3K9
during Drosophila spermatogenesis. Generally it is known that SU(VAR)3-9
catalyzes di- and trimethylation of H3K9 (Seum et al., 2007b). Although
SU(VAR)3-9 has not been fully characterized enzymologically, it should be
noted that substrate specificity of DmSETDB1 changes depending on
associated regulatory proteins. This may also be the case with SU(VAR)3-9
showingaspecificrole

inmonomethylation

of

H3K9

duringspermatogenesis.
In the heterozygous DmSetdb1 mutant, signals for both mono- andtrimethylated histone H3K9 were largely decreased (Table 2). However, it isreported that DmSETDB1 specifically catalyzes trimethylation of histone
H3K9 during oogenesis (Seum et al., 2007a). It is also reported that
DmSETDB1 catalyzes monomethylation of histone H3K9 in euchromatic
region of polytene chromosome and dimethylation of H3K9 in chromosome 4(Seum et al., 2007a). Furthermore overexpression of DmSETDB1 increases
levels of mono-, di, and trimethylation of histone H3K9 (Seum et al., 2007a).
Therefore substrate specificity of DmSETDB1 may change depending ontypes of cells and tissues. It should also be noted that some H3K9-specific50demethylases convert trimethylated H3K9 to monomethylated H3K9 (Shiand Whetstine, 2007). Therefore decrease of trimethylated histone H3K9
may also contribute to decrease of monomethylated histone H3K9. In flies
homozygous for Su(var)3-917 or heterozygous for DmSetdb110,1a, no apparent
defect was observed in X-Y chromosome disjunction during male meiosis.
Furthermore

neither

doublemutantmales

carrying

dG9aRG5andSu(var)3-917/+, nor those carrying dG9aRG5 and DmSetdb110,1a/+, orSu(var)3-917/+ and DmSetdb110,1a/+ showed any significant enhancement of
X-Y nondisjunction as compared to males with each single mutation.
Therefore, in Drosophila the reduced methylation of histone H3K9 inpremeiotic spermatocytes may not be critical for X-Y chromosome disjunction
in male meiosis.
In dG9a, Su(var)3-9 and DmSetdb1 mutants dimethylation status ofhistone H3K9 was only marginally changed. These results may indicate that
dG9a, SU(VAR)3-9 and DmSETDB1 have complementary overlapping
functions to each other in dimethylation of histone H3K9 during
spermatogenesis. Analyses with double and triple mutants may be necessary
to clarify these points. Our observations also suggest that dimethylated
histoneH3K9 may be more important for Drosophila spermatogenesis thaneither mono- or trimethylated forms.

51

Figure legends

Fig. 1 Western immunoblot analysis of testes extracts from wild type andhomozygous dG9aRG5 mutant flies with anti-dG9a antibody. Note the single
band corresponding to 180kDa dG9a detected in extracts from the wild type
(Canton S) testes (lane 1) but not from the dG9a RG5 mutant (w,dG9aRG5; +; +)(lane 2). α-tubulin was used as a loading control.

52

Fig. 2 Anti-dG9a immunofluorescence analysis of wild type and dG9a RG5mutant testes from growth to canoe stages. (A-H) Staining of testes with
DAPI. (A’-D’) Immunostaining of testes with the anti-dG9a antibody. (A”-H”)
Merged images are shown. (A to A” and D to D”) Canton S. (E to E” and H toH”) w, dG9aRG5; +; +. The stages are indicated on the left. (I) Quantificationof dG9a fluorescent signals. Means of ten independent measurements areshown. Error bars represent SD. Relative intensities to those of Canton S (+)
are shown in percentage. **P<0.01, ***P<0.001.

53

Fig. 3 Localization of methylated histone H3K9 in testes in the growthstage.
(A-L) Staining with DAPI. Immunostaining of testes with anti-monomethyl
H3K9 (A’, G’, D’ and J’), anti-dimethyl H3K9 (B’, H’, E’ and K’) andanti-trimethyl H3K9 (C’, I’, F’ and L’) antibodies. Note possible cross-reactionof the anti-trimethyl H3K9 antibody with nucleoli. (A”-L”) Merged images
are shown. (A to A”, B to B” and C to C”) Canton S. (D to D”, E to E” and F toF”) w, dG9aRG5; +; +. (G to G”, H to H” and I to I”) w; DmSetdb110,1a/ CyO. (J54

to J”, K to K” and L to L”) wm4; Su(var)3-917.

Fig. 4 Localization of the methylated histone H3K9 in testes in the onion
stage.
(A-L) Staining with DAPI. Immunostaining of testes with anti-monomethyl
H3K9 (A’, G’, D’ and J’), anti-dimethyl H3K9 (B’, H’, E’ and K’) andanti-trimethyl H3K9 (C’, I’, F’ and L’) antibodies. (A”-L”) Merged images areshown. (A to A”, B to B” and C to C”) Canton S. (D to D”, E to E” and F to F”)

w, dG9aRG5; +; +. (G to G”, H to H” and I to I”) w; DmSetdb110,1a/ CyO. (J to J”,

55

K to K” and L to L”) wm4; Su(var)3-917.

Fig. 5 Localization of the methylated histone H3K9 in testes in theelongation stage.
(A-L) Staining with DAPI. Immunostaining of testes with anti-monomethyl
H3K9 (A’, G’, D’ and J’), anti-dimethyl H3K9 (B’, H’, E’ and K’) andanti-trimethyl H3K9 (C’, I’, F’ and L’) antibodies. (A”-L”) Merged images areshown. (A to A”, B to B” and C to C”) Canton S. (D to D”, E to E” and F to F”)

w, dG9aRG5; +; +. (G to G”, H to H” and I to I”) w; DmSetdb110,1a/ CyO. (J to J”,

56

K to K” and L to L”) wm4; Su(var)3-917.

Fig. 6 Localization of the methylated histone H3K9 in testes in the canoestage.
(A-L) Staining with DAPI. Immunostaining of testes with anti-monomethyl
H3K9 (A’, G’, D’ and J’), anti-dimethyl H3K9 (B’, H’, E’ and K’) andanti-trimethyl H3K9 (C’, I’, F’ and L’) antibodies. (A”-L”) Merged images areshown. (A to A”, B to B” and C to C”) Canton S. (D to D”, E to E” and F to F”)

w, dG9aRG5; +; +. (G to G”, H to H” and I to I”) w; DmSetdb110,1a/ CyO. (J to J”,

57

K to K” and L to L”) wm4; Su(var)3-917.

Fig. 7 Quantification of methylated histone H3K9 fluorescent signals intestes.
The H3K9me1, H3K9me2 and H3K9me3 fluorescent signals in w, dG9aRG5;+; + (dG9aRG5), w; DmSetdb110,1a/ CyO (DmSetdb110,1a/+) and wm4;

Su(var)3-917 (Su(var)3-917) were quantified and shown as relative intensities
to those of Canton S at growth stage (A), onion stage (B), elongation stage (C)and canoe stage (D). Means of ten independent measurements are shown.
Error bars represent SD. Relative intensities to those of Canton S (+) are
shown in percentage. **P<0.01, ***P<0.001.

58

59