In addition, dJmj is excluded from regions stained with an antibody against Ser5-phosphorylated RNA polymerase II, suggesting a function of dJmj in transcriptionally inactive chromatin..
Trang 1nuclear protein that is required for metamorphosis
Nobuhiro Sasai1,2,3,*, Yasuko Kato2,3, Gaku Kimura2,3, Takashi Takeuchi4 and
Masamitsu Yamaguchi2,3
1 Venture Laboratory, Kyoto Institute of Technology, Japan
2 Department of Applied Biology, Kyoto Institute of Technology, Japan
3 Insect Biomedical Research Center, Kyoto Institute of Technology, Japan
4 Mitsubishi Kagaku Institute of Life Sciences (MITILS), Machida, Japan
The basic unit of chromatin in eukaryotes is the
nucleosome, which consists of 146 bp of DNA
wrapped around an octamer of histones H2A, H2B,
H3 and H4 [1] Covalent modifications of histone
tails, such as acetylation, methylation,
phosphoryla-tion and ubiquitinaphosphoryla-tion, modulate interacphosphoryla-tion affinities
for chromatin-associated proteins, leading to the
formation of either transcriptionally active or silent
chromatin structures [2] For example, methylation
at Lys9 of histone H3 (H3-K9) by the su(var)3-9,
enhancer of zeste, trithorax (SET) domain-containing
protein SUV39H1 creates binding sites for the chromo-domain-containing protein HP1, resulting in the establishment of heterochromatin [3] In addition, methylation of H3-K27 and H4-K20 and hypoacetyla-tion of histones are associated with transcriphypoacetyla-tionally silenced chromatin, whereas methylation of H3-K4 and hyperacetylation of histones are connected with active transcription [4]
The JmjC domain was initially characterized as
a conserved domain among jumonji (Jmj) family proteins, including Jmj, RBP2 and SMCX, and has
Keywords
euchromatin; JmjC domain; metamorphosis;
suppressor of PEV; transcriptional silencing
Correspondence
M Yamaguchi, Department of Applied
Biology, Kyoto Institute of Technology,
Matsugasaki, Sakyo-ku, Kyoto 606-8585
Japan
Fax: +81 75 724 7760
Tel: +81 75 724 7781
E-mail: myamaguc@kit.ac.jp
*Present address
CNRS ⁄ UMR218, Institute Curie, Paris,
France
(Received 25 July 2007, revised 4 October
2007, accepted 10 October 2007)
doi:10.1111/j.1742-4658.2007.06135.x
Jumonji (Jmj) is a transcriptional repressor that plays important roles in the suppression of cell proliferation and development of various tissues in the mouse To further clarify the roles of Jmj during development and gain insight into mechanisms of Jmj-mediated transcriptional regulation, we have taken advantage of Drosophila as a model organism Drosophila Jmj (dJmj) shares high homology with mammalian Jmj in the JmjN, JmjC and AT-rich interaction domains, as well as in the N-terminal repression domain dJmj localizes to hundreds of euchromatic sites but not to chro-mocenter heterochromatin on salivary gland polytene chromosomes In addition, dJmj is excluded from regions stained with an antibody against Ser5-phosphorylated RNA polymerase II, suggesting a function of dJmj in transcriptionally inactive chromatin Loss of djmj results in larval and pupal lethality with phenotypes similar to those observed in mutants of ecdysone-regulated genes, implying the involvement of dJmj in the repres-sion of gene expresrepres-sion in the ecdysone pathway Transgenic mouse Jmj mostly colocalizes with dJmj and partially rescues the phenotypes of djmj mutants, indicating that dJmj is a functional homolog of mammalian Jmj Furthermore, mutation in djmj suppresses position effect variegation of the T(2;3)SbV rearrangement These findings suggest that dJmj controls expression of developmentally important genes through modification of chromatin into a transcriptionally silenced state
Abbreviations
ARID, AT-rich interaction domain; DAPI, 4¢,6-diamidino-2-phenylindole; dJmj, Drosophila Jmj; GST, glutathione S-transferase; Jmj, jumonji; Lid, little imaginal disks; mJmj, mouse jumonji; PolII, RNA polymerase II.
Trang 2subsequently been identified in more than 100 proteins
in prokaryotic and eukaryotic organisms [5–7]
JmjC-containing proteins have been shown to play important
roles in various biological processes, including cellular
differentiation, DNA repair and regulation of
hetero-chromatin [8–10] These JmjC-containing proteins are
considered to regulate chromatin or transcription, as
they are generally associated with chromatin- or
DNA-binding domains, such as the plant
homeo-domain (PHD) finger, the TUDOR homeo-domain, the
AT-rich interaction domain (ARID) and the zinc finger
motif [11–13] Recent studies revealed that the
JmjC-containing proteins are histone demethylases and that
the JmjC domain is responsible for their enzymatic
activity [14–19] However, as several JmjC-containing
proteins are predicted to be enzymatically inactive
[11,20], additional mechanisms might be involved in
JmjC-mediated regulation of chromatin or
transcrip-tion
The jmj gene was originally identified by a gene trap
strategy in the mouse and shown to be required for
the appropriate development of various tissues,
includ-ing brain, liver, thymus and heart [7,21,22] jmj
encodes a transcriptional repressor containing the
JmjC domain, JmjN domain and ARID The latter
two mediate the interaction of Jmj with A⁄ T-rich
DNA sequences [23] Although the N-terminal region
of Jmj itself is known to be responsible for its
repres-sor activity [23,24], the mechanisms remain unknown
The JmjC domain of Jmj is predicted to be
enzymati-cally inactive as a histone demethylase [11,12] and its
function remains to be clarified
Jmj appears to have an important role in
suppres-sion of cellular proliferation In the developing heart,
Jmj binds to the promoter and represses the expression
of cyclinD1, which is essential for G1⁄ S phase
transi-tion, thereby suppressing cell proliferation and
regulat-ing morphogenesis of cardiac cells [24] Jmj also
represses E2F activity and reduces cell cycle
progres-sion by associating with the Rb protein [25]
Further-more, it represses expression of ANF, which encodes a
hormonal mediator that is required for heart
develop-ment, by counteracting the function of ANF activators
Nkx2.5 and GATA4 [26] As jmj is widely expressed
and is required for the correct development of various
tissues, involvement in the regulation of a diverse
range of developmental programs, not limited to
car-diac cells, is likely
To further clarify the roles of Jmj during
develop-ment and gain insight into mechanisms of
Jmj-medi-ated chromatin regulation, we have taken advantage of
Drosophila melanogaster as a model organism We
show here that loss of the Drosophila jumonji (djmj)
gene results in larval and pupal lethality with pheno-types similar to those with ecdysone-regulated genes
On salivary gland polytene chromosomes, Drosophila Jmj (dJmj) localizes to euchromatic sites excluded from highly transcribed regions that are stained with an antibody against RNA polymerase II (PolII), suggest-ing a function of dJmj in transcriptionally inactive chromatin Moreover, a djmj mutant suppresses the position effect variegation (PEV) of the T(2;3)SbV rearrangement These observations suggest that dJmj controls expression of developmentally important genes through modification of chromatin into a trans-criptionally silenced state
Results
The CG3654 gene encodes a Drosophila ortholog
of mammalian Jmj JmjC-containing proteins are classified into subgroups
on the basis of their protein structures [11,17] Jmj belongs to the JARID family, which is characterized
by possession of the conserved domains, JmjN, JmjC and ARID [13] Drosophila contains two JARID fam-ily proteins, little imaginal disks (Lid) and a novel pro-tein CG3654 (Fig 1A) Lid has been identified as a gene that enhances the phenotype of ash1 mutants, and is classified as a trithorax group gene [27] Lid is considered to be a sole ortholog of mammalian JARID1 proteins, including RBP2, PLU-1, SMCX and SMCY, as all of them contain additional PHD fingers [12,13]
Mouse Jmj (mJmj) and Drosophila CG3654 share 40%, 45% and 37% identities in the JmjN domain, JmjC domain and ARID, respectively (Fig 1A) In addition to these conserved domains, mJmj contains a zinc finger motif at its C-terminus, whereas CG3654 possesses two AT-hook motifs (Fig 1A) The N-termi-nal repression domain of Jmj is also conserved in CG3654 (Fig 1B), but not in Lid Therefore, we con-cluded that CG3654 is a Drosophila counterpart of mammalian Jmj and designated it as Drosophila jum-onji (dJmj) Jmj proteins are also found in various spe-cies, from insects to mammals, but not in worms and yeasts Importantly, all the Jmj proteins share high homology in the N-terminal region (data not shown), suggesting that this is important for Jmj function, probably acting as a repression domain
djmje03131is a loss of function allele of djmj The djmj gene localizes in the 67B9-10 cytological region and is composed of four exons, including
Trang 37053 bp of an ORF (Fig 2A) To confirm the
expres-sion of dJmj protein, we generated a polyclonal
anti-body to dJmj by immunizing rabbits with the
C-terminal region of dJmj (amino acids 1635–2351) as
an antigen Western blot analysis with affinity purified
antibody to dJmj recognized a protein corresponding
to the calculated molecular mass of dJmj (252 kDa)
from embryo to adult stages, indicating continuous
expression of dJmj throughout development (Fig 2B,
lanes 1–7) The lower band (120 kDa) detected by antibody to dJmj is evident in extracts of embryos (Fig 2B, lanes 1 and 2) and embryo-derived Kc cells (Fig 2B, lane 8) dsRNA-mediated knockdown of dJmj in Kc cells reduced the amount of the 250 kDa dJmj protein to an undetectable level at 4 days after dsRNA treatment, whereas that of the 120 kDa band was unchanged throughout dsRNA treatment (Fig 2B, lane 9) Therefore, we concluded that the 120 kDa
A
B
Fig 1 Identification of the Drosophila Jmj
protein (A) Schematic structures of mouse
Jmj, Drosophila Jmj and Lid The locations
of the JmjN domain, JmjC domain, ARID,
PHD, AT-hook domain and C5HC2 zinc
fin-ger domain are shown (B) Amino acid
align-ment of the N-terminal repression domain
of mouse and Drosophila Jmj Identical and
similar residues are shaded in black and
gray, respectively.
D
Fig 2 Characterization of transposon-inserted djmj mutants (A) The structure of djmj and the location of the transposon insertion in e03131 (piggyBac) is shown The noncoding and coding regions of the djmj transcript are depicted as open and filled boxes, respectively (B) Devel-opmental western blot analysis of dJmj Protein extracts from various develDevel-opmental stages were probed with polyclonal antibody to dJmj Anti-a-tubulin antibody was used to compare the amount of protein loading An asterisk shows nonspecific bands Lane 1: 0–12 h embryo Lane 2: 12–24 h embryo Lane 3: third larva Lane 4: early pupa Lane 5: late pupa Lane 6: adult male Lane 7: adult female Lane 8: Kc cells Lane 9: Kc cells treated with dsRNA (C) Protein extracts from third instar larvae were subjected to western blotting with antibody to dJmj (upper) The same blot was reprobed with antibody to a-tubulin to compare protein loading (lower) Lane 1: wild type Lane 2: djmj e03131 Lane 3: djmj e03131 ⁄ Df(3L)AC1 (D) RT-PCR analysis of expression of djmj in third instar larvae from wild-type and djmj e03131
mutants Rp49 was used as an internal control (E) Immunostaining for dJmj in whole salivary gland cells in wild-type and djmj e03131 mutant larvae DNA was visualized with DAPI (F) Semiquantitative RT-PCR analysis of cell cycle regulators in wild-type and djmje03131third instar lar-vae Expression of rp49 was used as an internal control.
Trang 4band is a nonspecific protein that is cross-reactive
with the antibody It should be noted that this
cross-reactive 120 kDa band is undetectable in extracts
from flies at later developmental stages
To clarify the in vivo roles of djmj, we analyzed
transposon-inserted djmj mutants Two fly strains that
contain the P or piggyBac transposons in the djmj gene
locus were identified The djmjEY02717 allele is an
inser-tion of the EY element [28] in the 5¢-UTR of djmj
However, this insertion does not affect djmj expression,
and homozygous djmjEY02717 flies proved to be viable
and fertile (data not shown) The djmje03131 allele
car-ries the insertion of the piggyBac construct RB, which
contains the splice acceptor and an FLP recombination
target (FRT) site [29], in the first intron of the djmj
gene (Fig 2A), and djmje03131 homozygotes, in
con-trast, showed a lethal phenotype The dJmj protein
was found to be absent in larval extracts of djmje03131
homozygotes or heterozygotes with the deficiency
chro-mosome, Df(3L)AC1, which lacks a genomic region
including the entire djmj locus (Fig 2C) RT-PCR
analysis also indicated a decrease of djmj transcripts in
djmje03131 homozygotes (Fig 2D) Immunostaining of
whole salivary gland cells from third instar larvae
showed predominant localization of dJmj protein in
the nuclei of wild-type but not of djmje03131
homozy-gous cells (Fig 2E)
As it has been reported that mammalian Jmj
represses cyclinD1 expression via binding to its
pro-moter [24], we investigated whether dJmj also represses
the expression of cyclinD, the sole ortholog of
mam-malian cyclinD genes in Drosophila [30]
Semiquantita-tive RT-PCR analysis showed that cyclinD is not
misregulated in djmje03131 mutant third instar larvae
(Fig 2F) The expression of other cell cycle regulators,
including cyclinE, cdk4, E2Fs, Rbfs and stg, was also
unaltered by loss of djmj (Fig 2F and data not
shown) These results suggest that dJmj does not play
a dominant role in the repression of cell cycle
regula-tors in Drosophila
dJmj localizes to euchromatic regions on
polytene chromosomes
The JmjC-containing proteins are thought to regulate
chromatin or transcription [11,12] To gain insight into
the roles of dJmj in chromatin regulation, we analyzed
its chromosomal localization by immunostaining of
polytene chromosomes of salivary glands from third
instar larvae (Fig 3) DNA was visualized with
4¢,6-di-amidino-2-phenylindole (DAPI), which stains brightly
at condensed DNA regions on euchromatic arms that
are divided into bands and interbands and at
chromo-center heterochromatin (Fig 3A,D) Immunostaining
of chromosomes with antibody to dJmj showed dJmj
at hundreds of euchromatic sites with 10–20 bright signals (Fig 3B,C) In contrast, no dJmj signals were detected in chromosomes of djmje03131 mutants (Fig 3E,F) Higher magnification of merged images of dJmj and DAPI staining showed that dJmj was local-ized mostly to bands, but it was also observed in inter-bands and at band–interband boundaries, and no correlation was observed between dJmj localization and DNA density (Fig 3G–I) dJmj was not localized
in chromocenter heterochromatin, as confirmed by co-immunostaining of chromosomes with antibodies for dJmj and HP1, a marker of heterochromatin (Fig 3J– L) These findings suggest that dJmj is involved in the regulation of specific target genes at euchromatin
dJmj is excluded from highly transcribed chromatin regions
Given that mammalian Jmj functions as a transcrip-tional repressor [23,24], dJmj is likely to be associated with transcriptionally inactive chromatin To investi-gate the correlation between dJmj localization and transcriptional activity, we performed coimmunostain-ing of polytene chromosomes with antibodies for dJmj (Fig 4A,D) and PolII (Fig 4B,E) Immunostaining with an antibody against Ser5-phosphorylated PolII detected numerous euchromatic bands in actively tran-scribed regions of the genome Merged images of dJmj and PolII staining revealed no overlap in the distribu-tions of these two proteins (Fig 4C,F), suggesting that dJmj is associated with transcriptionally inactive chro-matin
djmj is a suppressor of position effect variegation
To address whether dJmj regulates the organization of chromatin structure, we examined the effect of djmj on position effect variegation (Table 1) In chromosomes with T(2;3)SbV rearrangement, the dominant Stubble mutation (Sb1), which results in a short bristle pheno-type, is relocated close to pericentromeric hetero-chromatin, resulting in heterochromatin-induced silencing of Sb1 and a wild-type bristle phenotype [31] Female flies of wild-type, djmje03131⁄ TM6B and SUV4-20BG00814, a known suppressor of SbV variega-tion [32], were each crossed with T(2;3)SbV⁄ TM3 males, and the bristles of the progeny were scored for
Sb expression On the wild-type genetic background, 29.7% of bristles showed the Sb phenotype As a posi-tive control, we confirmed that on the background of
Trang 5SUV4-20BG00814, Sb bristles were increased to 54.7%.
In the djmje03131 mutant background the Sb bristles
were significantly increased to 52.6%, indicating that
djmje03131 acts as a suppressor of PEV Similar results
were obtained for the Df(3L)AC1 chromosome, which
lacks a djmj locus in the genome These results suggest
the involvement of dJmj in the establishment and⁄ or
maintenance of the closed chromatin structure
djmj is required for metamorphosis
To investigate in more detail the lethal phenotypes
and lethal phases associated with djmj mutants, the
djmje03131 allele was balanced with the green fluores-cent protein-expressing balancer chromosome, and via-ble larvae were counted in each developmental stage Almost all nonfluorescent djmje03131 homozygous lar-vae developed to the end of the third instar larlar-vae, similarly to control animals Approximately 95% of djmje03131 homozygous animals initiated pupation, but this was delayed for 2–3 days as compared to control animals, whereas the remaining animals continued to wander and did not undergo pupation Of pupated djmje03131 homozygotes, 23% died in the early pupal stage (Fig 5A,C) Other animals developed to the late pupal stage or pharate adults, with a few escapers that
G
H
I
Fig 3 dJmj localizes to euchromatic regions on polytene chromosomes (A–I) Polytene chromosomes of third instar larvae from wild-type (A–C, G–I) and djmj e03131 mutants (D–F) were immunostained with antibody to dJmj (B, E, H) DNA was counterstained with DAPI (A, D, G) (C, F, I) Merged images of dJmj and DAPI staining (G–I) Higher-magnification images of dJmj localization on polytene chromosomes of another spread (J–L) Higher magnification of dJmj staining at chromocenter heterochromatin Polytene chromosomes were coimmuno-stained with antibodies for HP1 (J) and dJmj (K) (L) Merged image of dJmj and HP1 staining.
Trang 6died shortly after eclosion (Fig 5A) Precise excision
of the piggyBac transposon reversed the lethality,
indicating that the transposon insertion was indeed
responsible for the phenotype (data not shown)
Hemi-zygous djmje03131⁄ Df(3L)AC1 animals also exhibited
larval and pupal lethality and displayed similar
pheno-types as homozygous djmje03131 mutants (Fig 5A and
data not shown), confirming that djmje03131 is a loss of function allele of djmj
Phenotypic characterization of pharate adults revealed some mutants to have defects in leg elonga-tion and to show a crooked leg phenotype (Fig 5D,F) These phenotypes are similar to those with loss of function of the genes involved in the ecdysone pathway [33,34], suggesting the participation of dJmj in ecdy-sone signaling
The jmj gene is functionally conserved from flies
to mammals
To investigate whether djmj is a functional homolog of mammalian jmj, we tested the chromosomal distribu-tion of mJmj and its ability to rescue the phenotypes
of the djmj mutants To this end, transgenic flies that
Table 1 The djmj gene is a suppressor of position effect
variega-tion of the T(2;3)Sb V rearrangement.
Genotype
Number
of flies
Total bristles
Number
djmj e03131 ⁄ Sb V 77 1078 567 52.6
Df(3L)AC1 ⁄ Sb V
E
F
Fig 4 dJmj is excluded from highly transcribed chromatin regions (A–F) Polytene chromosomes from wild-type third larvae were stained with antibodies for dJmj (A, D) and PolII (B, E) Higher-magnification images of dJmj (D) and PolII (E) staining of another spread are also shown (C, F) Merged images of dJmj and PolII staining.
C
D
E
F
Fig 5 The djmj gene is required for metamorphosis (A) Lethal phases were determined in animals with the following genotypes: + ⁄ +, djmj e03131 and djmj e03131 ⁄ Df(3L)AC1 (B–F) Lethal phenotypes of djmj e03131 homozygotes (B) Wild-type control animal 4 days after pupation (C, D) djmje03131mutant animals 5 days after pupation (E, F) djmje03131mutants show a crooked leg phenotype Third legs dissected from wild-type (E) and djmj e03131 pharate adults (F) are shown.
Trang 7express FLAG-tagged full-length mJmj (FLAG–mJmj)
under the control of the GAL4–UAS system [35] were
established To minimize the expression of FLAG–
mJmj, the hsp70–GAL4 driver line was used without
heat shock treatment, which results in leaky expression
of FLAG–mJmj that is barely detected by western
blotting with antibody to FLAG (Fig 6A)
Immuno-staining of polytene chromosomes from
FLAG–mJmj-expressing salivary gland cells detected numerous
euchromatic bands (Fig 6C,F), whereas no FLAG
sig-nals were detected in chromosomes without hsp70–
GAL4 (Fig 6H–J) Coimmunostaining of
chromo-somes with antibodies for dJmj (Fig 6B,E) and FLAG
(Fig 6C,F) showed that most, but not all, mJmj sites
colocalize with endogenous dJmj (Fig 6D,G),
suggest-ing that mJmj has similar function as dJmj on
chroma-tin The number of mJmj-binding sites was much
greater than that for dJmj This could be due to higher
expression of FLAG–mJmj on transgenic lines as
compared to endogenous dJmj or to stronger affinity
of the antibody for FLAG
We then expressed mJmj under the background of djmje03131 and investigated the lethal phases of the res-cued flies (Table 2) As most djmje03131 homozygotes develop to the pupal stage (Fig 5), third larvae with the desired genotype were picked up and tested for their lethal phases and phenotypes during pupal stages
Of the control flies that contain the either FLAG–mjmj (line 35) transgene or the hsp70–GAL4 driver under the background of the djmj mutation, 10.7–14.7% of pupae showed the abnormal leg phenotype and 0.6– 7.1% of animals eclosed, which is similar to what was seen with djmje03131 homozygous mutants In contrast, when mJmj was ubiquitously and modestly expressed
by the hsp70–GAL4 driver, the abnormal leg pheno-type was restored and 21.2% of rescued animals eclosed, indicating that mJmj can partially compensate for loss of djmj The FLAG–mjmj transgene inserted in
Fig 6 Transgenic mouse Jmj mostly colocalizes with endogenous dJmj (A) Western blot analysis of FLAG–mJmj expression in larval extracts with the indicated genotypes using antibody to FLAG (upper) The same blot was reprobed with antibody to tubulin to compare pro-tein loading (lower) Lane 1: FLAG–mjmj ⁄ + Lane 2: FLAG–mjmj ⁄ hsp70–GAL4 (B–J) Polytene chromosomes from FLAG–mjmj ⁄ hsp70–GAL4 (B–G) or FLAG–mjmj ⁄ + (H–J) larvae were coimmunostained with antibodies to dJmj (B, E, H) and FLAG (C, F, I) (D, G, J) Merged images of dJmj and FLAG–mJmj staining (E–G) Higher-magnification images of each staining.
Table 2 Transgenic mJmj partially rescues the phenotypes of djmj e03131 mutants.
Genotype
Lethal phase
FLAG–mjmj(19) ⁄ + djmj e03131 ⁄ djmj e03131
FLAG–mjmj(35) ⁄ hsp70–GAL4; djmj e03131 ⁄ djmj e03131 0 (0.0%) 2 (3.8%) 39 (75.0%) 11 (21.2%) 52 FLAG–mjmj(19) ⁄ hsp70–GAL4; djmj e03131 ⁄ djmj e03131 0 (0.0%) 2 (4.9%) 37 (90.0%) 2 (4.9%) 41
a The number of late pupae that show the crooked leg phenotype.
Trang 8the independent genomic locus (line 19) showed
simi-lar, but less pronounced, effects on the rescue
experi-ment It is not possible to draw definitive conclusions
regarding the degree to which mJmj can rescue the
djmj mutant phenotype, as we have not yet succeeded
in cloning the full-length cDNA for djmj to make
djmj-expressing flies, due to its large size However, these
findings strongly suggest the functional conservation of
the jmj gene from flies to mammals
Discussion
Although the Drosophila genome contains at least 13
genes encoding JmjC domain-containing proteins [11],
little is known about their biological roles and their
contributions to chromatin regulation In this study,
we showed that a novel JmjC-containing protein, dJmj,
a Drosophila homolog of mammalian Jmj, is associated
with euchromatic sites excluded from highly
tran-scribed regions on polytene chromosomes and is
required for metamorphosis during development
The mjmj gene appears to be involved in many
devel-opmental pathways, as clarified by analysis of mutant
mice that show various developmental abnormalities
[7,21,22] In the present study, loss of djmj function
caused lethality during larval and pupal stages (Fig 5),
indicating that djmj is also important in Drosophila
development Jmj plays critical roles in suppression of
cellular proliferation via repression of cyclinD1 [24]
However, dJmj is not likely to regulate Drosophila
cyc-linD, as the expression of cyclinD was unchanged in
djmj mutant larvae (Fig 2F) and in dJmj-depleted Kc
cells (data not shown) It is important to note that,
unlike mammalian D-type cyclin proteins, Drosophila
cyclin D is not required for G1⁄ S phase transition but
instead plays a role in cellular growth, whereas cyclin E
plays an essential role in G1⁄ S phase progression [36]
However, cyclinE and several other cell cycle-related
genes were not misregulated in djmj mutant larvae
(Fig 2F and data not shown) Furthermore, dJmj
depletion did not affect cell growth in Kc cells (data not
shown) Therefore, cyclinD repression and subsequent
suppression of cellular proliferation might be a
mam-mal-specific event However, these data do not rule out
the possibility that dJmj might repress cyclinD
expres-sion in restricted tissues, which would not be detected
by expression analysis of extracts of whole animals In
addition, although relatively high expression of dJmj
was observed during embryonic stages (Fig 2B), it
remains unclear whether dJmj is required for the
repres-sion of cell cycle regulators during early development,
as maternally deposited dJmj protein might contribute
to embryogenesis in djmj mutants Further studies are
required to investigate the involvement of dJmj in cell cycle regulation during early embryonic development The detailed mechanism by which Jmj represses tran-scription remains to be clarified Although it has been shown to counteract the function of DNA-binding tran-scription factors [25,26], Jmj directly binds to the cyclinD1 promoter to repress its expression [24] As our data do not show direct evidence that dJmj has a transcriptional repression activity, we cannot conclude that dJmj is indeed a transcriptional repressor like mammalian Jmj However, the observation that dJmj localizes on specific chromatin domains excluded from PolII sites on polytene chromosomes suggests that dJmj mediates transcriptional repression through modifica-tion of chromatin In addimodifica-tion, djmj is not likely to affect global modification of histone tails that are associated with transcriptional activity (supplementary Fig S1) Therefore, our findings suggest that dJmj is involved in the regulation of specific target genes at spe-cific chromosomal loci in response to developmental signals rather than acting as a global regulator of chro-matin
The finding that the phenotypes of djmj mutants resemble those of Drosophila lacking ecdysone-regu-lated genes [33,34] suggests the involvement of dJmj in the ecdysone pathway Expression of early and late puff genes are regulated in a direct or indirect manner
by a subset of chromatin-modifying proteins, including NURF, p66, dGcn5, dAda2a, Bonus, Rpd3 and dG9a [37–43] In addition, one property of JmjC-containing proteins is to associate with chromatin modification enzymes, such as the NCoR corepressor and histone deacetylase (HDACs) [8,44,45] Investigation of whether dJmj links with these proteins to control metamorphosis is clearly warranted The possible inter-action domain of dJmj for these factors is the N-termi-nal repression domain, which is evolutionarily conserved among Jmj proteins (Fig 1) Detailed analy-sis of the role of N-terminal and the JmjC domains in dJmj function may provide clues with which to address these issues
Several studies have clarified that JmjC-containing proteins act as histone demethylases [11] Lid, the clos-est protein to dJmj, was recently shown to be a histone demethylase that removes dimethyl and trimethyl K4
of H3 [46–48] Although our results showed that the mutation in the djmj gene does not affect global modi-fication of histone tails, including dimethyl K4 of H3 (supplementary Fig S1), we cannot rule out the possi-bility that dJmj might demethylate histones at specific chromosomal loci or target a nonhistone protein as
a substrate However, importantly, both mammalian and Drosophila Jmj proteins are predicted to be
Trang 9catalytically inactive as histone demethylases because
of the amino acid changes in the catalytic domain
[11,12] Several other JmjC-containing proteins are
considered to be enzymatically inactive as histone
demethylases [11] Epe1 has been shown to counteract
heterochromatin formation by interacting with Swi6, a
yeast homolog of HP1 This event requires an
enzy-matically inactive JmjC domain, suggesting a novel
function of the JmjC domain of Epe1 in
heterochro-matin formation [49] As the JmjC domain is also
found in bacteria, it might have diverse functions, and
its analysis in dJmj should provide novel insights
Despite the finding of djmj as a suppressor of PEV,
the detailed roles of dJmj in chromatin organization
remain unclear Several different genes are reported to
similarly act as suppressors, including Su(var)2-5,
Su(var)3-7 and Su(var)3-9, which encode structural
components of heterochromatin localizing to
chromo-center heterochromatin [50,51], and Z4, which encodes
a zinc finger protein that localizes to interbands of
euchromatin and regulates chromatin organization at
band–interband boundaries [52] In addition, JIL-1
his-tone kinase functions to maintain euchromatic regions
via antagonizing heterochromatinization by Su(var)3-9
[53,54] On polytene chromosomes, dJmj signals were
excluded from chromocenter heterochromatin, and
het-erochromatin components, including dimethyl K9-H3
and HP1, were not altered by loss of dJmj (data not
shown) In addition, dJmj does not affect PEV of the
whitem4 rearrangement (data not shown) Taken
together, these findings strongly suggest that dJmj is
not a structural element in heterochromatin and acts
at particular domains rather than functioning as a
gen-eral modifier of chromatin
In conclusion, our data suggest that dJmj plays
important roles during metamorphosis by regulating
gene expression in response to developmental signals
As mJmj shows similar distributions to dJmj on
poly-tene chromosomes (Fig 6) and partially rescues the
phenotypes of djmj mutants (Table 2), the Drosophila
system could be a powerful tool with which to analyze
Jmj functions in chromatin regulation and development
Experimental procedures
Fly stocks
Fly stocks were raised at 25C on standard medium
Canton-S was used as the wild-type strain The
piggy-Bac-inserted djmje03131⁄ TM6B fly was obtained from the
Harvard stock center [29], and djmjEY02717, Df(3L)AC1
rnroe-1pp⁄ TM3, SUV4-20BG00814
and T(2;3)SbV, In(3R)Mo,
Sb1, sr1⁄ TM3Ser flies were from the Bloomington stock
center The hsp70–GAL4⁄ CyO and whitem4
flies were obtained from the Drosophila Genetic Resource Center at Kyoto Institute of Technology
Lethal phase analysis and phenotypic characterization
The djmje03131and Df(3L)AC1 alleles were rebalanced with TM6BGFP and TM3GFP balancer chromosomes, respec-tively Lethal phase analysis and phenotypic characteriza-tion were performed as previously described [34]
Generation of transgenic flies and rescue experiment
For constructing the pUAST–FLAG–mjmj vector, a cDNA for FLAG–mjmj in pBluescript was digested with ClaI, blunt-ended and inserted into the pUAST vector [35], which was blunt-ended after EcoRI digestion Transgenic fly lines were generated as described previously [55,56], and three independent fly lines carrying the transgene on the second chromosome were established The GAL4–UAS system [35] was used for ubiquitous expression of FLAG–mJmj using the hsp70–GAL4 driver
For the rescue experiment, FLAG–mjmj (line 35), djmje03131⁄ TM6B or FLAG–mjmj (line 19)⁄ CyOGFP, djmje03131⁄ TM6B females were crossed with hsp70– GAL4⁄ CyOGFP, djmje03131⁄ TM6B males at 25 C As con-trol crosses, djmje03131⁄ TM6BGFP females and males were mated with hsp70–GAL4⁄ CyOGFP, djmje03131⁄ TM6B males and FLAG-mjmj⁄ (CyOGFP), djmje03131⁄ TM6B females, respectively Nontubby and nonfluorescent third larvae were picked up, and their lethal phases and phenotypes during pupal development were analyzed
PEV analysis
To examine the effect of djmj on the whitem4 variegation,
wm4⁄ wm4 females were crossed with w⁄ Y, djmje03131⁄ TM6B males, and the eyes of wm4⁄ Y, djmje03131⁄ +males were scored and compared with those of wm4⁄ Y, TM6B ⁄ +males The effect of djmj on the SbVvariegation was studied by crossing SUV4-20BG00814, djmje03131⁄ TM6B, Df(3L)AC1 ⁄ TM3Ser-GFP or Canton S females with T(2;3)SbV⁄ TM3Ser males [31], and 14 defined bristles were scored as being wild type or
Sb Male and female scores were combined because no differ-ences between sexes were observed
Production of polyclonal antibody to dJmj
To construct an expression vector for the glutathione S-transferase (GST)-fused C-terminal region of the dJmj protein (dJmjC, amino acids 1635–2351), the djmj cDNA fragment was inserted into the SalI and NotI sites of
Trang 10the pGEX4T-1 vector GST–dJmjC was expressed in the
bacterial strain BL-21(DE3), affinity purified with a
glutathi-one Sepharose column (GE Healthcare, Little Chalfont,
UK), and injected into rabbits The antiserum generated
was applied to GST-conjugated sepharose, and this was
fol-lowed by purification with GST–dJmj-conjugated sepharose
Cell culture and knockdown experiments
Kc cells were cultured at 25C in M3 medium (Sigma, St
Louis, MO, USA) supplemented with 2% fetal bovine
serum For dsRNA production, a 621 bp fragment spanning
from nucleotide 6485 to the 3¢-UTR (40 bp downstream of
the stop codon) of djmj were amplified using 5¢-CAC
GGGCGTATACCTCAAGC-3¢ and 5¢-TGTGCCTGA
ATCTTTCGTGC-3¢ primers and cloned into the pGEM-T
vector Sense and antisense RNAs were synthesized in vitro
and annealed For knockdown experiments, 1· 106cells
were plated on 6 cm dishes and transfected with 10 lg of
dsRNA using cellfectin transfection reagent (Invitrogen,
Carlsbad, CA, USA) according to the manufacturer’s
proto-col The cells were collected, directly suspended in SDS
sam-ple buffer, and subjected to western blotting
Western blotting
Protein extracts were prepared by homogenization of
ani-mals in ice-cold SDS sample buffer followed by boiling for
5 min After centrifugation at 12 000 g for 10 min at 4C,
protein samples were separated by SDS⁄ PAGE and
trans-ferred to poly(vinylidene difluoride) membranes (Millipore,
Billerica, MA, USA) Antibodies used were anti-dJmj
(1 : 2000), anti-a-tubulin (1 : 5000, Sigma), anti-FLAG
(M2, 1 : 2000; Sigma), anti-acetyl H3 (06–599, 1 : 5000),
anti-dimethyl K4-H3 (07-030, 1 : 2000),
anti-monometh-yl K9-H3 (07–450, 1 : 1000), anti-dimethanti-monometh-yl K9-H3 (07–212,
1 : 1000), and anti-trimethyl K27-H3 (07–449, 1 : 1000)
from Upstate (Lake Placid, NY, USA), and anti-H3
(1 : 1,000; Cell Signaling, Danvers, MA, USA) Horseradish
peroxidase-conjugated anti-rabbit and anti-mouse IgGs
(GE Healthcare) were used as secondary antibodies, and
proteins were detected with ECL-plus (GE Healthcare)
Immunostaining of polytene chromosomes and
whole salivary glands
For immunostaining of polytene chromosomes, salivary
glands from wandering third instar larvae were dissected in
0.7% NaCl, fixed for 5 min, and squashed in 45% acetic
acid⁄ 3.7% formaldehyde The slides were frozen in liquid
nitrogen and were then blocked in blocking buffer (5%
skimmed milk in NaCl⁄ Pi⁄ 0.1% Triton X-100) for 1 h at
25C Slides were incubated with primary antibodies for
16 h at 4C The antibodies used were anti-dJmj (1 : 400),
anti-FLAG (M2, 1 : 5,000; Sigma), anti-PolII (H-14, 1 : 100; Covance, Princeton, NJ, USA) and anti-HP1 (C1A9, 1 : 100; Developmental Studies Hybridoma Bank at the University
of Iowa) After being washed with NaCl⁄ Pi⁄ 0.1% Triton
X-100 twice for 15 min each, the slides were incubated with Alexa-488-conjugated anti-rabbit IgG, Alexa-488-conjugated anti-mouse IgM, or Alexa-594-conjugated anti-mouse IgG
or anti-rabbit IgG (1 : 400) from Invitrogen for 2 h at 25C DNA was visualized with DAPI Preparations were mounted
in FluoroGuard Antifade Reagent (Bio-Rad, Hercules, CA, USA), and images were obtained using an Olympus (Tokyo, Japan) BX-50 microscope equipped with a cooled CCD cam-era Each staining experiment was performed at least three times, and representative spreads are shown
For immunostaining of whole salivary glands, dissected glands were fixed in 4% formaldehyde⁄ 0.15% Triton X-100 for 20 min on ice After blocking in NaCl⁄ Pi containing 2% goat serum and 0.15% Triton X-100 for 30 min at
25C, the glands were incubated with antibody to dJmj (1 : 400) for 16 h at 4C, and this was followed by incuba-tion with Alexa-488-conjugated anti-rabbit IgG (1 : 400) for 2 h at 25C DNA was stained with DAPI
Semiquantitative RT-PCR Total RNA was extracted with Sepasol RNA I (Nacalai, Kyoto, Japan) First-strand cDNA was synthesized using oligo(dT)20 and Superscript III reverse transcriptase (Invi-trogen) PCR reactions were performed over a range of cDNA dilutions to ensure exponential amplification Primer sequences used were as follows: cycD-F, 5¢-GGGATCCCA CATTGTATTCG-3¢; cycD-R, 5¢-ACGGAGCTTTGAAG CCAGTA-3¢; cycE-F, 5¢-AAGGTGCAGAAGACGCA CTT-3¢; cycE-R, 5¢-AATCACCTGCCAATCCAGAC-3¢; cdk4-F, 5¢-TACAACAGCACCGTGGACAT-3¢; cdk4-R, 5¢-TGGGCATCGAGACTATAGGG-3¢; rp49-F, 5¢-CGG ATCGATATGCTAAGCTG-3¢; and rp49-R, 5¢-GAACG CAGGCGACCGTTGGGG-3¢
Acknowledgements
We would like to thank Haruki Shirato for providing the FLAG–mjmj plasmid and members of the Yamagu-chi laboratory for helpful comments and advice We also acknowledge the contribution of Malcolm Moore
in critical reading of the manuscript This work was supported in part by grants-in-aid from the Ministry
of Education, Sciences, Sports and Culture of Japan
References
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