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First published online 8 December 2005
doi: 10.1242/dev.02199
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Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.
* Author for correspondence (e-mail: kristen{at}iastate.edu)
Accepted 7 November 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: JIL-1 kinase, Chromatin, Histone modifications, HP1, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Formation of heterochromatin and repression of
transcription involves covalent modifications of histone tails and/or the
exchange of histone variants (Swaminathan
et al., 2005). Current evidence suggests that a major pathway in
the establishment of heterochromatin is initiated by the RNAi machinery that
marks prospective heterochromatic regions
(Volpe et al., 2002;
Pal-Bhadra et al., 2004;
Verdel et al., 2004). This
leads to the deacetylation of histone H3K9, followed by the dimethylation of
this residue and the recruitment of HP1
(Lachner et al., 2001;
Nakayama et al., 2001;
Ebert et al., 2004). Thus,
dimethylation of histone H3K9 and the presence of HP1 serve as major chromatin
modification markers for the presence of transcriptionally silenced chromatin
(Fischle et al., 2003;
Swaminathan et al., 2005).
However, it should be noted that HP1 recently has been demonstrated to also
play a role in euchromatic gene regulation that is not linked to histone H3K9
dimethylation (Piacentini et al.,
2003; Cryderman et al.,
2005).
Ebert et al. (Ebert et al.,
2004) recently identified the Su(var)3-1 mutations as
alleles of the JIL-1 locus that antagonize the expansion of
heterochromatin formation in Drosophila. JIL-1 is a tandem kinase
that localizes specifically to euchromatic interband regions of polytene
chromosomes (Jin et al.,
1999). Analysis of JIL-1 null and hypomorphic alleles
showed that JIL-1 is essential for viability, and that reduced levels
of JIL-1 protein lead to a global disruption of chromosome structure
(Jin et al., 2000;
Wang et al., 2001;
Zhang et al., 2003;
Deng et al., 2005). These
defects are correlated with severely decreased levels of histone H3S10
phosphorylation (pH3S10), providing evidence that JIL-1 is the predominant
kinase regulating the phosphorylation state of this residue at interphase
(Wang et al., 2001). However,
as the Su(var)3-1 alleles generate proteins with COOH-terminal
deletions that are dominant gain-of-function mutations, the experiments of
Ebert et al. (Ebert et al.,
2004) did not directly address the normal function of JIL-1. In
this study, we show that the reduction in JIL-1 protein levels and histone
H3S10 phosphorylation caused by hypomorphic or null loss-of-function alleles
of the JIL-1 locus results in the spreading of the major
heterochromatin markers dimethyl H3K9 (dmH3K9) and HP1 to ectopic locations on
the chromosome arms, with the most pronounced increase on the X chromosomes.
Furthermore, genetic interaction assays demonstrated that JIL-1 functions
antagonistically to Su(var)3-9, which is the major catalyst for dimethylation
of the histone H3K9 residue (Schotta et
al., 2002). These findings suggest a model where JIL-1 kinase
activity functions to mark euchromatic domains, and counteract
heterochromatization and gene silencing at ectopic locations by
Su(var)3-9-mediated histone H3K9 dimethylation and HP1 recruitment.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Canton-S was
used for wild-type preparations. The JIL-1z2,
JIL-1z60 and JIL-1h9 alleles have been
described by Wang et al. (Wang et al.,
2001) and by Zhang et al.
(Zhang et al., 2003). The
GFP-lacI transgenic stock 128.1, and the lac operator repeats
transgenic stock 4D5 were the generous gifts of Dr L. Wallrath (University of
Iowa, Iowa City). The application of the GFP-lacI and lac operator
repeat stocks for X chromosome identification in JIL-1 mutant
backgrounds is described by Deng et al.
(Deng et al., 2005).
Su(var)3-13/TM3 Sb Ser stocks were obtained from the
Bloomington Stock Center, whereas the Su(var)3-91 and
Su(var)3-92 stocks were from the Umeå Stock Center.
The molecular lesion of Su(var)3-13 was determined by PCR
mapping and sequencing, as described by Zhang et al.
(Zhang et al., 2003).
Recombinant JIL-1z60 Su(var)3-91 chromosomes
were identified by generating recombinants as described by Ji et al.
(Ji et al., 2005), except that
the dominant Su(var)3-91 phenotype was selected for in a
wm4 background, and the presence of
JIL-1z60 was confirmed by PCR as described by Zhang et al.
(Zhang et al., 2003). Balancer
chromosomes and markers were as described by Lindsley and Zimm
(Lindsley and Zimm, 1992).
Immunohistochemistry
Third instar salivary gland `smush' preparations of polytene nuclei were
prepared essentially as described by Wang et al.
(Wang et al., 2001). Polytene
chromosome squash preparations were performed as described by Kelley et al.
(Kelley et al., 1999), using
the 5-minute fixation and antibody-labeling protocol described by Jin et al.
(Jin et al., 1999). Primary
antibodies used include: affinity-purified Hope rabbit antiserum raised
against JIL-1 residues 886-1013 (Jin et
al., 1999); anti-tubulin mAb (Sigma); histone H3 goat antiserum
(Santa Cruz); phospho-histone H3S10 rabbit antiserum (Upstate Biotechnology);
dmH3K9 rabbit antiserum (Upstate Biotechnology); anti-HP1 mAb C1A9
(Developmental Studies Hybridoma Bank, University of Iowa); anti-MSL-1 rabbit
antiserum [generous gift of Drs M. Kuroda (Harvard Medical School, Boston, MA)
and R. Kelley (Baylor College of Medicine, Houston)]; and anti-GFP chicken IgY
(Aves Laboratory). DNA was visualized by staining with Hoechst 33258
(Molecular Probes) in PBS. The appropriate species- and isotype-specific Texas
Red-, TRITC- and FITC-conjugated secondary antibodies (Cappel/ICN, Southern
Biotech) were used (1:200 dilution) to visualize primary antibody labeling.
The final preparations were mounted in 90% glycerol containing 0.5%
n-propyl gallate. The preparations were examined using
epifluorescence optics on a Zeiss Axioskop microscope and images were captured
and digitized using a high resolution Spot CCD camera. Confocal microscopy was
performed with a Leica confocal TCS NT microscope system equipped with
separate Argon-UV, Argon and Krypton lasers, and the appropriate filter sets
for Hoechst, FITC, Texas Red and TRITC imaging. A separate series of confocal
images for each fluorophor of double-labeled preparations were obtained
simultaneously with z-intervals of, typically, 0.5 µm, using a PL
APO 100x/1.40-0.70 oil objective. A maximum projection image for each of
the image stacks was obtained using the ImageJ software
(http://rsb.info.nih.gov/ij/).
In some cases, individual slices or projection images from only two to three
slices were obtained. Images were imported into Adobe PhotoShop, where they
were pseudocoloured, image processed and merged. In some images, non-linear
adjustments were made for optimal visualization of the Hoechst labeling of
chromosomes.
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) and Zhang
et al. (Zhang et al., 2003),
using extracts from third-instar larvae of the specified genotype. For the
quantification of immunolabeling, digital images of exposures of immunoblots
on Biomax ML film (Kodak) were analyzed using the ImageJ software, as
previously described (Wang et al.,
2001). In these images, the grayscale was adjusted such that only
a few pixels in the wild-type lanes were saturated. The area of each band was
traced using the outline tool and the average pixel value determined. Levels
in mutant larvae were determined as a percentage relative to the level
determined for wild-type control larvae, using tubulin or histone H3 levels as
a loading control. |
RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Delattre et al.,
2004; Ebert et al.,
2004) (Fig. 1A-D).
In wild-type preparations, histone H3K9 dimethylation is found at the
chromocenter, in a banded pattern on the fourth chromosome, and in a few
locations on the chromosome arms (Delattre
et al., 2004; Ebert et al.,
2004) (Fig. 1A).
However, in the JIL-1z2/JIL-1h9 mutant
background there was a striking spreading of histone H3K9 dimethylation to the
chromosome arms that was most pronounced on the X chromosome
(Fig. 1B). The same
distribution was also observed in squash preparations from both male and
female JIL-1z2 homozygous null larvae
(Fig. 1C,D). In addition to the
changes in the distribution of the histone H3K9 dimethylation marker,
JIL-1 mutant polytene chromosomes exhibit a range of morphological
defects, as previously described by Wang et al.
(Wang et al., 2001) and Deng
et al. (Deng et al., 2005).
There is a shortening and folding of the chromosomes, with a non-orderly
intermixing of euchromatin and the compacted chromatin characteristic of
banded regions (Fig. 1B-D). The
extreme of this phenotype is exhibited by the male X polytene chromosome,
where no remnants of coherent banded regions can be observed
(Deng et al., 2005)
(Fig. 1B,C). In order to determine the degree of change in the levels of chromatin modification markers in JIL-1 mutant backgrounds, we analyzed immunoblots of protein lysates from wild-type, JIL-1z2/JIL-1h9 and JIL-1z2/JIL-1z2 third instar larvae. The immunoblots were probed with anti-pH3S10, anti-dmH3K9, anti-histone H3 and anti-tubulin antibodies. As illustrated in Fig. 1E, both JIL-1z2/ JIL-1h9 (15.2±4.0%, n=4) and JIL-1z2/JIL-1z2 (9.4%±5.0%, n=6) mutants showed greatly reduced levels of phosphorylated histone H3S10, when compared with that in wild-type larvae, indicating that JIL-1z2/JIL-1h9 has the properties of a strong hypomorph. By contrast, the levels of both dmH3K9 and HP1 were at or near wild-type levels (Fig. 1E). In the JIL-1z2/JIL-1z2 null background, the level of dmH3K9 was at 109.0±31.0% (n=9) of the wild-type level, and HP1 was at 99.3±17.8% (n=9). Thus, in JIL-1 mutant backgrounds the level of phosphorylated histone H3S10 was dramatically reduced, whereas the levels of the heterochromatin markers dmH3K9 and HP1 were unaffected.
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; Nakayama et al.,
2001; Schotta et al.,
2002). We therefore performed double labeling with antibodies to
these markers to determine whether HP1 also spreads to the same ectopic
locations as does dmH3K9 in JIL-1 mutant backgrounds. For these
experiments, we used the polytene salivary gland `smush' preparation that
employs the modified whole-mount staining technique first described by Wang et
al. (Wang et al., 2001). This
preparation does not afford the resolution of regular squash preparations;
however, it has an advantage in that it avoids the acetic acid treatment,
which, in our experience, can adversely affect the quality and reproducibility
of antibody labeling. Fig. 2A,B
show polytene nuclei from male and female wild-type and
JIL-1z2/JIL-1z2 third instar larvae labeled
with anti-HP1 and anti-dmH3K9 antibodies, and with Hoechst to visualize the
DNA. In wild-type nuclei, the dmH3K9 marker and HP1 were colocalized, with the
labeling largely confined to the chromocenter, whereas in
JIL-1z2/JIL-1z2 mutants both markers had spread
to ectopic locations in an overlapping pattern, with the highest levels being
found on the X chromosomes. To verify that it indeed was the X chromosome that
showed the most pronounced increase, we performed double labeling with
anti-HP1 and anti-MSL-1 antibodies. MSL-1 is a member of the MSL (male
specific lethal) dosage compensation complex and is found only on the male X
chromosome (Kuroda et al.,
1991; Kelly and Kuroda,
1995). Fig. 2C
shows that, apart from the chromocenter, there is no significant level of HP1
on the wild-type male X chromosome, whereas it is clearly distributed on the
male X chromosome in JIL-1z2/JIL-1z2 mutant
nuclei. In order to verify the distinct increase on the female X chromosome in
JIL-1z2/JIL-1z2 mutants, we labeled polytene
squash preparations from the transgenic line 4D5
(Danzer and Wallrath, 2004),
which contains lac operator repeats integrated onto the X chromosome
that can be detected by GFP-lacI binding
(Belmont, 2001;
Deng et al., 2005), with
anti-dmH3K9 and anti-GFP antibodies. The labeling shown in
Fig. 2D demonstrates that the
ectopic spreading of dmH3K9 in females is preferentially located on the X
chromosome.
JIL-1Su(var)3-1 alleles act differently from JIL-1 loss-of-function alleles
Ebert et al. (Ebert et al.,
2004) recently demonstrated that JIL-1 Su(var)3-1 alleles
are dominant gain-of-function mutations that are likely to antagonize the
expansion of heterochromatin. However, in contrast to JIL-1
loss-of-function alleles, JIL-1Su(var)3-1 homozygous
mutants exhibit no change in the distribution of the heterochromatin marker
dmH3K9 or in histone H3S10 phosphorylation
(Ebert et al., 2004). We
confirmed these results in heteroallelic mutants with only one copy of
JIL-1Su(var)3-1[3] together with the null
JIL-1z2 allele (Fig.
3). The JIL-1Su(var)3-1[3] allele is a point
mutation that introduces a premature in-frame stop codon, leading to a JIL-1
protein with a 166-amino acid truncation of the COOH-terminal domain. In order
to determine the distribution of the truncated JIL-1Su(var)3-1[3]
protein in relation to other chromatin constituents, we double-labeled
polytene squashes from
JIL-1z2/JIL-1Su(var)3-1[3] larvae with either
anti-dmH3K9 or anti-MSL-1 antibody, and with anti-JIL-1 antibody
(Fig. 3A-C).
Fig. 3A shows that one copy of
the JIL-1Su(var)3-1[3] allele is sufficient to prevent
ectopic spreading of the dmH3K9 marker, which is localized at the chromocenter
as in wild-type preparations. Interestingly, one copy of the
JIL-1Su(var)3-1[3] allele is also sufficient to largely
rescue the abnormal chromosome morphology of JIL-1 null mutants,
including the male X chromosome. This is despite the finding that the
JIL-1Su(var)3-1[3] protein is mislocalized on the chromosomes when
compared with wild-type JIL-1. Fig.
3A,B shows that although the JIL-1Su(var)3-1[3] protein
is associated with the chromosomes, the antibody labeling is diffuse, does not
define clear banded regions, and overlaps with Hoechst-labeled banded regions,
a distribution that was never observed for the wild-type JIL-1 protein
(Jin et al., 1999;
Wang et al., 2001).
Furthermore, the JIL-1Su(var)3-1[3] protein is not upregulated on
the male X chromosome (Fig. 3B)
and is present along the chromosome arms at ectopic locations. This is shown
at higher magnification in Fig.
3C, in a double labeling with JIL-1 and MSL-1 antibodies. In
wild-type preparations, MSL-1 and JIL-1 are colocalized on the male X
chromosome (except for the telomere) (Jin
et al., 2000); however, as indicated by the arrows in
Fig. 3C, the
JIL-1Su(var)3-1[3] protein is associated with many regions of the X
chromosome not labeled by MSL-1 antibody. Immunoblot analysis of protein
lysate from wild-type, JIL-1z2/+ and
JIL-1z2/JIL-1Su(var)3-1[3] larvae show that the
level of histone H3S10 phosphorylation in
JIL-1z2/JIL-1Su(var)3-1[3] mutants is
comparable to that of JIL-1z2/+ larvae, suggesting that
JIL-1Su(var)3-1[3] mutants have normal JIL-1 kinase
activity (see also Ebert et al.,
2004). Taken together, these data suggest that the COOH-terminal
domain of JIL-1 is required for proper chromosome localization, and indicate
that the dominant gain-of-function effect of the
JIL-1Su(var)3-1[3] allele
(Ebert et al., 2004) may be
attributable to JIL-1 kinase activity at ectopic locations.
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; Shotta et
al., 2002), Su(var)2-5, HP1 (Eissenberg and
Elgin, 2000), and Su(var)3-7
(Delattre et al., 2004), a 1169
amino acid zinc-finger protein, are all inherent components of heterochromatin
in Drosophila (Ebert et al.,
2004). These proteins physically interact and in a genetic
hierarchy Su(var)3-9 is epistatic to the other two genes
(Schotta et al., 2002;
Ebert et al., 2004).
Furthermore, Su(var)3-9 has been shown to catalyze most of the dimethylation
of the histone H3K9 residue (Schotta et
al., 2002). We therefore performed immunolocalization and genetic
interaction studies with the Su(var)3-91 and
Su(var)3-92 loss-of-function alleles
(Reuter et al., 1986;
Tschiersch et al., 1994) in
order to determine whether Su(var)3-9 also controls JIL-1
activity. However, in polytene squashes from
Su(var)3-91/Su(var)3-92 heteroallelic
larvae, JIL-1 localization in both male and female chromosomes was
indistinguishable from that observed in wild-type chromosomes
(Fig. 4A-D). By immunoblot
analysis of protein extracts from
Su(var)3-91/Su(var)3-92 heteroallelic
larvae, we verified that the dmH3K9 level was dramatically reduced
(Fig. 4E). The data further
indicate that histone H3S10 phosphorylation is at wild-type levels in this
mutant background (Fig. 4E).
These results suggest that JIL-1 activity and localization are not affected by
the absence of Su(var)3-9 activity, which is in contrast to the finding that
the distribution of Su(var)3-9 methyltransferase activity is altered in
JIL-1 null or hypomorphic mutants.
To determine whether Su(var)3-9 and JIL-1 genetically interact in the same
pathway in vivo, we explored interactions between mutant alleles of
Su(var)3-9 and JIL-1 by generating double-mutant
individuals. Because Su(var)3-9 and JIL-1 are both located
on the third chromosome, we first recombined the
Su(var)3-91 allele onto the JIL-1z60
chromosome. Subsequently, JIL-1z60 Su(var)3-91/TM6
Sb Tb males were crossed with JIL-1z2/TM6 Sb Tb
virgin females generating JIL-1z2/JIL-1z60
Su(var)3-91 progeny, identified as non-Sb. In control
experiments in which Su(var)3-9 activity was not altered, we crossed
JIL-1z60/TM6 Sb Tb males with JIL-1z2/TM6
Sb Tb virgin females generating
JIL-1z2/JIL-1z60 progeny.
JIL-1z2 is a null allele and JIL-1z60
is a strong hypomorph producing less than 0.3% of the levels of JIL-1
wild-type protein, resulting in semi-lethality of this heteroallelic
combination (Wang et al.,
2001; Zhang et al.,
2003). Consequently, in the control crosses, we observed only one
fly of the JIL-1z2/JIL-1z60 genotype out of a
total of 871 eclosed flies. However, in the double mutant combination
(JIL-1z2/JIL-1z60 Su(var)3-91) with
one copy of the Su(var)3-91 allele, the number of
surviving flies with the JIL-1z2/JIL-1z60
genotype increased dramatically to 243 out of a total of 789 eclosed flies. In
this cross, one-third of the eclosed flies would be expected to be of the
JIL-1z2/JIL-1z60 Su(var)3-91
genotype, assuming full rescue, indicating that the reduction of Su(var)3-9
activity in these animals resulted in a 92% viability rate compared with a
rate of 0.3% for JIL-1z2/JIL-1z60 flies without
the reduction in Su(var)3-9 activity. Consistent with the observed rescue of
viability, there was no detectable spreading of histone H3K9 dimethylation to
ectopic locations in polytene squashes from
JIL-1z2/JIL-1z60 Su(var)3-91 larvae,
and the morphology of the polytene chromosomes was markedly improved
(Fig. 4F).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We further provide evidence that JIL-1 activity and
localization are not affected by the absence of Su(var)3-9 activity,
suggesting that JIL-1 is upstream of Su(var)3-9 in this
pathway. Based on these findings, we propose a model where JIL-1 functions in
a novel pathway to establish or maintain euchromatic regions by antagonizing
Su(var)3-9-mediated heterochromatization.
According to the histone code hypothesis
(Strahl and Allis, 2000) and
the recently proposed binary switch model
(Fischle et al., 2003),
phosphorylation of a site adjacent to a methyl mark that engages an effector
molecule may regulate its binding. JIL-1 phosphorylates the histone H3S10
residue in euchromatic regions of polytene chromosomes
(Jin et al., 1999;
Wang et al., 2001), raising
the possibility that this phosphorylation at interphase prevents the
recruitment of Su(var)3-9 and/or the dimethylation of the neighboring K9
residue. This, in turn, would affect the binding of HP1, thus antagonizing the
formation of silenced heterochromatin at interbands. That different regions of
chromatin may have different combinations of posttranslational modifications
controlling effector/histone interactions, as predicted by the histone code
hypothesis (Strahl and Allis,
2000), is underscored by the finding that, in JIL-1 null
backgrounds, the level of the dmH3K9 marker and HP1 are preferentially
increased on the male and female X chromosomes. It is well documented that the
male X chromosome is unique because of the activity of the MSL dosage
compensation complex and the MOF histone acetyltransferase, which leads to
hyperacetylation of histone H4 (Bone et
al., 1994; Hilfiker et al.,
1997). However, comparable markers for the female X chromosome
have yet to be discovered, and our results are the first indication that
markers may exist that distinguish male and female X chromosomes from
autosomes, and that this difference may increase the affinity for Su(var)3-9.
That the spreading of heterochromatic markers in the absence of JIL-1 occurs
on both the male and female X chromosome further indicates that these changes
are independent of dosage compensation processes.
Unfortunately, in this study, we could not directly address the possibility
that the observed spreading of heterochromatin markers occurred preferentially
to specific euchromatic sites. In JIL-1 null and hypomorphic
backgrounds, chromosome morphology is greatly perturbed, and there is an
intermixing not only of euchromatin and the compacted chromatin characteristic
of banded regions, but also of non-homologous chromatid regions, which become
fused and confluent (Deng et al.,
2005). Thus, we cannot rule out as an alternative hypothesis that
JIL-1 activity may regulate boundary elements
(West et al., 2002) that
control the spreading of heterochromatic factors, or that the two mechanisms
may act in concert. However, the spreading of the dmH3K9 marker and HP1 to
ectopic locations on the chromosomes is likely to lead to heterochromatization
and repression of gene expression at these sites. Our results further suggest
the possibility that the lethality of JIL-1 null mutants may be due
to the repression of essential genes at these ectopic sites as a consequence
of the spreading of Su(var)3-9 activity. This hypothesis is supported by
genetic interaction assays that demonstrated that the lethality of a severely
hypomorphic JIL-1 heteroallelic combination could be almost
completely rescued by a reduction in Su(var)3-9 dosage that prevented
the ectopic dimethylation of histone H3K9.
It has recently been demonstrated that the Su(var)3-1 alleles of
JIL-1 consist of dominant gain-of-function alleles that antagonize
the expansion of heterochromatin formation
(Ebert et al., 2004). However,
we provide evidence that the underlying molecular mechanism of this antagonism
is different from that occurring in the loss-of-function null and hypomorphic
JIL-1 alleles described in this study.
JIL-1Su(var)3-1 alleles are characterized by deletions of
the COOH-terminal domain that do not affect JIL-1 kinase activity or the
spreading of heterochromatin markers (Ebert
et al., 2004) (this study). Furthermore, our results indicate that
the COOH-terminal domain of JIL-1 is required for proper chromosomal
localization and that JIL-1Su(var)3-1 proteins are mislocalized to
ectopic chromosome sites. Thus, we propose that the dominant gain-of-function
effect of the JIL-1Su(var)3-1 alleles may be attributable
to JIL-1 kinase activity at ectopic locations, possibly through the
phosphorylation of novel target proteins (see also
Ebert et al., 2004), or by
mis-regulated localization of the phosphorylated histone H3S10 marker.
Although the JIL-1Su(var)3-1 proteins are mislocalized, they still
associate with chromosomes and phosphorylate the histone H3S10 residue,
suggesting that other regions of the protein have a binding affinity for at
least some of the substrates and interaction partners of JIL-1. This is
supported by the finding that each of the two kinase domains of JIL-1 can
interact with the MSL-complex in vitro
(Jin et al., 2000).
In summary, we provide evidence that the JIL-1 kinase is a major regulator of histone modifications that affect gene activation, gene silencing and chromatin structure. Thus, it will be informative in future experiments to further explore the interaction of JIL-1 with genes controlling heterochromatin formation, in order to gain a better understanding of the molecular mechanisms of epigenetic gene regulation.
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ACKNOWLEDGMENTS |
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