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First published online 11 October 2006
doi: 10.1242/dev.02620
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Department of Developmental Genetics, National Institute of Genetics, and Department of Genetics, SOKENDAI, Mishima, Shizuoka-ken 411-8540, Japan.
Author for correspondence (e-mail:
shirose{at}lab.nig.ac.jp)
Accepted 7 September 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Supercoiling factor, Dosage compensation, Chromatin remodeling, MSL complex, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Bone et al.,
1994; Smith et al.,
2001). Although little is known about the molecular mechanisms
underlying the approximately twofold upregulation of X-linked genes, it is
likely that differences in chromatin structure play a key role in dosage
compensation.
At least five genes, msl-1, msl-2, msl-3, mle and mof,
have been identified in genetic screens for male-specific lethality
(Fukunaga et al., 1975;
Belote and Lucchesi, 1980;
Uchida et al., 1981;
Lucchesi et al., 1982;
Hilfiker et al., 1997). In
males, the products of these genes form an MSL complex that binds to numerous
sites along the X chromosome, but in females the complex fails to assemble as
a result of a translational block of the MSL-2 transcript by a protein called
Sex-lethal (Bashaw and Baker,
1995; Kelley et al.,
1995). In addition to these components, two noncoding RNAs,
rox1 and rox2, have also been identified as members of the
dosage compensation complex (Amrein and
Axel, 1997; Meller et al.,
1997; Franke and Baker,
1999). An essential histone H3 kinase, JIL-1, also associates with
the MSL complex (Jin et al.,
2000; Wang et al.,
2001).
Mutations in the genes that encode the Drosophila ISWI and NURF301
[E(bx) - FlyBase] components of the chromatin remodeling complex NURF cause an
X chromosome defect in males (the X chromosome is much less compact than
normal), and the phenotype can be alleviated by lowering acetylation levels on
the X chromosome. This suggests that ISWI ATPase activity and H4K16
acetylation counteract one another on the dosage-compensated X chromosome
(Deuring et al., 2000;
Corona et al., 2002;
Badenhorst et al., 2002). The
chromosome defect in the mutant strains is likely to be the result of an
imbalance caused by acetylation by MOF leading to a more open chromatin state,
without ISWI working toward a more condensed chromatin state
(Deuring et al., 2000).
However, it remains unclear whether the phenomenon is caused by functional
antagonism between the two factors already identified (MOF and ISWI), or
whether additional factors are also involved.
DNA supercoiling factor (SCF) was first identified in the silkworm as a
protein that generates negative supercoils in DNA in conjunction with
eukaryotic topoisomerase II (Ohta and
Hirose, 1990). A subsequent study revealed that a
Drosophila ortholog of SCF interacts with topoisomerase II in the
nucleus and localizes to puffs on polytene chromosomes
(Kobayashi et al., 1998),
suggesting a role for SCF in transcription on chromatin. Interestingly, recent
studies have shown that an ability to generate superhelical torsion in DNA is
shared by the ATP-dependent chromatin remodeling factors SWI/SNF, ISWI and
Mi-2 (Havas et al., 2000).
However, the supercoiling activities of these molecules, including that of
SCF, were demonstrated only in in vitro assay systems, and little is known of
their biological functions.
In this study, we analyzed the role of SCF in vivo. Although SCF may have a general function in the formation and maintenance of active chromatin, here we focused on the specific role of SCF in males. We chose this focus because knockdown of SCF resulted in male-specific reduction of X-linked gene expression and a male lethal phenotype. Our results illustrate a role for SCF in hypertranscription of X-linked genes via the alteration of chromatin structure after association of the MSL complex and acetylation of H4K16 along the male X chromosome. We propose that SCF counteracts ISWI action and forms (and/or maintains) transcriptionally active open chromatin.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
To construct Hsp83-SCF, the scf cDNA was amplified using a
5' SalI site-and FLAG-tag-containing primer
(5'-ACAATGTCGACAACATGGACTACAAGGACGACGATGACAAGATGCAGACTGTCTACGGCTTC-3'),
and a 3' NotI site-containing primer
(5'-TTCTAGGCGGCCGCCTAGAACTCATCGTGGCG-3'). The resulting fragment
was subcloned into pT7Blue and checked by sequencing. The scf cDNA
fragment was then excised with SalI and NotI, and cloned
into XhoI and NotI-digested pCaSpeR-Hsp83
(Horabin and Schedl,
1993).
Fly stocks and genetics
Flies were raised on standard agar/cornmeal/yeast medium.
UAS-IRscf and Hsp83-SCF transgenic flies were produced by
P-element-mediated transformation using the yw strain as a host, and
several independent lines were obtained. For each Hsp83-SCF
transgenic line, the levels of SCF were examined by immunoblotting. For rescue
experiments, a line expressing a similar level to endogenous SCF was selected.
More robust levels of expression were detected in another line, which exhibits
an X chromosome defect in males. The Act5C-GAL4 driver strain was a
gift from Yasushi Hiromi, National Institute of Genetics, Japan.
Df(3)Rac1 that lacks the scf locus
(Ng et al., 2002) was a gift
from Julian Ng. The msl-1
216 mutant line
was obtained from the Bloomington Stock Center. The [H83M2-6I]
transgenic line bearing the ectopic msl-2 gene under control of the
Hsp83 promoter, and the mof1 mutant line were
gifts from Yuji Kageyama, Nana Institute of Science and Technology, Japan.
P[ISWI+] (Deuring et
al., 2000) was a gift from J. Tamkun. The
P[ISWI+] chromosome was introduced into the
Hsp83-SCF line in order to overexpress both SCF and ISWI.
For RNAi assays, several independent UAS-IRscf lines were crossed to the Act5C-GAL4 driver strain at 25°C or 18°C. To generate control individuals that carry only Act5C-GAL4 for immunoblot, immunostaining and quantitative RT-PCR analyses, yw was crossed to yw;Act5C-GAL4/TM6B.
For immunostaining of mof mutant polytene chromosomes, mof1 male larvae were generated by crossing yw/Y males to mof1/mof1 females. To overexpress SCF in mof1, the P[Hsp83-SCF] transgene on the third chromosome was introduced into mof1; CyO, P[w+, mof+]/+ and F1 mof1/mof1; P[Hsp83-SCF] females were crossed to yw/Y; P[Hsp83-SCF] males.
Immunoblot analysis
Immunoblot analyses were performed using standard protocols
(Sambrook et al., 1989). To
prepare larval extracts, third instar larvae were homogenized in Laemmli
sample buffer. Extracts were separated by SDS-PAGE, transferred to a PVDF
membrane (Roche), probed with antibodies against SCF (1:1000), MSL-1 (1:500),
MSL-2 (1:500), MSL-3 (1:1000), MLE (1:500), MOF (1:500), ISWI (1:5000) or
-tubulin (1:1000) (all supplied by Sigma), followed by horseradish
peroxidase-linked anti-rabbit (1:5000), anti-mouse (1:3000) or anti-goat
(1:3000) IgG (Santa Cruz Biotechnology), and detected using SuperSignal
(Pierce).
Immunostaining of polytene chromosomes
Preparation of chromosomes and immunostaining were performed essentially as
described previously (Pile and Wassarman,
2002), with the following modifications. Salivary glands were
dissected in PBS and placed in a fixative containing 3.7% formaldehyde, 1%
Triton X-100 and PBS for 20 seconds, and then transferred to a fixative
containing 3.7% formaldehyde, 45% acetic acid for 90 seconds before squashing.
For spreads stained with anti-H4Ac16, salivary glands were dissected in PBS
containing 5 mM sodium butyrate as described by Turner et al.
(Turner et al., 1992).
Polytene chromosomes were then treated with purified antibodies against SCF
(1:25), MSL-1 (1:100), H4Ac16 (1:50, Serotec) or MSL-3 (1:1000), followed by
donkey anti-rabbit Cy2 (1:500) or anti-goat Cy3 (1:2000, Jackson
ImmunoResearch). We included appropriate controls in order to verify that the
signals are dependent on, and specific to, the primary antibody used, and that
there is no cross-reactivity of secondary antibodies under these conditions.
For staining with anti-SCF antibody, wash buffer containing 300 mM or 400 mM
NaCl, 0.2% Triton X-100, 0.2% NP-40 and PBS was used after primary antibody
treatment. Finally, the samples were mounted using VECTASHIELD mounting medium
containing DAPI (Vector Laboratories). At least six squash preparations were
analyzed for each antibody staining, and we verified that the staining pattern
was reproducible in the independent experiments.
Quantitative RT-PCR
Total RNA was isolated using Sepazol RNAI (Nakarai) from the six animals
collected at the time of puparium formation. The RNA was then treated with
RNase-free DNaseI (TaKaRa). To synthesize first strand cDNA, 1 µg of the
total RNA template was reverse transcribed with AMV-RTase (1st Strand cDNA
Synthesis Kit for RT-PCR; Roche). After the reverse transcription reaction,
the reaction mixture was diluted (from 1:10 to 1:50) and amplified by
quantitative PCR using the LightCycler-DNA Master SYBR Green I Kit (Roche) and
the following gene-specific primers:
ß1-tubulin (ßTub56D - FlyBase), 5'-AGTTCACCGCTATGTTCA-3' and 5'-CGCAAAACATTGATCGAG-3';
BR-C (br - FlyBase), 5'-ATGGACATGGTCTGCTCTAA-3' and 5'-GCTGCATGGAACATCTTGTT-3';
Pgd, 5'-GCCGGAGCTGTCTAATCTG-3' and 5'-CAGCAGCTCATAGGTGTG-3';
Rp49 (RpL32 - FlyBase), 5'-CCACCAGTCGGATCGATATG-3' and 5'-CACGTTGTGCACCAGGAACT-3';
Sgs4, 5'-GCGGATGTATTTTGAAGGAC-3' and 5'-CTTTTTGTGGCTGAGTCTTC-3';
Dspt4 (spt4 - FlyBase), 5'-AGTGGCAAAGATTGTCCC-3' and 5'-ATCGTTGACTTCTGTCCC-3';
Zw, 5'-TCTCATCCTCGACGTCTTCT-3' and 5'-ATTGTTCTCCTCGCACTTGC-3';
Gs2, 5'-TGCAGGAGAACATCGTTCAG-3' and 5'-TCCATCGTAGTTCCAAACGG-3';
mRpL16, 5'-GTATTTCGCTCCGCCTATTA-3' and 5'-CTTATGGAGCAGCGTGTTGT-3';
blue, 5'-TGGAGCAGCAGCGAAATGA-3' and 5'-TGTGTGCTCTGACTGCGTTGTA-3'; and
trio, 5'-GCCACCAAGTTCGCTCTGTA-3' and 5'-GGCCTTGATGATCTCCTTGA-3'.
The quantification value was then normalized to the value of the internal
standard ß1-tubulin as described previously
(Bhadra et al., 1999). Each
quantitative RT-PCR experiment was repeated at least three times using
independently prepared samples. For quantification of rox1 and
rox2 expression levels (Fig.
2A), the reverse transcription reaction products prepared as
described above were diluted (from 1:3 to 1:27) and amplified by PCR using
TaKaRa Ex Taq (TaKaRa) and the following gene-specific primers: rox1,
5'-CAAATGTCCTGCAGAAGAGG-3' and
5'-ATGGTTGGTTATTCGGGTGG-3'; rox2,
5'-AGATGTTGCGGCATTCGCGG-3' and
5'-TGCGACTTGTACAATGTTGCG-3'. The PCR products were resolved by gel
electrophoresis, stained with SYBR Green (Molecular Probes), and analyzed
using an LAS-1000 luminescent image analyzer (Fujifilm).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Kennerdell and Carthew, 2000;
Piccin et al., 2001). We
employed this method to knockdown scf function throughout
development. To do this, we generated transgenic fly lines carrying a
UAS-IRscf insert consisting of the GAL4-upstream activating sequence
(UAS) and an inverted repeat (IR) of a part of the scf gene. Several
independent transformants were crossed with flies carrying the yeast GAL4 gene
driven by the Actin5C promoter (Actin5C-GAL4) in order to
constitutively activate transcription from the hairpin-encoding transgene. To
estimate the effect of RNAi in the progeny, we examined expression levels of
SCF in late third-instar larvae by immunoblot analysis. When the transgenic
line 2-1 carrying the UAS-IRscf construct was crossed with the
Actin5C-GAL4 driver line, SCF protein levels were markedly reduced in
male and female progeny that carried both the GAL4 driver and the
UAS-IRscf transgene (Fig.
1A). Comparable results were obtained for transgenic lines 3-1 and
3-2 (data not shown). Next, we examined the effect of scf knockdown
on viability, and found a specific and dramatic reduction in the viability of
males carrying both GAL4 and UAS-IRscf
(Fig. 1B). Most of these males
died during the pupal stage, and the few individuals that did survive to
adulthood died shortly after eclosion. The effects on male viability do not
appear to be due to position effects, as the three independent transgenic
lines (2-1 on the second chromosome, and 3-1 and 3-2 on the third chromosome)
exhibited this phenotype. Whereas male viability was severely affected in all
three lines, the effects on female viability were less pronounced and varied
among the lines tested. No significant effect on females was observed for the
2-1 transgenic line (Fig. 1B).
In an experiment with the 2-1 line, we changed the temperature from 25°C
to 18°C because the activation levels in the GAL4/UAS system are
dramatically affected by temperature, and RNAi using this system at 18°C
is less effective than that at 25°C
(Fig. 1A, lanes 9-12). We found
that the reduction in male viability was significantly suppressed at 18°C
(Fig. 1B). These data show that
males are more sensitive than females to SCF knockdown. The similar decrease
in the amount of SCF protein in both sexes upon RNAi
(Fig. 1A) excludes the
possibility that the observed male lethality is due to a difference in the
extent of RNAi in the two sexes.
|
; Gorman et al.,
1995; Kelley et al.,
1995; Gu et al.,
2000). This led us to the idea that SCF might be involved in
dosage compensation. To test this possibility, we first examined the effect of
SCF knockdown on the expression levels of each MSL component
(Fig. 2A). Almost no difference
in the level of MSL-1, MSL-2, MSL-3, MLE or MOF protein or rox1 or
rox2 RNA was observed between the SCF-reduced background and the
control. This suggests that the male lethal phenotype caused by knockdown of
SCF is not due to reduced expression of the MSL components.
We next examined the possibility that SCF is involved in dosage
compensation via an interaction with the MSL complex. Two different analyses
were performed to test for genetic interactions between scf and the
msl alleles. First, we examined the ability of an scf
deficiency to prevent the lethality caused by the forced expression of MSL-2
protein in females (Fig. 2B).
The MSL-2 protein is not normally expressed in females and its ectopic
expression results in the appearance of the MSL complex on both X chromosomes,
leading to an abnormal regulation of X-linked genes and developmental delay or
decreased viability in females (Kelley et
al., 1995). Suppression of the misexpression phenotype provides
evidence that the mutation is involved in dosage compensation via function of
the MSL complex. Previous studies have shown that the abnormal development is
rescued in homozygotes for mle, msl-3 or rox mutant alleles,
and in heterozygotes for msl-1 or mof mutant alleles
(Kelley et al., 1995;
Hilfiker et al., 1997;
Meller and Rattner, 2002). We
found that the presence of the scf deficiency also significantly
rescued females ectopically expressing MSL-2
(Fig. 2B). Moreover, the rescue
was suppressed by introducing an Hsp83-driven scf cDNA
fragment. We also note that, in this case, the control sisters were more
severely affected by the misexpression of MSL-2 than lines carrying no
scf transgene.
Next, we examined the effect of combining the scf-deficient allele
and the msl-1 mutant allele on male viability
(Table 1). We observed a
significant reduction in male viability in double heterozygotes
(P<0.001, 
2 test; see
Table 1, row three) when
compared with each single allele (Table
1, rows one and two). Furthermore, the reduction in the viability
of these animals was also significantly suppressed by introducing the
Hsp83-driven scf cDNA fragment
(Table 1, row four). These data
demonstrate a functional interaction between SCF and the MSL complex.
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|
),
differences between the distribution of SCF in males and females, and between
the X-chromosome and autosomes remain to be addressed. We performed
immunostaining of SCF on polytene chromosomes prepared from both males and
females (Fig. 3A-E). We found many extra SCF signals along the male X chromosome that could be clearly identified by co-detection of MSL-3 (Fig. 3C), in comparison to what was observed for the female X chromosome (Fig. 3, compare B with E). On the male X chromosome, most of the SCF signals were detectable as fine bands or dots. These signals were weak when compared with the robust staining observed in discrete interbands and puffs of autosomes, and at some sites on the X chromosome. Whereas the intense staining pattern was the same in both males and females (e.g. see arrowheads in Fig. 3B,E), the `milky way'-like staining pattern consisting of many faint signals between the robust staining sites was specific to the male X chromosome (Fig. 3B,E). This suggests the presence of a relatively small amount of SCF on each of the many male X-specific faint signals. Furthermore, the male X-specific faint signals of SCF, but not the robust bands, colocalized with MSL-3, which is a component of the MSL complex (Fig. 3F-I). These observations support the idea that SCF regulates gene expression in the context of dosage compensation.
Knockdown of SCF does not disrupt localization of MSLs on the male X chromosome
To investigate the possible role of SCF in MSL complex-mediated dosage
compensation, we first examined whether the knockdown of SCF by RNAi affects
binding of the MSL complex to the X chromosome. We performed immunostaining of
polytene chromosomes with anti-MSL-1 antibody because MSL-1 plays a central
role in assembly of the MSL complex and is thus a good indicator of complex
formation (Scott et al.,
2000). The MSL-1 protein localized to the male X chromosome even
under scf RNAi conditions (Fig.
3, compare J with K). Consistent with this, immunostaining with
anti-acetylated H4K16 antibody showed that acetylation of H4K16 along the male
X chromosome was not affected by RNAi (Fig.
3, compare L with M). These results indicate that SCF is not
necessary for association of the MSL complex with chromatin or for the
subsequent H4K16 acetylation on the male X chromosome.
Histone acetyltransferase MOF is required for the proper localization of SCF along the male X chromosome
Taken together, the data presented thus far indicate that, if SCF
participates in dosage compensation, it does so only after association of the
MSL complex and H4K16 acetylation by MOF on the male X chromosome. To test
this, we looked at the distribution of SCF protein on polytene chromosomes in
a mof mutant background.
Compared with wild type (Fig. 3B), the many faint signals of SCF normally observed on the male X chromosome were significantly reduced in the mof mutant background (Fig. 3N,O). By contrast, the prominent SCF bands on the X chromosome and autosomes were not affected. These results strongly suggest that MOF activity is necessary for the male-specific pattern of localization of SCF on the male X chromosome.
|
;
Straub et al., 2005). If SCF
is truly involved in dosage compensation, the reduction of SCF function should
change or abolish the upregulation of X-linked genes. To test this, we
examined the effects of SCF RNAi on the expression levels of X-linked genes
using quantitative RT-PCR (Fig.
4). Six X-linked genes were examined: Pgd, Sgs-4, Zw,
Broad-Complex (BR-C), Gs2 and mRpL16. Each of
these genes has been shown to be dosage compensated in an MSL
complex-dependent manner (Belote and
Lucchesi, 1980; Smith et al.,
2001; Chiang and Kurnit,
2003; Hamada et al.,
2005; Straub et al.,
2005). The levels of transcripts from these genes, normalized to
the value of ß1-tubulin as an internal standard
(Bhadra et al., 1999), were
compared between individuals deficient in SCF function by RNAi and control
individuals for both sexes.
We found that the expression levels of all six X-linked genes were
significantly reduced in males but not in females deficient in the SCF
function (Fig. 4, X
chromosome). By contrast, the autosomal control genes Rp49, Dspt4,
blue and trio were not affected by the knockdown of SCF in
either sex (Fig. 4, Autosome).
The extent of male-specific reduction in X-linked gene expression upon SCF
RNAi was similar to that observed for MSL-2 RNAi
(Hamada et al., 2005;
Straub et al., 2005). These
results indicate that SCF is involved in the final step of the dosage
compensation; that is, hypertranscription of the male X chromosome
Overexpression of SCF causes a male X chromosome defect
In the larval salivary gland, overexpression of MSL-1 and MSL-2 causes
aberrant male X chromosome morphology (Oh
et al., 2003). The defective morphology is similar to that
observed for Iswi or nurf301 loss-of-function mutants in
that, in all cases, the male X chromosome appears to be much less compact than
the autosomes or female X chromosomes
(Deuring et al., 2000;
Corona et al., 2002;
Badenhorst et al., 2002),
suggesting an unusual chromatin organization. Interestingly, we found that the
male X chromosome is similarly affected in individuals overexpressing SCF from
the Hsp83-SCF transgene (Fig.
5A-F). The male X chromosome, identified by anti-MSL-1 staining,
was strikingly different in its appearance to the yw control,
exhibiting a bloated appearance and a partial loss of its banding pattern. By
contrast, the morphology of autosomes and the female X chromosomes appeared
normal. To see whether the morphological defect is accompanied by an increased
amount of SCF on the chromosome, we performed anti-SCF staining in this
transgenic line. Compared with the endogenous level, a strong staining was
observed along the aberrant male X chromosome
(Fig. 5E,F). However, the
disrupted banding pattern of the male X chromosome prevented us from
determining whether the staining pattern of SCF or MSLs was altered.
Immunoblot analyses confirmed that SCF was indeed overexpressed in the
Hsp83-SCF transgenic line, and revealed that the bloated appearance
of the male X chromosome was not due to reduced expression of ISWI
(Fig. 6). The defect was
specific to overexpression of SCF, as we were unable to observe it upon
overexpression of unrelated chromatin proteins, such as the GAGA factor,
SPT16, SSRP1 and SPT6 (M.N. and S.H., unpublished). Taken together, the
results suggest that SCF promotes opening of the chromatin structure.
To further understand the functional relationship between SCF and ISWI, we carried out a genetic study. The male X chromosome defect associated with overexpression of SCF was suppressed by simultaneous overexpression of ISWI (Fig. 5G), suggesting that SCF counteracts the ISWI function. Consistent with the observation that MOF activity is necessary for the proper loading of SCF on the male X chromosome, the overexpression phenotype of SCF was also suppressed by the loss-of-function mutation of mof (Fig. 5H) that abolishes acetylation of H4K16 (Fig. 5I). From these data, we conclude that SCF plays a role in the alteration of chromatin structure to accomplish hyperactivation of the male X chromosome.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Immunostaining of polytene chromosomes revealed that SCF is distributed along
the male X chromosome in the form of many faint signals that are weaker in
intensity than the discrete and prominent signals on autosomes or female X
chromosomes (Fig. 3). Most of
the faint signals of SCF colocalize with the MSL complex. Analysis using a
mof mutant showed that the histone acetyltransferase activity is
necessary for the proper loading of SCF on the male X chromosome. It is most
likely that the MSL complex per se, and/or its function, including the
acetyltransferase activity of MOF, creates many low-affinity sites that
promote SCF association on the male X chromosome. This idea is supported by
the weak interactions between SCF and the MSL components observed in
co-immunoprecipitation assays using embryonic nuclear extracts (H.F. and S.H.,
unpublished). Owing to its relatively weak affinity for the male X chromosome,
we speculate that the threshold for maintaining SCF function along this
chromosome is higher than that for maintaining SCF function at the other
genomic sites. Hence, males are thought to be more sensitive than females to a
reduction in SCF levels. However, we cannot exclude the formal possibility
that the relatively weak staining might be caused by other factors, such as
differences in epitope availability in each context, or loss of the antigen
upon fixation for reasons unrelated to true affinity.
|
), and further extend the model by suggesting underlying
mechanisms. It has been proposed that the generation of superhelical torsion
in DNA by chromatin remodeling activities could affect the formation of higher
order chromatin structure (Havas et al.,
2000). In addition, recent single-molecule analyses of chromatin
remodeling reactions have revealed that the SWI/SNF-type complexes introduce
negative supercoils into DNA, whereas the ISWI-type remodelers generate
positive supercoils as reaction intermediates
(Lia et al., 2006) (T.
Owen-Hughes, personal communication). Therefore, the negative supercoiling
activity of the SCF-topoisomerase II complex might function synergistically
with the SWI/SNF-type complexes and counteract ISWI-type remodeling factors.
This idea is supported by our finding that the bloated appearance of the male
X chromosome in the SCF-overexpressing line is similar to that observed in the
loss-of-function of Iswi, and is suppressed by overexpression of
ISWI. Moreover, acetylation of H4K16 has been shown to inhibit ISWI activity
(Corona et al., 2002). These
data suggest that SCF function and acetylation of H4K16 cooperate to form
and/or maintain transcriptionally active open chromatin on the male X
chromosome by counteracting ISWI action.
Another possible mechanism of SCF action is facilitation of transcription
elongation on chromatin. The X-linked dosage compensated genes Pgd
and Zw are known to be more highly acetylated in the coding region
than in the promoter region (Smith et al.,
2001). This suggests that the dosage-compensation machinery might
function in the transcription elongation step. As transcription proceeds, a
positively supercoiled domain should be formed in front of the RNA polymerase,
and a negatively supercoiled domain should be generated behind it
(Liu and Wang, 1987).
Transcription-coupled supercoiling has been documented using a yeast mutant
defective in topoisomerases (Giaever and
Wang, 1988). Recently, Matsumoto and Hirose observed the same
effect on Drosophila polytene chromosomes, even in the presence of
active topoisomerases (Matsumoto and
Hirose, 2004). The generation of negative supercoils by the action
of SCF may facilitate smooth tracking of the polymerase and the reassembly of
nucleosomes after the polymerase has passed. These processes might also be
coupled with replacement of histone H3 by its variant H3.3 during
transcription, culminating in maintenance of the active state through cell
division (Henikoff et al.,
2004; Mito et al.,
2005).
|
In summary, we found that SCF affects dosage compensation in Drosophila, and showed that SCF plays an important role in the alteration in chromatin structure required to execute hyperactivation of the male X chromosome. Further investigation to clarify the relationships between supercoiling activity and the topological status of the active chromosomal domain may provide new insights into the mechanisms of chromosome-wide gene regulation.
|
ACKNOWLEDGMENTS |
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Footnotes |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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scf/TM6B females (left) or
[Hsp83-SCF];
