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First published online 30 July 2008
doi: 10.1242/dev.024927
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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 3 July 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Pol II, Histone H3S10 phosphorylation, Transcription, Chromatin structure, JIL-1 kinase
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
JIL-1 is essential for viability and 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) as well as
to extensive ectopic spreading of heterochromatic factors
(Zhang et al., 2006). These
findings suggested a model in which maintenance of histone H3S10
phosphorylation levels at euchromatic chromatin regions is necessary to
counteract heterochromatization and gene silencing
(Wang et al., 2001;
Ebert et al., 2004;
Zhang et al., 2006;
Bao et al., 2007).
Recently, based on analyses of transcriptionally active regions during the
heat shock response (Nowak and Corces,
2000; Nowak et al.,
2003; Ivaldi et al.,
2007), an alternative model was proposed in which JIL-1 is
required for transcription by the RNA polymerase II (Pol II) machinery
(Ivaldi et al., 2007).
According to this model, rather than contributing to global chromosome
structure, JIL-1-mediated histone H3S10 phosphorylation maintains a local
chromatin environment that serves as a platform for the recruitment of the
Positive transcription elongation factor b (P-TEFb; Cdk9 - FlyBase) and the
consequent release of Pol II from promoter-proximal pausing
(Ivaldi et al., 2007). Ivaldi
et al. further suggested that histone H3S10 phosphorylation by JIL-1 is a
hallmark of early transcriptional elongation in Drosophila and that
this histone modification is required for the transcription of the majority,
if not all, genes in this organism (Ivaldi
et al., 2007). However, this model is contradicted by the findings
of Deng et al. (Deng et al.,
2007), which demonstrate that the lethality as well as some of the
chromosome morphology defects associated with the null JIL-1
phenotype can, to a large degree, be rescued by reducing the dose of the
Su(var)3-9 gene. Su(var)3-9 is a histone methyltransferase that is
necessary for pericentric heterochromatin formation
(Schotta et al., 2002;
Elgin and Reuter, 2007) and
that plays an important role in silencing of reporter genes by heterochromatic
spreading (reviewed by Weiler and
Wakimoto, 1995; Girton and
Johansen, 2008). If JIL-1 had a crucial role in promoting
transcription at a majority of genes by regulating transcriptional elongation,
it is difficult to envisage how lethality can be rescued to near wild-type
levels in the complete absence of JIL-1 and interphase histone H3S10
phosphorylation (Deng et al.,
2007). Furthermore, Deng et al. recently showed that
JIL-1-mediated ectopic histone H3S10 phosphorylation is sufficient to induce a
change in higher-order chromatin structure from a condensed
heterochromatin-like state to a more open, euchromatic state, and that these
changes are not associated with enhanced transcriptional activity
(Deng et al., 2008). Thus,
these findings are incompatible with the transcriptional elongation model for
JIL-1 function and we therefore attempted to repeat the experiments on which
it is based using three different histone H3S10 phosphorylation (H3S10ph)
antibodies, as well as a newly developed acid-free polytene chromosome squash
technique (DiMario et al.,
2006) that preserves the antigenicity of the H3S10
phospho-epitope. We show that many of the key findings of Nowak and Corces
(Nowak and Corces, 2000),
Nowak et al. (Nowak et al.,
2003) and Ivaldi et al.
(Ivaldi et al., 2007) are
likely to be artifacts caused by non-specific antibody cross-reactivity and by
fixation procedures that are not suitable for reliable antibody detection of
interphase phosphorylated histone H3S10. Taken together, the results of Deng
et al. (Deng et al., 2007;
Deng et al., 2008) and the
findings presented here are inconsistent with the model of Ivaldi et al.
(Ivaldi et al., 2007) that Pol
II-dependent transcription at active loci requires JIL-1-mediated histone
H3S10 phosphorylation, and instead support a model in which transcriptional
defects in the absence of histone H3S10 phosphorylation are the result of
structural alterations of chromatin.
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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 null allele
has been described (Wang et al.,
2001; Zhang et al.,
2003), as has the recombined JIL-1z2
Su(var)3-906 chromosome
(Deng et al., 2007). The
P-element insertion mutant allele tws02414 was obtained
from the Bloomington Stock Center and the twsP allele
(Mayer-Jaekel et al., 1993)
was the generous gift of Dr D. M. Glover (University of Cambridge, Cambridge,
UK). Balancer chromosomes and markers have been described
(Lindsley and Zimm, 1992). For
heat shock experiments, wandering third instar larvae were subjected to 25
minutes of heat shock treatment at 37°C as described previously
(Nowak et al., 2003).
Immunohistochemistry
Salivary gland nuclei smush preparations were made as described
(Wang et al., 2001), standard
polytene chromosome squash preparations were made
(Kelley et al., 1999) using
the 5-minute fixation protocol, and acid-free squash preparations followed the
procedure of DiMario et al. (DiMario et
al., 2006). Antibody labeling of these preparations was performed
as described (Jin et al.,
1999; Wang et al.,
2001). Primary antibodies used include rabbit anti-H3S10ph
(Epitomics, Upstate and Cell Signaling), mouse anti-H3S10ph (Upstate and Cell
Signaling), rabbit anti-histone H3 (Cell Signaling), mouse anti-lamin
Dm0 (Gruenbaum et al.,
1988), rabbit anti-Hsf (gift from Dr C. Wu, National Cancer
Institute, Bethesda, MD), mouse anti-Pol IIoser2 (H5, Covance),
rabbit anti-JIL-1 (Jin et al.,
1999), chicken anti-JIL-1 (Jin
et al., 2000) and anti-JIL-1 mAb 5C9
(Jin et al., 2000). 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. A separate series of confocal images
for each fluorophor of double labeled preparations was obtained simultaneously
with z-intervals of typically 0.5 µm using a PL APO 63x/1.40
oil objective. A maximum projection image for each of the image stacks was
obtained using ImageJ software
(http://rsb.info.nih.gov/ij/).
Images were imported into Photoshop (Adobe) where they were pseudocolored,
image processed, and merged. In some images, non-linear adjustments were made
to the channel with Hoechst labeling for optimal visualization of
chromosomes.
Immunoblot analysis
Protein extracts were prepared from salivary glands dissected from third
instar larvae (or, in some experiments, from whole larvae) homogenized in a
buffer comprising: 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM EDTA, 1 mM EGTA,
0.2% Triton X-100, 0.2% NP40, 2 mM Na3VO4, 1 mM PMSF,
1.5 µg/ml aprotinin. Proteins were separated by SDS-PAGE according to
standard procedures (Laemmli,
1970). Electroblot transfer was performed as described
(Towbin et al., 1979) with
transfer buffer containing 20% methanol and, in most cases, including 0.04%
SDS. For these experiments, we used the Bio-Rad Mini Protean II system,
electroblotting to 0.2 µm pore size nitrocellulose, and using anti-mouse or
anti-rabbit HRP-conjugated secondary antibody (Bio-Rad; 1:3000) for
visualization of primary antibody. A buffer comprising 150 mM NaCl, 100 mM
Tris pH 7.5, 0.1% Tween 20 and 5% BSA was used for blocking procedures in
experiments using the Pol IIoser2 mAb H5 as per the manufacturer's
instructions (Covance). Antibody labeling was visualized using
chemiluminescent detection methods (SuperSignal West Pico Chemiluminescent
Substrate, Pierce). The immunoblots were digitized using a flatbed scanner
(Epson Expression 1680).
Analysis of gene expression by qRT-PCR
Total RNA was extracted from 12 pooled whole third instar larvae of each
genotype [wild type, JIL-1z2/JIL-1z2,
and JIL-1z2/JIL-1z2,
Su(var)3-906] with or without heat shock using the
MicroPoly(A)Purist Small-Scale mRNA Purification Kit (Ambion) following the
manufacturer's instructions. cDNA derived from this RNA using SuperScript II
Reverse Transcriptase (Invitrogen) was used as template for quantitative
real-time (qRT) PCR performed with the Stratagene Mx4000 real-time cycler. In
addition, the PCR mixture contained Brilliant II SYBR Green QPCR Master Mix
(Stratagene) as well as the corresponding primers: rp49,
5'-AACGTTTACAAATGTGTATTCCGACC-3' and
5'-ATGACCATCCGCCCAGCATACAGG-3'; Hsp70,
5'-GTCATCACAGTTCCAGCCTACTTCAAC-3' and
5'-CTGGGTTGATGGATAGGTTGAGGTTC-3'. Cycling parameters were 10
minutes at 95°C, followed by 40 cycles of 30 seconds at 95°C, 60
seconds at 59°C, and 40 seconds at 72°C. Fluorescence intensities were
plotted against the number of cycles using an algorithm provided by
Stratagene. mRNA levels were quantified using a calibration curve based on
dilution of concentrated cDNA. mRNA values from the larvae were normalized to
that of rp49 (RpL32 - FlyBase).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Wei et al., 1998) and, as a
result, they are poorly characterized with regard to detection of interphase
histone H3S10 phosphorylation levels and distribution. Consequently, it is
important to verify the specificity and suitability of these antibodies for
experimental use at interphase to avoid artifacts. In this study, we have
adapted the `smush' preparation of Drosophila third instar salivary
gland nuclei as a rapid and sensitive screening procedure for such antibodies.
The smush preparation is a modified whole-mount staining technique in which
nuclei from dissected salivary glands are gently compressed beneath a
coverslip to flatten them before fixation in a standard paraformaldehyde/PBS
solution of physiological pH (Wang et al.,
2001). The procedure takes advantage of the finding that JIL-1 is
the kinase responsible for interphase histone H3S10 phosphorylation
(Wang et al., 2001) and that
both JIL-1 and H3S10 phosphorylation are upregulated on the male X chromosome
(Wang et al., 2001).
Consequently, reliable histone H3S10ph antibodies would be expected to show
co-localization with JIL-1, to show upregulation on the male X chromosome, and
all labeling should be absent from homozygous JIL-1 null nuclei as
illustrated in Fig. 1. The
upregulation of H3S10ph labeling on the male X chromosome is a particularly
useful indicator of proper antibody recognition. Furthermore, on immunoblots
of salivary gland protein extracts in JIL-1 null mutant backgrounds,
histone H3S10 labeling should be absent or greatly reduced as well. It should
be noted that whole larval extracts are not suitable for such immunoblots
because of the presence of a significant population of mitotic nuclei (>5%)
in which the H3S10 residue is phosphorylated by the Aurora B kinase
(Giet and Glover, 2001). Using
these criteria, we screened various monoclonal (mAb) and polyclonal (pAb)
antibodies from three different manufacturers. The results are summarized in
Table 1. We found that several
of the antibodies failed to fulfil one or more of the above criteria and that
different lots of the same antibody had, in some cases, different properties.
Of the suitable antibodies, the rabbit mAb from Epitomics, the rabbit pAb
(lots 2 and 3) from Cell Signaling, and the rabbit pAb (lot 32219) from
Upstate, were selected for further use in the present studies.
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), the highly acidic fixation conditions of conventional
squash protocols (Zink and Paro,
1989; Kelley et al.,
1999) prevent reliable antibody labeling of the histone H3S10
phospho-epitope. In such preparations, H3S10ph antibody labeling is extremely
weak and, except for rare cases, the upregulation of H3S10 phosphorylation on
the male X chromosome (Wang et al.,
2001) (data not shown) cannot be detected, indicating incomplete
or defective antibody recognition. In this study, to overcome these
difficulties we have adopted the acid-free squash technique of DiMario et al.
(DiMario et al., 2006), which
was originally developed to preserve the fluorescence of GFP-tagged proteins
in fixed preparations. As illustrated in
Fig. 2A-C, this technique also
preserves the antigenicity of the H3S10 phospho-epitope as indicated by the
robust antibody labeling in both male and female squash preparations by three
different H3S10ph antibodies, including upregulation on the male X chromosome.
The extensive co-localization of H3S10ph with JIL-1 is particularly evident in
the confocal images in Fig. 2A.
Furthermore, on immunoblots of protein extracts from third instar larval
salivary glands, H3S10ph labeling was greatly reduced in JIL-1 null
mutant backgrounds confirming that the antibodies recognized the H3S10ph
epitope. However, it should be noted that the Epitomics H3S10ph antibody, in
contrast to the other two antibodies, showed strong labeling of the
chromocenter (Fig. 2B,
asterisks). Although it was more difficult to properly spread the chromosomes
and the chromatin structure, as labeled by Hoechst, was slightly less
well-preserved in acid-free squashes than in conventional squash preparations,
our data strongly suggest that the acid-free squash procedure is the method of
choice in all antibody labeling studies of histone H3S10 phosphorylation in
polytene squash preparations.
JIL-1 and histone H3S10 phosphorylation are not upregulated at transcriptionally activated loci during heat shock
To determine the distribution of JIL-1 before and after heat shock, we
double labeled polytene chromosome squash preparations with the JIL-1 mAb 5C9
and with antibody to the elongating form of RNA polymerase II (Pol
IIoser2), which is phosphorylated at serine 2 in the C-terminal
domain and which serves as a marker for active transcription
(Weeks et al., 1993;
Boehm et al., 2003;
Ivaldi et al., 2007). In
non-heat shock preparations, both JIL-1 and Pol IIoser2 were
localized to a large number of euchromatic interband regions
(Fig. 3A, left panel). The
composite image in Fig. 3A
(left panel) further shows that although there may be some co-localization
between JIL-1 and Pol IIoser2 as previously reported
(Ivaldi et al., 2007),
relatively low levels of JIL-1 were observed at many sites where there were
especially high levels of Pol IIoser2 staining, such as at
developmental puffs. After 25 minutes of heat shock treatment, there was a
striking change in the distribution of Pol IIoser2 labeling,
whereas, by contrast, there was no appreciable redistribution of JIL-1. The
Pol IIoser2 labeling was reduced at most sites, while being
upregulated at heat shock puffs where transcription of heat shock-activated
genes was occurring. This was especially prominent at the heat shock loci
87A/C and 93D (Fig. 3B).
Notably, there were no indications of a concomitant upregulation of JIL-1 at
these sites (Fig. 3B). This
result was confirmed using two other JIl-1 antibodies (a chicken pAb and a
rabbit pAb; data not shown).
We next used the acid-free polytene squash technique to determine the distribution of H3S10ph before and after heat shock using three different H3S10ph antibodies (from Epitomics, Cell Signaling and Upstate). To mark heat shock puffs and other regions of enhanced transcription, the preparations were double labeled with antibody to Pol IIoser2. As illustrated in Fig. 4A-D, there was no obvious change in H3S10ph distribution before and after heat shock as detected by the Epitomics and Cell Signaling H3S10ph antibodies. Importantly, as also observed for JIL-1 (Fig. 3B), there was no upregulation at the 87A/C heat shock puffs, although they were robustly labeled by the Pol IIoser2 antibody (Fig. 4B,D). By contrast, we found that the Upstate H3S10ph antibody strongly labeled the 87A/C puffs after heat shock (Fig. 4G,F). However, contrary to the Cell Signaling and Epitomics H3S10ph antibodies, the Upstate pAb also labeled heat shock puffs in polytene chromosome squashes from JIL-1 null mutant larvae, which are devoid of histone H3S10ph phosphorylation (Fig. 5). Because similar results were obtained with two different lots of the Upstate H3S10ph pAb (Table 1), we conclude that the labeling of heat shock puffs by this antibody is due to non-specific cross-reactivity, possibly with proteins involved in the heat shock response. Furthermore, immunoblot analysis with all three H3S10ph antibodies of protein extracts from salivary glands before and after heat shock confirmed that there was no change in the overall level of histone H3S10 phosphorylation (Fig. 4G) as indicated by the polytene squash labelings (Fig. 4A,C,E). By contrast, there was a clear downregulation in the levels of Pol IIoser2 in response to heat shock (Fig. 4H).
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). Using a P-element insertion mutation into the regulatory
subunit of PP2A, twsP, that causes reduced catalytic
activity (Mayer-Jaekel et al.,
1993), Nowak et al. showed that on immunoblots of extracts from
twsP mutant larvae, there is a higher level of H3S10
phosphorylation than in wild-type larvae
(Nowak et al., 2003). This
difference was attributed to reduced PP2A phosphatase activity, indicating
that PP2A might function as a H3S10ph phosphatase at interphase
(Nowak et al., 2003) in
addition to its role as a mitotic H3S10ph phosphatase
(Mayer-Jaekel et al., 1993).
However, because whole larval extracts were used it remained a possibility
that the increased upregulation of H3S10ph levels was due solely to decreased
dephosphorylation of mitotic H3S10ph. Using a likely null PP2A regulatory
subunit P-element mutation, tws02414, we confirmed a
higher level of H3S10 phosphorylation in extracts from homozygous
tws02414 mutant larvae as compared with wild-type larvae
(Fig. 6A). However, when
extracts were compared from salivary glands, which do not contain mitotic
cells, there was no difference (Fig.
6B). Furthermore, in extracts from salivary glands of homozygous
twsP mutant larvae with or without heat shock there was no
difference in H3S10 phosphorylation levels as detected by the Epitomics
H3S10ph mAb (Fig. 6C). Taken
together, these results indicate that the PP2A phosphatase might play a role
in H3S10 dephosphorylation only at mitosis and not at interphase.
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). This suggests that the Pol II transcriptional machinery has
the capacity to function more or less normally in the complete absence of
JIL-1-mediated interphase histone H3S10 phosphorylation. We therefore
investigated the distribution of Pol IIoser2 labeling and heat
shock-induced transcription in JIL-1z2/JIL-1z2
null as well as in JIL-1z2/JIL-1z2
Su(var)3-906 double mutant backgrounds. In
JIL-1z2/JIL-1z2 Su(var)3-906 larvae,
the adult eclosion rate increases to 60% of that of wild-type larvae as
compared with 0% for JIL-1z2/JIL-1z2 null
larvae (Deng et al., 2007).
Fig. 7A shows robust antibody
labeling of Pol IIoser2 in polytene chromosome squashes from both
genotypes, even though the chromatin structure was greatly perturbed. This
included the characteristic `puffed' male X chromosome in JIL-1 null
larvae (Fig. 7A, upper panel).
Furthermore, on immunoblots of extracts from wild-type,
JIL-1z2/JIL-1z2, and
JIL-1z2/JIL-1z2 Su(var)3-906
salivary glands, there was no detectable difference in Pol IIoser2
levels (Fig. 7B). This
indicates that transcript elongation by the Pol II machinery is likely to be
functional in JIL-1 null mutant backgrounds. To further investigate
this possibility, we double labeled JIL-1 mutant polytene chromosome
squashes, after they were heat shocked, with Pol IIoser2 antibody
and with antibody to the heat shock transcription factor Hsf
(Fig. 8A). When inactive, Hsf
is diffusely distributed at very low levels; however, following heat shock,
Hsf redistributes very prominently to heat shock-induced puffs
(Westwood et al., 1991;
Ivaldi et al., 2007). As shown
in Fig. 8A, although the
chromosome morphology was greatly disrupted, puffed regions could be clearly
identified in JIL-1 null mutant backgrounds as defined by the
presence of decondensed chromatin and strong Hsf antibody labeling.
Importantly, these heat shock-induced puffs were also strongly labeled by Pol
IIoser2 antibody in a pattern coincident with that of the Hsf
antibody (Fig. 8A, arrows).
Furthermore, as also confirmed by immunoblot analysis
(Fig. 8B), Pol
IIoser2 levels were greatly reduced at non-heat shock sites
(Fig. 8A). This suggests that
Pol IIoser2 is actively involved in heat shock-induced
transcription in JIL-1 null mutants. To test this directly, we used
qRT-PCR to measure the transcription of the six, nearly identical, genes that
encode Hsp70, the major heat shock protein in Drosophila
(Gong and Golic, 2004), under
heat shock and non-heat shock conditions. Primers were designed that would
amplify transcripts from all six Hsp70 genes, and primers specific to
the gene encoding the ribosomal non-heat-shock-sensitive protein Rp49
[Ribosomal protein L32 (RpL32) - FlyBase] were used for normalization. We
performed two independent experiments in which total RNA was isolated from
wild-type, JIL-1z2/JIL-1z2, and
JIL-1z2/JIL-1z2 Su(var)3-906 third
instar larvae, and in which qRT-PCR determination of transcript levels was
performed in duplicate. As illustrated in
Fig. 8C, very low levels of
Hsp70 mRNA transcripts were detected in both wild-type and
JIL-1 mutant backgrounds under non-heat shock conditions. However, a
robust increase in Hsp70 mRNA transcript levels, relative to
rp49 transcript levels, was detected in response to heat shock
treatment in all three genotypes (Fig.
8C). The increase in
JIL-1z2/JIL-1z2 null mutant larvae was at least
two orders of magnitude greater than under non-heat shock conditions, although
this response was only about one-third that observed in wild-type larvae.
Interestingly, the heat shock-induced increase in Hsp70 mRNA levels
was enhanced considerably, to almost two-thirds of wild-type levels, in larvae
in which Su(var)3-9 levels were reduced by half (e.g.
JIL-1z2/JIL-1z2 Su(var)3-906
larvae).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and that histone H3S10 phosphorylation might play
an important role in specific transcriptional responses to signaling stimuli
(Mahadevan et al., 1991;
Lo et al., 2001;
Ivaldi et al., 2007). In this
study, we characterized three commercially available histone H3S10ph
antibodies and used an acid-free squash protocol to revisit the role of
histone H3S10 phosphorylation in transcription in Drosophila under
both heat shock and non-heat shock conditions. We show that there is no change
in the levels of the elongating form of RNA polymerase II in larvae from
JIL-1 null mutant backgrounds as compared with wild type.
Furthermore, we provide evidence that heat shock-induced puffs in
JIL-1 null mutant backgrounds are strongly labeled by Pol
IIoser2 antibody in a pattern coincident with that of Hsf antibody,
indicating that Pol IIoser2 is actively involved in heat
shock-induced transcription in the absence of H3S10 phosphorylation. That mRNA
of the six genes that encode Hsp70, the major heat shock protein in
Drosophila, is transcribed at robust levels in JIL-1 null
mutants was directly demonstrated by qRT-PCR. Thus, these data strongly
suggest that histone H3S10 phosphorylation by JIL-1 is not involved in
transcriptional elongation in Drosophila. The finding that there is
no redistribution of JIL-1 or H3S10 phosphorylation to transcriptionally
active puffs in wild-type polytene squash preparations during the heat shock
response further supports this conclusion. These results are contrary to those
reported previously (Nowak and Corces,
2000; Nowak et al.,
2003; Ivaldi et al.,
2007). However, the discrepancies might largely be due to the
reliance in these studies on the Upstate H3S10ph pAb, which our study
indicates is unsuitable for analysis of heat shock-induced transcription owing
to non-specific cross-reactivity at heat shock-induced puffs. Nonetheless, it
should be noted that the present study confirms the findings of Ivaldi et al.
(Ivaldi et al., 2007) that the
chromatin remodeling associated with heat shock puff formation still occurs in
JIL-1 null mutants, despite the disruption of chromatin structure and
the absence of H3S10 phosphorylation.
|
|
|
; Deng et al.,
2007). At interphase, JIL-1 phosphorylates the histone H3S10
residue in euchromatic regions of polytene chromosomes
(Jin et al., 1999;
Wang et al., 2001), suggesting
as a plausible model that this phosphorylation during interphase prevents
Su(var)3-9-mediated heterochromatization and gene repression at these sites
(Zhang et al., 2006;
Deng et al., 2007). However,
the present results clearly indicate that such repression is not global and
that the Pol II machinery is functional, as indicated by the robust
transcription of heat shock-induced genes in JIL-1 null mutants.
Furthermore, whereas dosage compensation of the white locus in males
is impaired in hypomorphic JIL-1 mutant backgrounds, white
expression in females is relatively unaffected
(Lerach et al., 2005). Thus,
the lethality might instead be caused by a severe repression of a few
essential genes and/or a more graded decrease in the expression of a larger
number of genes owing to the altered chromatin structure. The latter scenario
is supported by the finding that the expression of heat shock-induced genes in
JIL-1 null mutant backgrounds increases when chromatin structure is
partially rescued by reducing the levels of the heterochromatic factor
Su(var)3-9 (Deng et al., 2007)
(this study). That JIL-1 levels can directly affect gene expression was
recently demonstrated by experiments that showed that loss-of-function
JIL-1 alleles act as enhancers of position-effect variegation (PEV),
whereas the gain-of-function JIL-1Su(var)3-1 allele acts
as a suppressor of PEV at pericentric sites
(Bao et al., 2007). The
JIL-1Su(var)3-1 allele is one of the strongest suppressors
of PEV so far described (Ebert et al.,
2004) and it generates truncated proteins with C-terminal
deletions that mislocalize to ectopic chromosome sites
(Ebert et al., 2004;
Zhang et al., 2006). Thus, the
dominant gain-of-function effect of the JIL-1Su(var)3-1
alleles might be attributable to JIL-1 kinase activity at ectopic locations
leading to misregulated localization of the phosphorylated histone H3S10 mark
and thereby counteracting the spreading and gene repression of Su(var)3-9.
This is supported by the finding of Deng et al. that ectopic H3S10
phosphorylation at interphase can function as a causative regulator of
higher-order chromatin structure in vivo
(Deng et al., 2008).
Furthermore, studies of PEV of the whitem4 allele have
indicated that loss of JIL-1 function can also cause a change in the levels of
heterochromatic factors at the chromocenter that can indirectly affect gene
expression at nearby loci (Lerach et al.,
2006) (reviewed by Girton and
Johansen, 2008). In future experiments, it will be of interest to
further explore a possible mechanistic link between histone H3S10
phosphorylation and the regulation of chromatin structure and gene expression
in Drosophila.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Bao, X., Deng, H., Johansen, J., Girton, J. and Johansen, K. M. (2007). Loss-of-function alleles of the JIL-1 histone H3S10 kinase enhance position-effect-variegation at pericentric sites in Drosophila heterochromatin. Genetics 176,1355 -1358.
Boehm, A. K., Saunders, A., Werner, J. and Lis, J. T.
(2003). Transcription factor and polymerase recruitment,
modification, and movement on dhsp70 in vivo in the minutes following
heat shock. Mol. Cell. Biol.
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