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J. Biol. Chem., Vol. 275, Issue 30, 23267-23272, July 28, 2000
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From the Wadsworth Center, New York State Department of Health and
Department of Biomedical Sciences, State University of New York,
Albany, New York 12201-2002
Received for publication, December 20, 1999, and in revised form, April 13, 2000
Variant histones that differ in amino acid
sequence from S-phase histones are widespread in eukaryotes, yet the
structural changes they cause to nucleosomes and how those changes
affect relevant cellular processes have not been determined. H2A.F/Z is
a highly conserved family of H2A variants. H2Av, the H2A.F/Z variant of
Drosophila melanogaster, was localized in polytene chromosomes by indirect immunofluorescence and in diploid chromosomes by chromatin immunoprecipitation. H2Av was widely distributed in the
genome and not limited to sites of active transcription. H2Av was
present in thousands of euchromatic bands and the heterochromatic chromocenter of polytene chromosomes, and the H2Av antibody
precipitated both transcribed and nontranscribed genes as well as
noncoding euchromatic and heterochromatic sequences. The distribution
of H2Av was not uniform. The complex banding pattern of H2Av in
polytene chromosomes did not parallel the concentration of DNA, as did the pattern of immunofluorescence using H2A antibodies, and the density
of H2Av measured by immunoprecipitation varied between different
sequences. Of the sequences assayed, H2Av was least abundant on 1.688 satellite sequences and most abundant on the hsp70 genes.
Finally, transcription caused, to an equivalent extent, both H2Av and
H2A to be less tightly associated with DNA.
The basic unit of chromatin in eukaryotes is the nucleosome. A
nucleosome consists of 146 base pairs of DNA wrapped around an octamer
of histone proteins H2A, H2B, H3, and H4 (1). Although chromatin is a
highly reiterative structure, cells create heterogeneity in the
structure of nucleosomes to facilitate and regulate DNA-mediated processes such as transcription. Heterogeneity is created, in part, by
posttranslational modifications of histone proteins, including
acetylation, phosphorylation, methylation, ubiquitination, and
ADP-ribosylation (2-4). Acetylation status of the amino termini of
histones H3 and H4, in particular, plays a important role in transcriptional regulation (2, 5).
Heterogeneity in nucleosome structure also results from incorporation
of variant histone proteins into the nucleosome. In contrast to the
canonical histones, which are multicopy genes expressed during S-phase
of the cell cycle, variant histones are encoded by single copy genes
that differ in amino acid sequence from S-phase histones and whose
expression is not limited to S-phase (6, 7). Variant histones allow
specialization of nucleosome structure for specific purposes.
Sperm-specific variant histones, for example, facilitate the dramatic
compaction of DNA that occurs during spermatogenesis (4, 8). A specific
variant of histone H3 is incorporated specifically at centromeres
creating a specialized chromatin structure required for proper function
of the kinetochore (9-14), and macro-H2A, a histone H2A variant,
localizes preferentially to the inactive X chromosome in mammals and
may alter chromatin in a way that helps suppress transcription (15).
These and other variants have been identified for histones H2A, H2B,
and H3 but not H4. Histone variants are widespread, if not universal,
in eukaryotes, yet how variant histones change nucleosome structure and
how those changes affect relevant cellular processes have not been determined.
H2A.F/Z is a family of H2A variants that are highly conserved across
species and substantially divergent from S-phase H2A in any given
species (16, 17). An H2A.F/Z variant has been identified in a wide
variety of eukaryotes including Tetrahymena (hv1), budding
yeast (HTA3 and HTZ1), Drosophila (D2, H2A.2, H2AvD, His2AvD, His2Av, and H2Av), chickens (H2A.F), mice (H2A.Z), and humans
(H2A.Z) suggesting that incorporation of H2A.F/Z into chromatin is
evolutionarily ancient and conserved. H2A.F/Z typically constitutes 5-10% of total H2A proteins in the chromatin of cells (18, 19). H2A.F/Z is essential in Tetrahymena, Drosophila,
and mice, but its essential function is not known (20-22). H2A.F/Z may
play a role in transcriptional regulation since in
Tetrahymena its expression is associated with the
transcriptionally active macronucleus, and in Drosophila its
incorporation into chromatin during development is coincident with the
start of zygotic gene expression (23, 24). The chromosome loss
phenotype caused by mutation to the H2A.F/Z homolog in fission yeast,
pht1, suggests that H2A.F/Z could also play a role, directly
or indirectly, in chromosome segregation (25).
Determining the location of variant histones within chromosomes can
provide insight into their function (12, 14, 15). To this end, the
location of H2Av, the H2A.F/Z variant histone in Drosophila
melanogaster, was determined in both polytene and diploid
chromosomes in relation to transcriptionally active and inactive genes
as well as noncoding sequences, and the results were compared with the
localization of S-phase H2A.
Antibody Synthesis--
H2Av and H2A polyclonal antibodies were
generated by injecting rabbits with synthetic peptides homologous to
the carboxyl-terminal 15 amino acids of H2Av or H2A conjugated to
keyhole limpet hemocyanin via an added amino-terminal cysteine
(Pierce). Serum was used for all experiments. Affinity purified
antibody was generated for H2Av and gave identical results (data
not shown).
Immunofluorescence--
Immunolocalization of H2Av, H2A, and RNA
polymerase II (pol II)1 to
polytene chromosomes was done as described by Shopland and Lis (26).
Primary antibodies were H2Av or H2A rabbit antisera used at 1:1000
dilution and a mouse monoclonal antibody, 8WG16 (Babco), was used at a
dilution of 1:20. 8WG16 recognizes the heptapeptide repeat of the
carboxyl-terminal domain (CTD) of the large subunit of RNA pol II
primarily in an unphosphorylated state (27). In vivo 8WG16
can still bind phosphorylated pol II that is transcriptionally active
if the CTD retains some unphosphorylated repeats but may not bind pol
II when the CTD is completely phosphorylated (28, 29). Secondary
antibodies were goat anti-rabbit IgG coupled to rhodamine used at a
1:50 dilution (Jackson ImmunoResearch) and goat anti-mouse IgG coupled
to Cy2 used at a 1:150 dilution (The Jackson ImmunoResearch).
Chromosomes were viewed on a Zeiss Axioscope fluorescence microscope
with appropriate filters. Single and double exposure photographs were
taken, and the photographic slides were scanned into the computer and
prepared using Adobe Photoshop.
Chromatin Immunoprecipitation--
Chromatin
immunoprecipitations were done essentially as described by Orlando
et al. (30). S2 cells were maintained in
Drosophila serum-free medium (Life Technologies, Inc.) at
27 °C. 2-4 × 109 cells were cross-linked with
formaldehyde for 10 min at 23 °C followed by 50 min on ice. Heat
shock samples were heated to 36.5 °C for 1 h before being
cross-linked with formaldehyde for 10 min at 36.5 °C followed by 50 min on ice. The cross-linked DNA was sheared by sonication to an
average 1 kb in size and was purified by equilibrium sedimentation on a
cesium chloride gradient. 30-60 µg of cross-link DNA was used per
immunoprecipitation with 5 µl of H2Av antiserum, 5 µl of H2A
antiserum, or 10 µl of an affinity purified, rabbit polyclonal
antibody directed against the large subunit of pol II expressed in
bacteria (31). After reversing the cross-links, the DNA was purified,
quantitated using picogreen fluorescence (Molecular Probes), and
analyzed by slot-blot hybridization. Hybridization signals were
quantitated using stored phosphorimaging screens and ImagQuant software
(Molecular Dynamics). Levels of hybridization to immunoprecipitated DNA
were measured relative to the level of hybridization observed to the
same amount of total, non-precipitated DNA. The hsp70 probe
was a 0.9-kb BamHI-SalI fragment isolated from
plasmid 56H8 and is homologous to the 3'-half of all five
hsp70 genes (32). The hsp26 probe was a 2.2-kb
BamI-EcoRI fragment isolated from plasmid 202.7 and is homologous to the entire gene and 1.2 kb of 3'-flanking
sequences (32). The hsp83 probe was a 3.2-kb
BamHI-SalI fragment isolated from plasmid aDm4.46 and is homologous to the 5'-half of the gene and 0.8 kb of 5'-flanking sequences (32). The actin probe was a 0.9-kb SalI
fragment isolated from plasmid DmA2 and is homologous to the 3'-half of
both the 5C and 42A actin genes (32). The bicoid probe was a
3.2-kb XbaI fragment isolated from plasmid pUCHSNeo8.7 and
is homologous to the entire gene excluding 500 base pairs of the 5' end
(33). The s19 chorion probe was a 1.5-kb
EcoRV-EcoRI fragment isolated from plasmid pR7.7
and is homologous to the entire gene and 0.7 kb of 3'-flanking
sequences (34). The satellite probe was a 424-base pair AseI
fragment isolated from plasmid pHD-BPDp and is homologous to 1.688 satellite sequences on Dp(1;f)1187 (35).
Distribution of H2Av in Polytene Chromosomes by
Immunofluorescence--
A polyclonal antibody was generated against
Drosophila H2Av to determine where this H2A.F/Z histone
variant is located in chromosomes. The antibody recognized a 14.6-kDa
protein present in nuclei of embryos and S2 tissue culture cells as
well as in larval salivary glands (Fig.
1). The protein was absent from larval imaginal disc cells homozygous for the His2Av810
null allele of the His2Av gene (20) demonstrating that the 14.6-kDa protein is H2Av (Fig. 1).
The antibody was used to localize H2Av in polytene chromosomes of third
instar larval salivary glands by indirect immunofluorescence (Fig.
2). H2Av was widely distributed in
polytene chromosomes, being present in thousands of bands throughout
the euchromatin, and was also present in the heterochromatic
chromocenter (Fig. 2a). Some level of fluorescence was
detectable along virtually the entire length of each chromosome arm.
Low levels of fluorescence were unlikely to be due to nonspecific
background, since control antibodies, such as those to acetylated
isoforms of histone H4, gave no detectable signal between strong bands
of fluorescence.2 Therefore,
even low levels of immunofluorescence probably reflected the
presence of H2Av. Earlier studies had also reported the banded appearance of H2Av localization in polytene chromosomes (20, 23).
Immunocytological studies of Drosophila histone H2A.2, which
was likely to have been the same protein we now know to be H2Av, suggested that H2Av localization was limited to interband regions of
the polytene chromosome, and therefore, H2Av might play a general role
in determining the band-interband structure of polytene chromosomes (36). This hypothesis was directly tested by double labeling chromosomes with the H2Av antibody and the DNA stain
4',6-diamidine-2'-phenylindole dihydrochloride (DAPI), which reveals
the band-interband structure of the chromosome. H2Av localization was
not limited to interband regions and showed a complex pattern of
staining that did not correlate with the banded structure of the
polytene chromosome (Fig. 2b). Localization of H2Av,
therefore, does not simply parallel the concentration of DNA along the
polytene chromosome.
To determine if H2Av localization was correlated with transcriptional
activity, the pattern of H2Av staining was compared with sites of
transcription by co-localizing pol II and H2Av (Fig. 2c).
Loci of elevated pol II immunofluorescence were found throughout the
euchromatin with a limited number of loci having conspicuously higher
pol II immunofluorescence identifying sites where pol II density and
transcriptional activity were particularly high. It is likely that not
all sites of transcriptional activity were detected because the
antibody used, 8WG16, preferentially binds hypophosphorylated pol II
(see "Experimental Procedures"). The distribution of H2Av
immunofluorescence was generally more widespread than pol II with many
loci containing H2Av but little or no pol II, and some loci contained
equivalent levels of pol II and H2Av immunofluorescence (Fig.
2c). H2Av immunofluorescence was reduced at those loci
containing the most pol II and presumably the highest levels of
transcription (arrowheads in Fig. 2c). Reductions
in H2Av immunofluorescence were also observed at the hsp70
heat shock loci after a 1-h heat shock of larvae, which produces high
densities of pol II concomitant with transcriptional induction of the
hsp70 genes (Fig. 2d). No obvious reduction in
H2Av immunofluorescence was observed at heat shock loci under nonheat
shock conditions (data not shown), and the reduction in H2Av
immunofluorescence observed after heat shock is likely to be transient,
since no persistent change in H2Av immunofluorescence was observed at
heat shock loci in larvae subject to daily heat shocks during earlier development (data not shown).
Distribution of H2Av in Diploid Chromosomes by Chromatin
Immunoprecipitation--
Levels of H2Av immunofluorescence observed in
polytene chromosomes might reflect not only the density of H2Av but
also the influence of chromatin structure, such as the state of DNA
condensation or decondensation associated with chromosome puffing. To
avoid such complications and provide an alternative methodology for determining the distribution of H2Av in Drosophila
chromosomes, chromatin immunoprecipitations (ChIP) were used to measure
the association of H2Av with different types of DNA sequences in the diploid chromosomes of tissue culture cells.
DNA:protein cross-links were generated by formaldehyde treatment of
cells, and complexes were immunoprecipitated using H2Av antibodies. The
immunoprecipitated DNA was analyzed by slot-blot hybridization, and the
hybridization signal was compared with the hybridization signal for a
comparable mass of total, nonprecipitated DNA. A ChIP/total value of 1 means the sequence was neither enriched nor depleted relative to the
abundance of the same sequence in totals. Negligible DNA was
precipitated with preimmune sera or in no-antibody controls (Fig.
3a).
Immunoprecipitations were initially done using an antibody to pol
II and probing for the coding sequences of the hsp70,
hsp26, and hsp83 heat shock genes to establish
the validity of the protocol. Association of pol II with
hsp70, hsp26, and hsp83 increased 44-, 22-, and 6-fold, respectively, after a 1-h heat shock (Fig. 3, a and b), consistent with earlier observations
(32). mRNA levels for these genes also increased 43-, 23-, and
4-fold, respectively, after a 1-h heat shock (data not shown).
Immunoprecipitations were then done using the H2Av antibodies, and the
association of H2Av with a variety of gene sequences was measured (Fig.
3c). H2Av was associated with the constitutively expressed
hsp83 and cytoplasmic actin genes, the uninduced
and induced hsp70 and hsp26 genes, and the
nonexpressed developmental bicoid and chorion s19
genes (Fig. 3c). H2Av was also associated with noncoding
sequences 5' and 3' of both bicoid and hsp70
(data not shown). Levels of H2Av on all sequences tested were similar with ChIP/total ratios around 1, suggesting that H2Av is fairly uniformly distributed in chromatin irrespective of whether a sequence is transcribed, potentially transcribed, or noncoding. H2Av was even
associated with heterochromatic satellite sequences, although at lower
levels (Fig. 3c), suggesting that some amount of H2Av is
present even in heterochromatin, a conclusion consistent with localization of H2Av to the heterochromatic chromocenter of polytene chromosomes (Fig. 2a).
Comparison of H2Av immunoprecipitations from normal versus
heat shock cells suggested that transcription causes H2Av to cross-link less frequently to DNA (Fig. 3c). In uninduced cells, H2Av
was associated with all three heat shock genes (Fig. 3c).
After transcription was induced by heat shock, immunoprecipitation
levels for all three genes decreased as follows: 1.7-fold for
hsp70, 1.5-fold for hsp26, and 1.4-fold for
hsp83 (Fig. 3c). Immunoprecipitation levels for
the actin genes appeared to increase after heat shock, which would be
consistent with the decrease in transcription that occurs to these
genes (32). These results suggest that transcription causes H2Av to be
less frequently or less tightly associated with DNA, a conclusion
consistent with the reduced immunofluorescence observed at actively
transcribed loci in polytene chromosomes (Fig. 2c).
The Distribution of S-phase H2A in Polytene and Diploid
Chromosomes--
S-phase H2A is likely to be uniformly distributed in
chromosomes and therefore provides a good control for the pattern of H2Av localization. A polyclonal antibody was generated against the
carboxyl-terminal 15 amino acids of H2A. The antibody recognized a
single nuclear protein on Western blots that migrated at the position
of bulk H2A protein (Fig. 4a).
The antibody was used to localize H2A in polytene chromosomes of third
instar larval salivary glands by indirect immunofluorescence. H2A
immunofluorescence paralleled precisely the banded structure of the
chromosome revealed by DAPI staining (Fig. 4b). This result
agrees with earlier studies (37) and is consistent with a uniform
distribution of H2A-containing nucleosomes along the chromosome
paralleling the density of DNA. The H2A result contrasts markedly with
the complex immunofluorescence pattern observed for H2Av, which did not
correlate with the banded structure of the chromosome (compare Figs.
2b and 4b).
The distribution of H2A was also determined in diploid chromosomes by
chromatin immunoprecipitation. H2A and H2Av immunoprecipitations were
done in parallel on the same preparations of cross-linked DNA (Fig.
4c). Nearly identical amounts hsp70 sequences
were precipitated by both antibodies, and the same
transcription-induced reduction in DNA association was observed between
uninduced and heat shock-induced samples. In contrast, H2A and H2Av
antibodies precipitated different amounts of s19 and
satellite sequences (Fig. 4c). The H2A antibody precipitated
about the same amount of DNA from both the unexpressed s19
gene and 1.688 satellite sequences, whereas the H2Av antibody precipitated 1.6-fold less s19 and 5-fold less satellite
DNA. These differences suggest that H2Av is less abundant on these sequences.
The distribution of H2Av appears to be widespread in the
Drosophila genome. Some level of H2Av immunofluorescence was
detectable along virtually the entire length of each polytene
chromosome (Fig. 2). In addition, every DNA sequence tested was
immunoprecipitated by the H2Av antibody at levels significantly above
background irrespective of the transcriptional status or coding
capacity of the sequence (Fig. 3).2 Thus, the
immunofluorescence and immunoprecipitation results suggest that
H2Av-containing nucleosomes are widespread, if not ubiquitous, in the
genome. The distribution of H2Av along the length of the chromosome,
however, also appears to vary. The banded pattern of H2Av
immunofluorescence was complex and did not simply parallel the
concentration of DNA, as was the case for H2A (Figs. 2b and
4b), and significantly different amounts of s19
and satellite sequences were precipitated by the H2Av and H2A
antibodies (Fig. 4c). Thus, the immunofluorescence and
immunoprecipitation results also suggest that the density of
H2Av-containing nucleosomes is different on different sequences.
H2A.Z variant histones are likely to be involved in transcriptional
regulation. H2A.Z is associated with transcriptionally active nuclei in
both Tetrahymena and Drosophila (23, 24), and
recent genetic evidence suggests that H2A.Z regulates transcription in
budding yeast.3 The
distribution of H2Av in Drosophila chromosomes appeared to correlate with transcriptional potential, in general, but not necessarily with specific sites of active transcription. For example, by comparison to H2A, noncoding satellite sequences had the least H2Av,
the coding but nonexpressed s19 gene had more H2Av, and the
inducible hsp70 gene had the most H2Av (Fig. 4c).
In contrast, there was little difference in H2Av immunoprecipitation
levels between the constitutively expressed hsp83 and
actin genes and the nonexpressed bicoid and
s19 genes (Fig. 3c). In addition, sites of active
transcription in polytene chromosomes were not sites of elevated H2Av
immunofluorescence (Fig. 2c). So even if H2Av is involved in
transcriptional regulation, its incorporation into chromatin does not
appear to be transcription-dependent. H2Av is therefore
unlikely to act as a replacement histone that replaces H2A lost from
actively transcribed genes (38). Similarly, mammalian H2A.Z also fails
to accumulate in chromatin of nondividing rat neurons as would be
expected of a replacement histone (39).
If transcription is not responsible for the incorporation of H2Av into
chromatin, then what is the origin of H2Av's widespread but nonrandom
pattern of localization? H2Av could be localized throughout the genome,
including heterochromatic sequences, if it were incorporated into
nucleosomes during DNA replication. If this were the case, however,
H2Av-containing nucleosomes would need to be preferentially assembled
onto some sequences, like euchromatic genes, versus other
sequences, like satellite repeats, to create the observed variations in
H2Av density. Alternatively, H2Av incorporation during S-phase could be
stochastic if mechanisms exist that could subsequently change the level
of H2Av on specific sequences in the context of pre-existing chromatin.
The function of H2Av is likely to involve conformational changes to the
structure of the nucleosome. The We thank the Wadsworth Center Peptide
Synthesis Core Facility for assistance with peptide synthesis, John Lis
for a gift of the RNA pol II antibody used for immunoprecipitation,
Randall Morse and John Lis for critical reading of the manuscript, and Mitch Smith for the sharing of unpublished results.
*
This work was supported by National Institutes of Health
Grant GM53476.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Published, JBC Papers in Press, May 2, 2000, DOI 10.1074/jbc.M910206199
2
T. J. Leach, M. Mazzeo, H. L. Chotkowski, J. P. Madigan, M. G. Wotring, and R. L. Glaser, unpublished observations.
3
M. Smith, personal communication.
The abbreviations used are:
pol II, RNA
polymerase II;
CTD, carboxyl-terminal domain;
ChIP, chromatin
immunoprecipitation;
DAPI, 4',6-diamidine-2'-phenylindole
dihydrochloride;
kb, kilobase pair.
Histone H2A.Z Is Widely but Nonrandomly Distributed in
Chromosomes of Drosophila melanogaster*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (66K):
[in a new window]
Fig. 1.
An antibody to Drosophila
H2Av. Equal amounts of protein from embryo nuclei
(lane 1), S2 cell nuclei (lane 2), and from
larval salivary glands (lane 3) were fractionated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and assayed by
Western blot hybridization. The H2Av antiserum detected a 14.6-kDa
protein in all samples. The 14.6-kDa protein was detected in wing discs
from wild type larvae (lane 4) but not from larvae
homozygous for the His2Av810 null allele of
His2Av (lane 5). No signal was observed with
preimmune sera (data not shown).
View larger version (32K):
[in a new window]
Fig. 2.
Immunolocalization of H2Av in polytene
chromosomes. a, H2Av antibodies were bound to salivary
gland polytene chromosomes and detected with a secondary antibody
conjugated to rhodamine. No fluorescence was observed with preimmune
sera (data not shown). b, after localizing H2Av
(red), the chromosomes were counterstained with DAPI
(green). The merged image demonstrates the incongruity of
the H2Av and DAPI patterns. Four areas that differ in their relative
intensities of DAPI and H2Av fluorescence are highlighted by
vertical lines. c, chromosomes were double
labeled with antibody to RNA pol II (PolII)
(green) and to H2Av (red). Regions of highest pol
II density were regions of low H2Av density (arrowheads).
d, larvae were heat-shocked at 36.5 °C for 1 h and
their chromosomes double-labeled with RNA pol II (green) and
H2Av (red). The high RNA pol II density and the low H2Av
density at the 87A and 87C loci, which contain the hsp70
genes, are evident.
View larger version (33K):
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Fig. 3.
H2Av localization to diploid chromosomes by
ChIP. a, slot-blot hybridization results are shown for
hsp70 sequences precipitated with RNA pol II
(PolII) or H2Av antibodies from non-heat shock
(NHS) or heat shock (HS) cells. The
immunoprecipitation (IP), no-antibody control
(c), and non-precipitated totals are shown for each sample
with the amount of DNA blotted indicated in nanograms (ng).
Relative levels of enrichment or depletion can be seen by comparing the
amount of hybridization signal in the immunoprecipitation slot to the
amount of hybridization in the slot containing the same mass of total
DNA. b, quantitative slot-blot hybridization results are
shown for hsp70 (70), hsp26 (26),
hsp83 (83), and actin gene sequences
immunoprecipitated with RNA pol II antibodies from cells that were not
heat-shocked (light) or were heat-shocked (dark)
prior to cross-linking. ChIP/total values above 1 indicated enrichment
of the sequence in the precipitate relative to totals, and values below
1 indicate depletion. c, quantitative slot-blot
hybridization results are shown for hsp70 (70),
hsp26 (26), hsp83 (83), actin,
bicoid (bic), and chorion s19 (s19) genes and
1.688 satellite sequences (sat) immunoprecipitated with H2Av
antiserum from cells that were not heat-shocked (light) or
heat-shocked (dark) prior to cross-linking. ChIP/total
values are presented as described for b. Mean and S.D. were
calculated from results of at least three independent
immunoprecipitations.
View larger version (36K):
[in a new window]
Fig. 4.
Localization of Drosophila
H2A in polytene and diploid chromosomes. a, equal
amounts of protein from embryo nuclei were fractionated by
SDS-polyacrylamide gel electrophoresis and transferred to
polyvinylidene fluoride membrane. One lane was stained with Coomassie
Brilliant Blue to reveal histones H2A, H2B, H3, and H4, which are the
predominant proteins in nuclei preparations (lane 1). The
other lane was assayed by Western blot hybridization using the H2A
antiserum. A single protein was identified that co-migrated with H2A
(lane 2). No signal was observed with preimmune sera (data
not shown). b, H2A antibodies were bound to salivary gland
polytene chromosomes and detected with a secondary antibody conjugated
to rhodamine (red). The chromosome was then counterstained
with DAPI (green). The merged image demonstrates the
congruity of the H2A and DAPI patterns. c, chromatin
immunoprecipitations were done as described for Fig. 3 using H2Av
(light) and H2A (dark) antibodies done in
parallel on the same preparations of cross-linked DNA. Quantitative
results for the hsp70 (70), chorion s19, and
1.688 satellite sequences (sat) are shown. Hsp70
sequences were precipitated from both nonheat-shocked (NHS)
and heat-shocked (HS) cells. Chorion s19 and
1.688 satellite sequences were precipitated from nonheat-shocked cells.
Mean and S.D. were calculated from results of three independent
immunoprecipitations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
C helix region of H2Av is required
for H2Av function and is the only region of H2Av for which the amino
acid sequence of H2A cannot be substituted without loss of function
(40). The C helix of H2Av along with the 3 helix and
carboxyl-terminal tail forms a "docking" domain that interacts with
the carboxyl-terminal tail of H4 within the core of the nucleosome and
might be expected to influence the conformational stability of the
nucleosome (1). Transcription-induced changes in nucleosome structure
are likely to be the cause of reductions in hsp70
immunoprecipitation levels observed after heat shock (Figs
3c and 4c). An earlier cross-linking analysis of
histone associations with the hsp70 genes suggested that
transcription causes a conformation change in nucleosome structure that
results in reduced cross-linking of histone globular domains to DNA,
whereas cross-linking of tail domains is unaffected (41). This
particular transcription-induced alteration in nucleosome structure
appeared to be the same for H2Av-containing and H2A-containing
nucleosomes since equivalent reductions in immunoprecipitation levels
were observed using both antibodies (Fig. 4c). Although this
result did not reveal a unique aspect of H2Av function, it is still
possible that quantitative differences between the H2Av and H2A
immunoprecipitations were obscured by the very high levels of
hsp70 transcription induced by heat shock and that important
differences in the behavior of H2Av-containing and H2A-containing
nucleosomes might be revealed at lower levels of transcription or on
different, developmentally regulated genes.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Wadsworth Center, New
York State Dept. of Health and Dept. of Biomedical Sciences, State
University of New York, P. O. Box 22002, Albany, NY 12201-2002 Tel.:
518-473-4201; Fax: 518-474-3181; E-mail: glaser@wadsworth.org.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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