Transcriptional activation of a constitutive heterochromatic domain of the human genome in response to heat shock.

Abstract

Heat shock triggers the assembly of nuclear stress bodies that contain heat shock factor 1 and a subset of RNA processing factors. These structures are formed on the pericentromeric heterochromatic regions of specific human chromosomes, among which chromosome 9. In this article we show that these heterochromatic domains are characterized by an epigenetic status typical of euchromatic regions. Similarly to transcriptionally competent portions of the genome, stress bodies are, in fact, enriched in acetylated histone H4. Acetylation peaks at 6 h of recovery from heat shock. Moreover, heterochromatin markers, such as HP1 and histone H3 methylated on lysine 9, are excluded from these nuclear districts. In addition, heat shock triggers the transient accumulation of RNA molecules, heterogeneous in size, containing the subclass of satellite III sequences found in the pericentromeric heterochromatin of chromosome 9. This is the first report of a transcriptional activation of a constitutive heterochromatic portion of the genome in response to stress stimuli.

Full text

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Molecular Biology of the Cell
vbn Vol. 15, 543–551, February 2004
Transcriptional Activation of a Constitutive
Heterochromatic Domain of the Human Genome in
Response to Heat Shock□
D
Nicoletta Rizzi, Marco Denegri, Ilaria Chiodi, Margherita Corioni,
Rut Valgardsdottir, Fabio Cobianchi, Silvano Riva, and Giuseppe Biamonti*
Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, 27100 Pavia, Italy
Submitted July 11, 2003; Revised October 7, 2003; Accepted October 17, 2003
Monitoring Editor: Joseph Gall
Heat shock triggers the assembly of nuclear stress bodies that contain heat shock factor 1 and a subset of RNA processing
factors. These structures are formed on the pericentromeric heterochromatic regions of specific human chromosomes,
among which chromosome 9. In this article we show that these heterochromatic domains are characterized by an
epigenetic status typical of euchromatic regions. Similarly to transcriptionally competent portions of the genome, stress
bodies are, in fact, enriched in acetylated histone H4. Acetylation peaks at 6 h of recovery from heat shock. Moreover,
heterochromatin markers, such as HP1 and histone H3 methylated on lysine 9, are excluded from these nuclear districts.
In addition, heat shock triggers the transient accumulation of RNA molecules, heterogeneous in size, containing the
subclass of satellite III sequences found in the pericentromeric heterochromatin of chromosome 9. This is the first report
of a transcriptional activation of a constitutive heterochromatic portion of the genome in response to stress stimuli.
INTRODUCTION
The genomic material in eukaryotic nuclei can be roughly
partitioned in two cytologically distinct entities termed eu-
and hetero-chromatin. Heterochromatin was originally de-
fined as that portion of the genome that remains condensed
and intensely stained with DNA intercalating dyes through-
out the cell cycle. It represents a significant fraction of most
eukaryotic genomes and is generally associated with telo-
meres and pericentric regions of chromosomes. Contrary to
euchromatin, heterochromatic regions consist predomi-
nantly of repetitive DNA, including satellite sequences and
middle repetitive sequences related to transposable ele-
ments and retroviruses. Although not devoid of genes, these
regions are typically gene-poor (Hennig, 1999).
Establishment of heterochromatin depends on two basic
elements: the histone modification code (Strahl and Allis,
2000) and the interaction of nonhistone chromosomal pro-
teins (Nielsen et al., 2001). The covalent modifications of
histone tails by deacetylase and methyl-transferase activities
act in concert to establish the “histone code” essential for
assembly of silenced chromatin. In general, the histone tails
in heterochromatin are relatively hypo-acetylated, whereas
histone H3 and H4 tails found in euchromatin are generally
acetylated. Concerning the nonhistone chromosomal pro-
teins, one of the best characterized is heterochromatin pro-
tein 1 (HP1), which was originally identified as a protein
primarily concentrated in the pericentric heterochromatin.
HP1 homologues exist in evolutionary distant organisms
such as Schizosaccharomyces pombe (Swi6p) and Homo sapiens
(HP1?, HP1?, and HP1?; Eissenberg and Elgin, 2000) and
are characterized by an amino-terminal chromo domain that
mediates the interaction with chromatin (Nakayama et al.,
2001). Methylation of lysine 9 (K9) of histone H3 creates a
binding site for recruitment of Swi6/HP1 (Maison et al.,
2002; Peters et al., 2002).
The factors underlying the selection of a specific chromo-
somal locus for the assembly of heterochromatin are still
elusive; however, repetitive DNA rather than specific DNA
sequences are likely involved. Also the function of hetero-
chromatin is not yet completely understood. In addition to
serve important roles in chromosome mechanics, particu-
larly in the centromere function (Hennig, 1999), heterochro-
matic structures are involved in the inactivation of genes
normally resident in euchromatic domains. This is sug-
gested by two key observations. First, inactivation of X-chro-
mosome in mammals, a mechanism that leaves the inactive
X as a visibly stained structure, the Barr body (Brockdorff,
2002). The second indication came from the analysis of po-
sition effect variegation (PEV) in Drosophila, that is the mo-
saic pattern of expression exhibited by genes placed near
centric heterochromatin by chromosomal rearrangements or
transposition events.
Stress treatments such as heat shock or exposure to heavy
metals drastically affect the function and the structure of the
eukaryotic cell. Among the numerous effects triggered by
heat shock on the structure of the cell nuclei, one of the most
intriguing is the transitory appearance of novel nuclear dis-
tricts, termed stress bodies (Weighardt et al., 1999). Stress
bodies are exclusively detectable in human cells (Denegri et
al., 2002) and consist of clusters of perichromatin granules,
i.e., highly packed forms of ribonucleoprotein complexes
(Chiodi et al., 2000). They are sites of accumulation of heat
shock factor 1 (HSF1; Jolly et al., 1999) and of a subset of
RNA processing factors, including two proteins of the het-
erogeneous nuclear ribonucleoprotein complexes (hnRNP
Article published online ahead of print. Mol. Biol. Cell 10.1091/
mbc.E03–07–0487. Article and publication date are available at
www.molbiolcell.org/cgi/doi/10.1091/mbc.E03–07–0487.
□
D
Online version of this article contains supplementary figure
material. Online version is available at www.molbiolcell.org.
* Corresponding author. E-mail address: biamonti@igm.cnr.it.
© 2004 by The American Society for Cell Biology543

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HAP and hnRNP M), two members of the SR family of
splicing factors (SF2/ASF and SRp30c), and the RNA-bind-
ing protein Sam68 (Denegri et al., 2001). We have recently
shown that the assembly of stress bodies depends on the
presence of pericentromeric heterochromatic regions on hu-
man chromosomes 9, 12, and 15. These heterochromatic
regions most likely act as recruiting centers as suggested by
their colocalization with stress bodies (Denegri et al., 2002).
Moreover, the subclass of satellite III elements in the hetero-
chromatic pericentromeric q1.2 band of chromosome 9 con-
tains sequence motifs similar to the HSF1-binding site. It has
been proposed that the interaction between HSF1 and satel-
lite III sequences directs the recruitment of HSF1 to stress
bodies (Jolly et al., 2002).
In this article we show that after heat shock the hetero-
chromatic regions associated with stress bodies are charac-
terized by an higher-order organization typical of euchro-
matic portions of the genome. Moreover, heat shock triggers
the production of satellite III transcripts, highly heteroge-
neous in size, that are undetectable in unstressed cells. To
our knowledge this is the first indication that environmental
stresses such as heat shock can elicit a transient alteration of
the higher-order structure of specific heterochromatic re-
gions and induce the transcriptional activation of silent por-
tions of the genome.
MATERIALS AND METHODS
Cell Culture and Cell Treatments
HeLa cells were grown in DMEM medium (Sigma, St. Louis, MO), 10% fetal
calf serum (Sigma), 50 ?g/ml gentamicin, and 2 mM l-glutamine. For heat
shock experiments, cells grown in monolayers were incubated 1 h at 42°C in
complete medium made with 40 mM HEPES, pH 7.0, and allowed to recover
at 37°C as indicated in the text.
When required, HeLa cells were treated with different concentrations of
trichostatin A (TSA; Sigma) dissolved in ethanol at a concentration of 1
mg/ml.
Indirect Immunofluorescence
HeLa cells grown on coverslips were washed once with phosphate-buffered
saline (PBS), fixed for 7 min in 4% formaldehyde, and subsequently perme-
abilized in 0.5% Triton X-100 for 7 min on ice. Primary antibodies were
diluted to working concentration in PBS containing 5% skimmed milk (Difco,
Detroit, MI) and then added to the coverslips. Primary antibodies used were
as follows: affinity-purified rabbit anti-HAP polyclonal antibody (Weighardt
et al., 1999), mouse anti-HAP monoclonal antibody (mAb) 16C3 (Weighardt et
al., 1999), rabbit antihyperacetylated histone H4 (Upstate, Lake Placid, NY),
rabbit acetyl-histone H4 antibody set (Ac K5; Ac K8; Ac K12; Upstate), rat
anti-HSF1 mAb 10H8 (NeoMarker, Freemont, CA), rat anti-HP1? mAb
MAC353 and rat anti-HP1? mAb MAC385 (Serotec, Cergy Saint-Christophe,
France) and mouse anti-HP1? mAb 3446 (Chemicon, International, Temecula,
CA), mouse anti-SF2/ASF mAb-96 (Zymed, San Francisco, CA), and rabbit
anti-4x-methil-histone H3(Lys9) polyclonal antibody (kindly provided by Dr.
Thomas Jenuwein, Research Institute for Molecular Pathology, Vienna). After
1 h at 37°C in a humid chamber, coverslips were washed three times with PBS.
Secondary antibodies used were rhodamine-conjugated anti-rabbit, anti-
mouse, or anti-rat immunoglobulin (IgG) goat antibodies (Jackson Immu-
noResearch Laboratories, West Grove, PA) and fluorescein isothiocyanate
(FITC)-conjugated anti-rabbit or anti-mouse IgG goat antibodies (Jackson
ImmunoResearch Laboratories). Secondary antibodies were diluted at the
final concentration recommended by the supplier in PBS-made 5% skimmed
milk and were added to coverslips. After 1 h at 37°C in a humid chamber,
coverslips were washed three times with PBS, rinsed, and mounted in 90%
glycerol in PBS. Confocal microscopy was performed with a TCS-NT digital
scanning confocal microscope (Leica, Deerfield, IL) equipped with a 63
?/NA ? 1.32 oil immersion objective. We used the 488-nm laser line for
excitation of FITC (detected at 500 nm ? FITC ?540 nm) and the 543-nm laser
line for the rhodamine fluorescence (detected at ?590 nm). The pinhole
diameter was kept at 1 ?m. Images were exported to Adobe Photoshop
(Adobe Systems, Mountain View, CA).
RNA Extraction and Northern Blotting
Total RNAs were isolated from HeLa cells using the guanidinium thiocyanate
solubilization method (Chirgwin et al., 1979). Northern blotting was per-
formed as described (Sambrook and Russel, 2001). Prehybridization and
hybridization solutions: 4? SSC (1? SSC: 0.15 M NaCl, 0.015 M Na citrate),
4? Denhardt’s solution, 50% formamide, 0.5% SDS, 7.5 mM Na4P2O7, 12.5
mM NaH2PO4, and 0.3 mg/ml tRNA. Probes were labeled with the Mega-
prime DNA labeling System (Amersham, Buckinghamshire, UK). After an
overnight hybridization, membranes were washed twice in 0.2? SSC, 1% SDS
at 60°C. Probes used were as follows: pHuR98 (ATCC, Manassas, VA),
containing a monomer of the subclass of satellite III sequences on chromo-
some 9, pAL1 pBR12, pMC15 pDMX1 specific for the alphoid DNA on
chromosomes 1, 12, 15, and X. In addition we used the ?-actin cDNA (Clon-
tech Laboratories, Palo Alto, CA) as a control of loaded material.
In Situ Hybridization to RNA
Cells were grown on coverslips and heat-shocked as described above. After
3 h of recovery at 37°C, cells were fixed in 4% paraformaldehyde in PBS for
15 min, washed thoroughly in PBS, and permeabilized in 0.5% Triton X-100
on ice for 5 min. Cells were then washed in PBS and 2? SSC and incubated
overnight at 42°C in hybridization buffer (1 ?g/ml tRNA [Sigma-Aldrich], 2?
SSC, 10% dextran sulfate [Sigma-Aldrich], 5? Denhardt’s solution, 25% for-
mamide) containing 5 ng/ml 5? biotinylated oligonucleotide probe. The fol-
lowing morning, cells were washed in 2? SSC and the biotinylated probe was
detected with fluorescein-avidin (Vector Laboratories, Burlingame, CA). The
signal was amplified by incubation with antiavidin D antibody (Vector Lab-
oratories) followed by fluorescein-avidin. Cells were counterstained with
anti-HAP rabbit antibodies and rhodamine-conjugated anti-rabbit antibodies.
The oligonucleotide probes used were as follows: Direct (5?-GGA ATG GCA
TGG ATT GGA AT-3?), and Reverse (5?-ATT CCA ATC CAT GCC ATT
CC-3?). Both oligonucleotides were 5? biotinylated and are complementary to
nucleotides 97–106 of the satellite III sequence of human chromosome 9
(accession number: X06137). Cells were also hybridized to a control oligo
(5?-CCGGGAAGCTAGAGTAAGTAG-3?) complementary to a pBR322 se-
quence.
RESULTS
Trichostatin A Prevents the Formation of Nuclear Stress
Bodies
We have previously reported that nuclear stress bodies form
on heterochromatic regions of specific human chromosomes
(Denegri et al., 2002). This raises the possibility that the
chromatin packaging status could be directly involved in
this process. We decided to investigate this possibility by
means of TSA, an inhibitor of histone deacetylases (Yoshida
et al., 1995). HeLa cells were incubated 6 h with increasing
concentrations of TSA and, after drug withdrawal, were
heat-shocked 1 h at 42°C. After 1 h of recovery at 37°C, cells
were fixed and stained with antibodies directed against
hnRNP HAP, HSF1, or splicing factor SF2/ASF, three com-
ponents of stress bodies (Denegri et al., 2001). The results of
this analysis are reported in Figure 1. We observed that
doses higher than 500 ng/ml drastically reduced the fraction
of cells in which HAP and SF2/ASF were recruited to stress
bodies. This fraction dropped to ?10% of the control value
when cells were treated with 1000 ng/ml TSA. At this high
concentration a full effect was already detectable after 1 h of
incubation (unpublished data). Although the distribution of
HSF1 was less drastically perturbed, this factor also dis-
played an homogenous distribution in most of the cell nuclei
at the highest TSA dose. It is worth noticing that these
treatments did not increase the fraction of apoptotic cells
measured by DAPI staining after 48 h of recovery (unpub-
lished data). The effect of the drug was completely reversible
and an interval of 3 h between TSA withdrawal and heat
shock was sufficient to rescue the ability of the cells to form
stress bodies.
Stress Bodies Contain Acetylated Histone H4
The result reported above prompted us to investigate in
more detail the relationship between chromatin and stress
bodies. A standard marker of heterochromatin domains,
from yeast to mammals, is the presence of underacetylated
histone H4 (Turner, 2002). On the other hand, TSA, an
inhibitor of histone deacetylases that induces a drastic in-
N. Rizzi et al.
Molecular Biology of the Cell 544

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crease in the level of acetylated histones, prevents the as-
sembly of stress bodies. A reasonable prediction is that
stress bodies might correspond to nuclear districts particu-
larly poor of acetylated histones. We decided to verify this
prediction by assessing the relative distribution of acetylated
histone H4 (Ac-H4), hnRNP HAP, and HSF1 in unstressed
and heat-shocked HeLa cells. Surprisingly, immunofluores-
cence analysis showed a perfect colocalization of Ac-H4
with hnRNP HAP and HSF1 in stress bodies that, contrary to
our expectations, were the nuclear districts most intensely
decorated by the anti-(Ac-H4) antibodies (Figure 2). This
staining pattern suggests that stress bodies are assembled on
large chromatin territories characterized by a high density of
Ac-H4. Notably, anti-(Ac-H4) antibodies do not decorate
structures similar to stress bodies in unstressed cells (Figure
2), indicating that acetylation of histone H4 bound to these
chromosomal domains is specifically triggered by heat
shock.
Histone H4 can be acetylated in vivo on four lysine resi-
dues (K5, K8, K12, and K16) all located in the N-terminal tail.
Lysine-specific H4 acetylation has been linked to the regu-
lation of mammalian chromatin (Johnson et al., 1998). How-
ever the details of this control mechanism are still mostly
undefined. A few evidences have recently linked acetylation
of K8 and K16 with transcription (Johnson et al., 1998; Agali-
oti et al., 2002). Acetylation of K5 and K12, on the other hand,
identifies newly synthesized histone H4 (Sobel et al., 1995). It
is generally assumed that deposition of histone H4 acety-
lated on K5 and K12 during DNA replication is followed by
rapid deacetylation to preserve the underacetylated pattern
of histone H4, a critical parameter for the maintenance of the
silenced state of heterochromatin (Taddei et al., 1999). To
decipher the pattern of histone H4 modification in stress
bodies, we stained unstressed and heat-shocked HeLa cells
with commercially available antibodies able to distinguish
between the four acetylated lysines. The immunofluores-
cence analysis in Figure 3 shows that stress bodies lie in
nuclear districts devoid of Ac-K5 and Ac-K12. On the con-
trary, they are recognized by antibodies specific for Ac-K8
and Ac-K16. The association with Ac-K8 is particularly ev-
ident and, in fact, stress bodies are the nuclear regions most
intensely stained by antibodies against this acetylated lysine.
In this respect, the staining pattern observed with anti-
Ac-K8 is highly similar to that obtained with antibodies
directed to the hyper-acetylated form of histone H4. Collec-
tively, these results indicate that the chromatin regions as-
sociated to stress bodies are enriched in histone H4 isoforms
characterized by acetylation of K8 and K16, a posttransla-
tional modification that distinguishes transcriptionally com-
petent portions of the genome.
Stress Bodies Lack Distinguishing Features of
Heterochromatin
Although formed by arrays of satellite sequences and cyto-
logically defined as heterochromatin, chromosomal domains
associated to stress bodies are characterized by the presence
of Ac-H4, a marker of euchromatic portions of the genome.
We decided to explore this aspect more in detail by deter-
Figure 1.
were treated for 6 h with increasing concentrations of TSA. After
removal of the drug, cells were heat-shocked 1 h at 42°C and then
allowed to recover 1 h at 37°C before fixation with 4% formalde-
hyde. Cells were then independently stained with (1) rabbit poly-
clonal antibodies against hnRNP HAP, (2) mAb-96 against SF2/
ASF, or (3) rat mAb 10H8 against HSF1. Primary antibodies were
revealed with FITC-conjugated anti-rabbit, anti-mouse, or anti-rat
goat antibodies. The fraction of cells with stress bodies was mea-
sured in three independent experiments counting 500 cells per
experiments. This fraction was expressed as a percentage of the
value measured in cells treated only with the solvent of TSA (eth-
anol). White bars, hnRNP HAP; gray bars, SF2/ASF; black bars,
HSF1.
TSA inhibits the assembly of stress bodies. HeLa cells
Figure 2.
were heat-shocked 1 h at 42°C and then allowed to recover 1 h at
37°C. Cells were then fixed in 4% formaldehyde and costained with
a rabbit polyclonal antibody against the hyper-acetylated histone
H4 and with either anti-HAP 16C3 mAb or the rat anti-HSF1 10H8
mAb. The distribution of hnRNP HAP and Ac-H4 in unstressed
cells was also determined (37°C). Primary antibodies were revealed
with FITC-conjugated anti-rabbit and with rhodamine-conjugated
anti-rat or anti-mouse goat antibodies. Cells were then analyzed by
confocal laser microscopy. Images of the same cells are shown.
Stress bodies contain acetylated histone H4. HeLa cells
Activation of Heterochromatic Domains
Vol. 15, February 2004 545

Page 4

mining whether some features typical of heterochromatin
domains were still present in these regions.
Pericentromeric heterochromatin is assembled in a spe-
cific higher-order structure, characterized by the presence of
hypo-acetylated histones and of nonhistone proteins such as
HP1. In accordance with the notion that HP1 and Ac-H4
label, respectively, hetero- and euchromatin domains, anti-
bodies directed against HP1? failed to recognize stress bod-
ies stained by anti-hnRNP HAP antibodies (Figure 4). An
identical pattern was observed with antibodies against the
other two members of the HP1 family, HP1? and ? (Supple-
mentary Figure 1). Moreover, no colocalization of HP1?
with hnRNP HAP was detectable in unstressed cells (Figure
4). The absence of colocalization with stress bodies was
unlikely to originate from massive degradation of HP1 in
response to heat shock since the steady state level of these
proteins remained constant throughout the experiment
(Supplementary Figure 2).
Another marker of heterochromatin is histone H3 meth-
ylated on K9 (H3-mK9). Two antibodies against H3-mK9 are
available. The first one was raised against a linear K9-dim-
ethylated peptide and detects any silenced chromatin do-
mains with no particular preference for pericentromeric het-
erochromatin. The second antibody was raised against a
branched peptide presenting four fingers of the K9-dimethy-
lated TARSK consensus sequence of the H3 terminus (Mai-
son et al., 2002). It has recently been shown that the structure
recognized by the latter antibody is part of the binding site
of HP1 and is sensitive to TSA treatment (Maison et al.,
2002). In unstressed cells hnRNP HAP and the antigen rec-
ognized by antibody against the branched peptide occupy
mutually exclusive nuclear sites (Figure 5), consistently with
the fact that methylated histone H3 marks trascriptionally
silent portions of the genome, whereas hnRNP HAP labels
ribonucleoprotein complexes. No colocalization between the
two antigens was also observed after 1 h of heat-shock at
42°C. However, during recovery from stress a more complex
pattern was detectable in a subset of cells. After 3 h of
recovery, in ?10% of the cells, methylated H3 accumulated
in large structures adjacent to stress bodies. These structures
were more evident, always in subset of cells, after an addi-
tional 3 h of recovery when hnRNP HAP, as previously
Figure 3.
H4 associated to stress bodies. Unstressed
(37°C) or heat-shocked HeLa cells (1 h at
42°C followed by 1 h of recovery at 37°C)
(42°C) were stained with rabbit polyclonal
antibodies directed against different acety-
lated lysine residues of histone H4 as indi-
cated. Primary antibodies were revealed
with a FITC-conjugated anti-rabbit goat an-
tibody. To reveal the localization of stress
bodies, heat-shocked cells were costained
with anti-HAP mAb 16C3 and with a rhoda-
mine-conjugated anti-mouse goat antibody.
Confocal laser microscopy images of repre-
sentative cells are shown.
Pattern of acetylation of histone
Figure 4.
HP1?. HeLa cells either unstressed (37°C) or heat-shocked 1 h at
42°C followed by 1 h at 37°C (42°C) were fixed in 4% formaldehyde.
Cells were costained with a rabbit polyclonal antibody against
hnRNP HAP, to reveal stress bodies, and with the rat mAb MAC353
specific for HP1?. Primary antibodies were revealed with FITC-
conjugated anti-rabbit and rhodamine-conjugated anti-rat goat an-
tibodies. Confocal laser microscopy images of the same fields are
shown.
Stress bodies are not major sites of accumulation of
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Molecular Biology of the Cell546

Page 5

described (Weighardt et al., 1999), was already redistributed
throughout the nuclear volume. Finally, these structures
were no longer visible after 10 h of recovery. The relevance
of this finding for the dynamics of stress bodies is still matter
of investigation.
Kinetics of Histone H4 Acetylation in Stress Bodies
We have previously described the kinetics of the recruitment
of HSF1 and hnRNP HAP to stress bodies. Although HSF1 is
already detectable in these structures after a very short
incubation at 42°C, the recruitment of HAP is temporally
delayed and peaks after 3 h of recovery at 37°C (Weighardt
et al., 1999). To determine the kinetics of H4 acetylation,
HeLa cells were heat-shocked 1 h at 42°C and allowed to
recover for increasing time intervals at 37°C. Cells were then
analyzed by indirect immunofluorescence with antibodies
against Ac-H4. As shown in Figure 6, an altered distribution
of Ac-H4 was already observed after 15 min at 42°C. In these
cells a number of small dots was detectable, some of which
appeared to be clustered in the proximity of the nucleoli.
Structures comparable to stress bodies were clearly visible
after 30 min at 42°C, but the intensity of the signals and the
size of these structures kept growing and peaked at 6 h of
recovery. At longer times (10 h of recovery) the fraction of
cells displaying stress bodies started to diminish, and stress
bodies had a fuzzier appearance. A normal distribution of
Ac-H4 was detectable after 20 h of recovery (unpublished
data). Altogether this analysis indicates that heat shock in-
duces a transient modification of the higher-order structure
of the heterochromatic regions associated with stress bodies.
Heat Shock Induces Transcription of Satellite III DNA
Sequences
After heat shock the heterochromatic domains associated to
stress bodies have a higher-order structure competent for
transcription. An open possibility is that heat shock can
Figure 5.
of accumulation of histone H3 methylated
on K9 (H3-mK9). HeLa cells either un-
stressed (37°C) heat-shocked 1 h at 42°C
(42°C) or allowed to recover for the indi-
cated time intervals were fixed in 4% form-
aldehyde. Cells were stained with the anti-
HAP mAb 16C3 and with the rabbit
antibody raised against
?-methH3-K9 peptide. Primary antibodies
were revealed with FITC-conjugated anti-
mouse and rhodamine-conjugated anti-rab-
bit goat antibodies. Confocal laser micros-
copy images of the same fields are shown.
Stress bodies are not major sites
the branched
Activation of Heterochromatic Domains
Vol. 15, February 2004547