3262
Research Article
P-STAT1 mediates higher-order chromatin remodelling
of the human MHC in response to IFN␥
Rossitza Christova1,2,*, Tania Jones1,2,*, Pei-Jun Wu1,*, Andreas Bolzer1, Ana P. Costa-Pereira3,4,
Diane Watling3,4, Ian M. Kerr3 and Denise Sheer1,2,‡
1
Human Cytogenetics Laboratory, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London, WC2A 3PX, UK
Institute of Cell and Molecular Science, Queen Mary’s School of Medicine and Dentistry, 4 Newark St, London, E1 2AT, UK
3
Biochemical Regulatory Mechanisms Laboratory, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London,
WC2A 3PX, UK
4
Imperial College London, Faculty of Medicine, Department of Oncology/SORA, Hammersmith Hospital, Du Cane Road, London, W21 ONN, UK
2
*These authors contributed equally to this work
‡
Author for correspondence (e-mail: d.sheer@qmul.ac.uk)
Journal of Cell Science
Accepted 13 July 2007
Journal of Cell Science 120, 3262-3270 Published by The Company of Biologists 2007
doi:10.1242/jcs.012328
Summary
Transcriptional activation of the major histocompatibility
complex (MHC) by IFN␥ is a key step in cell-mediated
immunity. At an early stage of IFN␥ induction, chromatin
carrying the entire MHC locus loops out from the
chromosome 6 territory. We show here that JAK/STAT
signalling triggers this higher-order chromatin remodelling
and the entire MHC locus becomes decondensed prior to
transcriptional activation of the classical HLA class II
genes. A single point mutation of STAT1 that prevents
phosphorylation is sufficient to abolish chromatin
remodelling, thus establishing a direct link between the
JAK/STAT signalling pathway and human chromatin
architecture. The onset of chromatin remodelling
corresponds with the binding of activated STAT1 and the
Introduction
The human major histocompatibility complex (MHC) located
on chromosome band 6p21 is the most important genomic
region with respect to immunity to infectious agents,
autoimmunity and transplantation (Horton et al., 2004). The
products of the classical MHC class I (HLA-A, HLA-B, HLAC) and class II (HLA-DR, HLA-DP, HLA-DQ) genes present
processed antigens to cytotoxic and helper T-cells, respectively.
The MHC class III region encodes complement proteins and
inflammatory cytokines. Classical MHC class II genes are
expressed constitutively only in antigen-presenting cells such
as B-lymphocytes, macrophages and dendritic cells. Treatment
of other cell types with interferon-␥ (IFN␥), however, induces
expression of the classical class II and several other genes in
the MHC via the JAK/STAT signalling pathway (Boehm et al.,
1997; Stark et al., 1998).
IFN␥ binding to its receptor at the cell membrane leads to
phosphorylation of JAK1 and JAK2. These proteins then
phosphorylate the transcription factor STAT1 at Tyr701.
Phosphorylated STAT1 (P-STAT1) dimerises and moves
rapidly into the nucleus, where it binds to the GAS (gamma
activating sequence) element of the promoters to initiate
transcription of IFN␥ primary response genes. In the MHC,
these include TAP1 (Min et al., 1996), Hsp70/90 (HSPA1)
(Stephanou et al., 1999) and tapasin (TAPBP) (Herberg et al.,
chromatin remodelling enzyme BRG1 at specific sites
within the MHC, and is followed by RNA-polymerase
recruitment and histone hyperacetylation. We propose that
the higher-order chromatin remodelling of the MHC locus
is an essential step to generate a transcriptionally
permissive chromatin environment for subsequent
activation of classical HLA genes.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/120/18/3262/DC1
Key words: JAK/STAT signalling, MHC, Chromatin architecture,
Decondensation, Looping
1998). The primary response genes on other chromosomes
include IRF1 (Hobart et al., 1997) and the class II
transactivator gene (CIITA) (van den Elsen et al., 2004), which
are required for subsequent activation of the HLA genes (Reith
et al., 2005). HLA class I and II genes share well-conserved
promoter modules to which the constitutively expressed
transcription factors, RFX, X2BP and NF-Y, bind
cooperatively to form the enhanceosome. In response to IFN␥,
CIITA is synthesised, and then binds and stabilises the
enhanceosome (Gobin et al., 1999; Krawczyk et al., 2004).
Histone hyperacetylation of HLA class II genes, including
HLA-DRA, is detected 4 hours after the start of IFN␥ treatment,
and is followed by transcription 2 hours later (Spilianakis et
al., 2003).
The cell-type-specific and inducible expression of the MHC
makes it a powerful model system for analysing the
relationship between transcription and chromatin architecture.
We previously found that transcriptional activation by IFN␥ of
the human HLA class II genes in fibroblasts is preceded by
massive remodelling of the chromatin fibre, which manifests
as a rapid looping-out from the chromosome 6 territory (CT6)
(Volpi et al., 2000). Similar giant chromatin loops have been
observed for the human epidermal differentiation complex
(Williams et al., 2002), the human and mouse ␤-globin loci
(Ragoczy et al., 2003) and the mouse Hox gene cluster
STAT mediates MHC chromatin remodelling
Journal of Cell Science
(Chambeyron and Bickmore, 2004). The significance of this
chromatin remodelling over large genomic regions is not
known. However, important insights into the relationship
between chromatin movement and gene expression have been
obtained from studies on artificial transgene arrays where
targeting of strong transcriptional activators initiates
recruitment of histone acetyl transferases (HATs) and other
chromatin modifiers, such as BRG1, accompanied by largescale chromatin decondensation (Carpenter et al., 2005; Muller
et al., 2007).
To understand the mechanism and significance of higherorder chromatin remodelling of the MHC, we examined the
architecture of the locus together with DNA-protein
interactions and histone acetylation at the early stages of IFN␥
induction. We provide evidence for a direct link between the
JAK/STAT signalling pathway and higher-order chromatin
remodelling across the MHC. We further show that the
conformational changes reflect a statistically significant level
of decondensation in the MHC but not in the surrounding
genomic regions. Taken together, our findings suggest that the
induction of an ‘open’ or transcriptionally competent
chromatin conformation is a crucial early step in cytokinemediated activation of the MHC.
3263
domain towards the MHC signal. Each chromosome
homologue was counted separately and a score derived for the
percentage of loci that were located on an external chromatin
loop. These criteria were applied to all cell types studied and
all FISH experiments were repeated at least twice, and
analysed by two independent researchers to ensure objective,
reproducible results. The frequency with which the MHC locus
was present on an external chromatin loop in untreated
HT1080 cells, 13% of CT6s (n=306), almost doubled within
the first 10 minutes of IFN␥ treatment, reaching a peak of 34%
at 24 hours (n=219, P<0.05) (Fig. 1A, supplementary material
Fig. S1). This time-course is identical to our previous findings
on MRC5 fibroblasts treated with IFN␥ (Volpi et al., 2000),
indicating that HT1080 cells provide a suitable model system
for studying higher-order chromatin architecture in the MHC.
The role of STAT1 in higher-order chromatin remodelling of
the MHC was then examined in HT1080-derived U3A cells,
which are STAT1-negative and not responsive to IFN␥
(Chatterjee-Kishore et al., 2000; Muller et al., 1993). No
higher-order chromatin remodelling of the MHC was detected
in U3A cells in response to IFN␥ (MHC locus on an external
chromatin loop before treatment in 16% of CT6s, n=493; and
after treatment in 15% of CT6s, n=481; P=0.59; Fig. 1B). A
Results
We previously found a clear difference in the conformation of
the chromatin fibre carrying the MHC in cells with different
profiles of HLA class II expression (Volpi et al., 2000). In Blymphoblastoid cells, in which the HLA class II genes are
constitutively expressed, the MHC is present on a giant
external chromatin loop in ~35% of CT6s. By contrast, in
fibroblasts that do not express HLA class II genes, the
frequency is ~10%, increasing after IFN␥ treatment to ~35%.
It is of particular interest that this conformational change is
seen in fibroblasts after only 10 minutes exposure to IFN␥,
several hours before the HLA class II gene cluster is
transcribed.
To understand the significance of these observations, we
addressed the following questions. Is the massive chromatin
conformational change induced through the JAK/STAT
pathway, which is required for subsequent HLA class II
transcription? Which DNA-protein interactions are significant
in this process? Does histone acetylation play a role? Finally,
does the visible conformational change reflect an altered level
of chromatin condensation?
STAT1 is essential for IFN␥-induced higher-order
chromatin remodelling
To determine whether higher-order chromatin remodelling
occurs through the JAK/STAT signal transduction pathway, we
analysed a well-characterised set of STAT1-mutant cell lines
derived from the fibrosarcoma cell line HT1080 (ChatterjeeKishore et al., 2000; Muller et al., 1993). We first verified that
IFN␥ was able to induce looping-out of the MHC in HT1080
cells by analysing the location of MHC-specific probes relative
to the chromosome-6-territory paint by FISH. As in our
previous work, ‘external chromatin loops’ were defined as
configurations where the MHC probe signal was outside the
painted chromosome 6 domain, without touching the border of
the domain. This included observations in which a faint stalklike projection was seen extending from the chromosome
Fig. 1. P-STAT1 is essential for the induction of external chromatin
loops carrying the MHC locus in response to 24-hour IFN␥
treatment. FISH signals for HLA-DRA are detected in red and for the
CT6 in green. (A) IFN␥ induction of external chromatin loops
carrying the HLA-DRA gene. (B) No IFN␥ induction of external
chromatin loops carrying the HLA-DRA gene in this STAT1-null cell
line. U3A cells are tetraploid and contain four copies of chromosome
6. (C) IFN␥ induction of external chromatin loops carrying the HLADRA gene in U3A cells complemented with STAT1. (D) No IFN␥
induction of external chromatin loops carrying the HLA-DRA gene in
U3A cells complemented with a STAT1 point mutant (Y701F) that
cannot be phosphorylated by JAKs.
Journal of Cell Science
3264
Journal of Cell Science 120 (18)
complemented U3A cell line (U3A/STAT1) that stably
expresses STAT1 (Kumar et al., 1997; Muller et al., 1993) was
then examined. STAT1 is phosphorylated in these cells to
normal levels and HLA class II genes are expressed in response
to IFN␥ (Improta et al., 1994). IFN␥ treatment was able to
induce MHC looping-out from the CT6 in U3A/STAT1 cells
(MHC locus on an external chromatin loop before treatment in
18% of CT6s, n=782, and after treatment in 26% of CT6s,
n=449, P<0.05; Fig. 1C). However, complementation of U3A
cells with a Tyr to Phe point mutant of STAT1 (STAT1Y701F)
that cannot be phosphorylated by JAKs, did not restore MHC
looping-out in response to IFN␥ (MHC locus on an external
chromatin loop before treatment in 17% of CT6s, n=393, and
after treatment in 15% of CT6s, n=448; P=0.45; Fig. 1D).
These findings implicate P-STAT1 in both transcriptional
upregulation of HLA class II genes and higher-order chromatin
remodelling of the MHC in response to IFN␥.
The rapid induction of higher-order chromatin remodelling
in the MHC by IFN␥ suggested that CIITA is not involved. To
test this and to distinguish a possible direct role for STAT1 in
chromatin remodelling from its role in activating CIITA
transcription, U3A cells were transfected with a constitutively
expressed CIITA vector (pEBS-PL-CIITA form III). Although
CIITA was detected at both mRNA and protein levels (see
supplementary material Fig. S2A), neither HLA-DRA
expression (see supplementary material Fig. S2B) nor loopingout of the MHC were observed (MHC locus on an external
chromatin loop in 17% of CT6s, n=314, P=0.80). These
findings indicate that expression of CIITA alone is not
sufficient for IFN␥ induced changes in chromatin structure
across the MHC locus.
P-STAT1 binds in vivo to the primary IFN␥-activated
genes in the MHC
To determine the order of transcription factor interactions at
individual genes, chromatin immunoprecipitation (ChIP)
experiments were performed on HT1080 and STAT1-null U3A
cells at different times after the start of IFN␥ treatment. In vivo
recruitment of STAT1, RNAP II, TFIIB, BRG1, and histone
acetylation changes to promoters of the TAP1, HSPA1 and
HLA-DRA genes located in the MHC (see supplementary
material Fig. S3), and the IRF1 gene located on chromosome
5, were then determined by quantitative real-time (RT)-PCR.
Since the classical MHC class II genes are coordinately
regulated, HLA-DRA was used, as elsewhere, as a model for
changes that occur at all of the class II genes. GAPDH
promoter sequences were amplified from the same
immunoprecipitated material as a control for the efficiency of
immunoprecipitation. IFN␥-induced changes were calculated
relative to non-induced levels for each time point.
Phosphorylation of STAT1 and its translocation to the
nucleus, where it binds to the target sequences with very high
specificity, occurs within 5 minutes of IFN␥ treatment (Haspel
et al., 1996). We found that, during IFN␥ treatment of HT1080
cells, P-STAT1 becomes associated with the promoters of the
primary response genes TAP1, HSPA1 and IRF1, reaching a
maximum at 30 minutes and decreasing after 1 hour. P-STAT1
did not associate with the GAPDH promoter, which has no
STAT1-response element (Fig. 2). As expected, the same
experiments performed on U3A cells showed no P-STAT1
binding. The slight delay in the detection of P-STAT1 in the
ChIP assay, as compared with FISH observations, is probably
owing to the characteristics of the ChIP method, which detects
the average promoter occupancy in a population of cells at any
given time.
These findings are consistent with the activationdeactivation cycle of STAT1 revealed by EMSA (Haspel and
Darnell, 1999). We used for our study a highly specific
antibody that recognises only the IFN␥-activated form of
STAT1, phosphorylated on the Tyr701. Therefore, we cannot
exclude the possibility that STAT1 is dephosphorylated on the
target sequence and then continues to play a role in the
activation of other genes, such as LMP2 (PSMB9) (ChatterjeeKishore et al., 2000). When combined with the observation that
higher-order chromatin architecture of the MHC is unaffected
by IFN␥ in the U3A cells complemented with the
phosphorylation-defective STAT1Y701F protein, these data
strongly suggest that P-STAT1 is the factor that transmits the
signal for chromatin modification to specific sites.
RNAP II is recruited to the IFN␥ primary response
genes in a STAT1-dependent manner, whereas TFIIB is
present constantly
A significant increase in RNAP II recruitment was found at the
TAP1, HSPA1 and IRF1 promoters in HT1080 cells at 1 hour
of IFN␥ treatment, and the level escalated thereafter (Fig. 2).
By contrast, there was no enrichment of RNAP II at the HLADRA promoter during the first 6 hours of IFN␥ induction, as
expected from the lack of transcriptional activity of CIITAdependent MHC genes during this time period (Spilianakis et
al., 2003). RNAP II was not found at the promoters tested at
any time in STAT1-null U3A cells, consistent with the absence
of TAP1, IRF1 and HLA-DRA expression (Chatterjee-Kishore
et al., 1998; Muller et al., 1993).
TFIIB was found to be associated with the promoter regions
of the primary response genes TAP1, HSPA1 and IRF1, and the
CIITA-dependent HLA-DRA gene at the same levels before and
after IFN␥ treatment (data not shown). The enrichment at these
promoters was well above (20-30 times) that for the ␤-globin
gene, which is not expressed in fibroblasts (see supplementary
material Table S1), and did not differ between HT1080 and
U3A cells, indicating that the binding of TFIIB to MHC
promoters is not dependent on STAT1. The presence of TFIIB
and other general transcription factors (Spilianakis et al., 2003)
at the promoters of these genes before they are expressed might
reflect the presence of partially assembled pre-initiation
complexes to help maintain a transcriptionally poised
chromatin conformation.
BRG1 is recruited to the promoters of the TAP1 and
IRF1 genes after IFN␥ induction
ATP-dependent chromatin remodelling complexes play a
significant role in altering chromatin structure during
mammalian differentiation, cell cycle and recombination
(reviewed in de la Serna et al., 2006). They act by disrupting
histone-DNA interactions, leading to the exposure of DNA
sequences to regulatory proteins. BRG1, a catalytic subunit of
the chromatin-remodelling SWI/SNF complex, has been
implicated in transcriptional activation of the heat shockinduced mouse gene hsp70 (homologous to human HSPA1)
(Corey et al., 2003) and certain human IFN␣-induced genes
(Huang et al., 2002). Analysis of BRG1 recruitment during
Journal of Cell Science
STAT mediates MHC chromatin remodelling
3265
Fig. 2. ChIP analysis of transcription factor recruitment upon IFN␥ induction. HT1080 and STAT1-null U3A cells were treated with 200 IU/ml
IFN␥ at the times shown, and crosslinked with formaldehyde. Immunoprecipitated DNA and serial dilutions of genomic DNA were then
subjected to quantitative RT-PCR. The fold enrichment was calculated relative to the non-induced levels. ChIP experiments were performed
using antibodies recognizing P-STAT1, RNAP II, BRG1 and acetylated Histone H3. The mean values of at least three experiments are shown,
together with standard error bars.
IFN␥ treatment in HT1080 cells revealed enrichment relative
to non-induced levels at the TAP1 and IRF1 promoters reaching
a maximum at 30 minutes after the start of IFN␥ treatment
(Fig. 2). The levels then decreased at 1 hour, similar to the
kinetics of P-STAT1 recruitment. BRG1 was not associated
with the HSPA1 or HLA-DRA promoters in the first 6 hours of
IFN␥ treatment, indicating that its recruitment to HSPA1 might
be activator- or species-dependent. BRG1 was not enriched at
any of the promoters examined in STAT1-negative U3A cells.
These findings suggest that BRG1 is involved in STAT1dependent remodelling of the TAP1 and IRF1 promoters in
response to IFN␥.
To determine whether BRG1 participates in IFN␥-induced
higher-order chromatin remodelling of the MHC, we analysed
the cell line SW13, which lacks BRG1 and BRM, another
catalytic subunit of the SWI/SNF complex (Pattenden et al.,
2002). SW13 cells do not express HLA class II genes but have
an intact JAK/STAT pathway. The induction of at least one
IFN␥ response gene, CIITA, can be restored after introduction
of exogenous BRG1 (Pattenden et al., 2002). Expression of
TAP1, HSPA1, IRF1, HLA-DRA and CIITA was found by RTPCR to be unaffected in SW13 by treatment with IFN␥ (see
supplementary material Fig. S4A). FISH analysis showed that
IFN␥ treatment also had no significant effect on higher-order
chromatin conformation of the MHC (MHC locus on an
external chromatin loop before treatment in 19% of CT6s,
n=762, and after treatment in 16% of CT6s, n=743, P=0.10;
Fig. 3). However, complementation of SW13 cells with BRG1
(retroviral expression vector pBabe-IRESpuroBRG1) restored
induction of TAP1, IRF1, HLA-DRA and CIITA in response to
IFN␥ (see supplementary material Fig. S4A). In these cells,
IFN␥ induced looping-out of the MHC (MHC locus on an
external chromatin loop before treatment in 15% of CT6s
(n=294), and after treatment in 29% CT6s; n=294, P<0.05; Fig.
3), as in HT1080 cells. SW13 cells stably infected with BRM
or an empty vector showed no increase in the number of
external chromatin loops. These findings suggest that BRG1 is
involved in higher-order chromatin remodelling of the MHC
upon IFN␥ induction.
Histone acetylation
Histone acetylation is recognised as a hallmark of
transcriptionally competent chromatin. At 4-8 hours after the
start of IFN␥ treatment, histone acetylation is reported to
increase at the promoter of the HLA-DRA gene in a CIITAdependent manner (Beresford and Boss, 2001; Spilianakis et
al., 2003). Since STAT1 interacts with CBP/p300, which has
histone acetylase activity (Zhang et al., 1996), we set out to
determine whether higher-order chromatin remodelling arose
from earlier histone hyperacetylation within the MHC. ChIP
experiments on HT1080 cells and MRC5 fibroblasts following
IFN␥ treatment revealed a progressive increase in histone H3
acetylation at the promoters of the primary response genes
TAP1 and HSPA1, reaching a maximum at 2-4 hours of IFN␥
treatment (Fig. 2). At the HLA-DRA promoter, an increase in
histone H3 acetylation was observed after 2 hours of IFN␥
treatment, increasing further within 24 hours. The same
experiments performed on STAT1-deficient U3A cells showed
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Journal of Cell Science 120 (18)
Journal of Cell Science
Fig. 3. Analysis of the role of BRG1 and BRM in large-scale chromatin remodelling of the MHC. SW13 cells (BRG1-BRM-negative) were
treated with IFN␥ for 24 hours where indicated. HLA-DRA is detected in red and the CT6 in green. (A) SW13 cells without IFN␥. (B) SW13
cells treated with IFN␥. No external chromatin loops are observed. (C) SW13 cells, complemented with BRG1 and treated with IFN␥. External
chromatin loops carrying the MHC are clearly observed.
no IFN␥ induced acetylation at any of the promoters tested.
Histone H3 acetylation occurring at both primary and CIITAdependent promoters within the first 4 hours of IFN␥ treatment
is therefore STAT1-dependent. As no significant histone
hyperacetylation was observed at the genes examined
immediately after the start of IFN␥ treatment, these
experiments indicate that histone hyperacetylation is unlikely
to play a role in the onset of higher-order chromatin
remodelling in the MHC.
Next, we investigated whether unspecific hyperacetylation
by histone deacetylase (HDAC) inhibitors could induce higherorder chromatin remodelling in the MHC. MRC5 fibroblasts
were treated with 10 mM sodium butyrate (SB) for 1 hour in
order to avoid induction of apoptosis and cell cycle arrest
associated with prolonged exposure to HDAC inhibitors. ChIP
analysis of HLA-DRA indicated that treatment with SB for 1
hour leads to the maximum level of acetylation in H4 histones
and two-thirds of the maximum acetylation in H3 histones
(data not shown). No transcriptional activation of HLA genes
was observed.
SB was found to induce looping-out of the MHC. Treatment
with SB resulted in an increase in the number of external loops
across the entire MHC locus. External chromatin loops
increased from 11% (n=163) to 22% (n=127) P=0.02, for the
class I region; from 11% (n=135) to 32% (n=228) P<0.05, for
the class II region; and from 12% (n=125) to 24% (n=109)
P=0.02, for the class III region (Fig. 4A,B). Only a small
increase of loop induction from 5% (n=108) to 8% (n=109)
P=0.44, of CT6s was found in the gene-poor 6p24 region.
Treatment of HT1080 with SB showed identical results. A
Fig. 4. Chromatin remodelling can be induced
in MRC5 fibroblasts by IFN␥ and sodium
butyrate (SB). (A) FISH image showing HLADRA (red) extending from the painted CT6
(green) in MRC5 cells after treatment with SB
for 1 hour. (B) Comparison of external
chromatin loops for the MHC and the 6p24
region in untreated control cells, in cells
treated with IFN␥ for 24 hours and in cells
treated with SB for 1 hour (n⭓100). A
significant increase in the percentage of
external chromatin loops was observed for all
three MHC regions in IFN␥ and SB-treated
fibroblasts compared with untreated cells.
(C) External chromatin loops carrying HLADRA induced by SB treatment over a 1-hour
time course. (D) Comparison of external
chromatin loops induced by IFN␥ over a 24hour time course in control cells (without
IFN␥) and cells treated with SB for the last 1
hour of the IFN␥ treatment. The mean
percentage of signals on external chromatin
loops is represented on the y-axis, error bars
show the standard deviation. The value at the
0-minute (0’) time point for MRC5+SB+IFN␥
is high because the cells have been exposed to
SB for 1 hour.
STAT mediates MHC chromatin remodelling
Journal of Cell Science
time-course analysis of SB treatment of MRC5 cells showed
that external chromatin loops were formed within 10 minutes
of the start of treatment (Fig. 4C, n=232, P=0.01).
Furthermore, treatment with IFN␥ and SB together did not
enhance the number of external chromatin loops (Fig. 4D;
P>0.07), suggesting that once external loops have formed
SB cannot induce additional higher-order chromatin
modifications.
Chromatin in the MHC becomes decondensed upon
treatment with IFN␥
Chromatin decondensation has been associated with induction
of transcription in both transgene arrays and gene clusters
(Carpenter et al., 2005; Sproul et al., 2005). To examine
whether IFN␥ induces alterations in chromatin decondensation
in MRC5 cells, interphase distances were measured from a
series of six probe pairs in the MHC separated by genomic
distances ranging from 0.63-3.4 Mb (see supplementary
material Fig. S3). The mean values of the interphase distances
were in the range of 0.9-1.94 ␮m in untreated cells and 1.212.52 ␮m in IFN␥-treated cells (see supplementary material
Table S2). IFN␥ treatment led to an increase in interphase
distances in the MHC of up to 25%, indicating that the
chromatin becomes decondensed, as observed previously for
part of the MHC class II region (Müller et al., 2004). No
statistically significant increase was observed for probe pairs
in the control gene-poor 6p24 region, values were 0.52 ␮m and
0.54 ␮m (P>0.05) for untreated and IFN␥ treated cells,
respectively. We then measured the interphase distances in the
genomic regions flanking the MHC, using probe pairs with
genomic separations up to ~3Mb. No significant change was
observed with IFN␥ treatment (supplementary material Table
S2), indicating that decondensation induced by IFN␥ treatment
is limited to the MHC.
A linear relationship was found in the MHC between the
mean square interphase distance and genomic distance
between probes tested for both IFN␥-treated and -untreated
cells (Fig. 5A). The slope of the regression line for IFN␥treated cells (2.26 ␮m2/Mb) was found to be ~1.5 times greater
than for the untreated cells (1.50 ␮m2/Mb), demonstrating that
the chromatin in the MHC becomes decondensed after IFN␥
treatment. Evaluating the shape of the statistical distribution
measured for each probe pair gave us ratios for the standard
deviation to its mean (s.d. : mean) and for the median to its
3267
mean (median : mean), close to the ideal values of a Raleigh
distribution before and after IFN␥ treatment (data not shown).
These findings demonstrate that chromatin behaves as a
random polymer (van den Engh et al., 1992).
The distance measurements described above were taken
irrespective of their position relative to the chromosome
territory, i.e. we did not discriminate whether a probe signal
was located on an external loop. Therefore, we compared
chromatin within the painted chromosome domain and within
loops. In untreated cells, comparison of the slope of the
regression line for probe pairs within a painted chromosome
domain (1.23 ␮m2/Mb), with that for probe pairs on an
external chromatin loop (3.49 ␮m2/Mb) reveals a 2.7 times
increase in decondensation. Comparison of the slope of the
regression line for probe pairs within a painted chromosome
domain before (1.23 ␮m2/Mb) and after IFN␥ treatment (1.88
␮m2/Mb) revealed a moderate 1.5 times increase in
decondensation (Fig. 5B). No significant difference was
found in the slope of the regression line for probe pairs on an
external chromatin loop before (3.26 ␮m2/Mb) and after
IFN␥ treatment (3.49 ␮m2/Mb) (Fig. 5C). Since the level of
chromatin condensation in external loops was relatively
unchanged after IFN␥ treatment, which suggests that once an
external loop has formed no further decondensation can
occur.
Discussion
The MHC is a large gene cluster whose physiological
activation by IFN␥ is a crucial step in the cell-mediated
immune response. The classical HLA class I and class II genes
are highly inducible by IFN␥ in almost all cell types, yet the
mechanism by which this occurs is different for these two sets
of genes. Nevertheless, within a few minutes of IFN␥
treatment, chromatin carrying the entire MHC undergoes
massive higher-order chromatin remodelling (Volpi et al.,
2000). Here, we assess the role of transcriptional activators and
chromatin remodelling factors in chromatin organization
across the MHC, taking advantage of well-characterised
STAT1-deficient cell lines. We integrate FISH, to visualise
chromosome architecture, with ChIP, to examine the ordered
assembly of the transcription machinery and the acetylation
changes at the early time points of transcriptional activation by
IFN␥. Our findings demonstrate that the higher-order
chromatin remodelling observed over the entire MHC in
Fig. 5. Chromatin in the MHC becomes decondensed by IFN␥ treatment. Relationship between mean square interphase distance and genomic
separation (Mb) of the probe pairs within the MHC. Black diamonds: control cells without IFN␥. Grey squares: cells treated with IFN␥ for
24 h. The slope of each regression line is indicated by y, r is the correlation coefficient. (A-C) Distances were measured between probe pairs,
regardless of whether they were (A) on an external loop or within the CT6, (B) located within the CT6 or, (C) located on an external loop.
Journal of Cell Science
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Journal of Cell Science 120 (18)
response to IFN␥ is mediated by STAT1, and that it reflects
decondensation of the chromatin.
IFN␥ treatment induced visible chromatin changes of the
MHC in HT1080 fibrosarcoma cells with the same dynamics
as found previously in fibroblasts (Volpi et al., 2000). The basal
level (~10%) of external loops carrying the MHC in cells that
do not express the HLA class II genes rises to 35% after IFN␥
induction. Even within a population of cells that is expressing
the locus at a very high level we still detect a maximum of
35%. Hence, external loops might not be an absolute
requirement for transcription per se, but may enhance
transcriptional competence of the locus. They might also
reflect the intermittent transcription of a locus at any given
moment in time. The significance of these observations is
likely to be understood only when techniques are developed to
visualise higher-order chromatin alterations in live cells.
Remarkably, higher-order chromatin remodelling was not
detected in U3A, the STAT1-null derivative of HT1080.
Complementation of U3A cells with wild-type STAT1 restores
chromatin remodelling, strongly supporting a role of STAT1 in
chromatin decondensation of the locus. Furthermore, a single
point mutation in STAT1 that prevents phosphorylation also
abolishes higher-order chromatin remodelling, providing a
clear indication that the cytokine signal is indeed transmitted
through the JAK/STAT signalling cascade. In HT1080 cells
treated with IFN␥, P-STAT1 binds to the promoters of the
primary response genes TAP1 and HSPA1 in the MHC, and
IRF1 on chromosome 5. Recent genome-wide profiling of
STAT1 recruitment in HeLa cells reveals high-affinity binding
to predicted target sites located predominantly at the promoter
and enhancer regions (Heintzman et al., 2007; Robertson et al.,
2007). Our data are the first to demonstrate a direct role of the
JAK/STAT signalling pathway in altering higher-order
chromatin architecture in mammalian cells. A role for
JAK/STAT signalling in chromatin architecture has recently
been reported in Drosophila, where overactivation of the
pathway disrupts the stability of heterochromatin leading to
transcription (Shi et al., 2006).
The STAT family of transcriptional activators is implicated
in the regulation of a variety of cellular processes far beyond
the IFN␥ response (Levy and Darnell, 2002). Cell-typespecific responses are achieved in cooperation with a variety
of transcription factors, co-activator proteins and chromatinremodelling complexes (reviewed in Platanias, 2005). In
response to IFN␥, the SWI/SNF chromatin remodelling
component BRG1 is recruited to TAP1 and IRF1 around the
same time as P-STAT1. IFN␥-dependent BRG1 recruitment
does not occur in STAT1-null cells, suggesting that the
sequence-specific binding of STAT1 to the GAS elements
brings BRG1 to the TAP1 and IRF1 promoters in response to
IFN␥. BRG1 and STAT1 are reported to cooperate during
induction of IFN␥-responsive genes (Ni et al., 2005). We
suggest that STAT1 and BRG1 interact with each other, as
shown for STAT2 and BRG1 (Huang et al., 2002). A possible
role for BRG1 in higher-order chromatin unfolding of the
MHC is suggested by our finding that complementation of the
BRG1-BRM-deficient cell line SW13 with BRG1 restores
higher-order chromatin remodelling across the MHC locus in
response to IFN␥. The activator-dependent binding of BRG1
does not exclude further recruitment of SWI/SNF complexes
by acetylated histones or HATs at later stages of MHC
induction. Although the role of ATP-dependent chromatinremodelling complexes in modulating higher-order chromatin
structure is still not defined, components of these complexes
have been shown to be involved in regulating chromatin
structure and gene expression over large distances in T-cell
differentiation (Yasui et al., 2002).
Comparison of the kinetics of P-STAT1, BRG1 and RNAP
II recruitment reveals a slight delay of RNAP II binding to the
target promoters. Since there is no evidence for a direct
interaction between STAT1 and RNAP II subunits, this delay
might be explained by an intermediate interaction of P-STAT1
with the TRAP-mediator complex (Zakharova et al., 2003)
and/or CBP, which tethers RNAP II to the promoters of STAT1
activated genes. The amount of RNAP II increases further with
time after induction despite the decrease in P-STAT1. This
finding indicates that although the initial recruitment of RNAP
II is STAT1-dependent, it is probably maintained at the
promoters by other factors. The presence of TFIIB at the
promoters of the classical HLA genes before they are expressed
might reflect the presence of a preinitiation complex that helps
retain an open chromatin conformation even without
transcription.
The higher-order chromatin changes in response to IFN␥ are
followed by an increase of histone H3 acetylation at the
promoters of primary response genes as well as HLA-DRA,
which is only expressed several hours later. Interestingly, we
find that inhibition of deacetylation by SB in the absence of
IFN␥ induces external chromatin loops carrying the MHC. The
inducible genes within the MHC have high basal levels of
histone acetylation and, hence, inhibition of histone
deacetylase inhibitors results in hyperacetylation, which might
maintain an open chromatin structure. Similar chromatin
conformational changes and an increase in acetylated histone
H3 and dimethylated H3 have been found in other cell types
upon treatment with HDAC inhibitors (Bartova et al., 2005).
As the developmentally regulated Hox genes do not show
chromatin decondensation in response to the deacetylase
inhibitor TSA (Chambeyron and Bickmore, 2004), inducible
and developmentally regulated genes may have different levels
of basal histone acetylation.
The timing of recruitment of transcription factors and
chromatin modifying activities to the MHC, as observed here
by ChIP, agrees well with immuno-fluorescence studies of
VP16-induced transcriptional activation and decondensation of
artificial lac-operator arrays (Carpenter et al., 2005). Local
chromatin changes induced by IFN␥ at specific positions
within the MHC, a natural array of coordinately regulated
genes, appear to be multiplied rapidly throughout the locus
leading to an altered chromatin configuration. Using distance
measurements between probe pairs in the MHC, we show here
a linear correlation between interphase distance and genomic
separation over a range of 3.4 Mb. Our measurements provide
quantitative evidence that IFN␥ leads to decondensation of the
MHC region both within the chromosome territory and, even
more so, when the locus is present on an external chromatin
loop where maximum decondensation is achieved. We have
demonstrated previously that the MHC class II region and other
gene-rich, transcriptionally active regions are closely
associated with PML nuclear bodies (Wang et al., 2004). It
remains to be determined whether decondensed chromatin
carrying the MHC is directed towards transcription factories
STAT mediates MHC chromatin remodelling
3269
Journal of Cell Science
Fig. 6. Model for chromatin changes during activation of the MHC by IFN␥. The MHC is located within the CT6 before induction. GAS
elements (䉫) and CIITA-binding sites (䊉) are shown. Binding of P-STAT1 (䉬) leads to the release of the entire MHC, which loops outside the
CT6 domain. After 2-4 hours the locus becomes hyperacetylated and accessible to transcription factors. CIITA (✹) is synthesised and binds to
the HLA genes 4-6 hours after IFN␥ induction to activate transcription.
(Cook, 2002; Osborne et al., 2004) or towards other coregulated genes, as shown for genes involved in T-cell
differentiation (Spilianakis and Flavell, 2004).
In conclusion, our findings suggest that large-scale
chromatin remodelling represents an important early step in
transcriptional activation of the MHC (Fig. 6). IFN␥ activates
the JAK-STAT cascade, which signals to the chromatin and
RNAP II machinery. Transcriptional upregulation of primary
response genes in the MHC coincides with remodelling of the
entire locus within minutes of IFN␥ treatment. It is striking that
the entire MHC locus becomes decondensed, including the
classical HLA genes, which are not direct targets for STAT1.
Although it is unclear how the higher-order chromatin
modification spreads so rapidly across the MHC, it seems
possible that the acquisition of a decondensed chromatin state
generates a transcriptionally permissive environment for the
subsequent HLA class II response. The human transcriptome
map reveals a clustering of highly expressed genes in specific
chromosomal regions, suggesting that this arrangement is
important for the regulation of certain genes (Caron et al.,
2001). The clustering of MHC genes has been suggested to
provide a selective advantage in a number of respects,
including the co-inheritance of advantageous haplotypes
(Trowsdale, 2002). We propose that, because the entire locus
is subject to a rapid higher-order chromatin modification in
response to STAT1, clustering is also beneficial in facilitating
an efficient immune response to infection.
Virus infection and transfection experiments
Retroviral vectors pBabe-IRESpuro, pBabe-hBRG1-IRESpuro and pBabe-hBRMIRESpuro were obtained from Hideo Iba (Mizutani et al., 2002). Stable populations
of transfected human SW13 cells were generated by retroviral infection as described
previously (Costa-Pereira et al., 2005). U3A cells were transfected with 20 ␮g
CIITA (pEBS-CIITA form III, obtained from Walter Reith, Université de Genève,
Geneva, Switzerland) using GeneJuise transfection protocol (Novagen). Stable
transformants were selected with 200 mg/ml hygromycin B. Western blot analysis
was performed with 10 ␮g nuclear extract using a standard protocol with anti-BRG1
(Upstate), anti-␤-Actin (Sigma) and anti-CIITA (Abcam) antibodies.
FISH and image analysis
FISH was performed using standard methods (Volpi et al., 2000). Preparations were
examined with a Zeiss Axiophot microscope equipped for epifluorescence using a
Zeiss plan-neofluar 100⫻ objective and an optivar set at 1.25⫻ (for chromatin
conformation analysis) or 2⫻ (for chromatin condensation analysis). Separate greyscale images were recorded with a cooled CCD-camera (Photometrics). They were
then pseudocoloured and merged. SmartCapture 2.1.1 software (Digital Scientific,
Cambridge, UK) was used for image analysis and processing. A binomial test was
performed for statistical analysis using R software. P<0.05 was considered to be
significant.
For analysing the conformation of the chromatin fibre carrying the MHC, the
PAC probe RP1-172K2 for the MHC class II gene HLA-DRA was co-hybridised
with FITC-labelled chromosome 6 paint (Cambio) to fixed nuclear preparations and
detected with rhodamine-conjugated anti-digoxigenin (Vector). For analysing the
response of the chromatin fibre carrying the MHC to sodium butyrate (SB), the class
I cosmids P1454 and C0426 (Goldsworthy et al., 1996) and the class III cosmid
K101 (Kendall et al., 1990) were also used. Nuclei were counterstained with DAPI
(200 ng/ml) and mounted in Cityfluor antifade solution.
For analysing chromatin condensation, the distances between pairs of probes
within and outside the MHC were measured in randomly selected interphase nuclei
using the programme ImageJ (http://rsb.nih.gov) (Yokota et al., 1995). One-hundred
measurements were taken for each pair tested. Correlation analysis, linear regression
analysis and other statistical evaluations were performed with Microsoft Excel.
ChIP experiments and real-time PCR
Treatment protocols
ChIP experiments were performed as described (Christova and Oelgeschlager,
2002) with the following antibodies against P-STAT1 (Tyr701) from Cell Signalling,
RNA polymerase II (clone 8WG16) from Covance, TFIIB (C-18) from Santa Cruz,
Brg-1 (H-88) from Santa Cruz and acetyl-histone H3 and Histone H4 from Upstate.
Real-time (RT)-PCR was performed with SybrGreen master mix from Sigma on an
MJ Chromo 4 Robocycler (Bio-Rad) with immunoprecipitated samples and
corresponding input genomic DNA. The amounts of immunoprecipitated material
were normalised to the relevant genomic DNA to allow direct comparison between
different antibodies. The fold enrichment was calculated relative to the non-induced
levels and more than twofold enrichment counted as significant. GAPDH promoter
sequences were amplified from the same material as a control for the IP in each
sample and results were corrected accordingly. Primer sequences are shown in
supplementary material Table S1.
Cells were grown to 70% confluence, and 200 IU/ml of IFN␥ (recombinant human
IFN␥, R&D Systems) added to the culture medium for times between 10 minutes
and 24 hours. Untreated cells from the same culture were used as a control. Cells
were exposed to 10 mM SB (Sigma-Aldrich) for up to 1 hour prior to harvest. For
combined treatments, MRC5 cells were treated with SB for 1 hour and IFN␥
was added for the final 10 minutes for the first time point. For the final time
point, cells were treated with IFN␥ for 24 hours and SB added for the last hour.
Cell lines were harvested by standard techniques using methanol-acetic acid
fixation to produce nuclear preparations for fluorescence in situ hybridisation
(FISH).
We thank Walter Reith for the CIITA expression vector, Hideo Iba
and C. Muchardt for the BRG1 and BRM expression vectors, Gavin
Kelly for statistical evaluation of results, and Facundo Batista, Julie
Cooper, Stephan Beck, Alistair Newall and Petra Gross for critical
discussions. We thank the reviewers for their insightful and helpful
comments. A.B. was supported in part by a Marie Curie Research
Fellowship (QLG1-CT-2002-51704). This work was supported by
Cancer Research UK.
Materials and Methods
Cell lines
The human fibrosarcoma cell line HT1080, its STAT1-null derivative U3A, and
complemented U3A cells, U3A/STAT1 and U3A/STAT1(Y701F), were grown as
described previously (Muller et al., 1993). Normal embryonic fibroblast MRC5 cells
(CCL-171) and a BRG1-BRM-deficient cell line SW13 (CCL-105) derived from
the human small-cell carcinoma of the adrenal cortex were obtained from the ATCC
and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% foetal
calf serum, 2 mM L-glutamine, 50 U/ml penicillin and 50 mg/ml streptomycin, at
37°C in a 10% CO2 atmosphere.
3270
Journal of Cell Science 120 (18)
Journal of Cell Science
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