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TH cell differentiation is accompanied
by dynamic changes in histone
acetylation of cytokine genes
Orly Avni1, Dong Lee1, Fernando Macian1, Susanne J. Szabo2, Laurie H. Glimcher2 and Anjana Rao1
Published online: 10 June 2002, doi:10.1038/ni808
Naïve T cells differentiate into effector cells upon stimulation with antigen, a process that is
accompanied by changes in the chromatin structure of effector cytokine genes. Using histone
acetylation to evaluate these changes, we showed that T cell receptor (TCR) stimulation results in early
activation of the genes encoding both interleukin 4 and interferon-γ.We found that continued culture in
the presence of polarizing cytokines established a selective pattern of histone acetylation on both
cytokine genes; this correlated with restricted access of the transcription factor NFAT1 to these gene
regulatory regions as well as mutually exclusive gene expression by the differentiated T cells. Our data
point to a biphasic process in which cytokine-driven signaling pathways maintain and reinforce
chromatin structural changes initiated by the TCR. This process ensures that cytokine genes remain
accessible to the relevant transcription factors and promotes functional cooperation of the inducible
transcription factor NFAT with lineage-specific transcription factors such as GATA-3 and T-bet.
in the context of a human YAC transgene or the endogenous mouse locus
results in significant decreases in production of IL-4, IL-5 and IL-13 by
TH2 cells12,13. Similarly, deletion of DH sites V and VA from the endogenous Il4 locus in mice results in pronounced impairment of Il4 expression by two different IL-4–producing cell types: TH2 cells and bone marrow–derived mast cells14.
Decondensed or “open” chromatin is characterized by hyperacetylation of associated histones as well as by increased accessibility to
restriction enzymes, nucleases and transcription factors15–19. The NH2terminal tails of histones H3 and H4 are subject to diverse modifications,
including phosphorylation, methylation and acetylation; these covalent
modifications may alter the interaction of histone tails with DNA or
serve as docking sites for chromatin-associated proteins (the “histone
code” hypothesis)17. Core histone acetylation is a reversible post-translational modification, and transcriptional activators and repressors
recruit histone acetyltransferases (HATs) and histone deacetylases
(HDACs) respectively to gene promoters and enhancers15–19.
We show here that T cell differentiation is associated with a dynamic process of chromatin remodeling and histone acetylation at regulatory regions of IL4 and IFNG. Stimulation of naïve T cells for 17–40 h
resulted in a phase of nonselective histone hyperacetylation at both
cytokine genes: this process required T cell receptor (TCR) signals, did
not depend on the presence of polarizing cytokines and was associated
with increased accessibility for the IL4 enhancer to the restriction
enzyme SwaI. At later times, in T cells differentiated for 1 week under
TH1 or TH2 conditions, the nonselective pattern of histone hyperacetylation gave way to selective histone hyperacetylation at regulatory
regions of only the appropriate cytokine genes. This process was
When naïve T helper (TH) cells encounter antigen for the first time in
the periphery, they begin a process of differentiation that includes commitment to a specific pattern of cytokine production. The TH1 cell population is characterized by the production of interferon-γ (IFN-γ) upon
stimulation, whereas the TH2 cell population transcribes the linked
cytokine genes encoding interleukin 4 (IL-4), IL-5 and IL-13. Full
polarization can readily be achieved in vitro by manipulating the
cytokine milieu: IL-12 and IL-4 strongly potentiate TH1 and TH2 differentiation, respectively, via the signal transducers and activators of transcription 4 (STAT4) and STAT6. The lineage-specific transcription factors GATA-3 and T-bet are critical for TH2 and TH1 differentiation;
when ectopically expressed, these proteins not only promote expression
of the relevant cytokines but also suppress transcription of the inappropriate cytokine genes1–5.
Compared to naïve T cells, differentiated TH1 and TH2 cells show
marked changes in the chromatin structure and DNA methylation status
of IFNG and the linked genes IL4, IL5 and IL13 respectively6,7.
Specifically, naïve T cells have only two DNase I hypersensitive (DH)
sites in a ∼43-kb genomic interval that spans IL4 and IL13. Within 48 h
of primary stimulation in the presence of IL-4, ten additional clusters of
TH2-specific DH sites are observed8–10. All ten DH sites are stable markers of differentiated TH2 cells. Stimulation leads to the appearance of an
inducible hypersensitive site (VA) located 3′ of IL4; this site corresponds
to an IL4 enhancer that binds GATA-3 and the antigen-induced transcription factor NFAT111. The in vivo role played by the two clusters of
DH sites has been demonstrated by deletion in the context of the
endogenous mouse locus. The TH2-specific DH sites HSS1 and HSS2
are located in the intergenic region between IL13 and IL4; their deletion
1
The Center for Blood Research and the Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA. 2Department of Immunology
and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115, USA. Correspondence should be addressed to A. R.([email protected]).
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a
b
VA
IV
P
α-AcH4
V
IL-4 E
IL-4 P
DH
site IV
IFN-γ P
CD3ε P
-
+
-
+
-
+
-
+
E+P
1
2
3
4
5
6
7
8
9
Input
IV IFN- γ CD3ε
KIF3
C57BL/6
IL-4
Exon 1 2
3
4
CNS-2
5 kb
BALB/c
© 2002 Nature Publishing Group http://immunol.nature.com
P
IFN-γ
23
Exon 1
DO11.10
1 kb
4
10
11
12
Figure 1. No detectable histone acetylation at Il4 and Ifng regulatory regions of naïve T cells. (a) Diagram of the genes encoding IL-4 and IFN-γ.The Il4 and Ifng
promoters (P) are indicated.The last exon of the KIF3 gene encoding kinesin family member 3A is shown. (b) ChIP assay to assess histone H4 acetylation at the Il4 and Ifng
regulatory regions shown in a. CD4+ T cells were purified from the spleen and lymph nodes of 3-week-old C57BL/6, BALB/c and DO11.10 TCR–transgenic mice. Chromatin
complexes were immunoprecipitated with antibodies to acetylated histones H4 (+) or with nonimmune rabbit serum (–) (lanes 1–8). PCR primers specific for the Il4 promoter and enhancer, DH site IV, the Ifng promoter and the Cd3e promoter were used to amplify the precipitated DNA. Lanes 9–12 show the input control in each case.
The PCR product from the Cd3e promoter in lane 8 was of the expected size; the nonspecific bands in lane 12 reflect the fact that this PCR was done on unfractionated
genomic DNA. P, promoter; E, enhancer.
mice showed almost no detectable acetylation of histone H4 on the Il4
promoter and enhancer (Fig. 1b, lane 2), the Ifng promoter (lane 6) or
DH site IV, which exists as a DH site in naïve T cells9 (lane 4). The
Cd3e promoter, which is already active in naïve T cells, served as a positive control (Fig. 1b, lane 8). Histone H3 acetylation was barely
detectable in naïve T cells; it was much below that observed in differentiated T cells (data not shown).
strongly dependent on the continued presence of the appropriate polarizing cytokines and lineage-specific transcription factors (IL-4 and
STAT6 in the case of IL4, and IL-12 and T-bet in the case of IFNG). We
demonstrate here in vivo binding of STAT6 to the IL4 promoter and
enhancer, stimulation-dependent binding of GATA-3 to the IL4
enhancer and functional cooperation between GATA-3 and NFAT1 at
the IL4 enhancer. Our results emphasize the complexity and dynamic
nature of the mechanisms initiated through the TCR and maintained by
polarizing cytokines, which ultimately specify the appropriate pattern
of cytokine expression by differentiated T cells.
Histone hyperacetylation in differentiated T cells
To assess histone acetylation at the cytokine regulatory regions of fully
differentiated T cells, we cultured CD4+ T cells from C57BL/6 mice or
BALB/c mice for 8 days under TH1 or TH2 conditions and examined
histone acetylation both before and after stimulation (Fig. 2a).
Differentiated TH2 cells—whether unstimulated (Fig. 2a, lane 6) or
stimulated (lane 8)—showed strong hyperacetylation of histone H4 at
all Il4 regulatory regions examined (panels 1–3), but no detectable histone acetylation at the Ifng promoter (panel 4). Conversely, TH1 cells
(Fig. 2a, lanes 5 and 7) showed detectable hyperacetylation of histone
H4 at the Ifng promoter both before and after stimulation (panel 4), but
no detectable histone acetylation at the regulatory regions of Il4 (panels 1–3). This selective pattern of histone hyperacetylation correlated
completely with the restricted binding of NFAT1 to the Il4 and Ifng reg-
Results
Histone hypoacetylation in naïve T cells
We used chromatin immunoprecipitation (ChIP) to examine qualitatively the acetylation status of histones at the Ifng promoter and four
selected regulatory regions of Il4: the Il4 promoter, the inducible DH
site VA (referred to here as the Il4 enhancer), the constitutive TH2-specific DH site V, and the “common” DH site IV observed in naïve T cells
and differentiated TH1 and TH2 cells10,12,14 (Fig. 1a). We first assessed
histone acetylation in naïve T cells, which have the potential to differentiate into either TH1 or TH2 cells (Fig. 1b). Freshly isolated CD4+
T cells from young C57BL/6, BALB/c or D011.10 TCR–transgenic
a
b
α-AcH4
No Ab
T H1 T H2 T H1 T H2 T H1 T H2 T H1 T H2
-
-
+
+
-
-
+
+ Stimulation
1
IL-4 E
IL-4 P
2
Site IV
3
Site V
4
IFN-γ P
No Ab
α-NFAT1
-
-
+
+
Stimulation
IL-4 E
IL-4 P
T H2 cells
1
2
3
4
5
6
7
Figure 2. Selective histone acetylation at Il4 and Ifng regulatory regions in
differentiated T cells. (a) ChIP assay to assess histone H4 acetylation at Il4 and
Ifng regulatory regions. CD4+ T cells were purified from the spleen and lymph nodes
of 3-week-old C57BL/6 mice, differentiated for 8 days under TH1 or TH2 conditions,
then left unstimulated (–) or stimulated with PMA and ionomycin for 6 h (+).
Chromatin complexes were immunoprecipitated with beads alone (lanes 1–4) or
with antibodies to acetylated histone H4 (lanes 5–8). PCR primers specific for the Il4
promoter, the Il4 enhancer, DH sites IV and V and the Ifng promoter were used to
amplify the precipitated DNA. As a control, the PCR was done directly on input
DNA purified from chromatin before immunoprecipitation (lower panel). (b) NFAT1
binds the Il4 promoter and enhancer after activation. Chromatin from the same resting (–) and stimulated (+) TH2 samples in a was immunoprecipitated with antibodies
to NFAT1.The DNA was amplified with primers for the Il4 promoter and enhancer.
8
TH1 T H2 TH1 TH2
-
-
+
+ Stimulation
IL-4 E
IL-4 P
Input
C57BL /6 mice
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ulatory regions in TH2 and TH1 cells, respectively12. Histone hyperacetylation was apparent in resting differentiated TH2 cells that do not
actively transcribe the cytokine genes; thus, in this system, histone
acetylation correlated with the transcriptional competence of Il4 and
Ifng rather than overt gene transcription.
After activation, two changes were often, but not invariably, observed.
The first was increased histone acetylation at DH site IV (Fig. 2a, panel
2, lanes 6 and 8); this effect was consistently seen in C57BL/6 TH2 cells
but was less apparent in BALB/c T cells (data not shown). We also frequently observed altered histone acetylation at the Ifng promoter in
stimulated TH1 cells (Fig. 2a, panel 4, compare lanes 5 and 7). A control
ChIP assay with the same TH2 cell chromatin preparation showed
NFAT1 binding to the Il4 enhancer and promoter only after stimulation,
confirming that the cells were adequately stimulated (Fig. 2b).
The pattern of histone hyperacetylation at Ifng and Il4 recalls the
similarly selective and constitutive changes in DNase I hypersensitivity patterns observed at these genes during TH differentiation10–12. The
correlation between histone acetylation and DNase I hypersensitivity
was not absolute, however. DH site IV is a strong DH site in naïve
T cells9, but we were not able to detect histone acetylation at this
region; conversely, DH site VA—the inducible Il4 enhancer—appears
as a hypersensitive site only after stimulation12, but was associated with
hyperacetylated histones even in resting TH2 cells.
antibodies to acetylated histones H3 and H4 revealed similar amounts of
histone acetylation for all the Il4 regulatory regions (promoter, enhancer,
DH site IV and DH site V) under the two conditions of differentiation
(Fig. 3a, panels 1–3, lanes 3–6). To rule out the possibility that the nonselective hyperacetylation pattern reflected contamination of the TH1
population with differentiated TH2 cells, we repeated the experiment
with CD4+ T cells purified from the spleen and lymph nodes of young
BALB/c DO11.10 TCR–transgenic mice, which express a single TCR
specific for an ovalbumin peptide that is not encountered in the mouse
(Fig. 3b). T cells from these mice that had been left to differentiate for
48 h displayed a similar nonselective pattern of histone hyperacetylation
at the Il4 promoter and enhancer as was observed in non-TCR–transgenic BALB/c mice (compare Fig. 3a and b).
In contrast to the nonselective pattern of histone hyperacetylation on
Il4, a partially selective pattern was already established on the Ifng promoter by 48 h after the first stimulation (Fig. 3a, panel 4 and Fig. 3b,
lower panel). To determine whether the Ifng promoter might display a
less selective pattern at earlier times of stimulation, we differentiated
CD4+ T cells from young BALB/c mice for 17 h under TH1 and TH2
conditions or cultured them in media alone without stimulation and in
the absence of cytokines. Compared to unstimulated naïve T cells, the
Ifng promoter from 17 h–stimulated T cells was hyperacetylated under
both TH1 and TH2 conditions (Fig. 3c, lower panel, lanes 4–6).
Changes in chromatin structure have classically been assessed by
changes in accessibility to restriction enzymes. After stimulation, fully
differentiated TH2 cells develop an inducible DH site (VA) at the Il4
enhancer in a manner that parallels the inducible binding of NFAT1 to
the enhancer; this process is accompanied by increased accessibility of
a SwaI restriction site at the 3′ end of the enhancer region12. We used a
sensitive ligation-mediated polymerase chain reaction (LM-PCR) tech-
TCR signals induce early changes in chromatin
We next examined histone acetylation after 2 days of stimulation under
TH1 or TH2 conditions, the earliest time at which reproducible changes
in DNase I hypersensitivity have been documented for Il410 (Fig. 3).
CD4+ T cells from young BALB/c mice were differentiated for 48 h
under TH1 or TH2 conditions. Immunoprecipitation of chromatin with
a
α-AcH4
No Ab
TH1 T H2
TH1 T H2
b
α-AcH3
T H1
α-AcH4
No Ab
TH1
T H2
T H2 TH1
c
α-AcH3
T H2
TH1
TH1
T H2 N
TH1
T H2 N
IL-4 E
IL-4 P
IL-4 E
IL-4 P
IL-4 E
IL-4 P
1
α-AcH4
No Ab
T H2
IFN-γ P
IFN-γ P
1
Site IV
2
2
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1
5
6
TH1 T H2
Site V
4
IFN-γ P
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TH1
5
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Input
IFN-γ P
-
TH1
40h
TH
6h
Naïve
6
T H2
Input
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IFN-γ P
DO11.10
d
1
3
TH1T H2 N
Input
3
2
-
-
-
TH2
40h
-
Stimulation
SwaI
IL-4 E
(LM-PCR)
IFN-γ P
IL-4 P
(PCR)
BALB/c
1
2
3
4
5
6
7
8
9
10 11 12
DO11.10 TCR Tg-RAG
Figure 3. Nonselective histone acetylation and restriction enzyme accessibility at Il4 regulatory elements at an early stage of T cell differentiation.
(a) ChIP assay assessing histone acetylation at cytokine gene regulatory elements. CD4+ T cells, purified from the spleen and lymph nodes of 3-week-old BALB/c mice, were
differentiated for 48 h under either TH1 or TH2 conditions. Chromatin complexes were immunoprecipitated without antibodies (lanes 1 and 2) or with antibodies to acetylated histone H4 (lanes 3 and 4) or H3 (lanes 5 and 6). The lower panel shows the input control. (b) As in a except that the CD4+ T cells were purified from DO11.10
TCR–transgenic mice. (c) ChIP assay assessing histone acetylation at the Il4 enhancer, promoter and Ifng promoter from CD4+ T cells incubated for 17 h in medium alone
or under either TH1 or TH2 conditions. N, undifferentiated naïve cells.The lower panel shows the input control. (d) Restriction enzyme accessibility of the Il4 enhancer (DH
site VA). Nuclei derived from DO.11.10 TCR–transgenic RAG-2–/– cells were untreated (N), activated for 6 h or 40 h under TH1 and TH2 conditions and incubated with increasing amounts of the restriction enzyme SwaI. (Upper panel) The genomic DNA was isolated and subjected to LM-PCR with primers for the Il4 enhancer. (Lower panel) A
control PCR reaction for the Il4 promoter (see Methods).
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α-AcH4
TH1
-
T H2
-
T H0
-
Figure 4. The selective histone acetylation pattern is established by the
polarizing cytokines. ChIP assay assessing histone acetylation at gene regulatory
elements. CD4+ T cells from BALB/c mice were differentiated under TH1,TH2 or TH0
conditions for 8 days (see Methods), then left unstimulated (–) or stimulated with
PMA and ionomycin for 6 h (+). Chromatin complexes were immunoprecipitated
with antibodies to acetylated histone H4 (lanes 1–6) or H3 (lanes 7–12). No PCR
signal was observed in a control immunoprecipitation done without any antibodies
(data not shown).The lower panel shows the input control.
α-AcH3
T H1 T H2
+
+
T H0
+
T H1
-
T H2
-
T H0
T H1
-
+
T H2
+
T H0
+ Stimulation
IL-4 E
IL-4 P
© 2002 Nature Publishing Group http://immunol.nature.com
IFN-γ P
CD3ε P
1
TH1
-
2
T H2
-
3
4
5
Input
T H0 T H1
-
T H2
+
+
6
7
8
10
9
11
12
T H0
+
IL-4 E
IL-4 P
ble amounts of PCR product, especially when a higher concentration of
SwaI was used (Fig. 3d, upper panel, lanes 7–12). Control PCR reactions confirmed that the Il4 promoter, which lacks a SwaI site, was represented at equivalent amounts in all samples (Fig. 3d, lower panel).
These results showed that the earliest phase of TH differentiation was
accompanied by histone hyperacetylation at regulatory regions of Il4 and
Ifng as well as increased accessibility of the Il4 enhancer region to the
restriction enzyme SwaI. This phase was driven by TCR stimulation
because it occurred regardless of whether the cultures contain IL-4 or IL12, polarizing cytokines for TH2 and TH1 development, respectively. By 1
week of TH differentiation, the histone acetylation pattern became fully
selective (Fig. 2a) and the DH site VA that contained the SwaI restriction
site disappeared, to be observed again only in stimulated TH2 cells12.
nique to monitor accessibility of the Il4 enhancer to the restriction
enzyme SwaI in differentiating T cells (Fig. 3d). DO11.10 recombination activating gene 2–deficient (RAG-2–/–) naïve CD4+ T cells were
stimulated for 6 h or 40 h under TH1 or TH2 conditions, and nuclei were
prepared and incubated with increasing amounts of SwaI. The resulting
blunt-ended genomic DNA was isolated and ligated to a unidirectional
linker, after which primers for site VA were used in conjunction with a
linker primer to assess the extent of SwaI cleavage at the Il4 enhancer.
Little PCR product was detected in naïve cells that were either unstimulated (Fig. 3d, upper panel, lanes 1–3) or stimulated for 6 h (lanes 4–6);
this indicated that SwaI did not effectively gain access to and cleave its
restriction site in site VA under these conditions. In contrast, stimulation
for 40 h under either TH1 or TH2 conditions yielded high and compara-
a
b
α-AcH4
WT
TH1
_
T H2
_
TH1
T H2
+
+
TH1
_
STAT6 -/TH2 TH1
–
+
T H2
+
_
IL-4 E
IL-4 P
4
5
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7
+
TH 2
+
1
2
3
4
Input
T_H1 T_H2 T H1 T H2
+
+
CD3ε P
3
_
TH1
TH1
_
α-STAT6
T_H2 TH1 T H2
+
+ Stimulation
IL-4 E
IL-4 P
IFN-γ P
2
TH2
Stimulation
IL-4 E
IL-4 P
1
No Ab
TH1
c
input
_
WT
No Ab
STAT6 -/_ +
+
5
6
7
8
IL-4 E
IL-4 P
α-NFAT
α-AcH4
STAT6 -/WT
WT
_
_ +
_ Stimulation
+
8
IL-4 E
IL-4 P
Figure 5. Histone acetylation at gene regulatory elements in wild-type and
STAT6-deficient T cells, and in vivo binding of STAT6 and NFAT1 to Il4 regulatory regions. (a) Histone acetylation at gene regulatory elements in wild-type
Site IV
and STAT6-deficient T cells. CD4+ T cells from 3-week-old wild-type (lanes 1–4) or
STAT6-deficient (lanes 5–8) mice were differentiated for 8 days under TH1 or TH2
conditions, then left unstimulated (–) or stimulated with PMA and ionomycin for 6 h
8
1
2
3
4
7
9
5
6
(+). Chromatin complexes were immunoprecipitated with antibodies to acetylated
TH2 cells
histone H4. No PCR signal was observed in a control immunoprecipitation done
Input
without any antibodies (data not shown). (b) Direct binding of STAT6 to the Il4 promoter and enhancer in TH2 cells. CD4+ T cells, purified from 3-week-old C57BL/6
STAT6 -/WT
_
_
+
+
mice, were differentiated for 8 days under TH1 or TH2 conditions, then left unstimulated (–) or stimulated with PMA and ionomycin for 6 h (+). Chromatin complexes
IL-4 E
were immunoprecipitated without antibodies (lanes 1–4) or with anti-STAT6 (lanes
IL-4 P
5–8). PCR primers specific for the Il4 promoter and enhancer were used to amplify
the precipitated DNA. The lower panel shows the input control. (c) ChIP assay
assessing the binding of NFAT1 in wild-type and STAT6-deficient TH2 cells. CD4+ T cells from 4-week-old wild-type (lanes 1, 2, 5, 6 and 9) or STAT6-deficient (lanes 3, 4, 7
and 8) mice were differentiated for 8 days under TH2 conditions, then left unstimulated (–) or stimulated with PMA and ionomycin for 6 h (+). Chromatin complexes were
immunoprecipitated without antibodies (lanes 1–4), with antibodies to H4 (lane 9) or with NFAT1 antibodies (lanes 5–8).The lower panel shows the input control.
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Figure 6. Functional cooperation between
500
α-GATA3 α-AcH4
No Ab
GATA-3 and NFAT1 and inducible binding of
Resting
_
+ _
+
+ Stimulation
TH2 _
GATA-3 to the Il4 enhancer. (a) GATA-3 binds
PMA+Iono
400
IL-4 E
inducibly to the Il4 enhancer in TH2 cells, but does not
bind DH site IV. CD4+ T cells from 3-week-old
300
C57BL/6 mice were differentiated for 8 days under
Site IV
TH2 conditions, then left unstimulated (–) or stimulat200
ed with PMA and ionomycin for 6 h (+). Chromatin
1
2
3
4
5
6
complexes were immunoprecipitated with antibodies
100
C
N
C
N
to GATA-3 or acetylated histone H4. (b) Immunoblot
_
_
Stimulation
+ +
analysis assessing the expression of GATA-3 in resting
0
GATA3
and stimulated TH2 cells. CD4+ T cells from 3-weekBlot
old mice were differentiated for 7 days under TH2 conβ-actin
ditions, then left unstimulated (–) or stimulated with
1
2
3
4
PMA and ionomycin for 6 h (+).The lysates from 3 ×
106 cells were separated into nuclear (N) and cytosolic (C) fractions and immunoblot analysis was done
with GATA-3 and β-actin antibodies. (c) NFAT1 and GATA-3 synergistically promote transcription at the Il4 enhancer. Jurkat T cells were transiently cotransfected with a
luciferase reporter plasmid driven by the Il4 enhancer (DH site VA) and expression plasmids for GATA-3, NFAT1 and an AP-1 interaction mutant of NFAT1.The luciferase
activity of resting or PMA and ionomycin–stimulated cells from one of two representative experiments is shown.
Polarizing cytokines establish the selective patterns
We asked next whether STAT6 might act by binding directly to Il4
(Fig. 5b). ChIP assays showed clearly that STAT6 bound directly to
both the Il4 promoter and enhancer regions in differentiated TH2 cells
under both resting and stimulated conditions, most likely because these
cells were cultured in the constant presence of IL-4 (Fig. 5b, lanes 6
and 8). Notably, STAT6 was also required for NFAT binding to the Il4
promoter and enhancer (Fig. 5c). In agreement with published data12,
NFAT1 bound the Il4 promoter and enhancer in activated wild-type TH2
cells; however, this was not observed in STAT6-deficient TH2 cells (Fig.
5c, compare lanes 6 and 8). NFAT1 did not bind site IV (located ∼2 kb
upstream of the enhancer) in either cell type (Fig. 5c, lower panel, lanes
6 and 8); immunoprecipitation with H4 antibodies served as a positive
control (lane 9).
These results indicated that STAT6 mediates the ability of IL-4 to
maintain histone hyperacetylation at the Il4 promoter and enhancer.
This is done, in part, by inducing expression of TH2-specific transcription factors such as GATA-3 and Maf21–24, but also potentially by binding directly to the Il4 promoter and enhancer and recruiting histonemodifying enzymes. In addition, STAT6 facilitated NFAT1 binding to
the Il4 promoter and enhancer.
After a period of nonselective histone hyperacetylation, cell type–specific histone hyperacetylation at cytokine genes could be established
through maintained acetylation that was dependent on the polarizing
cytokines, active deacetylation driven by the opposing cytokines or both.
To distinguish between these possibilities, we differentiated precursor
CD4+ T cells in the absence of polarizing cytokines and in the presence
of neutralizing antibodies to IL-4, IL-12 and IFN-γ for 8 days (TH0 conditions). As expected, cells cultured under TH2 conditions showed histone H3 and H4 hyperacetylation at the Il4 promoter and enhancer (Fig.
4, upper panel, lanes 2, 5, 8 and 11), whereas cells cultured under TH1
(lanes 1, 4, 7 and 10) or TH0 (lanes 3, 6, 9 and 12) conditions showed
reduced histone acetylation at these regions. Conversely, cells cultured
under TH1 conditions showed histone hyperacetylation at the Ifng promoter, as expected (Fig. 4, middle panel, lanes 1, 4, 7 and 10), whereas
cells cultured under TH2 (lanes 2, 5, 8 and 11) or TH0 (lanes 3, 6, 9 and
12) conditions showed reduced acetylation. The histone acetylation status of the Cd3e promoter was unaffected (Fig. 4, lower panel). These
results showed that the correct polarizing cytokines were necessary to
maintain histone hyperacetylation at cytokine genes beyond the early
(17–48 h) period after antigen stimulation. We cannot rule out, however, that the opposing cytokines also contributed to establishing the selective hyperacetylation pattern, for example, by accelerating deacetylation
of the inappropriate locus in differentiating T cells.
GATA-3 inducibly binds the site VA enhancer
GATA-3 is a downstream target of STAT621 and maintains the differentiated TH2 phenotype by autoactivating its own expression21,22. We evaluated GATA-3 binding to Il4 regulatory regions in differentiated TH2
cells (Fig. 6a). ChIP assays showed that GATA-3 bound weakly to the
site VA enhancer in resting cells (Fig. 6a, upper panel, lane 3) and that
binding was much enhanced after stimulation (lane 4). However GATA3 did not bind detectably to the Il4 promoter12 or DH site IV (Fig. 6a,
lower panel) in either resting or stimulated TH2 cells. The inducible
binding of GATA-3 to the Il4 enhancer partly reflected up-regulation of
GATA-3 protein in nuclear extracts of stimulated TH2 cells, as shown by
immunoblotting (Fig. 6b, upper panel, compare lanes 2 and 4); the cytoplasmic protein β-actin served as a control for cell fractionation and
equivalent protein loading in the lanes (lower panel, lanes 1 and 3).
Using transient reporter assays in Jurkat T cells, we demonstrated a
strong functional cooperation between GATA-3 and NFAT1 at the Il4
enhancer (Fig. 6c). Cells transfected with a luciferase reporter plasmid
driven by the site VA enhancer11 showed low inducible activity, presumably due to endogenous NFAT (cluster 1). Ectopic expression of NFAT1
alone or GATA-3 alone did not lead to transcriptional activation above this
basal activity (clusters 2 and 3), whereas the combination of both proteins
STAT6 maintains histone hyperacetylation
STAT6 is a primary transcription factor activated by IL-4 receptor signaling, and STAT6 binding sites are present in both the Il4 promoter
and enhancer12, 20. We asked whether the ability of IL-4 to maintain histone hyperacetylation at Il4 was mediated via STAT6. We compared the
histone H4 acetylation status of Il4 regulatory elements in wild-type
and STAT6-deficient T cells cultured for 8 days under TH1- and TH2differentiation conditions (Fig. 5a). Wild-type TH2 cells showed the
expected selective pattern of histone hyperacetylation at the Il4 promoter and enhancer (Fig. 5a, upper panel, lanes 2 and 4). However,
under the same conditions, STAT6-deficient T cells were hypoacetylated at H4 histones in these regions (Fig. 5a, upper panel, lanes 6 and 8).
As expected, STAT6 deficiency did not affect histone acetylation at the
Ifng promoter in resting and stimulated TH1 cells (Fig. 5a, middle
panel, lanes 1, 3, 5 and 7); stimulation of TH2 cells resulted in some
hyperacetylation at the Ifng promoter (lane 8). Histone acetylation at
the Cd3e promoter was unaffected (Fig. 5a, lower panel).
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Figure 7. Histone acetylation in wild-type and T-bet–deficient T cells, and in vivo binding of NFAT1 to the Ifng promoter. (a) Histone acetylation at gene regulatory elements in wild-type and T-bet–deficient T cells. CD4+ T cells from 3-week-old wild-type (lanes 1–4) or T-bet–deficient (lanes 5–8) mice were differentiated for 8
days under TH1 or TH2 conditions, then left unstimulated (–) or stimulated with PMA and ionomycin for 6 h (+). Chromatin complexes were immunoprecipitated with antibodies to acetylated histone H3. No PCR signal was observed in a control immunoprecipitation done without any antibodies (data not shown). (b) As in a except that the
chromatin complexes were immunoprecipitated with antibodies to acetylated histone H4. (c) ChIP assay assessing the binding of NFAT1 in wild-type and T-bet–deficient
T cells. Chromatin complexes as in a and b were immunoprecipitated with antibodies to NFAT1. (d) The input control for a–c.
Discussion
led to strong synergistic activation (cluster 4). The region of NFAT1
involved in GATA-3 cooperation is different from that required for cooperation with the established NFAT partner AP-1 (Fos-Jun)25. This is
because a mutant NFAT1 incapable of Fos-Jun interaction25 synergized
with GATA-3 even more effectively than wild-type NFAT1 (compare
clusters 4 and 6). The stronger synergy could reflect the fact that the
mutant protein, unlike wild-type NFAT1, cannot be diverted away from
reporter DNA to genomic DNA elements at which it can bind cooperatively with endogenous AP-1. We concluded that increased GATA-3 binding occurred at the Il4 enhancer after stimulation, partly because GATA-3
protein concentrations increased and partly because GATA-3 cooperated
with NFAT1 at this regulatory element in stimulated TH2 cells.
We have shown here that differentiating T cells undergo dynamic
changes in histone acetylation of cytokine genes. TCR stimulation of
naïve T cells resulted in increased histone acetylation at regulatory
regions of Il4 and Ifng, in a manner that was independent of the
cytokine milieu. However this phase was transient: continued culture in
the presence of IL-4 or IL-12 was required to maintain increased histone acetylation at the Il4 and Ifng regulatory regions, respectively.
These results provide a molecular correlate for published findings
showing that IFN-γ–producing T cells pass through an early stage of Il4
expression28 and that initial Il4 expression is TCR-dependent but independent of the IL-4 signaling pathway29–31. More recently, sensitive
assays have documented early Il4 and Ifng transcription by naïve
T cells stimulated under both TH1 and TH2 conditions32, consistent with
the early increases in histone acetylation we observed at each of these
cytokine genes. In the absence of polarizing cytokines or key transcription factors, both early histone hyperacetylation (our data) and early
cytokine gene expression32 are reduced to the low basal amounts
observed in naïve T cells. TCR stimulation activates an immediate and
global chromatin derepression program in naïve T cells33; this might be
sufficient to permit binding of TCR-induced transcription factors to the
promoter regions of cytokine genes, thus initiating localized histone
modification in parallel with gene transcription. NFAT itself is an
attractive candidate for recruiting histone acetyltransferases34,35 and driving early transcription, and it will be useful to determine whether
NFAT1 binds in vivo to the Il4 and Ifng regulatory elements soon after
primary stimulation.
Our data suggest that the chromatin structure of Il4 and Ifng in naïve
T cells is different from that of the silenced genes in terminally differentiated T cells. In naïve T cells, the genes are maintained in a state that
is permissive for gene transcription, so that the first TCR stimulation
leads to histone acetylation of regulatory regions and limited gene transcription. In contrast, in differentiated TH1 and TH2 cells, cytokine loci
with similar low amounts of histone H4 acetylation are maintained in a
silenced, nonpermissive state; in this state, the histones remain deacety-
T-bet maintains histone hyperacetylation
T-bet is a TH1-specific transcription factor that is necessary for IFN-γ
expression in CD4+ TH1 cells26. To determine whether T-bet participated in
maintaining histone hyperacetylation at Ifng in differentiated TH1 cells, we
compared the histone acetylation status of the Ifng promoter in wild-type
and T-bet–deficient27 CD4+ T cells cultured for 8 days under TH1 and TH2
conditions. Wild-type T cells showed the expected TH1-selective pattern of
histone H3 and H4 hyperacetylation at the Ifng promoter (Fig. 7a,b, upper
panels, lanes 1 and 3). Under the same conditions, resting T-bet–deficient
TH1 cells showed lower amounts of histone acetylation (lane 5), which
increased upon stimulation (lane 7). This suggested the participation of a
stimulation-responsive transcription factor whose effect was unmasked in
the T-bet–deficient cells. This factor was not NFAT, as NFAT1 was unable
to bind the Ifng promoter in T-bet–deficient T cells (Fig. 7c, upper panel,
compare lanes 3 and 7), even though its binding to the Il2 promoter was
not impaired (lower panel, lanes 3 and 7). Fig. 7d confirms that input
DNA was equivalent in all lanes. The Il4 promoter and enhancer were
hyperacetylated in T-bet–deficient TH1 cells (Fig. 7a,b, lower panels,
compare lanes 5 and 7 to 1 and 3), most likely reflecting the suppressive
activity of T-bet on Il426. Together these results suggested that T-bet was
critical for robust acetylation at the Ifng promoter and facilitated NFAT1
binding to the promoter after activation.
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lated after activation and gene transcription does not occur. The difference between naïve and differentiated T cells is emphasized by the fact
that inactive cytokine genes in naïve T cells are located away from centromeric heterochromatin, whereas a large fraction of the silenced
genes in differentiated T cells are repositioned to such regions32. It is
likely, therefore, that in naïve T cells, TCR stimulation initiates permissive chromatin modifications that facilitate early gene expression.
In contrast, this may not occur in differentiated T cells because of
inhibitory modifications, previously established by the polarizing
cytokines, that down-regulate the expression of inappropriate genes.
H3 Lys10 methylation provides a particularly attractive mechanism by
which genes may be simultaneously silenced and repositioned to heterochromatin; this modification is recognized by the chromodomaincontaining heterochromatin-associated protein HP-136, which colocalizes with Ikaros in the vicinity of silenced genes37.
It is likely that increased histone acetylation is a major mechanism
through which cytokine signaling pathways maintain cytokine genes in
a transcriptionally permissive state. After the initial TCR-induced
increase in histone acetylation, polarizing cytokines—as well as STAT6
in the case of TH2 cells and T-bet in the case of TH1 cells—are needed
to maintain histone hyperacetylation at Il4 and Ifng; early histone
hyperacetylation declines both in the absence of polarizing cytokine
and in the presence of the inappropriate cytokine. Another well known
effect of cytokines, STAT factors and lineage-specific transcription factors such as GATA3 and T-bet is to repress expression of inappropriate
cytokine genes2–6. Possibly, this is done by increasing the kinetics or
efficiency with which histone acetylation declines at inappropriate
genes and/or by promoting inhibitory histone modifications as discussed above. Our data are consistent with published results showing
that the IL-4 signaling pathway is required for stable IL-4 expression
by differentiated T cells23,31,33,38–42. STAT6-deficient mice produce normal amounts of IL-4 in response to primary stimulation with several
immunogens, but are strongly impaired in their recall (memory)
responses31. Similarly, STAT6-deficient CD4+ T cells generate relatively normal IL-4 responses when stimulated in vitro for a few hours, or
even for 3 days, with anti-CD3 and anti-CD28, but fail to produce high
amounts of IL-4 in response to secondary stimulation31,32.
How does the IL-4 signaling pathway stabilize TCR-induced chromatin changes at Il4? We have shown that STAT6 binds in vivo to the
Il4 enhancer and promoter in recently differentiated TH2 cells, which
suggests that it may have a direct role in regulating Il4 expression.
Indeed, even STAT6-deficient T cells that exhibit stochastic expression
of GATA-3 express only half the amount of IL-4 expressed by wildtype T cells23. Given its ability to recruit the acetyltransferases p300 and
CBP43, STAT6 may directly promote histone acetylation at Il4. Indeed
STAT6 and NFAT may bind in concert to the Il4 promoter and
enhancer, cooperatively recruiting a stronger constellation of chromatin-remodeling and histone-modifying enzymes than NFAT alone.
The STAT6 target gene GATA-3, when retrovirally introduced into
developing TH1 cells, induces IL-4 expression as well as the entire DH
pattern of Il421–23,44–47. However, our results suggest that GATA-3 does
not directly maintain the accessible chromatin structure over the entire
Il4-Il5-Il13 locus. GATA-3 binds in vitro to several regulatory elements
in the locus47–50, but our ChIP experiments suggested that only a subset
of these sites binds GATA-3 in vivo. GATA-3 does not bind the Il4 promoter, which—by sequence inspection—possesses an adequate GATA3 site12, nor does it bind to DH site IV. In addition, despite the fact that
histone acetylation at this Il4 enhancer was similar before and after
stimulation, GATA-3 associated strongly with the VA enhancer only in
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stimulated cells. Although we cannot rule out the possibility that
GATA-3 is inaccessible to the immunoprecipitating antibodies at certain sites or time points, two other possibilities are pertinent. GATA-3
bound to a small number of specific sites might propagate the accessible chromatin structure to more distal regions of the locus; alternatively, some other TH2-specific factor, potentially induced by GATA-3,
might be stably DNA-bound in resting T cells and maintain chromatin
accessibility at the Il4-Il5-Il13 locus. A major role of GATA-3 might be
to induce such secondary factors, while also cooperating acutely with
NFAT and other inducible transcription factors at the Il4 enhancer.
Our data suggest that one function of lineage-specific transcription
factors is to facilitate, perhaps directly, the binding of NFAT proteins to
cytokine regulatory regions. STAT6-deficient T cells showed
hypoacetylation of histones and no binding of NFAT1 at the Il4 promoter and enhancer. Likewise, T-bet–deficient T cells showed no NFAT1
binding to the Ifng promoter, despite a stimulation-dependent increase of
histone acetylation at this region that may be secondary to binding of a
distinct inducible factor. By analogy with NFAT1-GATA-3 cooperation
at the Il4 enhancer, T-bet may directly cooperate with and stabilize
NFAT1 binding to the Ifng promoter. Because we have not detected
GATA-3 binding to the Il4 promoter, STAT6 itself—or a different lineage-specific transcription factor induced by STAT6 (for example, cMaf24)—may participate in stabilizing NFAT binding to the Il4 promoter.
In summary, we have shown that TCR-CD28–induced signaling pathways contribute to early cytokine expression by inducing histone modifications at Il4 and Ifng. However these pathways have only a limited
ability to maintain the locus in a state capable of supporting an efficient
recall response. In contrast, polarizing cytokines, STAT factors and lineage-specific transcription factors provide an efficient and stable mechanism for maintaining the appropriate locus in an active state. This is characterized by increased histone acetylation, even under resting conditions,
and the potential to bind acute transcription factors after activation.
Methods
Mice. B6J, BALB/c, STAT6–/– mice (Jackson laboratories, Bar Harbor, ME) and DO11.10
TCR–transgenic51, RAG-2–/– DO11.10 TCR–transgenic (provided by A. K. Abbas) and
T-bet–/–27 mice were maintained in pathogen-free conditions in barrier facilities in the Center
for Animal Resources and Comparative Medicine (Harvard Medical School). All mouse
protocols were approved by the Center for Blood Research and Harvard Medical School.
In vitro T cell differentiation. CD4+ T cells were purified from the spleen and lymph nodes
of young mice with the use of magnetic beads (Dynal, Oslo, Norway). For TH differentiation,
cells (106/ml) were stimulated with 1 µg/ml of anti-CD3ε and 1 µg/ml of anti-CD28
(145.2C11 and 37.51, respectively, Pharmingen, San Diego, CA) in a flask coated with 0.3
mg/ml of goat anti-hamster (ICN, Aurora, OH) in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with 10% fetal calf serum, L-glutamine, penicillin-streptomycin,
nonessential amino acids, sodium pyruvate, vitamins, HEPES and 2-mercaptoethanol. For
TH1 differentiation, the cells were stimulated in the presence of 5 ng/ml of recombinant
mouse IL-12 (courtesy of Wyeth Research, Cambridge, MA) and 10 µg/ml of purified
anti–IL-4 (11B11, a gift of the Biological Resources Branch, National Cancer Institute
Preclinical Repository). For TH2 differentiation, cells were stimulated in the presence of 1000
U/ml of mouse IL-4 (added as supernatant of the I3L6 cell line)52, 5 µg/ml of anti–IFN-γ
(XMG1.2 or R4-6A2) and 3 µg/ml of anti–IL-12 (courtesy of Wyeth Research). For TH0 conditions, the cells were cultured in the presence of anti–IL-4, anti–IL-12 and anti–IFN-γ. For
cultures longer than 3 days, cells were expanded (fourfold) in the absence of anti-TCR and
anti-CD28, but in the continued presence of cytokines and antibodies that included 10 U/ml
of IL-2; they were then expanded every other day. After 8 days, the cells were left unstimulated or were stimulated with PMA (20 nM) and ionomycin (1 µM) for 6 h.
ChIP. ChIP analysis was carried out essentially as described53. Cells (5 × 107–15 × 107) were
fixed for 20 min on ice with one-tenth the volume of 11% formaldehyde solution (in 0.1 M
NaCl, 1 mM EDTA, 0.5 mM EGTA and 50 mM HEPES at pH 8.0). After incubation,
glycine was added to a final concentration of 0.125 M for 5 min. Cells were rinsed with cold
PBS and resuspended in 10 ml of lysis buffer (50 mM HEPES at pH 7.5, 140 mM NaCl,
1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100 and the following protease
inhibitors: 1 mM phenylmethanesulfonyl fluoride, 25 µg/ml of aprotinin, 25 µg/ml of leu-
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trol plasmid. Twenty-four hours after transfection, cells were stimulated with 1 µM ionomycin and 20 nM PMA. Twelve hours after stimulation, cells were collected and cell
extracts were assessed for luciferase activity with the Dual Luciferase Assay System
(Promega, Madison, WI). Cotransfection of the pRL-TK vector (Promega) allowed normalization by Renilla luciferase activity.
peptin and 10 mM iodoacetamide) and incubated for 10 min on ice. Nuclei were pelleted,
resuspended and incubated at room temperature for 10 min in 0.2 M NaCl, 1 mM EDTA,
0.5 mM EGTA, 10 mM Tris HCl (pH 8.0) and protease inhibitors. The nuclei were pelleted again and resuspended in 10 ml of sonication buffer (1 mM EDTA, 0.5 mM EGTA and
10 mM Tris HCl at pH 8.0 and protease inhibitors). The suspension was sonicated eight to
ten times for 20 s with 1 min cooling on ice in-between. Samples were adjusted to 0.5%
Sarkosyl and DNA-protein complexes were purified by CsCl (567.8 mg/ml) centrifugation
(for 48–72 h). The samples that contained the DNA were dialyzed against 10 mM Tris HCl
(pH 8.0), 1 mM EDTA, 0.5 mM EGTA and 10% glycerol and kept frozen in 0.5 ml aliquots
or used for immunoprecipitation, after adjustment to RIPA buffer (1% Triton X-100, 0.1%
sodium deoxycholate, 0.1% SDS, 140 mM NaCl and protease inhibitors). Two hours of preclearing with the beads preceded overnight incubation at 4 °C with various antibodies.
Immunocomplexes were precipitated for 3 h with protein A or protein G sepharose beads
blocked with salmon sperm DNA. The precipitates were washed seven times for 5 min each
with 1 ml of RIPA buffer (supplemented with 0.5 M NaCl and 100 µg/ml of yeast tRNA)
and resuspended in 100 µl of TE buffer (10mM Tris at pH 8 and 1 mM EDTA). The samples were adjusted to 0.5% SDS and 200 µg/ml of proteinase K and incubated at 55 °C for
3 h. Incubation at 65 °C overnight reversed the formaldehyde cross-links. The DNA was
purified by phenol-chloroform extraction, precipitated in the presence of 20 µg of glycogen
and resuspended in 100 µl of TE buffer.
PCR was done with 20 µl of the immunoprecipitated DNA for 26 cycles (1 min at 95 °C,
1 min at 48 °C and 1 min at 72 °C, completed by 10 min in 72 °C) with various primers. As
a control, the PCR was done directly on input DNA purified from chromatin before
immunoprecipitation. PCR products were resolved on 3% NuSieve agarose gels and visualized with ethidium bromide. Except for Figure 4, all results are representative of two or
more similar experiments. The antibodies used were anti-NFAT1: anti-67.1 and anti-T2B1
against peptides from the NH2- and COOH-terminal regions of NFAT1, respectively54; antiGATA-3 (HG3-31, Santa Cruz Biotechnology, Santa Cruz, CA); anti-STAT6 (M-20 and M200, Santa Cruz); and anti-acetylated forms of H4 (lysines 5, 8, 12 and 16) and H3 (lysines
9 and 14) NH2-terminal tails (Upstate Biotechnology, Lake Placid, NY). The following
primers were used. IL-4 P, 5′-TTGGTCTGATTTCACAGG-3′ and 5′-AACAAT
GCAATGCTGGC-3′ (182-bp product); IL-4 E (VA), 5′-AGGGCACTTAAACATTGC-3′
and 5′-ACGCCTAAGCACAATTCC-3′ (239-bp product); DH site IV, 5′-GCCAGTTAGA
GATAACCC-3′ and 5′-ATGCTCAGCCTCTGATTC-3′ (257-bp products); DH site V, 5′TTGGGTTAGGCAATATCC-3′ and 5′-CGTCTTACCCAAACACCG-3′ (229-bp product);
IFN-γ P, 5′-GCTCTGTGGATGAGAAAT-3′ and 5′-AAGATGGTGACAGATAGG-3′ (250bp product); CD3ε P, 5′-CATTTCCAAGTGACGTGG-3′ and 5′-AACACACTGGCTG
CATGC-3′ (206-bp products).
Acknowledgments
We thank members of the laboratory for critical reading of the manuscript and valuable
discussions. Supported by grants from the National Institutes of Health (to A. R. and L. H.
G.), a gift from the G. Harold and Leila Y. Mathers Charitable Foundation (to L. H. G.), the
Cancer Research Institute (to O. A.), the National Institutes of Health (to D. L.) and the
Leukemia Society and a grant from the Burroughs Wellcome Fund (to S. J. S.).
Competing interests statement
The authors declare that they have no competing financial interests.
Received 7 January 2002; accepted 16 May 2002.
1. Abbas, A. K., Murphy, K. M. & Sher, A. Functional diversity of helper T lymphocytes. Nature 383,
787–793 (1996).
2. Wurster, A. L.,Tanaka,T. & Grusby, M. J.The biology of Stat4 and Stat6. Oncogene 19, 2577–2584
(2000).
3. Murphy, K. M. et al. Signaling and transcription in T helper development. Annu. Rev. Immunol. 18,
451–494 (2000).
4. Glimcher, L. H. & Murphy, K. M. Lineage commitment in the immune system: the T helper lymphocyte
grows up. Genes Dev. 14, 1693–1711 (2000).
5. Reiner, S. L. Helper T cell differentiation, inside and out. Curr. Opin. Immunol. 13, 351–355 (2001).
6. Avni, O. & Rao, A.T cell differentiation: a mechanistic view. Curr. Opin. Immunol. 12, 654–659 (2000).
7. O’Garra, A. & Arai, N.The molecular basis of T helper 1 and T helper 2 cell differentiation. Trends Cell
Biol. 10, 542–550 (2000).
8. Lee, D., Agarwal, S. & Rao, A.Th2 lineage commitment and efficient IL-4 production involves extended
demethylation of the IL-4 gene. Immunity 16, 649–660 (2002).
9. Agarwal, S. & Rao, A. Modulation of chromatin structure regulates cytokine gene expression during T
cell differentiation. Immunity 9, 765–775 (1998).
10. Takemoto, N. et al.Th2-specific DNAse I-hypersensitive sites in the murine IL-13 and IL-4 intergenic
region. Int. Immunol. 10, 1981–1985 (1998).
11. Agarwal, S., Avni, O. & Rao, A. Cell-type-restricted binding of the transcription factor NFAT to a distal IL-4 enhancer in vivo. Immunity 12, 643–652 (2000).
12. Mohrs, M. et al. Deletion of a coordinate regulator of type 2 cytokine expression in mice. Nature
Immunol. 2, 842–847 (2001).
13. Loots, G. G. et al. Identification of a coordinate regulator of interleukins 4, 13, and 5 by cross-species
sequence comparisons. Science 288, 136–140 (2000).
14. Solymar, D. C., Agarwal, S., Bassing, C. H., Alt, F.W. & Rao, A. A 3’ enhancer in the IL-4 gene regulates
cytokine production by Th2 cells and mast cells. Immunity, (in the press, 2002).
15. Jenuwein,T. & Allis, C. D.Translating the histone code. Science 293, 1074–1080 (2001).
16. Rice, J. C. & Allis, C. D. Histone methylation versus histone acetylation: new insights into epigenetic
regulation. Curr. Opin. Cell Biol. 13, 263–273 (2001).
17. Strahl, B. D. & Allis, C. D.The language of covalent histone modifications. Nature 403, 41–45 (2000).
18. Cheung, P., Allis, C. D. & Sassone-Corsi, P. Signaling to chromatin through histone modifications. Cell
103, 263–271 (2000).
19. Roth, S.Y., Denu, J. M. & Allis, C. D. Histone Acetyltransferases. Annu. Rev. Biochem. 70, 81–120 (2001).
20. Lederer, J. A. et al. Cytokine transcriptional events during helper T cell subset differentiation. J. Exp.
Med. 184, 397–406 (1996).
21. Kurata, H., Lee, H., O’Garra, A. & Arai, N. Ectopic expression of activated Stat6 induces the expression of Th2-specific cytokines and transcription factors in developing Th1 cells. Immunity 11,
677–688 (1999).
22. Lee, H. J. et al. GATA-3 induces T helper cell type 2 (Th2) cytokine expression and chromatin
remodeling in committed Th1 cells. J. Exp. Med. 192, 105–116 (2000).
23. Ouyang,W. et al. Stat6-independent GATA-3 autoactivation directs IL-4-independent Th2 development and commitment. Immunity 12, 27–37 (2000).
24. Ho, I.-C., Hodge, M. R., Rooney, J.W. & Glimcher, L. H.The proto-oncogene c-maf is responsible for
tissue-specific expression of interleukin-4. Cell 85, 973–983 (1996).
25. Macian, F., Garcia-Rodriguez, C. & Rao, A. Gene expression elicited by NFAT in the presence or
absence of cooperative recruitment of Fos and Jun. EMBO J. 19, 4783–4795 (2000).
26. Szabo, S. J. et al. A novel transcription factor,T-bet, directs Th1 lineage commitment. Cell 100,
655–669 (2000).
27. Szabo, S. J. et al. Distinct effects of T-bet in TH1 lineage commitment and IFN-γ production in CD4
and CD8 T cells. Science 295, 338–342 (2002).
28. Kamogawa,Y., Minasi, L.-A., Carding, S., Bottomly, K. & Flavell, R.The relationship of IL4 and IFNγ-producing T cells studied by lineage ablation of IL4-producing cells. Cell 75, 985–995 (1993).
29. Croft, M. & Swain, S. L. Recently activated naïve CD4 T cells can help resting B cells, and can produce sufficient autocrine IL-4 to drive differentiation to secretion of T helper 2-type cytokines.
J. Immunol. 154, 4269–4282 (1995).
30. Noben-Trauth, N., Hu-Li, J. & Paul,W. E. Conventional, naïve CD4+ T cells provide an initial source of
IL-4 during Th2 differentiation. J. Immunol. 165, 3620–3625 (2000).
31. Finkelman, F. D. et al. Stat6 regulation of in vivo IL-4 responses. J. Immunol. 164, 2303–2310 (2000).
32. Grogan, J. L. et al. Early transcription and silencing of cytokine genes underlie polarization of T helper
cell subsets. Immunity 14, 205–215 (2001).
33. Zhao, K. et al. Rapid and phosphoinositol-dependent binding of the SWI/SNF-like BAF complex to
chromatin after T lymphocyte receptor signaling. Cell 95, 625–636 (1999).
34. Garcia-Rodriguez, C. & Rao, A. Nuclear factor of activated T cells (NFAT)-dependent transactivation
regulated by the coactivators p300/CREB-binding protein (CBP). J. Exp. Med. 187, 2031–2036
(1998).
35. Avots, A. et al. CBP/p300 integrates Raf/Rac-signaling pathways in the transcriptional induction of
NF-ATc during T cell activation. Immunity 10, 515–524 (1999).
36. Lachner, M., O’Carroll, D., Rea, S., Mechtler, K. & Jenuwein,T. Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410, 116–120 (2001).
37. Brown, K. E. et al. Association of transcriptionally silent genes with Ikaros complexes at centromeric
heterochromatin. Cell 91, 845–854 (1997).
Restriction enzyme accessibility assay. Naïve T cells (6 × 106) were used for each condition (resting, 6 h–stimulated, TH1 and TH2). Nuclei were prepared as for DNase I HS analysis10, but resuspended in 600 µl of buffer F (100 mM NaCl, 50 mM Tris (pH 8), 5 mM
MgCl2, 0.1 mM EGTA and 1 mM 2-mercaptoethanol) with a wide-bore pipette tip and then
split into three 200-µl aliquots. SwaI (100 or 200 U, New England Biolabs, Beverly, MA)
was added to two aliquots and all samples were incubated at room temperature for 1 h with
periodic agitation. Purified DNA was digested to completion with BamHI, ethanol precipitated, resuspended in TE and the concentration was determined by 260 nm absorbance.
Preparation of unidirectional linker: a 20 µM unidirectional linker mix was prepared (20 µM
LM-1, 20 µM LM-2 and 250 mM Tris HCl at pH 7.9; LM-1: 5′-GCGGTGACCCGGGAGATCTGAATTC-3′; LM-2: 5′-GAATTCAGATC-3′). The linker mix was placed at 95 °C for
5 min, transferred to a 70 °C environment, allowed to cool to 25 °C and then transferred and
incubated at 4 °C overnight. The linker mix was stored at –20 °C and thawed on ice.
LM-PCR: first-strand synthesis reaction was not necessary because SwaI cleavage produces
blunt-ended double-stranded DNA that can ligate directly to the linker. Genomic DNA (200 ng)
and a final concentration of 1 µM unidirectional linker mix was set up in a 50-µl ligation reaction with 2 µl of T4 DNA ligase (New England Biolabs) for each sample. Ligation was done
at 16 °C for 17 h. Samples were ethanol-precipitated with glycogen, washed twice in 70%
ethanol and resuspended in water. A third was used in a 50-µl radioactive PCR with the final
conditions: 1× PCR buffer (Applied Biosystems, Foster City, CA), 1.5 mM MgCl2, 0.2 mM
dNTPs, 1 µM LM-1 primer, 1 µM VA-1 primer, 2 U of Taq polymerase and 0.2 µl of [32Pα]dCTP (10 µCi/µl) per reaction. Samples were heated to 95 °C for 5 min and amplification
was done for 40 cycles at 94 °C for 40 s, 60 °C for 40 s, 72 °C for 40 s and 72 °C for 7 min.
The reaction mixture (10 µl) was resolved on a 0.5× TBE nondenaturing 5% polyacrylamide
gel and subjected to autoradiography. The Il4 promoter control PCR reactions were done as
described above, except that 26 cycles at 94 °C for 40 s, 52 °C for 40 s and 72 °C for 40 s were
used. VA-1: 5′-GGACTGAGAACCCAACAGAGATGCTTG-3′ (250-bp product with LM-1);
pro-1: 5′-GGATCCACACGGTGCAAAGAGAGACCC-3′ and pro-2: 5′-TCGGCCTTTCAGACTAATCTTATCAGC-3′ (530-bp product).
Immunoblot analysis. Nuclear and cytosolic extracts were separated on 10% PAGE followed by immunoblot analysis with anti–GATA-3 and anti–β-actin.
Transient transfection assay. Jurkat cells were cultured in DMEM supplemented with
10% fetal calf serum, 10 mM HEPES and 2 mM L-glutamine. Jurkat cells were transfected
by electroporation in serum-free medium with pulses of 250 V and 960 µF. Typically, 107
cells were transfected with 5 µg of luciferase reporter plasmid12, 10 µg of expression plasmids encoding wild-type NFAT155 or a mutant NFAT1 incapable of Fos-Jun interaction
(R468A, I469A, T535G)25 and/or GATA-3 (a gift of S. Orkin, Harvard University) or a con-
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A RTICLES
38. Kaplan, M. H., Schindler, U., Smiley, S. & Grusby, M. Stat6 is required for mediating responses to IL-4
and for development of TH2 cells. Immunity 4, 313–319 (1996).
39. Shimoda, K. et al. Lack of IL-4-induced Th2 response and IgE class switching in mice with disrupted
Stat6 gene. Nature 380, 630–633 (1996).
40. Takeda, K. et al. Essential role of Stat6 in IL-4 signalling. Nature 380, 627–630 (1996).
41. Kopf, M. et al. Disruption of the IL-4 gene blocks Th2 cytokine responses. Nature 362, 245–248
(1993).
42. Jankovic, D. et al. Single cell analysis reveals that IL-4 receptor/Stat6 signaling is not required for the
in vivo or in vitro development of CD4+ lymphocytes with a Th2 cytokine profile. J. Immunol. 164,
3047–3055 (2000).
43. Gingras, S., Simard, J., Groner, B. & Pfitzner, E. p300/CBP is required for transcriptional induction by
interleukin-4 and interacts with Stat6. Nucleic Acids Res. 27, 2722–2729 (1999).
44. Zhang, D. et al. Inhibition of allergic inflammation in a murine model of asthma by expression of a
dominant-negative mutant of GATA-3. Immunity 11, 473–482 (1999).
45. Zheng,W. & Flavell, R. A.The transcription factor GATA-3 is necessary and sufficient for Th2
cytokine gene expression in CD4 T cells. Cell 89, 587–596 (1997).
46. Ouyang,W. et al. Inhibition of Th1 development mediated by GATA-3 through an IL-4-independent
mechanism. Immunity 9, 745–755 (1998).
http://immunol.nature.com
•
july 2002
47. Takemoto, N. et al. Cutting edge: chromatin remodeling at the IL-4/IL-13 intergenic regulatory region
for Th2-specific cytokine gene cluster. J. Immunol. 165, 6687–6691 (2000).
48. Henkel, G. & Brown, M. A. PU.1 and GATA: components of a mast cell-specific interleukin 4 intronic
enhancer. Proc. Natl. Acad. Sci. USA 91, 7737–7741 (1994).
49. Ranganath, S. et al. GATA-3-dependent enhancer activity in IL-4 gene regulation. J. Immunol. 161,
3822–3826 (1998).
50. Lee, G. R., Fields, P. E. & Flavell, R. A. Regulation of IL-4 gene expression by distal regulatory elements
and GATA-3 at the chromatin level. Immunity 14, 447–459 (2001).
51. Murphy, K. M., Heimberger, A. B. & Loh, D.Y. Induction by antigen of intrathymic apoptosis of
CD4+CD8+TCRlo thymocytes in vivo. Science 250, 1720–1723 (1990).
52. Tepper, R. I., Pattengale, P. K. & Leder, P. Murine interleukin-4 displays potent anti-tumor activity in
vivo. Cell 57, 503–512 (1989).
53. Parekh, B. & Maniatis,T.Virus infection leads to localized hyperacetylation of histones H3 and H4 at
the IFN-β promoter. Mol. Cell 3, 125–129 (1999).
54. Wang, D., McCaffrey, P. & Rao, A.The cyclosporin-sensitive transcription factor NFATp is expressed
in several classes of cells in the immune system. Ann. NY Acad. Sci. 766, 182–194 (1995).
55. Luo, C. et al. Recombinant NFAT1 (NFATp) is regulated by calcineurin in T cells and mediates transcription of several cytokine genes. Mol. Cell Biol. 16, 3955–3966 (1996).
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