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Loci Accompany Th1/Th2
the IL-4 and IFN-
Cutting Edge: Changes in Histone Acetylation at
Patrick E. Fields, Sean T. Kim and Richard A. Flavell
2002; 169:647-650; ;
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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Copyright © 2002 by The American Association of
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The Journal of Immunology
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Cutting Edge: Changes in Histone Acetylation at the
IL-4 and IFN-? Loci Accompany Th1/Th2
Patrick E. Fields,* Sean T. Kim,* and Richard A. Flavell1*†
Peripheral T cell differentiation is accompanied by chromatin
changes at the signature cytokine loci. Using chromatin immu-
noprecipitation we demonstrate that profound increases in hi-
stone acetylation occur at the IFN-? and IL-4 loci during Th1/
Th2 differentiation. These changes in histone acetylation status
are locus and lineage specific, and are maintained by the tran-
scription factors Tbet and GATA3 in a STAT-dependent man-
ner. Our results suggest a model of cytokine locus activation in
which TCR signals initiate chromatin remodeling and locus
opening in a cytokine-independent fashion. Subsequently, cy-
tokine signaling reinforces polarization by expanding and
maintaining the accessible state at the relevant cytokine locus
(IL-4 or IFN-?). In this model, GATA3 and Tbet serve as tran-
scriptional maintenance factors, which keep the locus accessi-
ble to the transcriptional machinery. The Journal of Immu-
nology, 2002, 169: 647–650.
by T cells is directed by the cytokine environment in which the T
cell encounters Ag. IL-12 and IL-4 can strongly drive differenti-
ation of Th1 and Th2 cells, respectively, via STAT4 and STAT6
(2, 3). GATA3, expressed predominantly in Th2 cells, is critical
for Th2 lineage development and may be the principal mediator of
STAT6 function (4–6). Tbet is expressed predominantly in Th1
cells and influences IFN-? production and Th1 development (7).
GATA3 and Tbet may be “master regulators” of Th lineage de-
termination. Both act as transactivators, can induce DNase I hy-
persensitivity changes in the cytokine loci, and possess negative
regulatory functions; GATA3 inhibits IFN-? expression, and Tbet
inhibits Th2 cytokine expression (7–10).
Epigenetic events are important determinants of cytokine gene
expression and lineage commitment. Selective changes in DNase I
hypersensitivity and methylation in the IL-4 and IFN-? loci ac-
company Th differentiation (11, 12). Also, repositioning of the
uch recent work has focused on the programs of dif-
ferentiation leading to the development and stability of
the Th1 and Th2 phenotypes (1). Lineage commitment
IL-4 or IFN-? loci to areas of silencing within the nucleus occurs
during Th1 or Th2 differentiation, respectively (13). A causative
relationship between chromatin remodeling and Th cell differen-
tiation has not yet been established. The study of other events more
closely tied to transcriptional regulation, such as enzymatic mod-
ification of histone proteins, might aid in our understanding of such
Histones undergo an array of posttranslational modifications on
tail domains, including acetylation, phosphorylation, and methyl-
ation (14). These modifications, as well as the primary sequence of
the histone tails themselves, are highly conserved from yeast to
man and have been closely linked to biological events (e.g., rep-
lication, transcription, and silencing). High levels of histone acet-
ylation at particular loci correlate with transcriptional activity (15),
whereas reduced levels correlate with silencing (16). Factors me-
diating acetylation and deacetylation serve as transcriptional co-
activators and corepressors, respectively, suggesting a causative
relationship between acetylation and transcription (17, 18).
We show that histones in the cytokine loci of naive T cells are
unacetylated. Upon TCR stimulation, the loci are rapidly and pro-
gressively acetylated on histones H3 and H4. Early acetylation
occurs in the IL-4 locus irrespective of polarizing conditions, cor-
relating with early transcription. The maintenance of acetylation
depends on cytokine/STAT signaling. Tbet and GATA3 also con-
tribute to the polarized acetylated state.
Materials and Methods
Four- to 8-wk-old C57BL/6J, BALB/c, STAT6?/?, and STAT4?/?mice
were purchased from The Jackson Laboratory (Bar Harbor, ME) and main-
tained in the Yale University Animal Resources Center (New Haven, CT).
In vitro T cell differentiation
Naive CD62LhighCD44lowCD4?T cells were purified by flow cytometric
cell (FACS) sorting as previously described (19). These cells were either
used directly in chromatin immunoprecipitation (ChIP2; naive samples) or
stimulated in 24-well plates under polarizing conditions as described with
1 ?g/ml anti-CD3 mAb (145-2C11; American Type Culture Collection,
Manassas, VA) plus 1 ?g/ml anti-CD28 (37.5.1; American Type Culture
Collection) (19). T cells were expanded under the same conditions as the
primary cultures. Cytokines were measured (ELISA) from 2 ? 105T cells
restimulated for 24 h by plate-bound anti-CD3 (1 ?g/ml) plus anti-CD28 (2
For ChIP analysis, 0.5–1.5 ? 107T cells were fixed with 1% formaldehyde
for 10 min at 37°C, washed with PBS, and lysed in ChIP lysis buffer
*Section of Immunobiology, Yale University School of Medicine, and†Howard
Hughes Medical Institute, New Haven, CT 06520
Received for publication March 13, 2002. Accepted for publication May 23, 2002.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1Address correspondence and reprint requests to Dr. Richard A. Flavell, Yale Uni-
versity School of Medicine and Howard Hughes Medical Institute, 330 Cedar Street,
Farnam Medical Building 430, New Haven, CT 06520. E-mail address:
2Abbreviations used in this paper: ChIP, chromatin immunoprecipitation; GFP, green
fluorescent protein; eGFP, enhanced GFP; HSS, hypersensitive site; IE, intronic
The Journal of Immunology
Copyright © 2002 by The American Association of Immunologists, Inc.0022-1767/02/$02.00
by guest on June 5, 2013
(Upstate Biotechnology, Lake Placid, NY). DNA was sonicated by pulsing
three to six times. Anti-acetylated histone H3 or H4 was added (4 ?l per
immunoprecipitation) and incubated overnight. Protein A-agarose beads (Up-
state Biotechnology) were added for 1 h then washed once each with low-salt
buffer, high-salt buffer, and LiCl buffer, and twice with TE (buffer ingredients
are available in the Upstate Biotechnology catalog). Beads were eluted with
0.1 M NaHCO3and 1% SDS, and cross-links were reversed at 65°C. DNA
was ethanol-precipitated in the presence of 20 ?g glycogen. Aliquots of equal
volume from each sample were used as input controls.
Polymerase chain reaction
PCR was performed on chromatin samples for 28 cycles under standard
reaction conditions. Real-time quantitative PCR was performed using an
icycler iQ (Bio-Rad, Hercules, CA). Samples were normalized against
standard sheared input DNA. Concentrations were determined using the
software provided by the manufacturer.
PCR primer sequences (Keck Oligonucleotide Synthesis Facility, Yale
University) were as follows: IL-4P, 5?-ACTCATTTTCCCTTGGTTTC
AGC-3? and 5?-GATTTTTGTCGCATCCGTGG-3?; IFN-?P, 5?-CGTA
ATCCCGAGGAGCCTTC-3? and 5?-CTTTCAATGACTGTGCCGTGG-
3?; intronic enhancer (IE), 5?-TCTGCTTGGACATCTCTCTTCCC and 5?-
ACCACCCCACAGGTCTTTGTTC-3?; hypersensitive site (HSS), 5?-TT
GGGGACAGAGGATGCCTTAC-3? and 5?-GCCTTGCTGAGAGTTTC
TTTTGC-3?; TCR? enhancer, 5?-AGATAGTGAATCAATAGCCAG-3?
company Th differentiation. Histones H3 (A) and H4 (B) acetylation of
naive, Th1, and Th2 day 6 effectors. PCR amplification was performed on
5 ?l from each 4-fold dilution (starting with undiluted sample). C, Control
samples. Shown is a representative of six separate experiments.
Locus- and lineage-specific histone acetylation changes ac-
loci. IL-4 (A) or IFN-? (B) locus acetylation over time. Quantitative, real-
time PCR was performed on samples at day 0 (naive cells), or 2, 6, and 12
days after initiation of the cultures. Cells were restimulated at day 6. Rel-
ative amounts of DNA in each sample were determined using a standard
curve of sonicated DNA of known concentration. Samples were normal-
ized to input DNA. Fold-change from naive samples is plotted. Shown is
a representative of three independent experiments.
Time course of histone acetylation in the IL-4 and IFN-?
loci is STAT dependent. Shown is H3 acetylation in wild-type (A),
STAT4?/?(B), and STAT6?/?(C) naive, Th1, and Th2 effector T cells.
ChIP was performed as described. D, Input controls for wild-type,
STAT4?/?, and STAT6?/?cells. E, Time course analysis of H3 acetyla-
tion in STAT4?/?(upper panels) and STAT6?/?(lower panels) T cells. At
different times after stimulation (as in Fig. 2), IL-4P and IFN-?P H3 acet-
ylation was examined by ChIP. Quantitative PCR was performed as in Fig.
2. Shown is a representative of three independent experiments. F, Cytokine
production by restimulated wild-type, STAT4?/?, and STAT6?/?T cells.
T cells were polarized for 6 days as described. Cells (2 ? 105) were re-
stimulated for 48 h with anti-CD3 plus anti-CD28. IL-4 (left panel) and
IFN-? (right panel) were measured in supernatants by ELISA.
Maintenance of histone acetylation in the IL-4 and IFN-?
648 CUTTING EDGE: HISTONE ACETYLATION OF CYTOKINE LOCI DURING Th DIFFERENTIATION
by guest on June 5, 2013
Real-time PCR sequences were as follows: IL-4P, 5?-TCTTGATAA
ACTTAATTGTCTCTCGTCAC-3? and 5?-GCAGGATGACAACTAGCT
GGG-3?; IFN-?P, 5?-TCAGCTGATCCTTTGGACCC-3? and 5?-CTCA
Fluorogenic probes (Biosearch Technologies, Novato, CA) were as fol-
lows: IL-4P, 5?-ACGGGACAGAGCTATTGATGGGTCTCA-3?; IFN-?P,
Retroviral transduction of T cells was performed as described (20). Ret-
roviral vectors (K. Murphy, Washington University, St. Louis, MO, and L.
Glimcher, Harvard University, Boston, MA), allowed expression of
GATA3 or Tbet plus enhanced green fluorescent protein (eGFP) (7, 21). At
24 h after stimulation, cells were infected with retroviral supernatant. At
days 5–6, cells were sorted into eGFP-negative and eGFP-positive popu-
lations, expanded for 4 days, and either restimulated or used in ChIP ex-
periments. The Phoenix-ECO packaging cell line was a gift of Dr. G. Nolan
(Stanford University, Palo Alto, CA).
Results and Discussion
Histone acetylation in the IL-4 and IFN-? loci in naive and effector
Th cells was assessed using ChIP with Abs specific for acetylated
histones H3 or H4. We analyzed the IL-4 and IFN-? promoter
regions (IL-4P and IFN-?P) as well as two IL-4 locus regulatory
regions, the DNase I HSS (CNS-1) and the IE (HS2) (11, 22).
These regions coordinately function as enhancers in the IL-4 locus
(19, 23). In naive cells, low levels of IL-4P, IFN-?P, HSS, or IE
were coimmunoprecipitated with acetylated histones H3 or H4. In
contrast, both H3 and H4 acetylation were substantially greater in
day 6 Th1 and Th2 effectors (Fig. 1, A and B). In Th1 cells, IFN-
?P, but not the IL-4 locus, was hyperacetylated. Conversely, in
Th2 cells, the IL-4 regulatory regions were hyperacetylated,
whereas IFN-?P was not. These acetylation changes persisted in
cells rested for 14 days, conditions where no cytokine transcription
occurred (data not shown). Acetylation changes appeared to affect
the entire IL-4 locus, because identical patterns were observed at
distal sites, including the region of the IL-5 gene (data not shown).
Input controls demonstrated that equivalent amounts of material
were added to each immunoprecipitation (Fig. 1C). As an immu-
noprecipitation control, TCR? enhancer acetylation was analyzed.
Identical results were observed in C57BL/6 (Fig. 1), BALB/c
(Figs. 2 and 3), and AND TCR-transgenic mice (data not shown),
establishing the generality of the findings. Identical patterns of H3
and H4 acetylation suggested that their regulation in the cytokine loci
might be similar. Subsequent studies focused on H3 acetylation.
Kinetics of H3 acetylation was examined by ChIP with quanti-
tative real-time PCR. In Th2-polarized cells, IL-4P acetylation in-
creased at day 2, reaching near-maximum levels by day 6 (Fig.
2A). IL-4P acetylation was also observed in Th1-cultured cells at
day 2, decreasing by day 6. This observation correlates with early
and transient differentiation condition-independent IL-4 transcrip-
tion (13, 24). Similar patterns at IE and HSS were observed (data
not shown). IFN-?P acetylation patterns resembled those of the
IL-4 locus (Fig. 2B). Acetylation increases were detectable under
both polarizing conditions as early as day 1 (data not shown).
Acetylation continued to increase after day 2 in Th1 cultures while
decreasing over time in Th2 cultures (Fig. 2B). Unlike the IL-4
locus, acetylation at the IFN-? locus in Th2 cells was substantially
less than that in Th1 cells at all time points examined. Early and
non-lineage-specific IFN-? transcription also occurs, but with ear-
lier and more transient kinetics (13). The “window” of acetylation
in the IFN-? locus may be transient and thus was less prominent in
these studies. The reduction in acetylation could reflect active
deacetylation under opposite polarizing conditions or selection of
a population that fails to maintain an acetylated locus. The earliest
phase of locus activation occurs independently of cytokine signaling
and might be mediated by nonspecific entities triggered by TCR/
CD28 stimulation, such as chromatin remodeling complexes (25).
IFN-?P and IL-4P acetylation was significantly lower in
STAT4- and STAT6-deficient T cells cultured under Th1 or Th2
conditions (Fig. 3, A–C). Acetylation never reached levels ob-
served in wild-type cells, even after restimulation (Figs. 2 and 3E).
Interestingly, IL-4P acetylation was significantly increased in Th1-
cultured, STAT4-deficient T cells (Fig. 3, B and E). Likewise,
IFN-?P was hyperacetylated in Th2-cultured, STAT6-deficient T
cells (Fig. 3, C and E). Thus, STAT signaling (i.e., IL-12, IL-4)
might negatively affect Th1 or Th2 development by inhibiting cy-
tokine loci accessibility. In the absence of STAT signaling, IL-4 or
IFN-? loci hyperacetylation and transcription proceeded under
normally suppressive conditions (Th1 or Th2, respectively). The
mechanism by which this cross-regulation occurs is unknown. One
possibility is that it is mediated by the coactivators, GATA3 and
Tbet, because extinction of their expression is mediated by IL-12
were introduced by retroviral transduction into STAT6?/?or STAT4?/?T cells stimulated under Th2 or Th1 conditions, respectively. H3 acetylation of
the IL-4 or IFN-? loci was determined by ChIP on sorted green fluorescent protein (GFP)?and GFP?populations. Real-time quantitative PCR analysis
of GATA3 (C) or Tbet (D) transduced cells was performed as in Fig. 2. Relative units were calculated by normalizing the value for each sample to that
of the empty vector/GFP-sample.
Changes in histone acetylation at the IL-4 and IFN-? loci induced by GATA3 and Tbet in STAT-deficient T cells. GATA3 (A) or Tbet (B)
649 The Journal of Immunology
by guest on June 5, 2013
and IL-4, respectively (8, 26). When restimulated, STAT6-defi-
cient T cells cultured under Th2 conditions and STAT4-deficient T
cells cultured under Th1 conditions produced detectable amounts
of IFN-? and IL-4, respectively (Fig. 3F), consistent with earlier
reports (2, 27). These results indicate that the polarizing cytokine
milieu can specify cytokine expression in two ways. First, STAT
function can maintain cytokine locus accessibility. Second, STATs
can inhibit locus accessibility of the opposing cytokine. Whether
these putative functions are mediated by direct STAT binding or
indirectly by STAT-induced factors remains to be determined.
GATA3 and Tbet are activators of IL-4 and IFN-? production,
respectively. To determine whether these factors affect cytokine
locus acetylation, we introduced by retroviral transduction GATA3
or Tbet into STAT6- or STAT4-deficient T cells cultured under
Th2 or Th1 conditions, respectively. Transduction of empty virus
into STAT6 knockout T cells had no effect on IL-4 locus acetyla-
tion (Fig. 4A). In contrast, GATA3 significantly enhanced acety-
lation in the locus (Fig. 4A, lower panel). These data correlate with
IL-4 transcription in GATA-transduced cells (20, 21). GATA3 in-
duced a slight decrease in acetylation of IFN-?P (Fig. 4A) while
also reducing IFN-? expression (8). Tbet induced a small but re-
producible increase (2- to 3-fold) in IFN-?P acetylation (Fig. 4B),
consistent with an ability to induce IFN-? expression (7, 9). In
contrast, Tbet did not affect IL-4P acetylation, despite a strong
inhibitory effect on IL-4 production (10- to 50-fold; Ref. 7 and data
not shown). These results were confirmed by real-time PCR anal-
ysis (Fig. 4, C and D). Thus, GATA3 and Tbet appear to have a
direct effect on acetylation of the IL-4 and IFN-? loci in the ab-
sence of STAT6 and STAT4 signaling. We have not addressed the
likely possibility that GATA3 and Tbet might cooperate with other
factors, such as NFAT or STATs, to induce optimal IL-4 and
IFN-? locus acetylation and transcription.
The relationship between acetylation and transcription in these cy-
tokine loci is distinguishable in two ways. First, acetylation is spread
over much of the loci, if not the entire loci. This contrasts with acet-
ylation patterns of other gene loci, such as IFN-?, where activation-
induced acetylation is restricted to promoter-proximal nucleosomes
(28). Second, IL-4 and IFN-? loci acetylation persists when there is
little if any transcription. In contrast, IFN-? locus acetylation occurs
during acute stimulation and rapidly dissipates upon loss of the stim-
ulus (28). Thus, acetylation in the IL-4 and IFN-? loci correlates more
closely with transcriptional competence than with acute transcrip-
tional activity, reminiscent of the ?-globin locus (15). We postulate
that this type of acetylation (widespread and persistent) is a mark of
differentiation rather than of transcriptional activation.
Our data support a multistep model of IL-4 and IFN-? gene
activation whereby locus accessibility (as measured by histone
acetylation) regulates cytokine expression (29). In the first step, T
cell activation results in acetylation (and transcription) of IL-4 and,
to a lesser degree, IFN-?, irrespective of the cytokine environment.
This phase is likely mediated by signals generated by TCR/CD28
stimulation. Maintenance and expansion of the accessibility of the
relevant cytokine locus, as well as suppressive events preventing
the expression of the opposing cytokine, combine to reinforce po-
larized Th1/Th2 populations. Positive and negative regulation is
mediated by STAT proteins as well as GATA3 and Tbet.
Thus, T cell differentiation is characterized by dynamic changes
in cytokine locus histone acetylation. Upon initial T cell stimula-
tion, these changes enable early transcription of cytokines, possi-
bly contributing to the cytokine milieu in which T cell differenti-
ation can proceed. Second, they help to establish heritable and
stable patterns of cytokine locus accessibility in effector cells, en-
abling rapid and robust secondary responses. Maintained acetyla-
tion would also provide locus and lineage specificity, because tran-
scription factor binding would highly favor an acetylated locus.
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650CUTTING EDGE: HISTONE ACETYLATION OF CYTOKINE LOCI DURING Th DIFFERENTIATION
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