TH Cell Differentiation Is Accompanied by Dynamic Changes in Histone Acetylation of Cytokine Genes Orly Avni1, Dong Lee1,Fernando Macian1, Susanne J

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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.

When naïve T helper (TH) cells encounter antigen for the first time in in the context of a human YAC transgene or the endogenous mouse locus the periphery, they begin a process of differentiation that includes com- results in significant decreases in production of IL-4, IL-5 and IL-13 by © http://immunol.nature.com Group 2002 Nature Publishing 12,13 mitment to a specific pattern of cytokine production. The TH1 cell pop- TH2 cells . Similarly, deletion of DH sites V and VA from the endoge- ulation is characterized by the production of interferon-γ (IFN-γ) upon nous Il4 locus in mice results in pronounced impairment of Il4 expres-

stimulation, whereas the TH2 cell population transcribes the linked sion by two different IL-4Ðproducing cell types: TH2 cells and bone mar- cytokine genes encoding interleukin 4 (IL-4), IL-5 and IL-13. Full rowÐderived mast cells14. polarization can readily be achieved in vitro by manipulating the Decondensed or “open” chromatin is characterized by hyperacetyla-

cytokine milieu: IL-12 and IL-4 strongly potentiate TH1 and TH2 differ- tion of associated histones as well as by increased accessibility to 15Ð19 entiation, respectively, via the signal transducers and activators of tran- restriction enzymes, nucleases and transcription factors . The NH2- scription 4 (STAT4) and STAT6. The lineage-specific transcription fac- terminal tails of histones H3 and H4 are subject to diverse modifications,

tors GATA-3 and T-bet are critical for TH2 and TH1 differentiation; including phosphorylation, methylation and acetylation; these covalent when ectopically expressed, these proteins not only promote expression modifications may alter the interaction of histone tails with DNA or of the relevant cytokines but also suppress transcription of the inappro- serve as docking sites for chromatin-associated proteins (the “histone priate cytokine genes1Ð5. code” hypothesis)17. Core histone acetylation is a reversible post-trans-

Compared to naïve T cells, differentiated TH1 and TH2 cells show lational modification, and transcriptional activators and repressors marked changes in the chromatin structure and DNA methylation status recruit histone acetyltransferases (HATs) and histone deacetylases of IFNG and the linked genes IL4, IL5 and IL13 respectively6,7. (HDACs) respectively to gene promoters and enhancers15Ð19. Specifically, naïve T cells have only two DNase I hypersensitive (DH) We show here that T cell differentiation is associated with a dynam- sites in a ∼43-kb genomic interval that spans IL4 and IL13. Within 48 h ic process of chromatin remodeling and histone acetylation at regulato- of primary stimulation in the presence of IL-4, ten additional clusters of ry regions of IL4 and IFNG. Stimulation of naïve T cells for 17Ð40 h 8Ð10 TH2-specific DH sites are observed . All ten DH sites are stable mark- resulted in a phase of nonselective histone hyperacetylation at both

ers of differentiated TH2 cells. Stimulation leads to the appearance of an cytokine genes: this process required T cell receptor (TCR) signals, did

inducible hypersensitive site (VA) located 3′ of IL4; this site corresponds not depend on the presence of polarizing cytokines and was associated to an IL4 enhancer that binds GATA-3 and the antigen-induced tran- with increased accessibility for the IL4 enhancer to the restriction scription factor NFAT111. The in vivo role played by the two clusters of enzyme SwaI. At later times, in T cells differentiated for 1 week under

DH sites has been demonstrated by deletion in the context of the TH1 or TH2 conditions, the nonselective pattern of histone hyperacety-

endogenous mouse locus. The TH2-specific DH sites HSS1 and HSS2 lation gave way to selective histone hyperacetylation at regulatory are located in the intergenic region between IL13 and IL4; their deletion regions of only the appropriate cytokine genes. This process was

1The 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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IL-4 E DH a b γ ε VA IL-4 P site IV IFN- P CD3 P Input α γ ε P IV V -AcH4 -+ -+ -+ -+E+P IV IFN- CD3 KIF3 C57BL/6 IL-4 Exon 1 2 34 CNS-2 5 kb P BALB/c

IFN-γ DO11.10 Exon 1 23 4 1 kb

1234 5 67 8910 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.

strongly dependent on the continued presence of the appropriate polar- mice showed almost no detectable acetylation of histone H4 on the Il4 izing cytokines and lineage-specific transcription factors (IL-4 and promoter and enhancer (Fig. 1b, lane 2), the Ifng promoter (lane 6) or STAT6 in the case of IL4, and IL-12 and T-bet in the case of IFNG). We DH site IV, which exists as a DH site in naïve T cells9 (lane 4). The demonstrate here in vivo binding of STAT6 to the IL4 promoter and Cd3e promoter, which is already active in naïve T cells, served as a pos- enhancer, stimulation-dependent binding of GATA-3 to the IL4 itive control (Fig. 1b, lane 8). Histone H3 acetylation was barely enhancer and functional cooperation between GATA-3 and NFAT1 at detectable in naïve T cells; it was much below that observed in differ- the IL4 enhancer. Our results emphasize the complexity and dynamic entiated T cells (data not shown). nature of the mechanisms initiated through the TCR and maintained by polarizing cytokines, which ultimately specify the appropriate pattern Histone hyperacetylation in differentiated T cells of cytokine expression by 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

Results BALB/c mice for 8 days under TH1 or TH2 conditions and examined Histone hypoacetylation in naïve T cells histone acetylation both before and after stimulation (Fig. 2a). © http://immunol.nature.com Group 2002 Nature Publishing We used chromatin immunoprecipitation (ChIP) to examine qualita- Differentiated TH2 cells—whether unstimulated (Fig. 2a, lane 6) or tively the acetylation status of histones at the Ifng promoter and four stimulated (lane 8)—showed strong hyperacetylation of histone H4 at selected regulatory regions of Il4: the Il4 promoter, the inducible DH all Il4 regulatory regions examined (panels 1Ð3), but no detectable his-

site VA (referred to here as the Il4 enhancer), the constitutive TH2-spe- tone acetylation at the Ifng promoter (panel 4). Conversely, TH1 cells cific DH site V, and the “common” DH site IV observed in naïve T cells (Fig. 2a, lanes 5 and 7) showed detectable hyperacetylation of histone 10,12,14 and differentiated TH1 and TH2 cells (Fig. 1a). We first assessed H4 at the Ifng promoter both before and after stimulation (panel 4), but histone acetylation in naïve T cells, which have the potential to differ- no detectable histone acetylation at the regulatory regions of Il4 (pan- + entiate into either TH1 or TH2 cells (Fig. 1b). Freshly isolated CD4 els 1Ð3). This selective pattern of histone hyperacetylation correlated T cells from young C57BL/6, BALB/c or D011.10 TCRÐtransgenic completely with the restricted binding of NFAT1 to the Il4 and Ifng reg-

α a No Ab -AcH4 b No Ab α -NFAT1 TH1TH2TH1 TH2 TH1TH2TH1TH2 - + - + Stimulation --+ + --++Stimulation IL-4 E IL-4 E IL-4 P 1 IL-4 P TH2 cells 2 Site IV 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 3 Site V 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 (+). 4 IFN-γ P Chromatin complexes were immunoprecipitated with beads alone (lanes 1–4) or 12 3 4 5678 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 T 1T2T 1 T2 amplify the precipitated DNA. As a control, the PCR was done directly on input H H H H Stimulation - - + + DNA purified from chromatin before immunoprecipitation (lower panel). (b) NFAT1 IL-4 E binds the Il4 promoter and enhancer after activation. Chromatin from the same rest- Input IL-4 P ing (–) and stimulated (+) TH2 samples in a was immunoprecipitated with antibodies C57BL /6 mice to NFAT1.The DNA was amplified with primers for the Il4 promoter and enhancer.

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12 ulatory regions in TH2 and TH1 cells, respectively . Histone hyper- antibodies to acetylated histones H3 and H4 revealed similar amounts of

acetylation was apparent in resting differentiated TH2 cells that do not histone acetylation for all the Il4 regulatory regions (promoter, enhancer, actively transcribe the cytokine genes; thus, in this system, histone DH site IV and DH site V) under the two conditions of differentiation acetylation correlated with the transcriptional competence of Il4 and (Fig. 3a, panels 1Ð3, lanes 3Ð6). To rule out the possibility that the non-

Ifng rather than overt gene transcription. selective hyperacetylation pattern reflected contamination of the TH1

After activation, two changes were often, but not invariably, observed. population with differentiated TH2 cells, we repeated the experiment The first was increased histone acetylation at DH site IV (Fig. 2a, panel with CD4+ T cells purified from the spleen and lymph nodes of young

2, lanes 6 and 8); this effect was consistently seen in C57BL/6 TH2 cells BALB/c DO11.10 TCRÐtransgenic mice, which express a single TCR but was less apparent in BALB/c T cells (data not shown). We also fre- specific for an ovalbumin peptide that is not encountered in the mouse quently observed altered histone acetylation at the Ifng promoter in (Fig. 3b). T cells from these mice that had been left to differentiate for

stimulated TH1 cells (Fig. 2a, panel 4, compare lanes 5 and 7). A control 48 h displayed a similar nonselective pattern of histone hyperacetylation

ChIP assay with the same TH2 cell chromatin preparation showed at the Il4 promoter and enhancer as was observed in non-TCRÐtrans- NFAT1 binding to the Il4 enhancer and promoter only after stimulation, genic BALB/c mice (compare Fig. 3a and b). confirming that the cells were adequately stimulated (Fig. 2b). In contrast to the nonselective pattern of histone hyperacetylation on The pattern of histone hyperacetylation at Ifng and Il4 recalls the Il4, a partially selective pattern was already established on the Ifng pro- similarly selective and constitutive changes in DNase I hypersensitivi- moter by 48 h after the first stimulation (Fig. 3a, panel 4 and Fig. 3b, 10Ð12 ty patterns observed at these genes during TH differentiation . The lower panel). To determine whether the Ifng promoter might display a correlation between histone acetylation and DNase I hypersensitivity less selective pattern at earlier times of stimulation, we differentiated + was not absolute, however. DH site IV is a strong DH site in naïve CD4 T cells from young BALB/c mice for 17 h under TH1 and TH2 T cells9, but we were not able to detect histone acetylation at this conditions or cultured them in media alone without stimulation and in

region; conversely, DH site VA—the inducible Il4 enhancer—appears the absence of cytokines. Compared to unstimulated naïve T cells, the as a hypersensitive site only after stimulation12, but was associated with Ifng promoter from 17 hÐstimulated T cells was hyperacetylated under

hyperacetylated histones even in resting TH2 cells. both TH1 and TH2 conditions (Fig. 3c, lower panel, lanes 4Ð6). Changes in chromatin structure have classically been assessed by TCR signals induce early changes in chromatin changes in accessibility to restriction enzymes. After stimulation, fully

We next examined histone acetylation after 2 days of stimulation under differentiated TH2 cells develop an inducible DH site (VA) at the Il4

TH1 or TH2 conditions, the earliest time at which reproducible changes enhancer in a manner that parallels the inducible binding of NFAT1 to in DNase I hypersensitivity have been documented for Il410 (Fig. 3). the enhancer; this process is accompanied by increased accessibility of CD4+ T cells from young BALB/c mice were differentiated for 48 h a SwaI restriction site at the 3′ end of the enhancer region12. We used a

under TH1 or TH2 conditions. Immunoprecipitation of chromatin with sensitive ligation-mediated polymerase chain reaction (LM-PCR) tech-

TH1TH2 TH1TH2TH1TH2 TH1TH2 TH1TH2 TH1TH2 TH1TH2 N TH1TH2 N IL-4 E IL-4 E IL-4 E IL-4 P 1 IL-4 P IL-4 P IFN-γ P IFN-γ P 13452 6 Site IV 12 3 4 5 6 2 TH1TH2 N TH1TH2 Input IFN-γ P Input IFN-γ P DO11.10 3 Site V

d Naïve TH TH1 TH2 4 IFN-γ P - 6h 40h 40h Stimulation ---- SwaI 12 3 4 5 6 TH1TH2 IL-4 E (LM-PCR) Input IFN-γ P BALB/c IL-4 P (PCR)

12 3 45 678910 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 α-AcH3 Figure 4. The selective histone acetylation pattern is established by the polarizing cytokines. ChIP assay assessing histone acetylation at gene regulatory TH1TH2TH0TH1TH2TH0TH1TH2TH0TH1TH2TH0 + - - -+ + + - - - + ++Stimulation 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 IL-4 E IL-4 P 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 IFN-γ P (data not shown).The lower panel shows the input control.

CD3ε P

123456 78910 11 12 Input

TH1TH2TH0TH1TH2TH0 - --+ + + IL-4 E IL-4 P

nique to monitor accessibility of the Il4 enhancer to the restriction ble amounts of PCR product, especially when a higher concentration of enzyme SwaI in differentiating T cells (Fig. 3d). DO11.10 recombina- SwaI was used (Fig. 3d, upper panel, lanes 7Ð12). Control PCR reaction activating gene 2Ðdeficient (RAG-2Ð/Ð) naïve CD4+ T cells were tions confirmed that the Il4 promoter, which lacks a SwaI site, was rep-

stimulated for 6 h or 40 h under TH1 or TH2 conditions, and nuclei were resented at equivalent amounts in all samples (Fig. 3d, lower panel).

prepared and incubated with increasing amounts of SwaI. The resulting These results showed that the earliest phase of TH differentiation was blunt-ended genomic DNA was isolated and ligated to a unidirectional accompanied by histone hyperacetylation at regulatory regions of Il4 and

linker, after which primers for site VA were used in conjunction with a Ifng as well as increased accessibility of the Il4 enhancer region to the linker primer to assess the extent of SwaI cleavage at the Il4 enhancer. restriction enzyme SwaI. This phase was driven by TCR stimulation Little PCR product was detected in naïve cells that were either unstim- because it occurred regardless of whether the cultures contain IL-4 or IL-

ulated (Fig. 3d, upper panel, lanes 1Ð3) or stimulated for 6 h (lanes 4Ð6); 12, polarizing cytokines for TH2 and TH1 development, respectively. By 1

this indicated that SwaI did not effectively gain access to and cleave its week of TH differentiation, the histone acetylation pattern became fully

restriction site in site VA under these conditions. In contrast, stimulation selective (Fig. 2a) and the DH site VA that contained the SwaI restriction 12 for 40 h under either TH1 or TH2 conditions yielded high and compara- site disappeared, to be observed again only in stimulated TH2 cells .

b a α-AcH4 No Ab α-STAT6 © http://immunol.nature.com Group 2002 Nature Publishing -/- T 1T2 T 1T2 T 1 2 1T2 WT STAT6 __H H H H H_ T_H TH H T 1T2 T 1T2 T 1 T 2 T 1T2 ++++ Stimulation __H H H H _H H H H ++++ – Stimulation IL-4 E IL-4 E IL-4 P IL-4 P 12 3456 78 Input TH1TH2TH1TH2 IFN-γ P _ _ ++ IL-4 E IL-4 P CD3ε P c No Ab α-NFAT α-AcH4 IL-4 E input WT STAT6 -/- WT STAT6 -/- WT IL-4 P _ + _ + _ ++_ _ Stimulation 123456 78 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 and STAT6-deficient T cells. CD4+ T cells from 3-week-old wild-type (lanes 1–4) or Site IV

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 12 34 56 7 8 9 (+). Chromatin complexes were immunoprecipitated with antibodies to acetylated TH2 cells histone H4. No PCR signal was observed in a control immunoprecipitation done without any antibodies (data not shown). (b) Direct binding of STAT6 to the Il4 pro- Input + moter and enhancer in TH2 cells. CD4 T cells, purified from 3-week-old C57BL/6 WT STAT6 -/- __ mice, were differentiated for 8 days under TH1 or TH2 conditions, then left unstimu- + + lated (–) 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 a No Ab α-GATA3 α-AcH4 c 500 GATA-3 and NFAT1 and inducible binding of Resting T 2 _ + _ + _ + Stimulation GATA-3 to the Il4 enhancer. (a) GATA-3 binds H PMA+Iono )

3 400 inducibly to the Il4 enhancer in TH2 cells, but does not IL-4 E 10 bind DH site IV. CD4+ T cells from 3-week-old x C57BL/6 mice were differentiated for 8 days under 300 Site IV TH2 conditions, then left unstimulated (–) or stimulated with PMA and ionomycin for 6 h (+). Chromatin 12 3 4 56 200 complexes were immunoprecipitated with antibodies Luciferase units ( to GATA-3 or acetylated histone H4. (b) Immunoblot b CNN C 100 Stimulation _ _ + + analysis assessing the expression of GATA-3 in resting + GATA3 0 and stimulated TH2 cells. CD4 T cells from 3-week- Blot old mice were differentiated for 7 days under TH2 con- β -actin NFAT ditions, then left unstimulated (–) or stimulated with GATA3 NFAT* × 1234 PMA and ionomycin for 6 h (+).The lysates from 3 Untransfected 106 cells were separated into nuclear (N) and cytoso- NFAT+GATA3 NFAT*+GATA3 lic (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 After a period of nonselective histone hyperacetylation, cell typeÐspe- (Fig. 5b). ChIP assays showed clearly that STAT6 bound directly to

cific histone hyperacetylation at cytokine genes could be established both the Il4 promoter and enhancer regions in differentiated TH2 cells through maintained acetylation that was dependent on the polarizing under both resting and stimulated conditions, most likely because these cytokines, active deacetylation driven by the opposing cytokines or both. cells were cultured in the constant presence of IL-4 (Fig. 5b, lanes 6 To distinguish between these possibilities, we differentiated precursor and 8). Notably, STAT6 was also required for NFAT binding to the Il4 CD4+ T cells in the absence of polarizing cytokines and in the presence promoter and enhancer (Fig. 5c). In agreement with published data12,

of neutralizing antibodies to IL-4, IL-12 and IFN-γ for 8 days (TH0 con- NFAT1 bound the Il4 promoter and enhancer in activated wild-type TH2

ditions). As expected, cells cultured under TH2 conditions showed his- cells; however, this was not observed in STAT6-deficient TH2 cells (Fig. tone H3 and H4 hyperacetylation at the Il4 promoter and enhancer (Fig. 5c, compare lanes 6 and 8). NFAT1 did not bind site IV (located ∼2 kb

4, upper panel, lanes 2, 5, 8 and 11), whereas cells cultured under TH1 upstream of the enhancer) in either cell type (Fig. 5c, lower panel, lanes

(lanes 1, 4, 7 and 10) or TH0 (lanes 3, 6, 9 and 12) conditions showed 6 and 8); immunoprecipitation with H4 antibodies served as a positive reduced histone acetylation at these regions. Conversely, cells cultured control (lane 9). © http://immunol.nature.com Group 2002 Nature Publishing under TH1 conditions showed histone hyperacetylation at the Ifng pro- These results indicated that STAT6 mediates the ability of IL-4 to moter, as expected (Fig. 4, middle panel, lanes 1, 4, 7 and 10), whereas maintain histone hyperacetylation at the Il4 promoter and enhancer.

cells cultured under TH2 (lanes 2, 5, 8 and 11) or TH0 (lanes 3, 6, 9 and This is done, in part, by inducing expression of TH2-specific transcrip- 12) conditions showed reduced acetylation. The histone acetylation sta- tion factors such as GATA-3 and Maf21Ð24, but also potentially by bind- tus of the Cd3e promoter was unaffected (Fig. 4, lower panel). These ing directly to the Il4 promoter and enhancer and recruiting histone- results showed that the correct polarizing cytokines were necessary to modifying enzymes. In addition, STAT6 facilitated NFAT1 binding to maintain histone hyperacetylation at cytokine genes beyond the early the Il4 promoter and enhancer. (17Ð48 h) period after antigen stimulation. We cannot rule out, howev-

er, that the opposing cytokines also contributed to establishing the selec- GATA-3 inducibly binds the site VA enhancer tive hyperacetylation pattern, for example, by accelerating deacetylation GATA-3 is a downstream target of STAT621 and maintains the differen- 21,22 of the inappropriate locus in differentiating T cells. tiated TH2 phenotype by autoactivating its own expression . We eval-

uated GATA-3 binding to Il4 regulatory regions in differentiated TH2 STAT6 maintains histone hyperacetylation cells (Fig. 6a). ChIP assays showed that GATA-3 bound weakly to the

STAT6 is a primary transcription factor activated by IL-4 receptor sig- site VA enhancer in resting cells (Fig. 6a, upper panel, lane 3) and that naling, and STAT6 binding sites are present in both the Il4 promoter binding was much enhanced after stimulation (lane 4). However GATA- and enhancer12, 20. We asked whether the ability of IL-4 to maintain his- 3 did not bind detectably to the Il4 promoter12 or DH site IV (Fig. 6a,

tone hyperacetylation at Il4 was mediated via STAT6. We compared the lower panel) in either resting or stimulated TH2 cells. The inducible histone H4 acetylation status of Il4 regulatory elements in wild-type binding of GATA-3 to the Il4 enhancer partly reflected up-regulation of

and STAT6-deficient T cells cultured for 8 days under TH1- and TH2- GATA-3 protein in nuclear extracts of stimulated TH2 cells, as shown by

differentiation conditions (Fig. 5a). Wild-type TH2 cells showed the immunoblotting (Fig. 6b, upper panel, compare lanes 2 and 4); the cyto- expected selective pattern of histone hyperacetylation at the Il4 pro- plasmic protein β-actin served as a control for cell fractionation and moter and enhancer (Fig. 5a, upper panel, lanes 2 and 4). However, equivalent protein loading in the lanes (lower panel, lanes 1 and 3). under the same conditions, STAT6-deficient T cells were hypoacetylat- Using transient reporter assays in Jurkat T cells, we demonstrated a ed at H4 histones in these regions (Fig. 5a, upper panel, lanes 6 and 8). strong functional cooperation between GATA-3 and NFAT1 at the Il4 As expected, STAT6 deficiency did not affect histone acetylation at the enhancer (Fig. 6c). Cells transfected with a luciferase reporter plasmid 11 Ifng promoter in resting and stimulated TH1 cells (Fig. 5a, middle driven by the site VA enhancer showed low inducible activity, presum-

panel, lanes 1, 3, 5 and 7); stimulation of TH2 cells resulted in some ably due to endogenous NFAT (cluster 1). Ectopic expression of NFAT1 hyperacetylation at the Ifng promoter (lane 8). Histone acetylation at alone or GATA-3 alone did not lead to transcriptional activation above this the Cd3e promoter was unaffected (Fig. 5a, lower panel). basal activity (clusters 2 and 3), whereas the combination of both proteins

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a WT T bet-/- b WT T bet-/- TH1TH2 TH1TH2 TH1TH2 TH1TH2 TH1TH2 TH1TH2 TH1TH2 TH1 TH2 __++++ __ Stimulation __++++ __ Stimulation

IFN-γ P IFN-γ P α -AcH3 α-AcH4 IL-4 E IL-4 E IL-4 P IL-4 P 12 345678 12 345678

c WT T bet-/- d WT T bet-/- TH1TH2 TH1TH2 TH1TH2 TH1TH2 TH1TH2 TH1TH2 TH1TH2 TH1TH2 __++++ __ Stimulation __++++ __ Stimulation

IFN-γ P Input IFN-γ P α -NFAT1 12 345678 IL-2 P

12 345678

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.

led to strong synergistic activation (cluster 4). The region of NFAT1 Discussion involved in GATA-3 cooperation is different from that required for coop- We have shown here that differentiating T cells undergo dynamic eration with the established NFAT partner AP-1 (Fos-Jun)25. This is changes in histone acetylation of cytokine genes. TCR stimulation of because a mutant NFAT1 incapable of Fos-Jun interaction25 synergized naïve T cells resulted in increased histone acetylation at regulatory with GATA-3 even more effectively than wild-type NFAT1 (compare regions of Il4 and Ifng, in a manner that was independent of the clusters 4 and 6). The stronger synergy could reflect the fact that the cytokine milieu. However this phase was transient: continued culture in © http://immunol.nature.com Group 2002 Nature Publishing mutant protein, unlike wild-type NFAT1, cannot be diverted away from the presence of IL-4 or IL-12 was required to maintain increased his- reporter DNA to genomic DNA elements at which it can bind coopera- tone acetylation at the Il4 and Ifng regulatory regions, respectively. tively with endogenous AP-1. We concluded that increased GATA-3 bind- These results provide a molecular correlate for published findings ing occurred at the Il4 enhancer after stimulation, partly because GATA-3 showing that IFN-γÐproducing T cells pass through an early stage of Il4 protein concentrations increased and partly because GATA-3 cooperated expression28 and that initial Il4 expression is TCR-dependent but inde- 29Ð31 with NFAT1 at this regulatory element in stimulated TH2 cells. pendent of the IL-4 signaling pathway . More recently, sensitive assays have documented early Il4 and Ifng transcription by naïve 32 T-bet maintains histone hyperacetylation T cells stimulated under both TH1 and TH2 conditions , consistent with

T-bet is a TH1-specific transcription factor that is necessary for IFN-γ the early increases in histone acetylation we observed at each of these + 26 expression in CD4 TH1 cells . To determine whether T-bet participated in cytokine genes. In the absence of polarizing cytokines or key transcrip-

maintaining histone hyperacetylation at Ifng in differentiated TH1 cells, we tion factors, both early histone hyperacetylation (our data) and early compared the histone acetylation status of the Ifng promoter in wild-type cytokine gene expression32 are reduced to the low basal amounts 27 + and T-betÐdeficient CD4 T cells cultured for 8 days under TH1 and TH2 observed in naïve T cells. TCR stimulation activates an immediate and 33 conditions. Wild-type T cells showed the expected TH1-selective pattern of global chromatin derepression program in naïve T cells ; this might be histone H3 and H4 hyperacetylation at the Ifng promoter (Fig. 7a,b, upper sufficient to permit binding of TCR-induced transcription factors to the panels, lanes 1 and 3). Under the same conditions, resting T-betÐdeficient promoter regions of cytokine genes, thus initiating localized histone

TH1 cells showed lower amounts of histone acetylation (lane 5), which modification in parallel with gene transcription. NFAT itself is an increased upon stimulation (lane 7). This suggested the participation of a attractive candidate for recruiting histone acetyltransferases34,35 and dri- stimulation-responsive transcription factor whose effect was unmasked in ving early transcription, and it will be useful to determine whether the T-betÐdeficient cells. This factor was not NFAT, as NFAT1 was unable NFAT1 binds in vivo to the Il4 and Ifng regulatory elements soon after to bind the Ifng promoter in T-betÐdeficient T cells (Fig. 7c, upper panel, primary stimulation. compare lanes 3 and 7), even though its binding to the Il2 promoter was Our data suggest that the chromatin structure of Il4 and Ifng in naïve not impaired (lower panel, lanes 3 and 7). Fig. 7d confirms that input T cells is different from that of the silenced genes in terminally differ- DNA was equivalent in all lanes. The Il4 promoter and enhancer were entiated T cells. In naïve T cells, the genes are maintained in a state that

hyperacetylated in T-betÐdeficient TH1 cells (Fig. 7a,b, lower panels, is permissive for gene transcription, so that the first TCR stimulation compare lanes 5 and 7 to 1 and 3), most likely reflecting the suppressive leads to histone acetylation of regulatory regions and limited gene tran- 26 activity of T-bet on Il4 . Together these results suggested that T-bet was scription. In contrast, in differentiated TH1 and TH2 cells, cytokine loci critical for robust acetylation at the Ifng promoter and facilitated NFAT1 with similar low amounts of histone H4 acetylation are maintained in a binding to the promoter after activation. silenced, nonpermissive state; in this state, the histones remain deacety-

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lated after activation and gene transcription does not occur. The differ- stimulated cells. Although we cannot rule out the possibility that ence between naïve and differentiated T cells is emphasized by the fact GATA-3 is inaccessible to the immunoprecipitating antibodies at cer- that inactive cytokine genes in naïve T cells are located away from cen- tain sites or time points, two other possibilities are pertinent. GATA-3 tromeric heterochromatin, whereas a large fraction of the silenced bound to a small number of specific sites might propagate the accessi- genes in differentiated T cells are repositioned to such regions32. It is ble chromatin structure to more distal regions of the locus; alternative-

likely, therefore, that in naïve T cells, TCR stimulation initiates per- ly, some other TH2-specific factor, potentially induced by GATA-3, missive chromatin modifications that facilitate early gene expression. might be stably DNA-bound in resting T cells and maintain chromatin In contrast, this may not occur in differentiated T cells because of accessibility at the Il4-Il5-Il13 locus. A major role of GATA-3 might be inhibitory modifications, previously established by the polarizing to induce such secondary factors, while also cooperating acutely with cytokines, that down-regulate the expression of inappropriate genes. NFAT and other inducible transcription factors at the Il4 enhancer. H3 Lys10 methylation provides a particularly attractive mechanism by Our data suggest that one function of lineage-specific transcription which genes may be simultaneously silenced and repositioned to hete- factors is to facilitate, perhaps directly, the binding of NFAT proteins to rochromatin; this modification is recognized by the chromodomain- cytokine regulatory regions. STAT6-deficient T cells showed containing heterochromatin-associated protein HP-136, which colocal- hypoacetylation of histones and no binding of NFAT1 at the Il4 pro- izes with Ikaros in the vicinity of silenced genes37. moter and enhancer. Likewise, T-betÐdeficient T cells showed no NFAT1 It is likely that increased histone acetylation is a major mechanism binding to the Ifng promoter, despite a stimulation-dependent increase of through which cytokine signaling pathways maintain cytokine genes in histone acetylation at this region that may be secondary to binding of a a transcriptionally permissive state. After the initial TCR-induced distinct inducible factor. By analogy with NFAT1-GATA-3 cooperation increase in histone acetylation, polarizing cytokines—as well as STAT6 at the Il4 enhancer, T-bet may directly cooperate with and stabilize

in the case of TH2 cells and T-bet in the case of TH1 cells—are needed NFAT1 binding to the Ifng promoter. Because we have not detected to maintain histone hyperacetylation at Il4 and Ifng; early histone GATA-3 binding to the Il4 promoter, STAT6 itself—or a different lin- hyperacetylation declines both in the absence of polarizing cytokine eage-specific transcription factor induced by STAT6 (for example, c- and in the presence of the inappropriate cytokine. Another well known Maf24)—may participate in stabilizing NFAT binding to the Il4 promoter. effect of cytokines, STAT factors and lineage-specific transcription fac- In summary, we have shown that TCR-CD28Ðinduced signaling path- tors such as GATA3 and T-bet is to repress expression of inappropriate ways contribute to early cytokine expression by inducing histone modi- cytokine genes2Ð6. Possibly, this is done by increasing the kinetics or fications at Il4 and Ifng. However these pathways have only a limited efficiency with which histone acetylation declines at inappropriate ability to maintain the locus in a state capable of supporting an efficient genes and/or by promoting inhibitory histone modifications as dis- recall response. In contrast, polarizing cytokines, STAT factors and lin- cussed above. Our data are consistent with published results showing eage-specific transcription factors provide an efficient and stable mecha- that the IL-4 signaling pathway is required for stable IL-4 expression nism for maintaining the appropriate locus in an active state. This is char- © http://immunol.nature.com Group 2002 Nature Publishing by differentiated T cells23,31,33,38Ð42. STAT6-deficient mice produce nor- acterized by increased histone acetylation, even under resting conditions, mal amounts of IL-4 in response to primary stimulation with several and the potential to bind acute transcription factors after activation. immunogens, but are strongly impaired in their recall (memory) responses31. Similarly, STAT6-deficient CD4+ T cells generate relative- Methods ly normal IL-4 responses when stimulated in vitro for a few hours, or Mice. B6J, BALB/c, STAT6Ð/Ð mice (Jackson laboratories, Bar Harbor, ME) and DO11.10 even for 3 days, with anti-CD3 and anti-CD28, but fail to produce high TCRÐtransgenic51, RAG-2Ð/Ð DO11.10 TCRÐtransgenic (provided by A. K. Abbas) and Ð/Ð27 31,32 T-bet mice were maintained in pathogen-free conditions in barrier facilities in the Center amounts of IL-4 in response to secondary stimulation . for Animal Resources and Comparative Medicine (Harvard Medical School). All mouse How does the IL-4 signaling pathway stabilize TCR-induced chro- protocols were approved by the Center for Blood Research and Harvard Medical School. matin changes at Il4? We have shown that STAT6 binds in vivo to the In vitro T cell differentiation. CD4+ T cells were purified from the spleen and lymph nodes Il4 enhancer and promoter in recently differentiated TH2 cells, which of young mice with the use of magnetic beads (Dynal, Oslo, Norway). For TH differentiation, suggests that it may have a direct role in regulating Il4 expression. cells (106/ml) were stimulated with 1 µg/ml of anti-CD3ε and 1 µg/ml of anti-CD28 Indeed, even STAT6-deficient T cells that exhibit stochastic expression (145.2C11 and 37.51, respectively, Pharmingen, San Diego, CA) in a flask coated with 0.3 of GATA-3 express only half the amount of IL-4 expressed by wild- 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, 23 type T cells . Given its ability to recruit the acetyltransferases p300 and nonessential amino acids, sodium pyruvate, vitamins, HEPES and 2-mercaptoethanol. For 43 CBP , STAT6 may directly promote histone acetylation at Il4. Indeed TH1 differentiation, the cells were stimulated in the presence of 5 ng/ml of recombinant STAT6 and NFAT may bind in concert to the Il4 promoter and mouse IL-12 (courtesy of Wyeth Research, Cambridge, MA) and 10 µg/ml of purified antiÐIL-4 (11B11, a gift of the Biological Resources Branch, National Cancer Institute enhancer, cooperatively recruiting a stronger constellation of chro- Preclinical Repository). For TH2 differentiation, cells were stimulated in the presence of 1000 matin-remodeling and histone-modifying enzymes than NFAT alone. U/ml of mouse IL-4 (added as supernatant of the I3L6 cell line)52, 5 µg/ml of antiÐIFN-γ µ The STAT6 target gene GATA-3, when retrovirally introduced into (XMG1.2 or R4-6A2) and 3 g/ml of antiÐIL-12 (courtesy of Wyeth Research). For TH0 conditions, the cells were cultured in the presence of antiÐIL-4, antiÐIL-12 and antiÐIFN-γ. For developing TH1 cells, induces IL-4 expression as well as the entire DH cultures longer than 3 days, cells were expanded (fourfold) in the absence of anti-TCR and pattern of Il421Ð23,44Ð47. However, our results suggest that GATA-3 does anti-CD28, but in the continued presence of cytokines and antibodies that included 10 U/ml not directly maintain the accessible chromatin structure over the entire of IL-2; they were then expanded every other day. After 8 days, the cells were left unstimu- µ Il4-Il5-Il13 locus. GATA-3 binds in vitro to several regulatory elements lated or were stimulated with PMA (20 nM) and ionomycin (1 M) for 6 h. 47Ð50 in the locus , but our ChIP experiments suggested that only a subset ChIP. ChIP analysis was carried out essentially as described53. Cells (5 × 107Ð15 × 107) were of these sites binds GATA-3 in vivo. GATA-3 does not bind the Il4 pro- fixed for 20 min on ice with one-tenth the volume of 11% formaldehyde solution (in 0.1 M moter, which—by sequence inspection—possesses an adequate GATA- 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 12 3 site , nor does it bind to DH site IV. In addition, despite the fact that PBS and resuspended in 10 ml of lysis buffer (50 mM HEPES at pH 7.5, 140 mM NaCl, histone acetylation at this Il4 enhancer was similar before and after 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- stimulation, GATA-3 associated strongly with the VA enhancer only in

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peptin and 10 mM iodoacetamide) and incubated for 10 min on ice. Nuclei were pelleted, trol plasmid. Twenty-four hours after transfection, cells were stimulated with 1 µM iono- resuspended and incubated at room temperature for 10 min in 0.2 M NaCl, 1 mM EDTA, mycin and 20 nM PMA. Twelve hours after stimulation, cells were collected and cell 0.5 mM EGTA, 10 mM Tris HCl (pH 8.0) and protease inhibitors. The nuclei were pellet- extracts were assessed for luciferase activity with the Dual Luciferase Assay System ed again and resuspended in 10 ml of sonication buffer (1 mM EDTA, 0.5 mM EGTA and (Promega, Madison, WI). Cotransfection of the pRL-TK vector (Promega) allowed normal- 10 mM Tris HCl at pH 8.0 and protease inhibitors). The suspension was sonicated eight to ization by Renilla luciferase activity. 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 Acknowledgments (for 48Ð72 h). The samples that contained the DNA were dialyzed against 10 mM Tris HCl We thank members of the laboratory for critical reading of the manuscript and valuable (pH 8.0), 1 mM EDTA, 0.5 mM EGTA and 10% glycerol and kept frozen in 0.5 ml aliquots discussions. Supported by grants from the National Institutes of Health (to A. R. and L. H. or used for immunoprecipitation, after adjustment to RIPA buffer (1% Triton X-100, 0.1% G.), a gift from the G. Harold and Leila Y. Mathers Charitable Foundation (to L. H. G.), the sodium deoxycholate, 0.1% SDS, 140 mM NaCl and protease inhibitors). Two hours of pre- Cancer Research Institute (to O.A.), the National Institutes of Health (to D. L.) and the clearing with the beads preceded overnight incubation at 4 ¡C with various antibodies. Leukemia Society and a grant from the Burroughs Wellcome Fund (to S. J. S.). 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 Competing interests statement with 1 ml of RIPA buffer (supplemented with 0.5 M NaCl and 100 µg/ml of yeast tRNA) The authors declare that they have no competing financial interests. 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 Received 7 January 2002; accepted 16 May 2002. 3 h. Incubation at 65 ¡C overnight reversed the formaldehyde cross-links. The DNA was 1. Abbas,A. K., Murphy, K. M. & Sher,A. Functional diversity of helper T lymphocytes. Nature 383, purified by phenol-chloroform extraction, precipitated in the presence of 20 µg of glycogen 787–793 (1996). and resuspended in 100 µl of TE buffer. 2. Wurster,A. L.,Tanaka,T. & Grusby, M. J.The biology of Stat4 and Stat6. 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