Functionally distinct Gata3/Chd4 complexes coordinately establish T helper 2 (Th2) cell identity

Abstract

GATA binding protein 3 (Gata3) is a GATA family transcription factor that controls differentiation of naïve CD4 T cells into T helper 2 (Th2) cells. However, it is unknown how Gata3 simultaneously activates Th2-specific genes while repressing those of other Th lineages. Here we show that chromodomain helicase DNA-binding protein 4 (Chd4) forms a complex with Gata3 in Th2 cells that both activates Th2 cytokine transcription and represses the Th1 cytokine IFN-γ. We define a Gata3/Chd4/p300 transcriptional activation complex at the Th2 cytokine loci and a Gata3/Chd4–nucleosome remodeling histone deacetylase repression complex at the Tbx21 locus in Th2 cells. We also demonstrate a physiological role for Chd4 in Th2-dependent inflammation in an in vivo model of asthmatic inflammation. Thus, Gata3/Chd4 forms functionally distinct complexes, which mediate both positive and negative gene regulation to facilitate Th2 cell differentiation.

The regulation of gene expression is fundamental to cell fate decisions and lineage specification during developmental processes. Lineage commitment of T lymphocytes in the immune system involves the activation of specific gene programs and the concomitant suppression of multipotential and alternate lineage gene profiles. These events are regulated in large part by a limited set of lineage-specific master transcription factors that functionally cross-antagonize one another.

The GATA family transcriptional factors (Gata1–6) selectively bind GATA sites in vertebrate genomes to regulate specific gene expression programs. Each GATA family member possesses two highly conserved type IV zinc fingers, referred to as the N-terminal and C-terminal zinc fingers, both of which are involved in DNA binding and protein–protein interactions (1). Among the six vertebrate homologs, Gata1–3 play key roles in the development and maintenance of hematopoietic and immune cells (2). Gata3 plays a critical role in T-cell development in the thymus; it has roles in the CD4 vs. CD8 lineage choice and at the β-selection checkpoint (3), as well as in T helper 2 (Th2) cell differentiation through the activation and repression of transcription (4, 5). After antigenic stimulation in a particular cytokine milieu, naïve CD4 T cells differentiate into one of several Th cell subsets including Th1 and Th2 cells (6). Differentiation of Th2 cells requires IL-4 stimulation, which leads to Stat6 phosphorylation and the up-regulation of Gata3 transcription (7, 8). The deletion of Gata3 in peripheral CD4 T cells prevents their differentiation into the Th2 lineage, causing cells to differentiate toward a Th1 phenotype in the absence of polarizing cytokines (9, 10). Conversely, the overexpression of Gata3 in Th1 cells switches their polarity to a Th2 phenotype (11). Recent genome-wide analyses using chromatin immunoprecipitation and microarray analysis (ChIP–chip), ChIP sequence, and RNA sequence (4, 5, 12) have indicated that Gata3 can directly or indirectly control a large number of Th2 cell-specific genes, as well as other genes including transcription factors such as T-bet (encoded by Tbx21), a key regulator of Th1 cell fate determination (13). Despite its fundamental importance, little is known regarding how the Gata3 transcriptional complex positively and negatively regulates T-cell lineage gene profiles in a cell type-specific manner.

Dynamic changes in chromatin structure induced through ATP-dependent remodeling and covalent histone modifications play important roles in regulating gene expression (14). Several lines of evidence identify Gata3 as a regulator of histone modifications, including histone H3-K9 acetylation (H3–K9Ac); H3–K4me1, 2, and 3; and H3–K27me3 (4, 5, 15). Such modifications influence transcription by modulating interactions of transcription factors with chromatin (16). Indeed, we previously identified CGRE, a conserved GATA response element, 1.6 kb upstream of the Il13 gene, corresponding to the 5′ border of the long-range histone hyperacetylation region, and Gata3 was shown to bind to CGRE with histone acetyltransferase (HAT) complexes including CREB-binding protein (CBP)/p300 (15).

Numerous ATP-dependent chromatin-remodeling and histone-modifying enzymes have been identified, including those important for T-cell development (17). Among them is the 2-MDa nucleosome remodeling histone deacetylase (NuRD) complex (18), which is highly expressed in the thymus and associates with the Ikaros family of lymphoid-lineage regulating factors in differentiating and mature T cells. Chromodomain helicase DNA-binding protein 4 (Chd4) is an ATP-dependent chromatin remodeler and a major subunit of the repressive NuRD complex (18, 19). The Chd4–NuRD complex plays pivotal roles in transcriptional regulation, reorganization, and maintenance of chromatin structures and has recently been implicated in DNA damage repair (20). Other components of the complex include a catalytic subunit Hdac1/2 and the nonenzymatic proteins methyl-CpG binding domain 2/3 (Mbd2/3), retinoblastoma-associated 46/48 (RbAp46/48), metastasis-associated 1/2/3 (Mta1/2/3), and p66 α/β (19). The subunit composition of NuRD can vary depending on the cell type, altering the activity and localization of the complex. To date, the NuRD complex has been shown to mediate both transcriptional activation and repression programs by several distinct transcriptional factors, including p53, Ikaros, Bcl-6, and friend of GATA 1 (Fog-1) (20). Chd4 is highly expressed in thymocytes and lymphocytes, and it exerts a positive role in gene expression at the Cd4 locus through the recruitment of HATs—i.e., p300, Moz, and Taf1—to the Cd4 enhancer and silencer regions (21, 22).

We herein identify Chd4 as a central component of two functionally distinct Gata3 complexes. Genome-wide analysis using ChIP sequence revealed that Gata3 together with Chd4 binds to both the Th2 cytokine gene loci and the Tbx21 locus. We found that Gata3 organizes a Gata3/Chd4/p300 complex at the Th2 cytokine gene loci and a Gata3/Chd4–NuRD repression complex at the Tbx21 locus in Th2 cells, thus simultaneously regulating Th2 cytokine gene activation and Tbx21 repression. We also demonstrated a physiological role for Chd4 in Th2-dependent inflammation in an in vivo model of asthmatic inflammation. Together, our results support a model in which Gata3/Chd4 centrally regulates T-cell fate and Th2 cell differentiation by forming functionally distinct complexes.

Results

Identification of Chd4, a Major Subunit of the NuRD Complex, as a Gata3-Interacting Protein in Th2 Cells.

Recent genome-wide analyses suggest that Gata3 mediates both activating and repressive gene regulation (4, 5). We therefore reasoned that Gata3 might interact with different cofactors to perform appropriate regulatory functions. To test this idea and isolate Gata3 complexes in Th2 cells, extracts from the Th2 cell clone D10G4.1, expressing Flag-tagged Gata3 at physiological levels (Fig. S1A), were subjected to two-step affinity purification by using anti-Flag and -Gata3 mAbs followed by SDS/PAGE and silver staining. Mass spectrometry identified multiple subunits of the NuRD complex, including Chd4, Mta1, Mta2, p66α, and Mbd3, as Gata3-interacting proteins (Fig. 1A). To pursue this observation, we first examined the Gata3 association with Chd4. Flag-tagged Gata3 and Myc-tagged Chd4 were ectopically coexpressed in 293T cells, and then immunoprecipitation (IP) was performed with an anti-Flag mAb. A specific complex containing Gata3 and Chd4 was detected by immunoblotting (IB) with anti-Myc mAb (Fig. S1B). The association of Gata3 with Chd4 persisted in the presence of ethidium bromide, suggesting that the association is DNA-independent (Fig. S1C). To confirm that endogenous Gata3 and Chd4 interact, cell extracts from primary Th2 cells were subjected to IP with anti-Gata3 mAb. Endogenous Chd4 was reproducibly detected in Gata3 immunoprecipitates (Fig. S1D). Further, purified recombinant Gata3 and Chd4 interacted in GST pull-down assays (Fig. S1E), and partial colocalization of the two proteins was detected by confocal microscopy (Fig. S1F). These results indicate that Chd4 is a bona fide Gata3-interacting protein in Th2 cells.

Fig. 1.

Identification of Chd4 in functionally distinct Gata3 complexes. (A) Total extracts from Flag–Gata3-expressing D10G4.1 cells were subjected to tandem affinity purification by using anti-Flag and -Gata3 mAbs followed by SDS/PAGE and silver staining. Several specific polypeptides were identified by mass spectrometry as described in SI Materials and Methods. (B) The 293T cells were transfected with expression plasmids encoding Myc-tagged Chd4, Flag-tagged Gata3, and HA-tagged p300 for 2 d. Extracts were immunoprecipitated with anti-Myc mAb, and elutes were then subjected to ultracentrifugation through a 15–40% (wt/vol) sucrose gradient. The collected gradient fractions were subjected to IB with anti-Myc, -Flag, -HA, -Mta2, -Hdac2, and anti-Mbd3 antibodies. The specific HAT and HDAC activities in each fraction were determined as described in SI Materials and Methods. The relative activity is shown with SDs. (C) A control or Chd4 siRNA was transfected into naive CD4 T cells, and the cells were stimulated with immobilized anti–TCR-β and anti-CD28 mAbs under Th1 or Th2 conditions for 4 d. (Left) Representative IFN-γ/IL-4 profiles are shown with percentages of cells in each quadrant. (Right) A summary of the percentage of IL-4–producing and IFN-γ–producing cells in Chd4 KD Th2 cells is presented. n = 5. **P < 0.01 (Student t test). (D) A portion of the same cultured cells used in C (Th2 conditions) were stimulated with immobilized anti–TCR-β mAb for 24 h, and the concentrations of cytokines in the culture supernatant were determined by ELISA. **P < 0.01 (Student t test). All data are representative of two or more independent experiments.

Gata3/Chd4 Complex Forms Functionally Distinct Assemblies with HAT or Histone Deacetylase Activity.

To address the functional role of the Gata3/Chd4 complex in chromatin regulation, 293T cells were cotransfected with Myc-tagged Chd4, Flag-tagged Gata3, and HA-tagged p300, because we have previously shown that Gata3 HAT complexes include p300 at the Th2 cytokine gene loci (15). Chd4 complexes were immunopurified, eluted by using a Myc-peptide, and then subjected to sucrose gradient ultracentrifugation and size fractionation (Fig. 1B). Chd4 and Gata3 were detected broadly throughout the gradient, indicating that Gata3/Chd4 are a part of multiple complexes of different sizes. Interestingly, HA-tagged p300 was detected in the slowly sedimenting fractions at a peak of ∼670 kDa, whereas endogenous subunits of the NuRD complex, Hdac2, Mta2, and Mbd3, were detected in the rapidly sedimenting fractions eluting with an apparent mass of >1 MDa (Fig. 1B Upper). This finding suggests that Chd4 forms distinct types of multiprotein complexes, including Gata3/Chd4/p300 and Gata3/Chd4–NuRD. We then measured HAT and histone deacetylase (HDAC) activity in each fraction, and importantly the peak pattern of HAT and HDAC activity segregated with p300 and Hdac2, respectively (Fig. 1B Lower). These data suggest the existence of functionally distinct Gata3/Chd4 complexes, which may differentially regulate gene expression in Th2 cells.

Activating and Repressive Functions of Chd4 in Th2 Cell Differentiation.

To determine whether Chd4 is required for efficient Gata3-mediated regulation of Th1 and Th2 cytokine expression, we engineered Chd4-knockdown (KD) D10G4.1 cells stably expressing a shRNA against Chd4 via a lentivirus system. Chd4 KD had no effect on the expression level of Gata3 mRNA (Fig. S2A). Interestingly, although Chd4 is known as a component of the NuRD repressor complex, its KD significantly inhibited the induction of mRNA for the Th2 cytokines Il4 and Il5. In contrast, the expression of Ifng was up-regulated in the Chd4-KD D10G4.1 cells (Fig. S2B), suggesting that Chd4 may have biphasic functions—Th2 cytokine gene activation and IFN-γ repression in Th2 cells. To further examine the role of Chd4 in Th2 cell differentiation in a more physiological experimental setting, naïve CD4 T cells from C57BL/6 mice were transfected with Chd4 siRNA and cultured under Th1 or Th2 conditions in vitro. Although Chd4 mRNA expression was silenced efficiently (Fig. S2C), Chd4 KD had no impact on the differentiation of naïve CD4 T cells into IFN-γ–producing Th1 cells (Fig. 1C). Intriguingly, however, the number of IL-4–producing cells cultured under Th2 conditions was decreased substantially by KD of Chd4 (18.3% vs. 7.9%), whereas the number of IFN-γ–producing cells was increased (3.7% vs. 13.8%) (Fig. 1C). Furthermore, quantitative RT-PCR (RT-qPCR) and ELISA analysis revealed that Chd4 KD during Th2 cell differentiation inhibited the expression of IL-4, -5, and -13, without altering Gata3 expression, whereas the expression of IFN-γ was significantly increased (Fig. 2D and Fig. S2D). These results indicate that, together with Gata3, Chd4 participates in both up-regulation of Th2 cytokines and repression of IFN-γ in developing Th2 cells.

Fig. 2.

Gata3 and Chd4 are necessary for the recruitment of p300 at the Th2 cytokine gene loci in developing Th2 cells. (A) Naïve CD4 T cells were stimulated under Th1 or Th2 conditions for 3 d, and then ChIP-seq for Gata3 and Chd4 was performed as described in SI Materials and Methods. The binding pattern of Gata3 and Chd4 at the Th2 cytokine gene loci is shown. Each bar represents the average base coverage at the corresponding position. The asterisk (*) indicates a fivefold greater peak value compared with input DNA. (B) CD4 T cells from WT or Gata3-deficient mice were cultured under Th1 or Th2 conditions for 3 d. The binding of Chd4, Hdac2, and Mbd3 to the Th2 cytokine gene loci including the CGRE or the Ifng promoter (indicated at bottom) in Th1 WT (filled bars), Th2 WT (open bars), or Th2 Gata3 KO (gray bars) cells was determined by ChIP assay with a qPCR analysis. The relative intensities (/input) are shown with SDs. (C) The binding of p300 to the Th2 cytokine gene loci was determined and analyzed as in B. (D) A control or Chd4 siRNA was transfected into naive CD4 T cells, and the cells were stimulated under Th1 or Th2 conditions for 3 d. The binding of p300 to the Th2 cytokine gene loci was determined and analyzed as in B. (E) Developing Th1 and Th2 cells were transfected with 3.5 μg of the indicated reporter constructs in the presence of 0.5 μg of the pRL-TK vector as an internal control on day 3 of the culture. One day after transfection, cells were stimulated with immobilized anti–TCR-β mAb for 12 h, followed by measurement of luciferase activity. The data indicate the mean results of three independent experiments with SDs. **P < 0.01 (Student t test). (F) CD4 T cells were stimulated under Th2 conditions for 2 d, and then the cells were infected with a retrovirus vector containing E1A WT cDNA or E1A d2-36 cDNA. Three days later, the cells were stimulated with immobilized anti–TCR-β mAb for 6 h before IL-4/IFN-γ staining. All data are representative of two or more independent experiments.

Gata3/Chd4 Complex Recruits p300 to Th2 Cytokine Gene Loci and Facilitates Th2 Cytokine Transcription.

Next, we performed genome-wide analysis of Gata3 and Chd4 occupancy in developing Th1 and Th2 cells by ChIP followed by massive parallel sequencing (ChIP-Seq). We identified 611 target genes shared by Gata3 and Chd4 in Th2 cells (Fig. S3A), including several regions in the Th2 cytokine gene loci. Significant binding of Gata3 and Chd4 at the Th2 cytokine gene loci was observed specifically in Th2 cells (Fig. 2A, asterisks), and three out of six of significant peaks overlapped (Fig. 2A).

To determine whether the increased binding of Chd4 in Th2 cells was Gata3-dependent, wild-type (WT) and OX40–Cre-driven conditional Gata3-knockout (KO) (Gata3-deficient) CD4 T cells were cultured under Th1 or Th2 conditions for 3 d and then subjected to Chd4 ChIP. Chd4 binding at various regions in the Th2 cytokine gene loci was significantly increased in WT Th2 cells compared with WT Th1 cells, including the overlapping Gata3/Chd4 binding site (G3/C4BS), but this binding was abolished in Gata3-deficient Th2 cells (Fig. 2B Left). Interestingly, the increase in Th2-specific binding was not observed for other subunits of the NuRD complex Hdac2 and Mbd3 (Fig. 2B Right). Gata3 is thus required for accumulation of Chd4 at the Th2 cytokine gene loci, and the Gata3/Chd4 complex recruited to this loci does not appear to include the repressor components of the NuRD complex, such as Hdac2. Given that one of the highest enrichments of the Gata3 and Chd4 coassociation was observed at the CGRE region of the Th2 cytokine gene loci—and that Gata3, Chd4, and p300 appear to form a complex with HAT activity (Fig. 1B)—we were prompted to examine Th2-specific histone hyperacetylation and recruitment of p300 to these sites. H3-K9Ac at the Th2 cytokine gene loci was lower in Chd4-KD cells compared with those of control Th2 cells (Fig. S3B). Among various HAT(s), Th2-specific binding of p300 to the CGRE, Il13p, and Il4p regions was observed, whereas others examined were either unaffected (CBP) or produced very low signal (Taf1 and Moz) in the assay (Fig. S3C). Moreover, the recruitment of p300 to the Th2 cytokine gene loci was abrogated in Gata3-deficient Th2 cells (Fig. 2C) and was significantly impaired in Chd4-KD Th2 cells (Fig. 2D), although the binding of Gata3 to the CGRE region was unaffected in Chd4-KD Th2 cells (Fig. S3D).

To determine whether the common binding regions for Gata3 and Chd4 around the Th2 cytokine gene loci play a functional role in transcriptional activation, 260-bp fragments spanning the Gata3 and Chd4 binding regions either upstream of the Il13 gene (CGRE) or intron 2 of the Il4 gene (HS2) were examined in reporter assays. Introduction of the CGRE and HS2 fragments significantly enhanced the transcriptional activity of the Il4 promoter in Th2 cells, but not in Th1 cells (Fig. 2E). We also assessed the role of p300 in Th2 cell differentiation, using an adenovirus E1A protein that binds the acetyltransferase region of p300 and inhibits its activity (23). Introduction of WT E1A (E1A WT) dramatically decreased the number of IL-4–producing cells, whereas mutant E1A lacking the p300-binding sequence (E1A d2-36) had no effect (Fig. 2F). Together, these results indicate that the Gata3/Chd4 complex positively regulates Th2 cell differentiation through the recruitment of p300 and subsequent induction of histone H3-K9Ac at the Th2 cytokine gene loci.

Gata3 Recruits the NuRD Complex to the Tbx21 Locus and Facilitates Repression of T-Bet–Dependent IFN-γ Expression in Developing Th2 Cells.

To clarify the role of the Gata3/Chd4 complex in the repression of IFN-γ in Th2 cells, we investigated the effect of Chd4 silencing on various transcription factors important for IFN-γ expression or Th1 cell differentiation. Expression of Tbx21 mRNA was increased in Chd4-KD cells, whereas there was little or no impact on Eomes, Hlx, or Runx3 (Fig. 3A and Fig. S4A). Expression of Tbx21 mRNA was increased in Gata3-deficient Th2 cells compared with naïve CD4 or WT Th2 cells (Fig. 3B), and T-bet protein was increased in Gata3-deficient Th2 cells compared with WT Th2 cells (Fig. S4B). These results indicate that both Chd4 and Gata3 are required for repression of T-bet in Th2 cells.

Fig. 3.

Gata3 recruits the NuRD complex to the Tbx21 locus and represses Tbx21 expression in developing Th2 cells. (A) Naive CD4 T cells were transfected with control or Chd4 siRNA and cultured under Th2 conditions for 3 d, and then expression of Tbx21 was determined by RT-qPCR. **P < 0.01 (Student t test). (B) Naive CD4 T cells from either WT or Gata3-deficient mice were cultured under Th2 conditions for 5 d, and the expression levels of Tbx21 mRNA were determined by RT-qPCR. **P < 0.01 (Student t test). (C) ChIP-seq for Gata3 and Chd4 was performed as described in SI Materials and Methods. The binding patterns of Gata3 and Chd4 at the Tbx21 locus are shown as Fig. 2A. (D) Developing Th2 cells were transfected with the indicated reporter constructs shown in Fig. S4E. Cells were then stimulated with or without 10 ng/mL IL-12 and 100 ng/mL IFN-γ for 36 h, followed by measurement of luciferase activity. The data indicate the mean results of three independent experiments with SDs. **P < 0.01 (Student t test). (E) The binding of Chd4, Hdac2, Mbd3, and p300 at the Tbx21 locus in Th1 WT, Th2 WT, and Th2 Gata3 KO cells was determined by ChIP assay using qPCR analysis. (F) CD4 T cells were stimulated under Th2 conditions, and 10 ng/mL TSA was added on day 2 of the culture. Three days later, the cells were stimulated with immobilized anti–TCR-β mAb and subjected to IL-4/IFN-γ staining, followed by FACS analysis. (G) Naive CD4 T cells were stimulated under Th1 or Th2 conditions, and 10 ng/mL TSA was added on day 2 of the culture. Three days later, the Tbx21 mRNA levels were assessed by RT-qPCR. All data are representative of two or more independent experiments.

ChIP-seq analysis revealed that in Th2 cells, both Gata3 and Chd4 bind to intron1 of the Tbx21 locus (Fig. 3C, asterisks); however, we detected no obvious binding of Chd4 at the Ifng locus in Th2 cells (Fig. S4C). In addition, the significant increase in Ifng expression detected in Chd4-KD Th2 cells was not observed when Chd4 was knocked down in Tbx21 KO Th2 cells (Fig. S4D). These results suggest that the Gata3/Chd4 complex represses IFN-γ production by direct inhibition of Tbx21 gene expression.

To assess whether the binding site of both Gata3 and Chd4 at intron1 of the Tbx21 locus (Tbx21 G3/C4BS) plays a functional role in transcriptional repression, a 250-bp fragment spanning the Tbx21 G3/C4BS was placed between the Tbx21 enhancer and promoter (−500) regions, and luciferase reporter assays were performed (Fig. S4E). As expected, the introduction of the Tbx21 G3/C4BS fragment substantially repressed the transcriptional activity of the Tbx21 enhancer–promoter in Th2 cells but not in Th1 cells (Fig. 3D and Fig. S4F).

Next, we sought to address whether Gata3 recruits the NuRD complex to the Tbx21 locus. Subunits of the NuRD repressor complex, Chd4, Hdac2, and Mbd3, were associated with Tbx21 G3/C4BS in a Gata3-dependent manner (Fig. 3E). Importantly, we found no preferential recruitment of p300 to Tbx21 G3/C4BS in Th2 cells. Because the NuRD complex represses transcription primarily via histone deacetylation, we examined the effect of a HDAC inhibitor, trichostatin A (TSA), on the Gata3-mediated repression of Tbx21 expression in Th2 cells. CD4 T cells were cultured for 3 d in the absence or presence of TSA after stimulation for 2 d under Th2 conditions. The number of IFN-γ–producing cells was increased after TSA treatment (4.7% vs. 20.0%), whereas no effect on the number of IL-4–producing cells was observed (67.3% vs. 66.8%) (Fig. 3F). In accordance with induction of IFN-γ production by TSA treatment, Tbx21 mRNA was up-regulated in TSA-treated Th2 cells (Fig. 3G). Thus, in Th2 cells, HDAC activity plays a pivotal role in the repression of Tbx21 and subsequent inhibition of IFN-γ production, but it has little activity in regulating Th2 cytokines. These results suggest that direct binding of Gata3 to the Tbx21 locus is required for recruitment of Chd4 and HDACs, to repress T-bet–dependent IFN-γ expression in Th2 cells.

Classification of Gata3/Chd4 Target Genes Identified by Using ChIP-Seq and Transcriptional Start Site Sequencing Analyses.

Our results thus far suggested that Gata3 simultaneously organizes functionally distinct complexes in Th2 cells—the Gata3/Chd4/p300 complex at the Th2 cytokine gene loci and the Gata3/Chd4–NuRD complex at the Tbx21 locus. To generalize this concept, we searched for additional genes that were bound by both Gata3 and Chd4 and that were differentially regulated in Th2 cells in a Gata3-dependent manner. Massive parallel sequencing of mRNA transcribed from transcriptional start sites (TSS-seq) (24) was used to identify genes that were specifically expressed in Th2 cells compared with both Th1 and Gata3-deficient Th2 cells. Of the 611 genes cobound by Gata3 and Chd4, we identified 18 candidate target genes that were positively regulated (higher expression in Th2 cells compared with both Th1 and Gata3-deficient Th2 cells) and 5 target genes that were negatively regulated (lower expression in Th2 cells compared with both Th1 and Gata3-deficient Th2 cells) in Th2 cells (Fig. S5A). We then measured expression of these candidate genes by RT-qPCR in Th1, Th2, and Gata3-deficient Th2 cells (Fig. S5B) and in Th2 cells where Chd4 had been silenced by siRNA (Fig. S5C). This analysis showed that Epas1, Ccr8, and Myl6b were highly expressed and that Mb21d1 was suppressed in Th2 cells in a Gata3- and Chd4-dependent manner. The other molecules were either Gata3-independent and/or Chd4-independent. We then performed ChIP assays to confirm the recruitment of the Gata3/Chd4/p300 activating complex or the Gata3/Chd4–NuRD repressive complex at these differentially regulated gene loci. As observed at the CGRE region of the Th2 cytokine gene loci, Chd4 and p300 were recruited to the Gata3 binding sites of the Epas1 and Ccr8 loci in a Gata3-dependent manner (Fig. S5D Left), and Chd4 silencing by siRNA compromised the recruitment of p300 to the Gata3 binding sites of the Ccr8 locus (Fig. S5E Left). Conversely, like the Tbx21 locus, Chd4 and Hdac2 were associated with the Gata3 binding site of the Mb21d1 locus in a Gata3-dependent manner, and Chd4 KD by siRNA disrupted Hdac2 recruitment to this region (Fig. S5 D Right and E Right). Thus, our data support a hypothesis whereby functionally distinct Gata3/Chd4 complexes simultaneously activate or repress specific subsets of genes in Th2 cells through the recruitment of p300 or Hdac2.

Physiological Role of Chd4 in Th2-Mediated Allergic Responses in Vivo.

Finally, we investigated the in vivo physiological role of Chd4 using a Th2-cell–dependent airway inflammation model (25). Chd4 KD Th2 cells or control cells generated by in vitro culture from DO11.10 transgenic [ovalbumin (OVA)-specific TCR (T-cell receptor)-αβ Tg] mice were i.v. injected into normal BALB/c mice. The mice were challenged twice by inhalation with 1% OVA (Fig. 4A). A significant decrease in the infiltration of inflammatory cells, including eosinophils, in the bronchioalveolar lavage (BAL) fluid was observed in the Chd4 KD group in comparison with the control group (Fig. 4B). Histological analysis showed that mononuclear cell infiltration into the peribronchiolar regions of the lung was also modest in the Chd4 KD animals (Fig. 4C Upper). The levels of mucus hyperproduction and Goblet cell metaplasia assessed by PAS staining were lower in the bronchioles of the Chd4 KD group (Fig. 4C Lower). No obvious methacholine-induced airway hyperresponsiveness was induced in the Chd4 KD group (Fig. 4D). Together, these results indicate that Chd4 is involved in the regulation of Th2-cell–mediated allergic airway inflammation and airway hyperresponsiveness.

Fig. 4.

Chd4 regulates OVA-induced allergic airway inflammation. (A) Schematic outline of in vivo model for Th2-mediated allergic responses. In brief, DO11.10 Tg Th2 cells (1 × 10⁶ cells) were treated with control (si Cont.) or Chd4 (si Chd4) siRNA and i.v. transferred into BALB/c mice on day 4, before OVA inhalation twice on days 5 and 7. A no-cell-transfer group was also prepared as a negative control (No-transfer). All assays were performed on day 8. (B) The number of inflammatory cells in the BAL fluid was counted. The absolute cell numbers of eosinophils (Eos.), neutrophils (Neu.), lymphocytes (Lym.), and macrophages (Mac.) are shown with SEM. Five mice per group were used. **P < 0.01; *P < 0.05 (ANOVA and Bonferroni posttest). (C) The lungs were fixed and stained with hematoxylin and eosin (H&E) or with PAS. Representative staining patterns are shown. (Bars, 100 μm.) (D) Airway resistance (RL) was assessed as described in SI Materials and Methods. Mean values (five mice per group) are shown with SEM. **P < 0.01; *P < 0.05 (ANOVA and Bonferroni posttest). All data are representative of two or more independent experiments.

Discussion

We herein describe a mechanism whereby Gata3 is able to mediate both positive and negative effects on gene expression in Th2 cells. Chd4 was found to be a central component of two functionally distinct Gata3 complexes. The activating Gata3/Chd4 complex recruits p300 and binds to specific locus control regions in the Th2 cytokine gene loci, whereas the repressive Gata3/Chd4 complex interacts with Hdac2 and forms a Gata3/Chd4–NuRD complex at the Tbx21 locus; the former induces Th2 cytokine gene activation, and the latter represses the Tbx21 gene, thus suppressing Th1 differentiation and IFN-γ expression (Fig. S6). Because Chd4 binding to the target genes is abolished in the absence of Gata3 (Figs. 2B and 3E), Chd4-mediated positive and negative regulation is dependent on Chd4–Gata3 interaction. These two functionally distinct complexes exist in the same developing Th2 cells and coordinately establish Th2 cell identity.

Although a requirement for Gata3-mediated chromatin remodeling at the Th2 cytokine gene loci during Th2 cell differentiation has been well established (26), the molecular mechanisms underlying Gata3-mediated chromatin remodeling remain unknown. Discovery of the Chd4/Gata3 interaction and the critical role of Chd4 in the initial step of Gata3-mediated chromatin remodeling, including H3-K9 hyperacetylation of the Th2 cytokine gene loci, provides key mechanistic insight into this process (Fig. 2). A similar mode of positive regulation by the Gata3/Chd4/p300 complex was shown for Ccr8 providing evidence of the generalized nature of this process (Fig. S5). It is important to note, however, that two thirds of the identified Gata3 target genes (1,040 out of 1,651 genes) were not bound by Chd4 in Th2 cells (Fig. S3A), and thus the expression of these Gata3 target genes is likely controlled by Chd4-independent mechanisms.

Previously identified Gata3-regulators such as repressor of GATA (Rog), Fog, spleen focus forming virus proviral integration oncogene spi1 (PU.1), T-bet, lymphoid enhancer factor 1 (Lef1), Eomesodermin, and Sox4 appear to primarily regulate Gata3 through inhibition of its DNA binding (27–33). However, in this study we show that the Gata3/Chd4–NuRD complex mediated direct repression of transcription through chromatin modulation of the Gata3 target gene regulatory regions (Fig. 3).

Our data demonstrating activation or repression of transcription by functionally distinct Gata3/Chd4 complexes pose a question of binding-site selectively. Given that, we found a palindromic GATA binding site with the GATC sequence (underlined) in the Tbx21 G3/C4BS (TATCACGATC) but not in the CGRE region. Thus, the differences in the primary DNA sequence of each Gata3 target site may contribute to the selection of the locus for Gata3/Chd4–NuRD complex-mediated repression. Further contribution to selectivity may come from recognition of histone modification by the two plant homeodomain (PHD) fingers and two chromodomains of Chd4. The Chd4 N-terminal PHD finger preferentially binds unmodified Lys4 on histone H3, and the C-terminal PHD finger specifically recognizes unmodified Lys4 and trimethylated Lys-9 (34). This bivalent recognition of nucleosomes by the two PHD fingers of Chd4 is required for Chd4-mediated transcriptional repression (35). Therefore, it is possible that the Gata3/Chd4–NuRD complex may be able to recognize differences in the epigenetic code at target regions.

Finally, we have demonstrated the importance of Chd4-mediated regulation of Th2 cell function in vivo. Allergic eosinophilic inflammation and airway hyperresponsiveness were significantly suppressed in mice transferred with Chd4 KD Th2 cells (Fig. 4). In our experimental system, any alterations in inflammation observed originate from defects in the transferred Th2 cells, and consequently as a result of the loss of Chd4 function. It is therefore likely that Chd4 controls asthmatic inflammation through the activation of Th2 cytokines and repression of IFN-γ expression in Th2 cells. Further detailed biochemical studies on the Gata3/Chd4/p300 and Gata3/Chd4–NuRD complexes may lead to the discovery of novel therapeutic targets for the treatment of allergic asthma.

In summary, we have herein demonstrated that Gata3 concomitantly forms the Gata3/Chd4/p300 transcriptional activation complex at the Th2 cytokine gene loci and the Gata3/Chd4–NuRD transcriptional repression complex at the Tbx21 locus. Functioning cooperatively, these complexes orchestrate proper gene expression during Th2 cell differentiation by repressing subsets of genes while facilitating transcription at other loci.

Materials and Methods

Mice.

All mice were maintained under specific pathogen-free conditions and were used at 6–8 wk of age. All animal experiments were approved by the Chiba University Review Board for Animal Care.

Identification of Gata3 Complexes in the Th2 Cell Clone D10G4.1.

Extracts from Flag-Gata3-expressing D10G4.1 cells were subjected to tandem affinity purification using anti-Flag and -Gata3 mAbs, followed by LC-MS/MS analysis as described in SI Materials and Methods.

Detailed descriptions of all materials and methods are provided in SI Materials and Methods.