Critical YxKxHxxxRP Motif in the C-Terminal Region of GATA3 for Its DNA Binding and Function

Mice

C57BL/6 mice were purchased from CLEA Japan. All mice used in this study were maintained under specific-pathogen-free conditions. The animal care was conducted in accordance with the guidelines of Chiba University (Chiba, Japan).

Expression plasmids and transfection

Myc-tagged GATA3 mutants (pCMV Tag 3B-GATA3) were generated by PCR-based mutation. Human embryonic kidney 293 (HEK 293) T cells were transfected using FuGENE reagent (Roche) according to the manufacturer’s protocol.

Cell cultures and in vitro Th1/Th2 cell differentiation

CD4 T cells were purified using magnetic beads and an AutoMACS sorter (Miltenyi Biotec) that yielded purity of >98%. The purified CD4 T cells (1.5 × 10⁶) were stimulated for 2 days with an immobilized anti-TCR mAb (H57-597; 3 μg/ml) in the presence of IL-2 (25 U/ml), IL-12 (100 U/ml), and anti-IL-4 mAb (11B11; 25% culture supernatant) for Th1 conditions. The cells were cultured for another 3 days in the presence of cytokines present in the initial culture. The number of Th1/Th2 cells was determined using intracellular staining with anti-IL-4 and anti-IFN-γ (43, 44). The production of cytokines was determined by ELISA as described (45).

Retroviral vectors and infection

The pMX-IRES-hNGFR plasmid was generated from the pMX-IRES-GFP plasmid by replacing the enhanced GFP with the cytoplasmic region-deleted human nerve growth factor receptor p75 (hNGFR) cDNA. The method for the generation of virus supernatant and the infection was described previously (45). Infected cells were subjected to intracellular staining with anti-IL-4 and anti-IFN-γ, a chromatin immunoprecipitation (ChIP) assay, or cell sorting. To prepare a large number of infected cells for ELISA, the infected cells were enriched by MACS with anti-hNGFR (clone C40-1457; BD Pharmingen).

ChIP assay

ChIP was performed using the histone H3 assay kit (catalog no.17-245: Upstate Biotechnology) as described previously (28). Semiquantitative PCR was performed with DNA samples from 3 or 1 × 10⁴ cells at 28 cycles. The PCR products were resolved in an agarose gel and then visualized by ethidium bromide. The images were recorded and quantified using the Atto Lane and Spot analyzer (Atto Bioscience). The primers used have been described previously (28).

Luciferase reporter assay

A single copy of an IL-5 promoter (−1200 bp) and an IL-13 promoter (−254 bp) in the luciferase reporter plasmid pGL3 Basic (Promega) was used. M12 cells (2.5 × 10⁶; B cell line) were used for transfection by electroporation. In addition, 5 ng of a Renilla luciferase reporter vector with HSV thymidine kinase promoter (pRL-TK; Promega) was added into each transfection as an internal control for transfection efficiency as described (28). The transfected cells were stimulated with PMA (30 ng/ml) plus dibutyryl cAMP (1 mM) for the IL-5 reporter assay and PMA (30 ng/ml) plus ionomycin (500 nM) for the IL-13 reporter assay. The cell extracts were prepared and subjected to a luciferase assay using the manufacturer’s instructions for Dual luciferase reporter (Promega).

EMSA

EMSAs were performed using a gel shift assay system (Promega) as described previously (44). In brief, the nuclear extracts were incubated at 4°C with a ³²P-labeled, double-stranded oligonucleotide in DNA-binding buffer. Electrophoresis was conducted on 4% native polyacrylamide gel (acrylamide/bisacrylamide ratio 29:10 in 0.5× Tris-borate-EDTA), and the radioactivity was visualized by autoradiography. The oligonucleotides used in this experiment are as follows: GATA consensus, 5′-CACTTGATAACAGAAAGTGATAACTCT-3′; GATA mutant, 5′-CACTCTTAACAGAAAGTCTTAACTCT-3′; CNS1, 5′-CGAGAAATGAATGAAGATAATGAGGCCTC-3′; V_A enhancer, 5′-ATCAACAGATAACTAGATAAAGAATAT-3′; CGRE (M5), 5′-ACTGGCGCGGCGATGGCCCGCGGAGATAGAGGCGCGGCTTTTTTTTACGGGAGATGGGGTCGATAAGA-3′; IL-5 promoter, 5′-TGCTAACAATCAGATAGAGG-3′; and IL-13 promoter, 5-′ TTCAAGATGAGTAAAGATGTGGTTTTC-3′. Supershift analysis was performed using anti-Myc tag Ab (clone PL14; MBL).

The C-terminal region of GATA3 is required for Th2 cell differentiation

The aim of this study was to identify the roles of the C-terminal region of GATA3 in Th2 cell differentiation. The wild-type and truncated GATA3 mutant genes (Fig. 1⇓A) were introduced into CD4 T cells cultured under Th1 conditions (developing Th1 cells) using a retrovirus vector system, and IFN-γ/IL-4 production profiles of infected hNGFR-positive cells were determined using an intracellular staining method. Wild-type GATA3 (aa 1–444), dCT-1 (aa 1–407), dCT-2 (aa 1–380), and dCT-3 (aa 1–371) truncated GATA3 mutants succeeded in generating IL-4-producing Th2 cells, whereas dCT-4 (aa 1–345), dCT-5 (aa 1–315), and dCT-6 (aa 1–262) mutants failed to induce IL-4-producing cells (Fig. 1⇓B). Little effect on the inhibition of the generation of IFN-γ-producing cells was observed in dCT-4, dCT-5, or dCT-6 mutants. These results indicate that the C-terminal region of GATA3, particularly the region of aa 346–371, is essential for the induction of Th2 cell differentiation.

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FIGURE 1.

The C-terminal region of GATA3 is required for the GATA3 function. A, Schematic representation of Myc-tagged GATA3 mutants. Wild-type GATA3 (WT) and six mutants (dCT-1, dCT-2, dCT-3, dCT-4, dCT-5, and dCT-6) are shown with location of the Myc tag, transactivation domains (TA1 and TA2), and the N-terminal and C-terminal zinc finger domains (N and C). B, Induction of IL-4-producing Th2 cells under Th1 conditions by GATA3 mutants. Freshly prepared splenic CD4 T cells were stimulated under Th1 culture conditions and infected with a retrovirus encoding wild-type or mutant GATA3 bicistronically with hNGFR on day 2. Control represents infection of a mock vector virus. Three days after infection, cells were stimulated with anti-TCR and subjected to IL-4/IFN-γ staining. The numbers represent the percentages of cells in each quadrant. Three independent experiments were performed with similar results. C, Reporter assays with the IL-5 promoter and the IL-13 promoter were performed using an M12 cell line that expresses no GATA3 family protein. Wild-type GATA3 or mutant dCT-4 cDNA was introduced, and the luciferase activity was measured. The mean values with SD of relative luciferase activity of four different experiments are shown. D, Induction of histone H3 hyperacetylation at the Th2 cytokine gene loci in developing Th1 cells. Wild-type or mutant GATA3 cDNA was introduced by a retrovirus-mediated gene transfer system as described in B. Three days after infection, hNGFR-positive infected CD4 T cells were enriched by cell sorting. The control represents an infection with a mock vector virus. The acetylation status of histone H3 (K9/14) was determined by a ChIP assay. Three independent experiments were performed with similar results. The relative band intensities normalized with input DNA bands are shown in the lower panel. V_A enh., V_A enhancer.

We next assessed whether the GATA3 dCT-4 mutant showed a transcriptional activity for the IL-5 gene and the IL-13 gene. The introduction of wild-type GATA3 into an M12 B cell line resulted in the induction of reporter activity of the IL-5 promoter and the IL-13 promoter, whereas that of the dCT-4 mutant failed (Fig. 1⇑C). Our previous study revealed that the ectopic expression of GATA3 in developing Th1 cells induced histone hyperacetylation (H3-K9) at the Th2 cytokine gene loci (28). Consequently, the wild type and the dCT-4 mutant were introduced into developing Th1 cells and a ChIP assay was performed using anti-acetyl histone H3 (K9/14) Ab. Wild-type GATA3 but not the dCT-4 mutant induced histone hyperacetylation at the Th2 cytokine gene loci (CGRE, CNS1, V_A enhancer, IL-4p, IL-13p, and IL-5p) (Fig. 1⇑D). The acetylation of the RAD50 promoter was not affected by the introduction of either GATA3 molecule. These results indicate that the dCT-4 mutant failed to show either any transcriptional activity on the IL-5 promoter and the IL-13 promoter or the ability to induce chromatin remodeling of the Th2 cytokine gene loci.

Conserved NRPL residues, which are located in the downstream region of the C-terminal zinc finger, are required for GATA3 function

Because the C-terminal region (aa 346–371) of GATA3 appeared to be critical for GATA3 function, we searched conserved amino acid sequences among the GATA family members. As shown in Table I⇓, NRPL residues (aa 352–355) are conserved in human and mouse GATA1, GATA2, and GATA3. A similar conserved (N/S)RPL sequence was found downstream from the N-terminal zinc finger. The core RP residues are conserved in all GATA molecules in mice and humans and also in the nonvertebrate GATA family proteins including Drosophila melanogaster GATA (dGATA-b), Caenorhabditis elegans GATA (END-1), Arabidopsis thaliana GATA (AtGATA-1), Saccharomyces cerevisiae GATA (GZF3), and Aspergillus nidulans GATA (areA).

Consequently, we addressed the role of NRPL residues (aa 352–355) adjacent to the C-terminal zinc finger domain of GATA3 (C-NRPL) in Th2 cell differentiation, Th2 cytokine production, and chromatin remodeling of the Th2 cytokine gene. Seven amino acid residues from 349 to 355 are missing in the d349/355 mutant, and the C-NRPL residues are substituted for alanine in the C-NRPL/AAAA mutant (Fig. 2⇓A). Wild-type GATA3 and these mutants were introduced into developing Th1 cells to assess their ability to generate IL-4-producing Th2 cells. As we expected, both mutants failed to induce IL-4-producing cells in the culture (Fig. 2⇓B). The inhibitory effect of the generation of IFN-γ producing cells also decreased in these two mutants. The production of IL-4, IL-5, and IL-13 was not induced by the introduction of d349/355 or C-NRPL/AAAA (Fig. 2⇓C). The inhibitory effect on IFN-γ production was also impaired. These mutants failed to induce transcriptional activity for the IL-5 promoter (Fig. 2⇓D) and histone hyperacetylation at the Th2 cytokine gene loci (Fig. 2⇓E). The levels of histone hyperacetylation at the IFN-γ promoter decreased with wild-type GATA3 introduction, but the decrease was marginal in these two mutants. The acetylation of the RAD50 promoter was not affected by the introduction of wild-type or mutant GATA3 molecules. These results indicate that C-NRPL is indispensable for the GATA3 function.

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FIGURE 2.

Role of NRPL sequence downstream adjacent to C-terminal zinc finger in GATA3 function. A, Schematic representation of the Myc-tagged GATA3 mutants. Wild-type GATA3 (WT) and two mutants (d349/355 and C-NRPL/AAAA) are shown. B, Induction of IL-4-producing Th2 cells under Th1-conditions with two NRPL mutants. The experiments were performed as described in Fig. 1⇑B. Three independent experiments were performed, and we obtained similar results. C, Two GATA3 NRPL mutants failed to induce Th2 cytokine production. hNGFR-positive infected CD4 T cells were enriched by magnetic cell sorting and stimulated (Stim.) with immobilized anti-TCR mAb for 24 h. The cytokine concentration in culture supernatants was determined by ELISA. D, Two GATA3 NRPL mutants failed to transactivate the IL-5 promoter. A luciferase assay was performed as described in Fig. 1⇑C. The mean values with SD of relative luciferase activity from four different experiments are shown. Med, Medium; Stim, stimulation. E, GATA3 NRPL mutants failed to induce of histone H3 hyperacetylation at the Th2 cytokine gene loci in developing Th1 cells. ChIP assay was performed as in Fig. 1⇑D. Three independent experiments were performed with similar results. The relative band intensities normalized by input DNA bands are shown in the lower panel. V_A enh., V_A enhancer.

The core R353/P354 residues play a crucial in the GATA3 function

We next introduced a single amino acid substitution to alanine within the C-NRPL residues of GATA3. Each mutant (C-ARPL, C-NAPL, C-NRAL, and C-NRPA) was introduced into developing Th1 cells, and the generation of IL-4-producing Th2 cells was determined. As shown in Fig. 3⇓A, the generation of IL-4-producing cells decreased in C-NAPL and C-NRAL mutants but not substantially in C-ARPL or C-NRPA mutants (Fig. 3⇓A). Similar results were obtained by ELISA (Fig. 3⇓B). Substantial increases in the transcriptional activity on the IL-5 promoter and the IL-13 promoter were detected in C-ARPL, C-NRPA, and C-ARPA mutants but not in the C-NAPL, C-NRAL, or C-NAAL mutants (Fig. 3⇓C). These results indicate that R353/P354 residues are essential for GATA3 function.

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FIGURE 3.

R353 and P354 residues are essential for the GATA3 function. A, A single alanine substitution at the R353 or P354 residue of GATA3 abolished the induction of IL-4-producing cells. Wild-type (WT) and mutant GATA3 genes were introduced as shown in Fig. 1⇑. Three independent experiments were performed and similar results were obtained. B, R353A (C-NAPL) and P354A (C-NRAL) mutants failed to induce Th2 cytokine production. Experiments were performed as described in Fig. 2⇑C. C, R353 and P354 residues of GATA3 are essential for GATA3-dependent transactivation of the IL-5 promoter and the IL-13 promoter. M12 cells were transfected with wild-type or GATA3 mutant cDNA, and a luciferase assay was performed. The mean values with a SD of the relative luciferase activity of four different experiments are shown. Med, Medium; Stim., stimulated.

DNA binding activity was impaired in the C-NAPL and C-NRAL mutants

We next examined the DNA binding activity of each GATA3 mutant (C-ARPL, C-NAPL, C-NRAL, and C-NRPA) using EMSA (Fig. 4⇓). Six different GATA3 binding sequences were used: a GATA3 consensus sequence, a binding sequence existing in CNS1, and sequences from the V_A enhancer, CGRE, the IL-5 promoter, and the IL-13 promoter. HEK 293 T cells were transfected with Myc-tagged GATA3 mutant genes, and nuclear protein extracts were prepared. Immunoblotting analysis with anti-Myc tag mAb confirmed the equivalent protein expression of GATA3 mutants (see Fig. 4⇓A, Input). The subcellular localization was not found to change in these GATA3 mutants (data not shown). As shown in Fig. 4⇓, the binding activity was abrogated almost completely by a single alanine mutation at R353 (C-NAPL). The binding of C-NRAL was also substantially reduced in some GATA binding sequences, including a GATA consensus binding site (Fig. 4⇓A), CNS1 (Fig. 4⇓B), IL-5 promoter (Fig. 4⇓E), and IL-13 promoter (Fig. 4⇓F). These results indicate that the R353 residue plays a critical role in the DNA binding activity of GATA3 and that the P354 residue is important for DNA binding activity.

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FIGURE 4.

R353 residue is essential for the DNA-binding activity of GATA3. Myc-tagged wild-type (WT) and mutant GATA3 molecules were expressed in HEK 293 T cells. Nuclear extracts of the transfected cells were prepared and subjected to an EMSA with the following probes: a GATA consensus binding site (A), a GATA binding site within CNS1 (B), V_A enhancer (C), CGRE (D), an IL-5 promoter (E) or an IL-13 promoter (F), and a control mutant oligonucleotide sequence for the GATA consensus binding site (G). The expression levels of GATA3 protein in the nuclear extract were examined by immunoblotting with anti-Myc tag mAb (Input) as a loading control. A supershift assay was performed with a control mouse IgG and an anti-Myc tag Ab. Three independent experiments were performed with similar results. 

DNA binding activity and the generation of Th2 cells in a GATA3 R353K mutant (C-NKPL)

To address the role of positive charge for arginine (R353), we generated a GATA3 R353K mutant (C-NKPL). The C-NKPL mutant did not show the ability to generate IL-4 producing Th2 cells (data not shown). An EMSA revealed that the levels of DNA binding activity for C-NKPL were similar to those of C-NAPL (data not shown). These results suggest that the positive charge of R353 itself is not sufficient for either GATA3 to bind to DNA or for the generation of Th2 cells.

Y345 and H349 are critical for GATA3 functions

In addition to C-NRPL, Y345, K347, and H349 adjacent to the C-terminal zinc finger domain of GATA3 are conserved among all mouse and human GATA family members (Table I⇑). Consequently, we generated various single alanine mutants (Y345A, Y346A, L348A, and H349A) and tested their ability to induce IL-4-producing Th2 cells. The generation of IL-4-producing cells dramatically decreased in Y345A and H349A mutants but not in Y346A or L348A mutants (Fig. 5⇓B). Similarly as for the R353A mutant, the DNA binding activity for Y345A and H349A mutants was found to be marginal (Fig. 5⇓C). The K347A mutant also showed also moderately decreased Th2 cell differentiation (data not shown). These results indicate that Y345, K347, and H349 each play a critical role in enabling GATA3 to bind to DNA and thus induce Th2 cell generation.

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FIGURE 5.

Y345 and H349 are required for the generation of Th2 cells. A, Schematic representation of the amino acid alignment around Y345 and H349. The amino acid residues substituted to alanine are indicated as gray characters. B, Y345 and H349 residues are required for the induction of IL-4-producing Th2 cells. Wild-type (WT) and mutant GATA3 genes (Y345A, Y346A, L348A, H349A, R353A, and R354A) were introduced as shown in Fig. 1⇑B. Four independent experiments were performed with similar results. C, wild-type and mutant GATA3 genes were expressed in HEK 293 T cells. An EMSA was performed with indicated probes as described in Fig. 4⇑. Three independent experiments were performed with similar results.

Role of the NRPL residues adjacent to the N-terminal zinc finger domain in GATA3 function

Finally, the role of NRPL residues adjacent to the N-terminal zinc finger domain of GATA3 was assessed using a GATA3 mutant with an alanine substitution (N-NRPL/AAAA). The ability to induce IL-4 producing Th2 cells was well preserved in the N-NRPL/AAAA mutant (Fig. 6⇓A). The production of IL-4 was lower than that of wild type but significantly higher than that of C-NRPL/AAAA (Fig. 6⇓B). In contrast, the production of IL-5 and IL-13 was not significantly induced with the N-NRPL/AAAA mutant. The transcription activity on the IL-5 promoter was not detected, and that on the IL-13 promoter was moderately induced (Fig. 6⇓C). Regarding the induction of histone modifications at the Th2 cytokine gene loci, no obvious defect was observed in the N-NRPL/AAAA mutant in H3-K9 acetylation (Fig. 6⇓D) or H3-K4 dimethylation (data not shown). An EMSA revealed that the levels of DNA binding to various GATA motifs for N-NRPL/AAAA were significantly lower than that of the wild type but obviously higher than that of the C-NRPL/AAAA mutant (data not shown). Therefore, although the impact of N-NRPL on GATA3 function was not as dramatic as that of C-NRPL, the N-NRPL residues of GATA3 also appear to play important roles in some GATA3 functions such as the transcription and production of IL-5 and IL-13.

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FIGURE 6.

Role of N-NRPL motif in GATA3 function. The experiments were performed as shown in Fig. 2⇑. A, The induction of IL-4-producing cells was not affected by the alanine substitution of N-NRPL (N-NRPL/AAAA). Three independent experiments were performed and similar results were obtained. B, GATA3 N-NRPL/AAAA mutant failed to induce IL-5 and IL-13 production. Three independent experiments were performed and similar results were obtained. C, the GATA3 N-NRPL/AAAA mutant failed to induce the transactivation of the IL-5 and IL-13 promoters. The mean values with SD of relative luciferase activity from four different experiments are shown. Med, Medium. D, The normal induction of histone H3 hyperacetylation in the Th2 cytokine gene loci by the N-NRPL/AAAA mutant. Three independent experiments were done with similar results. Relative band intensities normalized with input DNA bands are shown in the lower panel. WT, wild type; V_A enh., V_A enhancer.