The Journal of Immunology J Immunol: first published as 10.4049/jimmunol.177.9.5801 on 18 October 2006. Downloaded from

Critical YxKxHxxxRP Motif in the C-Terminal Region of GATA3 for Its DNA Binding and Function1

Ryo Shinnakasu,* Masakatsu Yamashita,* Kenta Shinoda,* Yusuke Endo,* Hiroyuki Hosokawa,* Akihiro Hasegawa,* Shinji Ikemizu,† and Toshinori Nakayama2*

A zinc finger , GATA3, plays an essential role in the development of T cells and the functional differentiation into type 2 Th cells. Two transactivation domains and two zinc finger regions are known to be important for the GATA3 function, whereas the role for other regions remains unclear. In this study we demonstrated that a conserved YxKxHxxxRP motif (aa 345–354) adjacent to the C-terminal zinc finger domain of GATA3 plays a critical in its DNA binding and functions, including transcriptional activity, the ability to induce of the Th2 cytokine loci, and Th2 cell differentiation. A single point of the key (Y, K, H, R, and P) in the motif abrogated GATA3 functions. A computer simulation analysis based on the solution structure of the chicken GATA1/DNA complex supported the importance of this motif in GATA3 DNA binding. Thus, we identified a novel conserved YxKxHxxxRP motif adjacent to the C-terminal zinc finger domain of GATA3 that is indispensable for GATA3 DNA binding and functions. The Journal of Immunology, 2006, 177: 5801–5810.

fter antigenic stimulation, naive CD4 T cells differenti- GATA3 binding motifs are identified in the promoters of the ate into two distinct helper T cell subsets, Th1 and Th2 IL-5 and IL-13 (22–24), and GATA3 acts as a transcriptional A cells (1). Th1 cells produce IFN-␥ to control cell-medi- factor for IL-5 and IL-13 genes (25–27). In addition to the pro- ated immunity against intracellular pathogens, whereas Th2 cells moter regions, GATA3 also binds to various regulatory regions for produce IL-4, IL-5, and IL-13 and are involved in humoral immu- Th2 cytokine expression, including the conserved GATA3 re- nity and allergic reactions (2–4). IL-4-induced STAT6 activation 3 Ј sponse element (CGRE) (28), the 3 site of IL-4 (VA) (29), the is crucial for Th2 cell differentiation (5–7). Several transcription IL-4-IL-13 intergenic region (CNS1) (30), and the 3Ј end of factors that govern Th1/Th2 cell differentiation have been re- RAD50 gene (31). ported. Among them, GATA3 appears to be a key factor for Th2 Changes in the chromatin structure of the Th1 cytokine (IFN-␥) cell differentiation (8, 9), as is T-bet for Th1 differentiation (10). and the Th2 cytokine (IL-5/IL-4/IL-13) gene loci occur during GATA3 is abundantly expressed in T and the em- Th1/Th2 cell differentiation (32–34). The histone modifications of bryonic brain (11, 12). GATA3 is expressed throughout thymocyte the Th2 cytokine gene loci is primarily mediated through GATA3 development (13) and its expression is required for the develop- (28, 35, 36). The binding of GATA3 at the CGRE region appears http://www.jimmunol.org/ ment of T cells in the (14, 15) and for ␤ selection and single positive CD4 thymocyte development (16). The activation to initiate the long-range histone hyperacetylation accompanied by of STAT6 induces GATA3 transcription, and the ectopic expres- intergenic transcription within the IL-13/IL-4 gene loci in devel- sion of GATA3 results in Th2 cell differentiation even in the ab- oping Th2 and Tc2 cells (28, 35). sence of STAT6 (17, 18). The autoactivation of GATA3 transcrip- GATA3 belongs to a family of zinc finger transcription factors. tion was also reported (19, 20). In addition to the transcriptional The six mammalian GATA (GATA1 to GATA6) share regulation, the expression of GATA3 is also regulated by a post- related Cys-X2-Cys-X17-Cys-X2-Cys (where X represents any transcriptional mechanism. We recently reported that the Ras-ERK amino acid residue) and bind to the consensus motif 5Ј-(A/ MAPK cascade controls GATA3 stability through the ubiquitin/ T)GATA(A/G)-3Ј (37). GATA3 possesses N-terminal transactiva- on October 1, 2021 by guest. Protected copyright. proteasome-dependent pathway (21). tion domains and two zinc fingers, namely the N-terminal zinc finger and the C-terminal zinc finger. The C-terminal zinc finger is essential for DNA binding, whereas the N-terminal zinc finger *Department of Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan; and †Division of Structural Biology, Graduate School of Pharmaceutical Sci- stabilizes this binding and physically interacts with other zinc fin- ences, Kumamoto University, Kumamoto, Japan ger proteins such as the Friends of GATA (FOG) (38–41). The Received for publication June 9, 2006. Accepted for publication August 9, 2006. N-terminal zinc finger and C-terminal zinc finger thus play differ- The costs of publication of this article were defrayed in part by the payment of page ent roles in the induction of IL-4, IL-13, and IL-5 (42). charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The C-terminal region of GATA3 is highly conserved among 1 This work was supported by grants from the Ministry of Education, Culture, Sports, the GATA family proteins, whereas the role of the C-terminal Science and Technology of Japan (Grants-in-Aid for Scientific Research in Priority region has not been addressed. We herein identified a unique Areas 17016010 and 17047007; Scientific Research B 17390139, Scientific Research amino acid motif (YxKxHxxxRP) adjacent to C-terminal zinc fin- C 18590466; Grant-in-Aid for Young Scientists 17790318; and Special Coordination Funds for Promoting Science and Technology), the Ministry of Health, Labor and ger domain of GATA3. The YxKxHxxxRP motif is revealed to be Welfare (Japan), the Program for Promotion of Fundamental Studies in Health Sci- ences of the National Institute of Biomedical Immovation (Japan), The Japan Health Science Foundation, Kanae Foundation, Uehara Memorial Foundation, and Mochida Foundation. 2 Address correspondence and reprint requests to Dr. Toshinori Nakayama, Depart- 3 Abbreviations used in this paper: CGRE, conserved GATA3 response element; ment of Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, ChIP, chromatin immunoprecipitation; HEK 293, human embryonic kidney 293; Chuo-ku, Chiba 260-8670 Japan. E-mail address: [email protected] hNGFR, human nerve growth factor p75.

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 5802 CRITICAL MOTIF FOR GATA3 DNA BINDING AND FUNCTION J Immunol: first published as 10.4049/jimmunol.177.9.5801 on 18 October 2006. Downloaded from

FIGURE 1. The C-terminal region of GATA3 is required for the GATA3 function. A, Schematic representation of -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 http://www.jimmunol.org/ 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-␥ 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 and the IL-13 promoter were performed using an M12 cell line that expresses no GATA3 family . 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 on October 1, 2021 by guest. Protected copyright. relative band intensities normalized with input DNA bands are shown in the lower panel.VA enh., VA enhancer.

important for GATA3 DNA binding and GATA3 functions, in- Cell cultures and in vitro Th1/Th2 cell differentiation cluding the transcriptional activity and the ability to induce chro- CD4 T cells were purified using magnetic beads and an AutoMACS sorter matin remodeling of the Th2 cytokine gene loci leading to Th2 cell (Miltenyi Biotec) that yielded purity of Ͼ98%. The purified CD4 T cells differentiation. (1.5 ϫ 106) were stimulated for 2 days with an immobilized anti-TCR mAb (H57-597; 3 ␮g/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. Materials and Methods The cells were cultured for another 3 days in the presence of cytokines Mice present in the initial culture. The number of Th1/Th2 cells was determined ␥ C57BL/6 mice were purchased from CLEA Japan. All mice used in this using intracellular staining with anti-IL-4 and anti-IFN- (43, 44). The study were maintained under specific-pathogen-free conditions. The animal production of cytokines was determined by ELISA as described (45). care was conducted in accordance with the guidelines of Chiba University (Chiba, Japan). Retroviral vectors and infection Expression plasmids and transfection The pMX-IRES-hNGFR plasmid was generated from the pMX-IRES-GFP plasmid by replacing the enhanced GFP with the cytoplasmic region-de- Myc-tagged GATA3 mutants (pCMV Tag 3B-GATA3) were generated by leted human nerve growth factor receptor p75 (hNGFR) cDNA. The PCR-based mutation. Human embryonic kidney 293 (HEK 293) T cells method for the generation of virus supernatant and the infection was de- were transfected using FuGENE reagent (Roche) according to the manu- scribed previously (45). Infected cells were subjected to intracellular stain- facturer’s protocol. ing with anti-IL-4 and anti-IFN-␥, a chromatin immunoprecipitation The Journal of Immunology 5803 J Immunol: first published as 10.4049/jimmunol.177.9.5801 on 18 October 2006. Downloaded from

Table I. Conserved amino acid sequences in the downstream region of N-terminal and C-terminal zinc fingers among GATA family membersa

GATA Family Species Amino Acid Sequence

GATA3 CF Human 345YYKLH---NINRP LTMKKEGIQTRNRKMSS371 Mouse 344YYKLH---NINRP LTMKKEGIQTRNRKMSS370 GATA3 NF Human 291YHKMN---GQNRP LIKPKRRLSAARRAGTS317 Mouse 290YHKMN---GQNRP LIKPKRRLSAARRAGTS316 GATA1 CF Human 285YYKLH---QVNRP LTMRKDGIQTRNRKASG311 Mouse 285YFKLH---QVNRP LTMRKDGIQTRNRKASG311 GATA1 NF Human 231YHKMN---GQNRP LIRPKKRLIVSKRAGTQ257 Mouse 231YHKMN---GQNRP LIRPKKRMIVSKRAGTQ257 GATA2 CF Human 376YYKLH---NVNRP LTMKKEGIQTRNRKMSN402 Mouse 376YYKLH---NVNRP LTMKKEGIQTRNRKMSS402 GATA2 NF Human 322YHKMN---GQNRP LIKPKRRLSAARRAGTC348 Mouse 322YHKMN---GQNRP LIKPKRRLSAARRAGTC348 GATA4 CF Human 298YMKLH---GVPRP LAMRKEGIQTRKRKPKN324 Mouse 297YMKLH---GVPRP LAMRKEGIQTRKRKPKN323 GATA4 NF Human 244YHKMN---GINRP LIKPQRRLSASRRVGLS270 Mouse 243YHKMN---GINRP LIKPQRRLSASRRVGLS269 GATA5 CF Human 270YMKLH---GVPRP LAMKKESIQTRKRKPKT296 Mouse 277YMKLH---GVPRP LAMKKESIQTRKRKPEN303 GATA5 NF Human 216YHKMN---GVNRP LVRPQKRLSSSRRAGLC242 Mouse 223YHKMN---GVNRP LVRPQKRLSSSRRSGLC249 GATA6 CF Human 471YMKLH---GVPRP LAMKKEGIQTRKRKPKN497 Mouse 465YMKLH---GVPRP LAMKKEGIQTRKRKPKN491 GATA6 NF Human 417YSKMN---GLSRP LIKPQKRVPSSRRLGLS443 Mouse 411YSKMN---GLSRP LIKPQKRVPSSRRLGLS437 dGATA-b D. melanogaster 346YYKLH---SVPRP LTMKKDTIQKRKRKPKG372 END-1 C. elegans 167YFRKN---GIQRP AELCRKTIMKRNRRPRA193 AtGATA-1 A. thaliana 224RYKSGRLVPEYRP ANSPTFTAELHSNSHRK250 GZF3 S. cerevisiae 158FLKLH---GKPRP ISLKTDVIKSRNRKSNT184 areA A. nidulans 700FLKLH---GVVRP LSLKTDVIKKRNRNSAN726

a Highly conserved amino acids are highlighted in boldface. NF, N-terminal zinc finger; CF, C-terminal zinc finger.

(ChIP) assay, or cell sorting. To prepare a large number of infected cells for AGATAGAGGCGCGGCTTTTTTTTACGGGAGATGGGGTCGATAA ELISA, the infected cells were enriched by MACS with anti-hNGFR GA-3Ј; IL-5 promoter, 5Ј-TGCTAACAATCAGATAGAGG-3Ј; and IL-13 ( C40-1457; BD Pharmingen). promoter, 5-Ј TTCAAGATGAGTAAAGATGTGGTTTTC-3Ј. Supershift analysis was performed using anti-Myc tag Ab (clone PL14; MBL). ChIP assay http://www.jimmunol.org/ ChIP was performed using the histone H3 assay kit (catalog no.17-245: Results Upstate Biotechnology) as described previously (28). Semiquantitative The C-terminal region of GATA3 is required for Th2 cell PCR was performed with DNA samples from 3 or 1 ϫ 104 cells at 28 differentiation cycles. The PCR products were resolved in an agarose gel and then visu- alized by ethidium bromide. The images were recorded and quantified us- The aim of this study was to identify the roles of the C-terminal ing the Atto Lane and Spot analyzer (Atto Bioscience). The primers used region of GATA3 in Th2 cell differentiation. The wild-type and have been described previously (28). truncated GATA3 mutant genes (Fig. 1A) were introduced into Luciferase reporter assay CD4 T cells cultured under Th1 conditions (developing Th1 cells) using a retrovirus vector system, and IFN-␥/IL-4 production pro- on October 1, 2021 by guest. Protected copyright. A single copy of an IL-5 promoter (Ϫ1200 bp) and an IL-13 promoter files of infected hNGFR-positive cells were determined using an (Ϫ254 bp) in the luciferase reporter plasmid pGL3 Basic (Promega) was used. M12 cells (2.5 ϫ 106; B cell line) were used for transfection by intracellular staining method. Wild-type GATA3 (aa 1–444), electroporation. In addition, 5 ng of a Renilla luciferase reporter vector dCT-1 (aa 1–407), dCT-2 (aa 1–380), and dCT-3 (aa 1–371) trun- with HSV thymidine kinase promoter (pRL-TK; Promega) was added into cated GATA3 mutants succeeded in generating IL-4-producing each transfection as an internal control for transfection efficiency as de- Th2 cells, whereas dCT-4 (aa 1–345), dCT-5 (aa 1–315), and scribed (28). The transfected cells were stimulated with PMA (30 ng/ml) dCT-6 (aa 1–262) mutants failed to induce IL-4-producing cells 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 (Fig. 1B). Little effect on the inhibition of the generation of IFN- extracts were prepared and subjected to a luciferase assay using the man- ␥-producing cells was observed in dCT-4, dCT-5, or dCT-6 mu- ufacturer’s instructions for Dual luciferase reporter (Promega). tants. These results indicate that the C-terminal region of GATA3, particularly the region of aa 346–371, is essential for the induction EMSA of Th2 cell differentiation. EMSAs were performed using a gel shift assay system (Promega) as de- We next assessed whether the GATA3 dCT-4 mutant showed a scribed previously (44). In brief, the nuclear extracts were incubated at 4°C transcriptional activity for the IL-5 gene and the IL-13 gene. The with a 32P-labeled, double-stranded oligonucleotide in DNA-binding buffer. Electrophoresis was conducted on 4% native polyacrylamide gel introduction of wild-type GATA3 into an M12 B cell line resulted (acrylamide/bisacrylamide ratio 29:10 in 0.5ϫ Tris-borate-EDTA), and the in the induction of reporter activity of the IL-5 promoter and the radioactivity was visualized by autoradiography. The oligonucleotides IL-13 promoter, whereas that of the dCT-4 mutant failed (Fig. 1C). used in this experiment are as follows: GATA consensus, 5Ј-CACTT Our previous study revealed that the ectopic expression of GATA3 GATAACAGAAAGTGATAACTCT-3Ј; GATA mutant, 5Ј-CACTCTTAA CAGAAAGTCTTAACTCT-3Ј; CNS1, 5Ј-CGAGAAATGAATGAAGAT in developing Th1 cells induced histone hyperacetylation (H3-K9) Ј Ј at the Th2 cytokine gene loci (28). Consequently, the wild type and AATGAGGCCTC-3 ;VA enhancer, 5 -ATCAACAGATAACTAGATAA AGAATAT-3Ј; CGRE (M5), 5Ј-ACTGGCGCGGCGATGGCCCGCGG the dCT-4 mutant were introduced into developing Th1 cells and 5804 CRITICAL MOTIF FOR GATA3 DNA BINDING AND FUNCTION J Immunol: first published as 10.4049/jimmunol.177.9.5801 on 18 October 2006. Downloaded from a ChIP assay was performed using anti-acetyl histone H3 (K9/14) S)RPL sequence was found downstream from the N-terminal zinc Ab. Wild-type GATA3 but not the dCT-4 mutant induced histone finger. The core RP residues are conserved in all GATA molecules hyperacetylation at the Th2 cytokine gene loci (CGRE, CNS1, VA in mice and humans and also in the nonvertebrate GATA family enhancer, IL-4p, IL-13p, and IL-5p) (Fig. 1D). The acetylation of proteins including Drosophila melanogaster GATA (dGATA-b), the RAD50 promoter was not affected by the introduction of either Caenorhabditis elegans GATA (END-1), Arabidopsis thaliana GATA3 molecule. These results indicate that the dCT-4 mutant GATA (AtGATA-1), Saccharomyces cerevisiae GATA (GZF3), failed to show either any transcriptional activity on the IL-5 pro- and Aspergillus nidulans GATA (areA). moter and the IL-13 promoter or the ability to induce chromatin Consequently, we addressed the role of NRPL residues (aa 352– remodeling of the Th2 cytokine gene loci. 355) adjacent to the C-terminal zinc finger domain of GATA3 (C-NRPL) in Th2 cell differentiation, Th2 cytokine production, Conserved NRPL residues, which are located in the downstream and chromatin remodeling of the Th2 cytokine gene. Seven amino region of the C-terminal zinc finger, are required for GATA3 acid residues from 349 to 355 are missing in the d349/355 mutant, function and the C-NRPL residues are substituted for alanine in the C- Because the C-terminal region (aa 346–371) of GATA3 appeared NRPL/AAAA mutant (Fig. 2A). Wild-type GATA3 and these mu- to be critical for GATA3 function, we searched conserved amino tants were introduced into developing Th1 cells to assess their acid sequences among the GATA family members. As shown in ability to generate IL-4-producing Th2 cells. As we expected, both Table I, NRPL residues (aa 352–355) are conserved in human and mutants failed to induce IL-4-producing cells in the culture (Fig. mouse GATA1, GATA2, and GATA3. A similar conserved (N/ 2B). The inhibitory effect of the generation of IFN-␥ producing http://www.jimmunol.org/ on October 1, 2021 by guest. Protected copyright.

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. 1B. 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. 1C. 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. 1D. Three independent experiments were performed with similar results. The relative band intensities normalized by input DNA bands are shown in the lower panel.VA enh., VA enhancer. The Journal of Immunology 5805 J Immunol: first published as 10.4049/jimmunol.177.9.5801 on 18 October 2006. Downloaded from cells also decreased in these two mutants. The production of IL-4, but not in the C-NAPL, C-NRAL, or C-NAAL mutants (Fig. 3C). IL-5, and IL-13 was not induced by the introduction of d349/355 These results indicate that R353/P354 residues are essential for or C-NRPL/AAAA (Fig. 2C). The inhibitory effect on IFN-␥ pro- GATA3 function. duction was also impaired. These mutants failed to induce tran- scriptional activity for the IL-5 promoter (Fig. 2D) and histone hyperacetylation at the Th2 cytokine gene loci (Fig. 2E). The lev- DNA binding activity was impaired in the C-NAPL and C-NRAL els of histone hyperacetylation at the IFN-␥ promoter decreased mutants with wild-type GATA3 introduction, but the decrease was mar- We next examined the DNA binding activity of each GATA3 mutant ginal in these two mutants. The acetylation of the RAD50 pro- (C-ARPL, C-NAPL, C-NRAL, and C-NRPA) using EMSA (Fig. 4). moter was not affected by the introduction of wild-type or mutant Six different GATA3 binding sequences were used: a GATA3 con- GATA3 molecules. These results indicate that C-NRPL is indis- sensus sequence, a binding sequence existing in CNS1, and sequences pensable for the GATA3 function. from the VA enhancer, CGRE, the IL-5 promoter, and the IL-13 pro- moter. HEK 293 T cells were transfected with Myc-tagged GATA3 The core R353/P354 residues play a crucial in the GATA3 mutant genes, and nuclear protein extracts were prepared. Immuno- function blotting analysis with anti-Myc tag mAb confirmed the equivalent We next introduced a single amino acid substitution to alanine protein expression of GATA3 mutants (see Fig. 4A, Input). The sub- within the C-NRPL residues of GATA3. Each mutant (C-ARPL, cellular localization was not found to change in these GATA3 mutants C-NAPL, C-NRAL, and C-NRPA) was introduced into developing (data not shown). As shown in Fig. 4, the binding activity was abro- Th1 cells, and the generation of IL-4-producing Th2 cells was gated almost completely by a single alanine mutation at R353 (C- determined. As shown in Fig. 3A, the generation of IL-4-producing NAPL). The binding of C-NRAL was also substantially reduced in cells decreased in C-NAPL and C-NRAL mutants but not substan- some GATA binding sequences, including a GATA consensus bind- tially in C-ARPL or C-NRPA mutants (Fig. 3A). Similar results ing site (Fig. 4A), CNS1 (Fig. 4B), IL-5 promoter (Fig. 4E), and IL-13 were obtained by ELISA (Fig. 3B). Substantial increases in the promoter (Fig. 4F). These results indicate that the R353 residue plays transcriptional activity on the IL-5 promoter and the IL-13 pro- a critical role in the DNA binding activity of GATA3 and that the moter were detected in C-ARPL, C-NRPA, and C-ARPA mutants P354 residue is important for DNA binding activity. http://www.jimmunol.org/ on October 1, 2021 by guest. Protected copyright.

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. 2C. 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. 5806 CRITICAL MOTIF FOR GATA3 DNA BINDING AND FUNCTION J Immunol: first published as 10.4049/jimmunol.177.9.5801 on 18 October 2006. Downloaded from

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), VA 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 http://www.jimmunol.org/ anti-Myc tag Ab. Three independent experiments were performed with similar results.

DNA binding activity and the generation of Th2 cells in a each play a critical role in enabling GATA3 to bind to DNA and GATA3 R353K mutant (C-NKPL) thus induce Th2 cell generation. To address the role of positive charge for (R353), we generated a GATA3 R353K mutant (C-NKPL). The C-NKPL Role of the NRPL residues adjacent to the N-terminal zinc mutant did not show the ability to generate IL-4 producing Th2 finger domain in GATA3 function on October 1, 2021 by guest. Protected copyright. cells (data not shown). An EMSA revealed that the levels of DNA Finally, the role of NRPL residues adjacent to the N-terminal zinc binding activity for C-NKPL were similar to those of C-NAPL finger domain of GATA3 was assessed using a GATA3 mutant (data not shown). These results suggest that the positive charge of with an alanine substitution (N-NRPL/AAAA). The ability to in- R353 itself is not sufficient for either GATA3 to bind to DNA or duce IL-4 producing Th2 cells was well preserved in the N-NRPL/ for the generation of Th2 cells. AAAA mutant (Fig. 6A). The production of IL-4 was lower than that of wild type but significantly higher than that of C-NRPL/ Y345 and H349 are critical for GATA3 functions AAAA (Fig. 6B). In contrast, the production of IL-5 and IL-13 was In addition to C-NRPL, Y345, K347, and H349 adjacent to the not significantly induced with the N-NRPL/AAAA mutant. The C-terminal zinc finger domain of GATA3 are conserved among all transcription activity on the IL-5 promoter was not detected, and mouse and human GATA family members (Table I). Conse- that on the IL-13 promoter was moderately induced (Fig. 6C). quently, we generated various single alanine mutants (Y345A, Regarding the induction of histone modifications at the Th2 cyto- Y346A, L348A, and H349A) and tested their ability to induce kine gene loci, no obvious defect was observed in the N-NRPL/ IL-4-producing Th2 cells. The generation of IL-4-producing cells AAAA mutant in H3-K9 acetylation (Fig. 6D) or H3-K4 dimethy- dramatically decreased in Y345A and H349A mutants but not in lation (data not shown). An EMSA revealed that the levels of DNA Y346A or L348A mutants (Fig. 5B). Similarly as for the R353A binding to various GATA motifs for N-NRPL/AAAA were sig- mutant, the DNA binding activity for Y345A and H349A mutants nificantly lower than that of the wild type but obviously higher was found to be marginal (Fig. 5C). The K347A mutant also than that of the C-NRPL/AAAA mutant (data not shown). There- showed also moderately decreased Th2 cell differentiation (data fore, although the impact of N-NRPL on GATA3 function was not not shown). These results indicate that Y345, K347, and H349 as dramatic as that of C-NRPL, the N-NRPL residues of GATA3 The Journal of Immunology 5807 J Immunol: first published as 10.4049/jimmunol.177.9.5801 on 18 October 2006. Downloaded from

FIGURE 5. Y345 and H349 are required for the generation of Th2 cells. A, Schematic representation of the amino acid alignment around Y345 and http://www.jimmunol.org/ 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. 1B. 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.

also appear to play important roles in some GATA3 functions such gram “O” (48) (Fig. 7). The N␧2 of H349 and DNA phosphate as the transcription and production of IL-5 and IL-13. atoms formed two hydrogen bonds with distances of 2.49 and

2.73Å. The N␩1 of R353 and DNA phosphate also formed a weak on October 1, 2021 by guest. Protected copyright. Discussion hydrogen bond with a distance of 3.62 Å. The H349 and R353 In this report we identified a novel conserved amino acid motif, form stacking interaction with Y345 to stabilize these side chains, YxKxHxxxRP, which is adjacent to the C-terminal zinc finger do- which are actually suitable positions for interacting with the phos- main of GATA3 (C-finger YxKxHxxxRP) and is crucial for phate groups of DNA. Stabilization of the basic residues H349 and GATA3 functions, including its DNA binding, transcriptional ac- R353 appears to be important for the binding to DNA. These ob- tivity, and ability to induce chromatin remodeling of the Th2 cy- servations are highly consistent with our experimental results, i.e., tokine gene loci leading to Th2 cell differentiation. A single amino a of these conserved amino acid residues resulted in acid mutation in the motif resulted in the abrogation of most of the GATA3 function, indicating a critical role for these amino acids. a dramatic decrease in GATA3 DNA binding activity (Figs. 4 and ␨ A basic local alignment search tool (BLAST) search for 5C). The N atom of K347, which is 4.05 Å apart from the phos- hGATA3 (hGATA3; aa 313–368) region, which includes the C- phate of DNA both in the solution structure ( terminal zinc finger and the YxKxHxxxRP motif, listed chicken accession no. 2GAT) and our model, seems to have a very weak GATA1 (cGATA1; aa 159–214) with 82.1% (46 of 56 aa) identity electrostatic interaction with DNA phosphate. In line with this and 92.9% (52 of 56 aa) similarity without any gaps. This indicates finding, the K347A mutant also showed decreased levels in Th2 that the structure of the hGATA3 C-terminal zinc finger and its cell differentiation (T. Nakayama, unpublished observation). downstream region are almost identical with that of the cGATA1 Indeed, 700F and 704H residues in a fungal GATA homolog C-terminal zinc finger. Consequently, based on the solution struc- areA, which correspond to the GATA3 Y345 and H349 residues, ture of the cGATA1/DNA complex (Protein Data Bank accession have been reported to be involved in the contact sites with DNA code 2GAT) (46, 47), the structure of hGATA3 (aa 313–368)/ (49). Moreover, the Y345/H349 residues are located at the ␣-he- DNA complex model has been manually built on the graphic pro- lical regions, which is important for the recognition of the major 5808 CRITICAL MOTIF FOR GATA3 DNA BINDING AND FUNCTION J Immunol: first published as 10.4049/jimmunol.177.9.5801 on 18 October 2006. Downloaded from

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; VA enh., VA enhancer. groove surface of DNA (Fig. 7A). These findings all support the In contrast to C-NRPL, a mutation in N-NRPL did not interfere

importance of Y345/H349 residues for DNA binding. with the histone modifications (H3-K9 acetylation and H3-K4 http://www.jimmunol.org/ The P354A mutant impaired GATA3 functions. However, P354 methylation) at the Th2 cytokine (IL-5/IL-13/IL-4) gene loci (Fig. has no interaction with DNA in the complex model or the key 6D). This finding is consistent with the observation that the N- residues whose disrupted GATA3 functions. Proline has terminal zinc finger of GATA3 is dispensable for the induction of less structural flexibility of the backbone. Mutation at P354 with DNase I hypersensitive sites within the IL-4/IL-13 gene loci (42). alanine seems to induce flexibility around this area. Downstream We found that the cytokine production of IL-5 and IL-13 and the of P354 there is an essential DNA minor groove binding RxRK (aa transactivation of the IL-5 and IL-13 promoters were impaired in 365–368) motif (46). In the P354A mutant, because of the intro- the developing Th1 cells introduced with the N-NRPL/AAAA mu- duced backbone flexibility at position 354 the RxRK (aa 365–368) tant (Fig. 6, B and C). This finding is also consistent with the motif might not be able to bind to DNA or the downstream of P354 results reported by Takemoto et al. (42), where the of on October 1, 2021 by guest. Protected copyright. might cover the DNA binding area of the C-finger or the YxKx- N-terminal zinc finger compromised GATA3 binding to the IL-5 HxxRP motif. This kind of structural restraint seems to play an promoter. Therefore, N-NRPL appears to play a role in some important role in the P354 function. GATA3 functions that are dependent on the N-terminal zinc finger.

FIGURE 7. Structure model of the mGATA3 (aa 312–367)/DNA complex based on the solution structure of cGATA1/DNA complex. A, Two orthogonal views of the GATA3/DNA complex model. The C␣ representation of GATA3 is shown in light blue, and the YxKxHxxxRP motif is highlighted in red. dsDNA is shown in faint purple with ball-and-stick representation. B, Interaction between DNA and the YxKxHxxxRP motif. The orientation is as in the right hand view of A. The key residues of the motif, Y345, K347, H349, R353, and P354 are shown in red. The figures are produced using the Bobscript (54) and Raster3D (55). The Journal of Immunology 5809 J Immunol: first published as 10.4049/jimmunol.177.9.5801 on 18 October 2006. Downloaded from

Both the C-terminal and N-terminal zinc fingers are reported to be 12. George, K. M., M. W. Leonard, M. E. Roth, K. H. Lieuw, D. Kioussis, required for the high-affinity interaction between GATA1 and F. Grosveld, and J. D. Engel. 1994. Embryonic expression and cloning of the murine GATA-3 gene. Development 120: 2673–2686. DNA (50). In fact, we observed a decrease in the binding of N- 13. Hendriks, R. W., M. C. Nawijn, J. D. Engel, H. van Doorninck, F. Grosveld, and NRPL/AAAA mutant GATA3 to some GATA motifs (T. Na- A. Karis. 1999. Expression of the transcription factor GATA-3 is required for the development of the earliest T cell progenitors and correlates with stages of cel- kayama, unpublished observation). lular proliferation in the thymus. Eur. J. Immunol. 29: 1912–1918. The H349 in the C-finger YxKxHxxxRP residue corresponds to 14. Hattori, N., H. Kawamoto, S. Fujimoto, K. Kuno, and Y. Katsura. 1996. Involve- N295 in the N-finger region (YxKxNxxxRP). Although the H349A ment of transcription factors TCF-1 and GATA-3 in the initiation of the earliest step of T cell development in the thymus. J. Exp. Med. 184: 1137–1147. mutation completely abolished the GATA3 functions (Fig. 5), the 15. Ting, C. N., M. C. Olson, K. P. Barton, and J. M. Leiden. 1996. Transcription H349N mutation had no effect (T. Nakayama, unpublished obser- factor GATA-3 is required for development of the T-cell lineage. Nature 384: vation), thus suggesting that H349 and N295 residues are poten- 474–478. 16. Pai, S. Y., M. L. Truitt, and I. C. Ho. 2004. GATA-3 deficiency abrogates the tially able to play a similar role in the GATA3 DNA binding re- development and maintenance of T helper type 2 cells. Proc. Natl. Acad. Sci. quired for the GATA3 function. USA 101: 1993–1998. It is known that GATA3 is expressed in the developing para- 17. Kurata, H., H. J. Lee, A. O’Garra, and N. Arai. 1999. Ectopic expression of activated Stat6 induces the expression of Th2-specific cytokines and transcription thyroids, inner ear, and kidney (12, 51). GATA3 haploinsuffi- factors in developing Th1 cells. Immunity 11: 677–688. ciency is reported to be associated with the , 18. Lee, H. J., N. Takemoto, H. Kurata, Y. Kamogawa, S. Miyatake, A. O’Garra, and N. Arai. 2000. GATA-3 induces type 2 (Th2) cytokine expression deafness, and renal dysplasia syndrome (52). A mutation in the and chromatin remodeling in committed Th1 cells. J. Exp. Med. 192: 105–115. splicing acceptor site around the GATA3 intron 5/ 6 boundary 19. Ouyang, W., M. Lohning, Z. Gao, M. Assenmacher, S. Ranganath, A. Radbruch, was found in familial hypoparathyroidism, deafness, and renal and K. M. Murphy. 2000. Stat6-independent GATA-3 autoactivation directs IL- 4-independent Th2 development and commitment. Immunity 12: 27–37. dysplasia syndrome patients (53). This mutation results in a frame 20. Asnagli, H., M. Afkarian, and K. M. Murphy. 2002. Cutting edge: identification shift that produces mis-sense polypeptides from aa 351 to 367 with of an alternative GATA-3 promoter directing tissue-specific in a premature termination at position 367. Indeed, this mis-sense mouse and human. J. Immunol. 168: 4268–4271. 21. Yamashita, M., R. Shinnakasu, H. Asou, M. Kimura, A. Hasegawa, GATA3 protein misses R353 and P354 residues, which are key K. Hashimoto, N. Hatano, M. Ogata, and T. Nakayama. 2005. Ras-ERK MAPK amino acids in the C-finger YxKxHxxxRP motif. This finding in- cascade regulates GATA3 stability and Th2 differentiation through ubiquitin- dicated that the C-terminal zinc finger YxKxHxxxRP motif there- proteasome pathway. J. Biol. Chem. 280: 29409–29419. 22. Kishikawa, H., J. Sun, A. Choi, S. C. Miaw, and I. C. Ho. 2001. The cell type- fore plays an important pathophysiological role in human beings. specific expression of the murine IL-13 gene is regulated by GATA-3. J. Immu- In summary, we identified a novel conserved amino acid motif, nol. 167: 4414–4420. 23. Siegel, M. D., D. H. Zhang, P. Ray, and A. Ray. 1995. Activation of the inter- YxKxHxxxRP, adjacent to the C-terminal zinc finger domains of leukin-5 promoter by cAMP in murine EL-4 cells requires the GATA-3 and all GATA family proteins in mouse and human. A single amino CLE0 elements. J. Biol. Chem. 270: 24548–24555. acid mutation in the motif of GATA3 resulted in the abrogation of 24. Lavenu-Bombled, C., C. D. Trainor, I. Makeh, P. H. Romeo, and I. Max-Audit. 2002. Interleukin-13 gene expression is regulated by GATA-3 in T cells: role of most of the GATA3 function, thus indicating that each amino acid a critical association of a GATA and two GATG motifs. J. Biol. Chem. 277: plays a crucial role in the GATA3 binding to DNA, thereby lead- 18313–18321. ing to chromatin remodeling of the Th2 cytokine gene loci, Th2 25. Lee, H. J., A. O’Garra, K. Arai, and N. Arai. 1998. Characterization of cis- regulatory elements and nuclear factors conferring Th2-specific expression of the cell differentiation, and Th2 cytokine expression. IL-5 gene: a role for a GATA- binding protein. J. Immunol. 160: 2343–2352. 26. Zhang, D. H., L. Yang, and A. Ray. 1998. Differential responsiveness of the IL-5 Acknowledgments and IL-4 genes to transcription factor GATA-3. J. Immunol. 161: 3817–3821. 27. Schwenger, G. T., R. Fournier, C. C. Kok, V. A. Mordvinov, D. Yeoman, and http://www.jimmunol.org/ We thank Hikari Asou, Satoko Norikane, and Kaoru Sugaya for their ex- C. J. Sanderson. 2001. GATA-3 has dual regulatory functions in human inter- cellent technical assistance. leukin-5 transcription. J. Biol. Chem. 276: 48502–48509. 28. Yamashita, M., M. Ukai-Tadenuma, M. Kimura, M. Omori, M. Inami, M. Taniguchi, and T. Nakayama. 2002. Identification of a conserved GATA3 Disclosures response element upstream proximal from the interleukin-13 gene . J. Biol. The authors have no financial conflict of interest. Chem. 277: 42399–42408. 29. Agarwal, S., O. Avni, and A. Rao. 2000. Cell-type-restricted binding of the tran- scription factor NFAT to a distal IL-4 enhancer in vivo. Immunity 12: 643–652. References 30. Takemoto, N., Y. Kamogawa, H. Jun Lee, H. Kurata, K. I. Arai, A. O’Garra, 1. Mosmann, T. R., and R. L. Coffman. 1989. TH1 and TH2 cells: different patterns N. Arai, and S. Miyatake. 2000. Cutting edge: chromatin remodeling at the IL- of lymphokine secretion lead to different functional properties. Annu. Rev. Im- 4/IL-13 intergenic regulatory region for Th2-specific cytokine gene cluster. J. Im-

munol. 7: 145–173. munol. 165: 6687–6691. on October 1, 2021 by guest. Protected copyright. 2. Constant, S. L., and K. Bottomly. 1997. Induction of Th1 and Th2 CD4ϩ T cell 31. Fields, P. E., G. R. Lee, S. T. Kim, V. V. Bartsevich, and R. A. Flavell. 2004. responses: the alternative approaches. Annu. Rev. Immunol. 15: 297–322. Th2-specific chromatin remodeling and enhancer activity in the Th2 cytokine 3. Seder, R. A., and W. E. Paul. 1994. Acquisition of lymphokine-producing phe- locus control region. Immunity 21: 865–876. notype by CD4ϩ T cells. Annu. Rev. Immunol. 12: 635–673. 32. Ansel, K. M., I. Djuretic, B. Tanasa, and A. Rao. 2006. Regulation of Th2 dif- 4. Reiner, S. L., and R. M. Locksley. 1995. The regulation of immunity to Leish- ferentiation and Il4 locus accessibility. Annu. Rev. Immunol. 24: 607–656. mania major. Annu. Rev. Immunol. 13: 151–177. 33. Lee, G. R., S. T. Kim, C. G. Spilianakis, P. E. Fields, and R. A. Flavell. 2006. T 5. Murphy, K. M., W. Ouyang, J. D. Farrar, J. Yang, S. Ranganath, H. Asnagli, helper cell differentiation: regulation by cis elements and epigenetics. Immunity M. Afkarian, and T. L. Murphy. 2000. Signaling and transcription in T helper 24: 369–379. development. Annu. Rev. Immunol. 18: 451–494. 34. Lohning, M., A. Richter, and A. Radbruch. 2002. Cytokine memory of T helper 6. Nelms, K., A. D. Keegan, J. Zamorano, J. J. Ryan, and W. E. Paul. 1999. The lymphocytes. Adv. Immunol. 80: 115–181. IL-4 receptor: signaling mechanisms and biologic functions. Annu. Rev. Immunol. 35. Omori, M., M. Yamashita, M. Inami, M. Ukai-Tadenuma, M. Kimura, Y. Nigo, 17: 701–738. H. Hosokawa, A. Hasegawa, M. Taniguchi, and T. Nakayama. 2003. CD8 T 7. O’Garra, A. 1998. Cytokines induce the development of functionally heteroge- cell-specific downregulation of histone hyperacetylation and gene activation of neous T helper cell subsets. Immunity 8: 275–283. the IL-4 gene locus by ROG, repressor of GATA. Immunity 19: 281–294. 8. Zhang, D. H., L. Cohn, P. Ray, K. Bottomly, and A. Ray. 1997. Transcription 36. Inami, M., M. Yamashita, Y. Tenda, A. Hasegawa, M. Kimura, K. Hashimoto, factor GATA-3 is differentially expressed in murine Th1 and Th2 cells and con- N. Seki, M. Taniguchi, and T. Nakayama. 2004. CD28 costimulation controls trols Th2-specific expression of the interleukin-5 gene. J. Biol. Chem. 272: histone hyperacetylation of the interleukin 5 gene locus in developing Th2 cells. 21597–21603. J. Biol. Chem. 279: 23123–23133. 9. Zheng, W., and R. A. Flavell. 1997. The transcription factor GATA-3 is neces- 37. Orkin, S. H. 1992. GATA-binding transcription factors in hematopoietic cells. sary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89: Blood 80: 575–581. 587–596. 38. Tsang, A. P., J. E. Visvader, C. A. Turner, Y. Fujiwara, C. Yu, M. J. Weiss, 10. Szabo, S. J., B. M. Sullivan, C. Stemmann, A. R. Satoskar, B. P. Sleckman, and M. Crossley, and S. H. Orkin. 1997. FOG, a multitype zinc finger protein, acts as L. H. Glimcher. 2002. Distinct effects of T-bet in TH1 lineage commitment and a cofactor for transcription factor GATA-1 in erythroid and megakaryocytic dif- IFN-␥ production in CD4 and CD8 T cells. Science 295: 338–342. ferentiation. Cell 90: 109–119. 11. Yamamoto, M., L. J. Ko, M. W. Leonard, H. Beug, S. H. Orkin, and J. D. Engel. 39. Svensson, E. C., R. L. Tufts, C. E. Polk, and J. M. Leiden. 1999. Molecular 1990. Activity and tissue-specific expression of the transcription factor NF-E1 cloning of FOG-2: a modulator of transcription factor GATA-4 in cardiomyo- multigene family. Genes Dev. 4: 1650–1662. cytes. Proc. Natl. Acad. Sci. USA 96: 956–961. 5810 CRITICAL MOTIF FOR GATA3 DNA BINDING AND FUNCTION J Immunol: first published as 10.4049/jimmunol.177.9.5801 on 18 October 2006. Downloaded from

40. Tevosian, S. G., A. E. Deconinck, A. B. Cantor, H. I. Rieff, Y. Fujiwara, 47. Tjandra, N., J. G. Omichinski, A. M. Gronenborn, G. M. Clore, and A. Bax. 1997. G. Corfas, and S. H. Orkin. 1999. FOG-2: a novel GATA-family cofactor related Use of dipolar 1H–15N and 1H–13C couplings in the structure determination of to multitype zinc-finger proteins Friend of GATA-1 and U-shaped. Proc. Natl. magnetically oriented macromolecules in solution. Nat. Struct. Biol. 4: 732–738. Acad. Sci. USA 96: 950–955. 48. Jones, T. A., J. Y. Zou, S. W. Cowan, and M. Kjeldgaard. 1991. Improved 41. Fox, A. H., K. Kowalski, G. F. King, J. P. Mackay, and M. Crossley. 1998. Key methods for building protein models in electron density maps and the location of residues characteristic of GATA N-fingers are recognized by FOG. J. Biol. Chem. errors in these models. Acta Crystallogr. A 47: 110–119. 273: 33595–33603. 49. Starich, M. R., M. Wikstrom, S. Schumacher, H. N. Arst, Jr., A. M. Gronenborn, and G. M. Clore. 1998. The solution structure of the Leu22–ϾVal mutant AREA 42. Takemoto, N., K. Arai, and S. Miyatake. 2002. Cutting edge: the differential DNA binding domain complexed with a TGATAG core element defines a role for involvement of the N-finger of GATA-3 in chromatin remodeling and transacti- hydrophobic packing in the determination of specificity. J. Mol. Biol. 277: vation during Th2 development. J. Immunol. 169: 4103–4107. 621–634. 43. Yamashita, M., M. Katsumata, M. Iwashima, M. Kimura, C. Shimizu, T. Kamata, 50. Trainor, C. D., J. G. Omichinski, T. L. Vandergon, A. M. Gronenborn, T. Shin, N. Seki, S. Suzuki, M. Taniguchi, and T. Nakayama. 2000. T cell re- G. M. Clore, and G. Felsenfeld. 1996. A palindromic regulatory site within ver- ceptor-induced calcineurin activation regulates T helper type 2 cell development tebrate GATA-1 promoters requires both zinc fingers of the GATA-1 DNA- by modifying the receptor signaling complex. J. Exp. Med. 191: binding domain for high-affinity interaction. Mol. Cell. Biol. 16: 2238–2247. 1869–1879. 51. Debacker, C., M. Catala, and M. C. Labastie. 1999. Embryonic expression of the 44. Kimura, M. Y., H. Hosokawa, M. Yamashita, A. Hasegawa, C. Iwamura, human GATA-3 gene. Mech. Dev. 85: 183–187. H. Watarai, M. Taniguchi, T. Takagi, S. Ishii, and T. Nakayama. 2005. Regula- 52. Van Esch, H., P. Groenen, M. A. Nesbit, S. Schuffenhauer, P. Lichtner, tion of T helper type 2 cell differentiation by murine Schnurri-2. J. Exp. Med. 201: G. Vanderlinden, B. Harding, R. Beetz, R. W. Bilous, I. Holdaway, et al. 2000. 397–408. GATA3 haplo-insufficiency causes human HDR syndrome. Nature 406: 419–422. 45. Kimura, M., Y. Koseki, M. Yamashita, N. Watanabe, C. Shimizu, T. Katsumoto, 53. Nesbit, M. A., M. R. Bowl, B. Harding, A. Ali, A. Ayala, C. Crowe, A. Dobbie, T. Kitamura, M. Taniguchi, H. Koseki, and T. Nakayama. 2001. Regulation of G. Hampson, I. Holdaway, M. A. Levine, et al. 2004. Characterization of GATA3 Th2 cell differentiation by mel-18, a mammalian polycomb group gene. Immunity mutations in the hypoparathyroidism, deafness, and renal dysplasia (HDR) syn- 15: 275–287. drome. J. Biol. Chem. 279: 22624–22634. 46. Omichinski, J. G., G. M. Clore, O. Schaad, G. Felsenfeld, C. Trainor, E. Appella, 54. Esnouf, R. M. 1997. An extensively modified version of MolScript that includes S. J. Stahl, and A. M. Gronenborn. 1993. NMR structure of a specific DNA greatly enhanced coloring capabilities. J. Mol. Graph Model. 15: 132–134. complex of Zn-containing DNA binding domain of GATA-1. Science 261: 55. Merritt, E. A., and M. E. Murphy. 1994. Raster3D Version 2.0. A program for 438–446. photorealistic molecular graphics. Acta Crystallogr. D 50: 869–873. http://www.jimmunol.org/ on October 1, 2021 by guest. Protected copyright.