Both zinc finger domains of GATA-3 are important for the association with lymphoid enhancer factor 1 (LEF-1). (a) A picture of silver staining of the final sample for the identification of GATA-3-associated molecules. Lane 1: wild-type (WT) GATA-3. Lane 2: GATA-3d349. The arrow in lane 1 (50 kDa) indicates a band that was sliced and analysed by mass spectrometry. The numbers indicates the molecular mass markers. (b) A schematic representation of the wild type and GATA-3 mutants. The location of the serine (S)-proline (P)-rich transactivation domain (over the line) and zinc finger domains (Zn) are indicated. The approximate molecular weight (MW) of each mutant is also indicated. (c) The role of GATA-3 C-terminal and zinc finger regions for the association with LEF-1. 293 T cells were transfected with expression plasmids encoding Myc-tagged wild-type LEF-1 and Flag-tagged wild-type or mutant GATA-3 genes. Two days later, immunoprecipitates with anti-Flag monoclonal antibody (mAb) were subjected to immunoblotting with anti-Myc mAb (upper panel). The total lysates were also run in parallel and immunoblotted with anti-Myc mAb, anti-Flag mAb, or anti-tubulin-α mAb. (d) The N-terminal serine-rich region of GATA-3 is not essential for the binding to LEF-1. 293 T cells were transfected with expression plasmids encoding Myc-tagged wild-type LEF-1 and Flag-tagged wild-type or N-terminal deleted GATA-3. IB, immunoblotting; IgG, immunoglobulin G; IP, immunoprecipitation.

Association of endogenous GATA-3 with transfected lymphoid enhancer factor 1 (LEF-1) in a T-cell line. A mouse GATA-3-expressing T-cell line, TG40, was infected with retrovirus vector encoding wild-type (WT) Myc-tagged LEF-1 and the helix-1 mutant (dH1). Thereafter, the immunoprecipitates with control goat immunoglobulin G (IgG) antibody (lanes 1–3) or anti-GATA-3 antibody (lanes 4–6) were subjected to immunoblotting with anti-Myc monoclonal antibody (mAb) (left panel). The total lysates were also run in parallel as a loading control (right panel). IB, immunoblotting; IP, immunoprecipitation.

The high-mobility group (HMG) box of lymphoid enhancer factor 1 (LEF-1) is important for the association with GATA-3. (a) A schematic representation of the wild type (WT) and various LEF-1 mutants. The β-catenin-binding domain (βBD), context-dependent activation domain (CAD) and HMG DNA-binding domain are indicated. The numbers below the wild type indicate the amino acid positions of the respective protein domains. The approximate molecular weight (MW) of each mutant is also indicated. (b-d) The association of Flag-tagged wild-type GATA-3 with Myc-tagged wild type and various LEF-1 mutants was investigated as in Fig 1b. IB, immunoblotting; IP, immunoprecipitation.

Lymphoid enhancer factor 1 (LEF-1) suppresses the DNA-binding activity of GATA-3 and T helper 2 (Th2) cytokine expression in developing Th2 cells. (a) The expression levels of LEF-1 in freshly isolated splenic CD4 T cells after stimulation with immobilized anti-T-cell receptor (TCR) monoclonal antibody (mAb) in vitro were determined by a quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) analysis. (b) Splenic CD4 T cells were stimulated under Th2 culture conditions and infected with retrovirus vector encoding wild-type Myc-tagged LEF-1 and the helix-1 mutant (dH1). To examine the mRNA expression levels of Th2 cytokines in infected cells, the human nerve growth factor (NGF) receptor p75 (hNGFR)-positive cells were sorted and were stimulated with anti-TCR mAb for 4 hr and subjected to a quantitative RT-PCR analysis. (c) Th2 cytokine production in infected cells was determined by enzyme-linked immunosorbent assay (ELISA). The hNGFR⁺ cells were sorted and were stimulated with anti-TCR mAb for 24 hr. Thereafter, the culture supernatants were collected and subjected to ELISA. Three independent experiments were performed with similar results. (d) The levels of histone modification (H3-K9/14 acetylation and H3-K4 tri-methylation) at the Th2 cytokine gene loci were assessed in a chromatin immunoprecipitation (ChIP) analysis. (e) Flag-tagged GATA-3 and Myc-tagged LEF-1 were expressed in 293 T cells. Nuclear extracts from the transfected cells were prepared and subjected to an electrophoretic mobility shift assay (EMSA) with the GATA-binding sequence in the interleukin (IL)-5 promoter region. The relative amounts of LEF-1 in the nuclear lysates were indicated. The protein levels of GATA-3 and LEF-1 were determined by immunoblotting with anti-Flag mAb and anti-Myc mAb, respectively (lower panel). A super-shift assay was performed with control mouse immunoglobulin G (IgG) and an anti-Flag mAb (right panel). Arbitrary densitometric units are shown under each band. Three independent experiments were performed with similar results. HPRT, hypoxanthine phosphoribosyltransferase.