Of the GATA-binding proteins, only GATA-4 selectively regulates the human interleukin-5 gene promoter in interleukin-5-producing cells which express multiple GATA-binding proteins.
Interleukin-5 (IL-5) is produced by T lymphocytes and known to support B-cell growth and eosinophilic differentiation of the progenitor cells. Using ATL-16T cells which express IL-5 mRNA, we have identified a region within the human IL-5 gene promoter that regulates IL-5 gene transcription. This cis-acting sequence contains the core binding motif, (A/T)GATA(A/G), for GATA-binding family proteins and thus suggests the involvement of this family members. In this report, we describe the cloning of human GATA-4 (hGATA-4) and show that hGATA-4 selectively interacts with the -70 GATA site within the IL-5 proximal promoter region. By promoter deletion and mutation analyses, we established this region as a positive regulatory element. Cotransfection experiments revealed that both hGATA-4 and phorbol-12-myristate-13-acetate (PMA)-A23187 stimulation are necessary for IL-5 promoter activation. The requirement for another regulatory element called CLE0, which lies downstream of the -70 GATA site, was also demonstrated. ATL-16T cells express mRNAs of three GATA-binding proteins, hGATA-2, hGATA-3, and hGATA-4, and each of them has a potential to bind to the consensus (A/T)GATA(G/A) motif. However, using ATL-16T nuclear extract, we demonstrated that GATA-4 is the only GATA-binding protein that forms a specific DNA-protein complex with the -70 GATA site. An electrophoretic mobility shift assay with extracts of COS cells expressing GATA-binding proteins showed that GATA-4 has the highest binding affinity for the -70 GATA site among the three GATA-binding proteins. When the transactivation abilities were compared among the three, GATA-4 showed the highest activity. These results demonstrate the selective role of GATA-4 in the transcriptional regulation of the IL-5 gene in a circumstance where multiple members of the GATA-binding proteins are expressed.
Yamagata
T
T
Third Department of Internal Medicine, Tokyo University, Japan.
Nishida
J
J
Sakai
R
R
Tanaka
T
T
Honda
H
H
Hirano
N
N
Mano
H
H
Yazaki
Y
Y
Hirai
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D050989:GATA2 Transcription Factor; D050990:GATA3 Transcription Factor; D050991:GATA4 Transcription Factor; D015723:Gene Library; D017930:Genes, Reporter; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D015848:Interleukin-5 / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D050989:GATA2 Transcription Factor; C494711:GATA2 protein, human; D050990:GATA3 Transcription Factor; C494749:GATA3 protein, human; D050991:GATA4 Transcription Factor; D015848:Interleukin-5; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors