The crucial role of GATA-1 in CCR3 gene transcription: modulated balance by multiple GATA elements in the CCR3 regulatory region.
GATA-1, a zinc finger-containing transcription factor, regulates not only the differentiation of eosinophils but also the expression of many eosinophil-specific genes. In the current study, we dissected CCR3 gene expression at the molecular level using several cell types that express varying levels of GATA-1 and CCR3. Chromatin immunoprecipitation analysis revealed that GATA-1 preferentially bound to sequences in both exon 1 and its proximal intron 1. A reporter plasmid assay showed that constructs harboring exon 1 and/or intron 1 sequences retained transactivation activity, which was essentially proportional to cellular levels of endogenous GATA-1. Introduction of a dominant-negative GATA-1 or small interfering RNA of GATA-1 resulted in a decrease in transcription activity of the CCR3 reporter. Both point mutation and EMSA analyses demonstrated that although GATA-1 bound to virtually all seven putative GATA elements present in exon 1-intron 1, the first GATA site in exon 1 exhibited the highest binding affinity for GATA-1 and was solely responsible for GATA-1-mediated transactivation. The fourth and fifth GATA sites in exon 1, which were postulated previously to be a canonical double-GATA site for GATA-1-mediated transcription of eosinophil-specific genes, appeared to play an inhibitory role in transactivation, albeit with a high affinity for GATA-1. Furthermore, mutation of the seventh GATA site (present in intron 1) increased transcription, suggesting an inhibitory role. These data suggest that GATA-1 controls CCR3 transcription by interacting dynamically with the multiple GATA sites in the regulatory region of the CCR3 gene.
Division of Molecular and Life Sciences, College of Science and Technology, Hanyang University, Ansan, South Korea.
Byung Soo
BS
Kim
Tae Gi
TG
Uhm
Seol Kyoung
SK
Lee
Sin-Hwa
SH
Lee
Jin Hyun
JH
Kang
Choon-Sik
CS
Park
Il Yup
IY
Chung
D001483:Base Sequence; D002460:Cell Line; D045744:Cell Line, Tumor; D005091:Exons / Q000276:immunology; D005136:Eye Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D050980:GATA Transcription Factors / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D005786:Gene Expression Regulation / Q000276:immunology*; D006801:Humans; D007438:Introns / Q000276:immunology; D020014:K562 Cells; D008024:Ligands; D008969:Molecular Sequence Data; D017354:Point Mutation; D054397:Receptors, CCR3 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics / Q000276:immunology*; D020218:Response Elements / Q000276:immunology; D017384:Sequence Deletion / Q000276:immunology; D014158:Transcription, Genetic / Q000276:immunology*
C515350:CCR3 protein, human; D005136:Eye Proteins; D050980:GATA Transcription Factors; C464454:GATAD1 protein, human; D008024:Ligands; D054397:Receptors, CCR3