The Crucial Role of GATA-1 in CCR3 Gene Transcription: Modulated Balance by Multiple GATA Elements in the CCR3 Regulatory Region

Abstract

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.

CpG methylation at GATA elements in the regulatory region ofCCR3positively correlates withCCR3transcription

“…However, we observed the GATA-1 occupancy regardless of cell types (data not shown). Lack of the differential binding seems to be due to the fact that the current ChIP assay cannot discern, if any, differential binding of GATA-1 to these GATA sites, probably because the fourth and fifth GATA elements are positioned at only about 100 base pair away from the first GATA element that shows a high affinity binding for GATA-1, as seen in previous results (Kim et al, 2010). …”

“…The fourth and the fifth GATA sites in exon 1 are claimed to constitute a double-GATA site as a key element that dictates GATA-1-mediated transcription of many eosinophil-specific genes (Rothenberg and Hogan, 2006). However, our previous study using a reporter plasmid assay in K562 cells demonstrated that the first GATA site is solely responsible for GATA-1-mediated transactivation of CCR3 , while the fourth and fifth GATA sites play inhibitory roles in CCR3 transcription, with high affinity binding for GATA-1 comparable to that of the first GATA element (Kim et al, 2010). The differential contributions of these GATA sites to CCR3 transcription were almost exactly duplicated in A549 cells (Supplemental Data Figure S1), and GATA-1 bound to sequences in exon 1 of CCR3 genes, as analyzed by ChIP assay (Supplemental Data Figure S2).…”

“…When increasing concentrations of the exon 1 fragment of unmethylated CpG sites were ligated, transcription activity of the ligation mixture increased in proportion to the input amount of the exon 1 sequence used as an insert (Figure 6B). In addition, when the exon 1 fragment with a mutated version (mut1) of the first GATA site, which was shown to decrease transcription by more than one-half in K562 cells (Kim et al, 2010) and A549 cells (Supplemental Data Figure S1), was ligated to a pGL3 basic vector, its transcription activity was reduced by more than half compared to that of exon 1 with unmethylated CpG sites. These results ensure that the ligation mixture contains the proper recombinant plasmid for transcription activation.…”

“…Involvement of GATA-1 in CCR3 transcription has been demonstrated (Zimmerman et al, 2005) and subsequently leads to postulation of a double-GATA element as a key regulatory element of GATA-1-mediated transcription of eosinophil-specific genes, including human CCR3 , interleukin-5 receptor alpha, MBP and GATA-1 genes (Rothenberg and Hogan, 2006). We have recently analyzed GATA-1-mediated transcription of CCR3 at the molecular level (Kim et al, 2010). Of five GATA elements in exon 1 of CCR3 , the first GATA element is solely responsible for GATA-1-mediated transctivation.…”

“…Treatment with histone deacetylase inhibitors results in induction of CCR3 mRNA in myeloid cell lines (Tiffany et al, 1998; Ishihara et al, 2007; Kim et al, 2010). However, whether DNA methylation is involved in expression of the gene has not been reported.…”