Eosinophil-specific Regulation of gp91 Gene Expression by Transcription Factors GATA-1 and GATA-2

Abstract

The glycoprotein gp91phox is an essential component of the phagocyte NADPH oxidase and is expressed in eosinophils, neutrophils, monocytes, and B-lymphocytes. We previously suggested an eosinophil-specific mechanism of gp91 phox gene expression. To elucidate the mechanism, we performed functional assays on deletion mutants of the gp91 phox promoter in various types of gp91 phox -expressing cells. A 10-base pair (bp) region from bp ؊105 to ؊96 of the promoter activated transcription of the gene in eosinophilic cells, but not in neutrophilic, monocytic, or B-lymphocytic cells. A 2-bp mutation introduced into the GATA site spanning bp ؊101 to ؊96 (؊98GATA site) of the fragment abolished its activity. Gel shift assays using a GATA competitor and specific antibodies demonstrated that both GATA-1 and GATA-2 specifically bound to the ؊98GATA site with similar affinities. Individual transfection of GATA-1 and GATA-2 into Jurkat cells, which have neither endogenous GATA-1 nor GATA-2, activated the ؊105/؉12 construct in a ؊98GATA site-dependent manner. Combined transfection of GATA-1 and GATA-2 activated the promoter less than transfection of GATA-1 alone. These results suggest that GATA-1 is an activator and that GATA-2 is a relative competitive inhibitor of GATA-1 in the expression of the gp91 phox gene in human eosinophils.

Novel Combinatorial Interactions of GATA-1, PU.1, and C/EBPε Isoforms Regulate Transcription of the Gene Encoding Eosinophil Granule Major Basic Protein

“…We have shown that eosinophil-committed promyelocytic cell lines express GATA-1-3 mRNA in an inducible fashion, and peripheral blood eosinophils of mixed developmental maturity isolated from patients with the hypereosinophilic syndrome also express GATA-1 mRNA (21). Studies in the avian system using myb-ets-transformed myeloid progenitors have suggested that an intermediate level of GATA-1 expression in the context of C/EBP␤ and PU.1 is critical for eosinophil lineage-specific gene expression and differentiation (1,2,7,12,22). As well, GATA-1 expression is slowly down-regulated during eosinophilopoiesis in the avian system (3,7).…”

Cooperation of STAT-1 and IRF-1 in Interferon-γ-induced Transcription of the gp91 Gene

“…Constructs pϪ267/Luc, pϪ115/Luc, pϪ95/Luc, and pϪ84/Luc were prepared using the PCR method using pGV-267/Luc which had the fragment of the gene extending from its initiation codon to upstream bp Ϫ267 as the template as described previously in detail (21). A mutant of GAS (pϪ115GASm/Luc) with a two-point mutation (TC to AG) from the bp Ϫ103 to Ϫ102 and a mutant of ISRE (pϪ115IRFm/Luc) with a fivepoint mutation (AGGG to TCCC) from bp Ϫ87 to Ϫ84 were prepared from pϪ115/Luc by PCR using a mutated primer.…”

“…HAF-1 is a multiprotein complex, and its components are still not defined, but PU.1, IRF-1, ICSBP, and Elf-1 are picked out as candidates (19,20). GATA-1, GATA-2, and GATA-3 regulate transcription of the gp91 phox gene in eosinophilic cells (21,22). IFN-␥ is a cytokine that plays an important role in both innate and adaptive immunities (23).…”

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

“…However, it is not clear whether the double-GATA site is essential for the transcriptional activation of eosinophil-specific genes. Charcot-Leyden crystal protein (27) and gp91phox (40) are encoded by eosinophil-specific genes that contain a single-GATA site in their promoters, and mutation of these GATA sites significantly suppresses transcriptional activity. In addition, although the MBP P2 promoter contains a double-GATA site, mutation of one of the two GATA subsites is sufficient to inhibit transcriptional activity (26).…”