GATA Transcription Factors Regulate the Expression of the Human Eosinophil-derived Neurotoxin (RNase 2) Gene

Abstract

The transcription factors GATA-1 and GATA-2 have been implicated in promoting differentiation of eosinophilic leukocytes. In this study, we examined the roles of GATA-1 and GATA-2 in activating transcription of the secretory ribonuclease, the eosinophil-derived neurotoxin (EDN/RNase 2). Augmented expression of both GATA-1 and GATA-2 was detected in eosinophil promyelocyte HL-60 clone 15 cells in response to biochemical differentiation with butyric acid. Deletion or mutation of one or both of the two consensus GATA-binding sites in the extended 1000-bp 5 promoter of the EDN gene resulted in profound reduction in reporter gene activity. Antibody-augmented electrophoretic mobility shift and chromatin immunoprecipitation analyses indicate that GATA-1 and GATA-2 proteins bind to both functional GATA consensus sequences in the EDN promoter. Interestingly, RNA silencing of GATA-1 alone had no impact on EDN expression; silencing of GATA-2 resulted in diminished expression of EDN, and also diminished expression of GATA-1 in both butyric acid-induced HL-60 clone 15 cells and in differentiating human eosinophils derived from CD34 ؉ hematopoietic progenitors. Likewise, overexpression of GATA-2 in uninduced HL-60 clone 15 cells resulted in augmented transcription of both EDN and GATA-1. Taken together, our data suggest that GATA-2 functions directly via interactions with the EDN promoter and also indirectly, via its ability to regulate the expression of GATA-1 in differentiating eosinophils and eosinophil cell lines.

Fractionation of Mature Eosinophils in GATA-Reporter Transgenic Mice

“…These results thus demonstrate that G1-GFP is a useful marker for eosinophil isolation from the bone marrow, and that cells in the eosinophil lineage are highly enriched in the G1-R1 fraction. It is interesting to note that, in agreement with previous reports on eosinophils (Querfurth et al 2000;McNagny and Graf 2002;Qiu et al 2009), both Gata1 and Gata2 mRNA was expressed in the G1-R1 eosinophilic fraction.…”

“…3A) in LyE -/c-Kit -/CD71 -bone marrow cells. In addition, several reports have demonstrated simultaneous expression of the Gata1 and Gata2 genes in EoP fractionated with an anti-IL-5Rα antibody from mouse bone marrow (Iwasaki et al 2005(Iwasaki et al , 2006Mori et al 2009) or in eosinophils obtained by culturing progenitor cells (Qiu et al 2009). However, the major eosinophilic fraction in G1-Red:G2-GFP mouse bone marrow is the G1-Red + /G2-GFP -(G1G2-R3) fraction, in which the Gata2 mRNA expression level is very low or undetectable by quantitative RT-PCR analysis.…”

“…On the other hand, the balance between GATA2 and C/EBPα expression regulates the cell-fate decision of progenitor cells (Iwasaki et al 2006). RNA silencing and overexpression experiments demonstrate that GATA2 and C/EBPα , but not GATA1, drive eosinophil-specific gene expression (Fukushima et al 2009;Qiu et al 2009). These data suggest that GATA1 and GATA2 might regulate common but separate pathways of eosinophil development, and that expression of these genes might be used as a tool for isolating eosinophils.…”

“…CD16, Gr1,Mac1,etc) that are shared by other cells of hematopoietic origin and are normally present in inflammatory infiltrates, hampers the easy detection of eosinophils using flow cytometry (Hansel et al 1991;Gounni et al 2000;Stevens et al 2007;Fukushima et al 2009). To analyze the physiology and pathology of eosinophils, cytokine-induced differentiation systems of hematopoietic progenitor cells or cell lines are exploited (Clutterbuck and Sanderson 1988;Clutterbuck et al 1989;Caldenhoven et al 1998;Yamaguchi et al 1999;Querfurth et al 2000;Dyer et al 2008;Fukushima et al 2009;Qiu et al 2009). However, it is possible that cytokine-induced eosinophils are crucially different from eosinophils in vivo.…”

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

“…GATA-1 is expressed in human peripheral eosinophils and eosinophilic cell lines (24), and eosinophil progenitors are found exclusively within cells activating GATA-1 transcription (25). The promoters of the genes that encode the eosinophil-specific major basic proteins (MBPs), Charcot-Leyden crystal protein and eosinophil-derived neurotoxin (EDN), contain functional GATA elements at which GATA-1 interacts with other transcription factors to activate transcription (26)(27)(28). Zimmermann et al (19) showed that GATA-1 binds to a double-GATA element within exon 1 of the CCR3 gene.…”