Abstract
Regulation of the IL-5 receptor alpha (IL5RA) gene is complicated, with two known promoters (P1 and P2) driving transcription, and two known isoforms (transmembrane and soluble) dichotomously affecting the signaling potential of the protein products. Here, we sought to determine the patterns of P1 and P2 promoter usage and transcription factor occupancy during primary human eosinophil development from CD34+ hematopoietic stem cell progenitors. We found that during eosinophilopoiesis, both promoters were active but subject to distinct temporal regulation, coincident with combinatorial interactions of transcription factors, including GATA-1, PU.1, and C/EBP family members. P1 displayed a relatively constant level of activity throughout eosinophil development, while P2 activity peaked early and waned thereafter. The soluble IL-5Rα mRNA peaked early and showed the greatest magnitude fold-induction, while the signaling-competent transmembrane isoform peaked moderately. Two human eosinophilic cell lines whose relative use of P1 and P2 were similar to eosinophils differentiated in culture were used to functionally test putative transcription factor binding sites. Transcription factor occupancy was then validated in primary cultures by ChIP. We conclude that IL-5-dependent generation of eosinophils from CD34+ precursors involves complex and dynamic activity including both promoters, several interacting transcription factors, and both signaling and antagonistic protein products.
Highlights
The high-affinity interleukin (IL)-5 receptor is a heterodimeric protein consisting of an α subunit that binds to the ligand, IL-5, and a β common subunit that is shared with the receptors for IL-3 and granulocyte-macrophage colony stimulating factor (GM-CSF) [1,2]
Differential promoter usage and alternative splicing represent an elegant regulatory mechanism to provide tissue- and/or developmental stage-specific gene expression and transcript diversity from a single genetic locus. Such context-dependent transcription is critical during development, and aberrant promoter usage has been implicated in various diseases
Several genes important in eosinophil development are subject to gene expression modulation through alternative promoter usage
Summary
The high-affinity interleukin (IL)-5 receptor is a heterodimeric protein consisting of an α subunit that binds to the ligand, IL-5, and a β common (βc) subunit that is shared with the receptors for IL-3 and granulocyte-macrophage colony stimulating factor (GM-CSF) [1,2]. IL-5 signaling through its receptor is a critical event required for the differentiation, proliferation, recruitment and activation of the eosinophil [3,4]. The IL-5Rα subunit exists in two isoforms: transmembrane and soluble [12,13]. The transmembrane isoform with its cytoplasmic tail is required for signal transduction from the heterodimeric IL-5R complex [3,4], whereas the soluble isoform is thought to bind and sequester IL-5 to inhibit signaling [1,14]. Being the IL-5-binding subunit in the surface receptor [15,16], transmembrane IL-5Rα represents the rate-limiting component of the IL-5 signaling pathway, and its regulation may constitute an important aspect of primary eosinophil development
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