Identification and Characterization of a Functional Promoter Region in the Human Eosinophil IL-5 Receptor α Subunit Gene

Abstract

The molecular basis for the commitment of multipotential myeloid progenitors to the eosinophil lineage, and the transcriptional mechanisms by which eosinophil-specific genes are subsequently expressed and regulated during eosinophil development are currently unknown. Interleukin-5 (IL-5) is a T cell and mast cell-derived cytokine with actions restricted to the eosinophil and closely related basophil lineages in humans. The high affinity receptor for IL-5 (IL-5R) is composed of an alpha subunit (IL-5R alpha) expressed by the eosinophil lineage, that associates with a beta c subunit shared with the receptors for IL-3 and granulocyte-macrophage colony stimulating factor (GM-CSF). As a prerequisite to studies of the transcriptional regulation of the IL-5R alpha subunit gene, we used three different methods, including primer extension, RNase protection, and 5'-RACE to precisely map the transcriptional start site to a position 15 base pairs (bp) upstream of the 5' end of the published sequence of IL-5R alpha exon 1. To initially identify the IL-5R alpha promoter, 3.5 kilobases (kb) and 561 bp of the 5' sequence flanking the transcriptional start site were subcloned into the promoterless pXP2-luciferase vector. Transient transfection of these constructs into an eosinophil-committed HL-60 subline, clone HL-60-C15, induced the expression of approximately 240-fold greater luciferase activity than the promoterless vector, identifying a strong functionally active promoter region within the 561 bp of sequence proximal to the transcriptional start site and with activity equivalent to pXP2 constructs containing the entire 3.5 kb of upstream sequence. To more precisely localize the cis-acting regulatory elements in this region important for promoter activity, a series of 5' deletion mutants of the 561-bp region were generated in the pXP2-luciferase vector. Deletion of the region between bp -432 and -398 reduced promoter activity by more than 80% in the HL-60-C15 cell line. Further analyses of the activity of the IL-5R alpha promoter constructs in various other eosinophil, myeloid, and non-myeloid cell lines indicated that the promoter was relatively myeloid and eosinophil lineage-specific in its expression. Consensus sequences for known transcription factor binding sites were not present in the 34-bp region of the promoter required for maximal activity, suggesting unique myeloid- and possibly eosinophil-specific regulatory elements.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of a Novel Role of ZMIZ2 Protein in Regulating the Activity of the Wnt/β-Catenin Signaling Pathway

“…Reverse transcription was carried out following our previous report (37). Briefly, cDNA was synthesized from 1 g of total RNA with 9 units of avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI) using 0.1 M oligo(dT) primer in a total volume of 20 l. For quantitative PCR, cDNA samples were mixed with SYBR qPCR Super Mix Universal (Invitrogen) and specific primers in the MX 3005P thermocycler (Stratagene) according to the protocol of the manufacturer.…”

Engagement of the CrkL Adapter in Interleukin-5 Signaling in Eosinophils

“…The oligonucleotides were synthesized and purified by Integrated DNA Technologies, Inc. (Coralville, IA). To generate probes for electrophoretic mobility shift assays, 10 pmol of the double-stranded oligonucleotides were end-labeled with [ 32 P]ATP (PerkinElmer Life Sciences) using T4 polynucleotide kinase, and the double-stranded probes were purified on 15% polyacrylamide gels as described previously (60). For mobility shift assays, nuclear protein-DNA binding reactions were carried out at room temperature for 30 min in a final volume of 20 l containing the labeled oligonucleotide probe (10,000 cpm), 3 g of nuclear extract, and 2 g of poly(dI⅐dC) in 20 mM HEPES (pH 7.9) containing 50 mM KCl, 3 mM MgCl 2 , 1 mM dithiothreitol, 0.5 mM EDTA, and 5% glycerol.…”

“…Preparation of Whole Cell and Nuclear Extracts-Nuclear extracts were prepared using a rapid micropreparation technique for small numbers of cells (58) with minor modifications, including the use of protease inhibitor mixture tablets (Roche Molecular Biochemicals, Mannheim, Germany) and the addition of phenylmethylsulfonyl fluoride (0.5 mM) and diisopropyl fluorophosphate (1 mM) to the resuspension and lysis buffers (59,60).…”

The Aml14 and Aml14.3D10 Cell Lines: A Long‐Overdue Model for the Study of Eosinophils and More

“…Consensus sequences for known transcription factors were not found in the 34 bp region. Furthermore, gel shift experiments demonstrated the presence of a nuclear factor(s) that bound to the region only in myeloid cell lines [21,22]. Using methylation interference, the sequence responsible for nuclear protein binding was determined to be GTTGCCTAGG, extending from bp -430 to -421.…”