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 subunit (IL-5R) expressed by the eosinophil lineage, that associates with a 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 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 exon 1. To initially identify the IL-5R 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 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 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. Using electrophoretic mobility shift assays, we have identified a nuclear factor(s) that binds to the 34-bp functional region of the the promoter and that is expressed in the myeloid and eosinophilic cell lines in which the promoter is active, but not in non-myeloid or non-hematopoietic lines. This functional promoter segment likely serves as the binding site for a myeloid- and possibly eosinophil-specific transcription factor(s). Further study of the IL-5R promoter should elucidate unique transcriptional features of this gene whose expression is essential to the commitment and differentiation of multipotential myeloid progenitors to the eosinophil lineage and to the functional activation of the mature cell.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				J. Lee, J. Beliakoff, and Z. Sun The novel PIAS-like protein hZimp10 is a transcriptional co-activator of the p53 tumor suppressor Nucleic Acids Res., July 26, 2007; 35(13): 4523 - 4534. [Abstract] [Full Text] [PDF]

				X. Li, G. Thyssen, J. Beliakoff, and Z. Sun The Novel PIAS-like Protein hZimp10 Enhances Smad Transcriptional Activity J. Biol. Chem., August 18, 2006; 281(33): 23748 - 23756. [Abstract] [Full Text] [PDF]

				J. Du, M. J. Stankiewicz, Y. Liu, Q. Xi, J. E. Schmitz, J. A. Lekstrom-Himes, and S. J. Ackerman Novel Combinatorial Interactions of GATA-1, PU.1, and C/EBPepsilon Isoforms Regulate Transcription of the Gene Encoding Eosinophil Granule Major Basic Protein J. Biol. Chem., November 1, 2002; 277(45): 43481 - 43494. [Abstract] [Full Text] [PDF]

				K. Morotomi-Yano, K.-i. Yano, H. Saito, Z. Sun, A. Iwama, and Y. Miki Human Regulatory Factor X 4 (RFX4) Is a Testis-specific Dimeric DNA-binding Protein That Cooperates with Other Human RFX Members J. Biol. Chem., January 4, 2002; 277(1): 836 - 842. [Abstract] [Full Text]

				E. Nutku, Q. Zhuang, A. Soussi-Gounni, F. Aris, B. D. Mazer, and Q. Hamid Functional Expression of IL-12 Receptor by Human Eosinophils: IL-12 Promotes Eosinophil Apoptosis J. Immunol., July 15, 2001; 167(2): 1039 - 1046. [Abstract] [Full Text] [PDF]

				N. Zimmermann, B. L. Daugherty, J. L. Kavanaugh, F. Y. El-Awar, E. A. Moulton, and M. E. Rothenberg Analysis of the CC chemokine receptor 3 gene reveals a complex 5' exon organization, a functional role for untranslated exon 1, and a broadly active promoter with eosinophil-selective elements Blood, October 1, 2000; 96(7): 2346 - 2354. [Abstract] [Full Text] [PDF]

				J. Tavernier, J. Van der Heyden, A. Verhee, G. Brusselle, X. Van Ostade, J. Vandekerckhove, J. North, S. M. Rankin, A. B. Kay, and D. S. Robinson Interleukin 5 regulates the isoform expression of its own receptor alpha -subunit Blood, March 1, 2000; 95(5): 1600 - 1607. [Abstract] [Full Text] [PDF]

				A. Iwama, J. Pan, P. Zhang, W. Reith, B. Mach, D. G. Tenen, and Z. Sun Dimeric RFX Proteins Contribute to the Activity and Lineage Specificity of the Interleukin-5 Receptor alpha  Promoter through Activation and Repression Domains Mol. Cell. Biol., June 1, 1999; 19(6): 3940 - 3950. [Abstract] [Full Text] [PDF]

				M. A. Giembycz and M. A. Lindsay Pharmacology of the Eosinophil Pharmacol. Rev., June 1, 1999; 51(2): 213 - 340. [Abstract] [Full Text] [PDF]

				N. C. Wilkinson and J. Navarro PU.1 Regulates the CXCR1 Promoter J. Biol. Chem., January 1, 1999; 274(1): 438 - 443. [Abstract] [Full Text] [PDF]

				P. Wang, P. Wu, B. Cheewatrakoolpong, J. G. Myers, R. W. Egan, and M. M. Billah Selective Inhibition of IL-5 Receptor {alpha}-Chain Gene Transcription by IL-5, IL-3, and Granulocyte-Macrophage Colony-Stimulating Factor in Human Blood Eosinophils J. Immunol., May 1, 1998; 160(9): 4427 - 4432. [Abstract] [Full Text] [PDF]

				A.-M. Eades-Perner, J. Thompson, H. van der Putten, and W. Zimmermann Mice Transgenic for the Human CGM6 Gene Express Its Product, the Granulocyte Marker CD66b, Exclusively in Granulocytes Blood, January 15, 1998; 91(2): 663 - 672. [Abstract] [Full Text] [PDF]

				M. A. Baumann and C. C. Paul The Aml14 and Aml14.3D10 Cell Lines: A Long-Overdue Model for the Study of Eosinophils and More Stem Cells, January 1, 1998; 16(1): 16 - 24. [Abstract] [Full Text]

				A. Sturrock, K. F. Franklin, and J. R. Hoidal Human Proteinase-3 Expression Is Regulated by PU.1 in Conjunction with a Cytidine-rich Element J. Biol. Chem., December 13, 1996; 271(50): 32392 - 32402. [Abstract] [Full Text] [PDF]

				H. L. Tiffany, J. S. Handen, and H. F. Rosenberg Enhanced Expression of the Eosinophil-derived Neurotoxin Ribonuclease (RNS2) Gene Requires Interaction between the Promoter and Intron J. Biol. Chem., May 24, 1996; 271(21): 12387 - 12393. [Abstract] [Full Text] [PDF]

				J. Du, Y. M. Alsayed, F. Xin, S. J. Ackerman, and L. C. Platanias Engagement of the CrkL Adapter in Interleukin-5 Signaling in Eosinophils J. Biol. Chem., October 13, 2000; 275(42): 33167 - 33175. [Abstract] [Full Text] [PDF]