The human IL-3 locus is regulated cooperatively by two NFAT-dependent enhancers that have distinct tissue-specific activities.
The human IL-3 gene is expressed by activated T cells, mast cells, and eosinophils. We previously identified an enhancer 14 kb upstream of the IL-3 gene, but this element only functioned in a subset of T cells and not in mast cells. To identify additional mechanisms governing IL-3 gene expression, we mapped DNase I hypersensitive (DH) sites and evolutionarily conserved DNA sequences in the IL-3 locus. The most conserved sequence lies 4.5 kb upstream of the IL-3 gene and it encompassed an inducible cyclosporin A-sensitive DH site. A 245-bp fragment spanning this DH site functioned as a cyclosporin A-sensitive enhancer, and was induced by calcium and kinase signaling pathways in both T cells and mast cells via an array of three NFAT sites. The enhancer also encompassed AML1, AP-1, and Sp1 binding sites that potentially mediate function in both T and myeloid lineage cells, but these sites were not required for in vitro enhancer function in T cells. In stably transfected T cells, the -4.5-kb enhancer cooperated with the -14-kb enhancer to activate the IL-3 promoter. Hence, the IL-3 gene is regulated by two enhancers that have distinct but overlapping tissue specificities. We also identified a prominent constitutive DH site at -4.1 kb in T cells, mast cells, and CD34(+) myeloid cells. This element lacked in vitro enhancer function, but may have a developmental role because it appears to be the first DH site to exist upstream of the IL-3 gene during hemopoietic development before IL-3 expression.
Hawwari
Abbas
A
Hanson Center for Cancer Research, Institute of Medical and Veterinary Science, Adelaide, Australia.
Burrows
Joanna
J
Vadas
Mathew A
MA
Cockerill
Peter N
PN
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D017124:Conserved Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics*; D003850:Deoxyribonuclease I; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D006801:Humans; D007377:Interleukin-3 / Q000235:genetics*; D008407:Mast Cells / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D012689:Sequence Homology, Nucleic Acid; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D014018:Tissue Distribution; D014157:Transcription Factors / Q000235:genetics*; D014162:Transfection
D004268:DNA-Binding Proteins; D007377:Interleukin-3; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D014157:Transcription Factors; D004247:DNA; D003850:Deoxyribonuclease I