Identification of transcription factor binding sites important in the regulation of the human interleukin-5 gene.
This study identifies three regions of the human interleukin (IL)-5 promoter involved in binding nuclear factors from activated T cells. DNase I footprinting and mobility shift assays with nuclear proteins from the human T cell clone, SP-B21, demonstrated protein interactions with each of these response elements (REs), located between positions -79 and -45 (RE-I), -123 and -92 (RE-II), and -170 and -130 (RE-III). Two of these regions, RE-II and RE-III, have not previously been described to regulate IL-5 expression in T cells. The RE-II site was shown to be critical for inducible IL-5 promoter activity in transient transfection assays in D10.G4.1 T cells, while the RE-III site functions as a negative regulatory element. The activity of the RE-II site was specifically inhibited by cyclosporin A, and transfection assays with IL-5 constructs containing mutations in the RE-II site showed greatly reduced reporter gene activity. We have defined the sequence involved in stimulation-dependent transcription and have identified constitutive as well as inducible DNA-binding protein complexes that bind to RE-II. Antibodies against at least two members of the nuclear factor of activated T cells (NFAT) family of transcription factors are capable of binding to the IL-5 RE-II complexes, although they can be distinguished from previously identified NFAT-specific complexes by several characteristics.
Stranick
K S
KS
Department of Allergy, Schering-Plough Research Institute, Kenilworth, New Jersey 07033, USA. kimberly.stranick@spcorp.com
Zambas
D N
DN
Uss
A S
AS
Egan
R W
RW
Billah
M M
MM
Umland
S P
SP
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016572:Cyclosporine / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000502:physiology; D005786:Gene Expression Regulation; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics; D015848:Interleukin-5 / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000502:physiology*
D004268:DNA-Binding Proteins; D007376:Interleukin-2; D015848:Interleukin-5; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D014157:Transcription Factors; D016572:Cyclosporine; D004247:DNA