Abstract
A positive regulatory element in the interleukin-2 (IL-2) promoter, designated the antigen receptor response element-2, is essential for the induction of IL-2 gene expression upon the binding of an inducible multiprotein complex of proteins known as nuclear factor of activated T cells. In the current study, we demonstrated that the winged-helix transcription factor IL-2 enhancer binding factor (ILF) is constitutively expressed in both resting and activated Jurkat cells and binds to two adjacent sequence motifs immediately downstream of the binding site for NFAT. One of these elements has a high degree of homology with consensus binding sites for a variety of winged-helix DNA binding proteins, and the second site functions to modulate ILF binding. Mutagenesis of each of the two sequence elements required for ILF binding decreased IL-2 promoter activity when assayed in transfection assays. Although ILF bound constitutively to the IL-2 promoter, it was not detected as a component of the NFAT complex. These results suggest that important regulatory sequences in the IL-2 promoter are bound by ILF and that this binding may be involved in the control of IL-2 gene expression.
Highlights
Induction of IL-21 gene expression is a pivotal event in early T-lymphocyte activation
The 5Ј purine and core regions are bound in vivo by nuclear factor of activated cells (NFAT) and AP-1 transcription factors, respectively, and both of these elements have been demonstrated to be essential for maximal antigen receptor response element 2 (ARRE-2) transcriptional activity [6, 7, 11, 22]
Gel retardation analysis was performed with an IL-2 ARRE-2 probe to characterize the binding of constitutive and inducible factors that were present in nuclear extract prepared from nonactivated and phorbol myristate acetate (PMA) and ionomycin-activated Jurkat cells
Summary
Induction of IL-21 gene expression is a pivotal event in early T-lymphocyte activation. Winged-helix or forkhead proteins share a conserved 100-amino acid DNA binding domain and represent a family of transcription factors that participate in a number of processes that regulate cellular gene expression [25,26,27,28,29,30] Expressed factors such as ILF that bind to the ARRE-2 region may help to modulate the rapidity and tissue specificity of IL-2 mRNA induction. The 3Ј portion of the ILF binding site comprised of purine-rich sequences is not required for NFAT binding but is important for maximal IL-2 promoter activity These results suggest that ILF may have a positive role in regulating IL-2 gene expression, ILF is not a component of the multi-protein NFAT complex. ILF may be important both to prevent constitutive expression from the IL-2 promoter and to maintain the
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