Abstract
Elevated intracellular cyclic AMP levels, which suppress the proliferation of naive T cells and type 1 T helper (Th1) cells are a property of T helper 2 (Th2) cells and regulatory T cells. While cyclic AMP signals interfere with the IL-2 promoter induction, they support the induction of Th2-type genes, in particular of il-5 gene. We show here that cyclic AMP signals support the generation of three inducible DNase I hypersensitive chromatin sites over the il-5 locus, including its promoter region. In addition, cyclic AMP signals enhance histone H3 acetylation at the IL-5 promoter and the concerted binding of GATA-3 and NFATc to the promoter. This is facilitated by direct protein-protein interactions involving the C-terminal Zn(2+)-finger of GATA-3 and the C-terminal region of the NFATc1 DNA binding domain. Because inhibition of NFATc binding to the IL-5 promoter in vivo also affects the binding of GATA-3, one may conclude that upon induction of Th2 effector cells NFATc recruits GATA-3 to Th2-type genes. These data demonstrate the functional importance of cyclic AMP signals for the interplay between GATA-3 and NFATc factors in the transcriptional control of lymphokine expression in Th2 effector cells.
Highlights
DNA on chromosomal locus 5q31 in man and chromosome 11 in mouse [1]
These results indicate the importance of locus control region (LCR) in the commitment phase of T helper 2 (Th2) cell differentiation but suggest that the LCR is less important for lymphokine gene expression in Th2 effector cells (4 – 6)
While there are no data on a direct interaction between GATA-3 and NFATc factors in T cells so far, NFATc and GATA-3 were shown to bind in concert to the 3Ј enhancer of murine IL-4 gene in Th2 cells [22]
Summary
Transfections, and DNA Constructs—Murine EL-4 thymoma cells were grown in RPMI medium containing 5% FCS, and 293 human embryonic kidney (HEK) cells in Dulbecco’s modified Eagle’s medium containing 10% FCS. The proximal human IL-5 promoter spans the nucleotides from position ϩ44 to Ϫ120, the longer promoter version includes the nucleotides up to Ϫ507 Both were cloned as HindIII/XhoI fragments in front of the TATA-luciferase vector. For bacterial expression the Zn2ϩ finger domain of murine GATA-3 (amino acids 252–349) was amplified by PCR and cloned into the BamHI/SalI sites of pGEX-6P-1 (Amersham Biosciences). Immunoprecipitations from primary murine Th2 cells were performed using the nuclear complex Co-IP kit (54001, Active Motif) according to the manufacturer’s protocol. Nuclear extracts of 293 HEK cells were prepared 48 h after transfection with the empty expression vector or the plasmid encoding the HA-tagged RSD of human NFATc1 (amino acids 417–716, HA Nc1-RSD). ELISA was performed for detection of IL-5 from cell-free culture supernatants using mAbs according to the manufacturer’s instructions (BD Biosciences)
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