Functional analysis of a single nucleotide polymorphism in a potential binding site for GATA transcription factors in the ovine interleukin 2 gene

The transcription factor GATA-3 is one regulator of Th1/Th2 differentiation. In sheep, we recently discovered a putative GATA-binding site (WGATAR) in the second intron of the Th1-cytokine gene interleukin 2 (IL2), showing a single nucleotide polymorphism (G/C). As genetic variations in cytokine genes are thought to regulate cytokine production, we studied the significance of this polymorphism for IL2 transcription. Sheep with different IL2 genotypes were identified by single-strand conformation polymorphism (SSCP)-analysis and IL2 transcription levels in peripheral blood mononuclear cells (PBMC) isolated from these animals were compared. For this purpose, transcription of IL2 mRNA was quantified by real-time polymerase chain reaction in unstimulated PBMC and in PBMC incubated for 4 h in the presence of concanavalin A (ConA) or phorbol 12-myristate 13-acetate plus ionomycin (PMA/I). Compared to unstimulated cells, stimulation with ConA and PMA/I increased the IL2 mRNA transcription in average by 300- and 20-fold, respectively. Nevertheless, no significant differences in IL2 transcription between the genotypes could be detected. These findings were confirmed by band shift studies using different oligonucleotides containing variations of the potential binding motif, which showed no differences in the gel mobility after incubation with nuclear extract containing GATA-3. The obtained results argue against an impact of this polymorphism on the IL2 transcription and the genetic disease resistance in sheep.