Figure 2. Regulation of Grail expression in Th2 cells.

(a) Naïve CD4⁺ T cells from WT and Stat6 KO mice were cultured for 4 days under Th0 and Th2 conditions and analyzed for Grail mRNA expression by qRT-PCR. (b) ChIP analysis of histone H3 acetylation (AcH3),trimethyl histone H3 lysine 4 (H3k4) and trimethyl histone H3 lysine 27 (H3k27) at Grail promoter locus in CD4⁺ T cells from WT and Stat6 KO mice polarized under Th2 condition. The data from each replicate were normalized to the input control and the graphs represent fold enrichment of the indicated proteins to control antibody (rabbit IgG) at the designated locus. (c) ChIP assay to check binding of indicated transcription factors to the Grail promoter locus in naïve CD4⁺ T cells polarized under Th0 and Th2 conditions. (d) Grail and Il4 mRNA expression in naïve CD4⁺ T lymphocytes infected with bicistronic retroviruses expressing the indicated factors. (e) Luciferase assay in EL-4 cells transfected with Grail promoter (Grail-P) containing luciferase vector or with a mutated Gata3 site (GrailP-mu) promoter along with vectors containing the indicated factors. Luciferase activity is expressed relative to the expression of cotransfected renilla luciferase plasmid as a control for transfection efficiency. Relative luciferase units are expressed as a fold difference to the control (vector only, pGL3) value. (f) Grail mRNA expression in naïve CD4⁺ T lymphocytes from WT and Stat6 KO mice infected with bicistronic retroviruses expressing the indicated factors. The data shown in a, d and f were normalized by the expression of a reference gene Actb. The results shown are mean ± SEM. P values: *<0.05, **<0.01, Student’s t-test was performed to detect between-group differences.. The data are representative of least three independent experiments with consistent results.