Figure 7. RA increases histone acetylation at the Enhancer region containing NF-AT/Smad3 binding sites and facilitates increased Smad3 binding to Enhancer.

(A) Purified CD4⁺ cells from B6 mice were stimulated with plate bound anti-CD3 (10µg/ml), soluble anti-CD28 (2µg/ml) and TGF-β (5ng/ml) with or without retinoic acid (200nM) for 48 hours. CHIP assay was performed using anti-acetyi histone H4 antibody or rabbit IgG. Precipitated chromatins were subjected to real time PCR using primers targeting enhancerI region (upper) or promoter region (lower). Values were shown as the percentage of corresponding input. Data are representative of three independent experiments.

(B) Purified CD4⁺ cells from B6 mice were treated as in (A) with or without IL-27 (20ng/ml) for 2 hours. CHIP assay was performed using anti-Smad3 antibody or rabbit IgG. Precipitated chromatins were subjected to real time PCR using primers targeting enhancer I region. Values were shown as the percentage of corresponding input. Data are representative of two independent experiments.

(C) Purified CD4⁺ cells from B6 mice were stimulated with plate bound anti-CD3 (10µg/ml), soluble anti-CD28 (2µg/ml) and TGF-β (5ng/ml) with or without JNK kinase inhibitor (10µM) for 2 hours. CHIP assay was performed using anti-Smad3 antibody or rabbit IgG as described in (B).