RARα fails to bind the 5′ regulatory regions of the mouse CCR9 and Itg-α4 genes in BATF deficiency. WT or BATF KO CD4⁺ T cells were activated for 4–5 d with concanavalin A, IL-2, and RA. (A) Expression of nuclear RAR genes in BATF KO CD4⁺ T cells was examined by quantitative RT-PCR. Normalized values to β-actin levels are shown. (B and C) The binding of RARα and BATF and histone H4 acetylation on the CCR9 gene (B) or the Itg-α4 gene (C) were assessed by ChIP assay. Representative PCR data with duplicated measurements are shown. (D) WT or BATF KO CD4⁺ T cells were activated for 4–5 d with anti-CD3/CD28 in the presence of IL-2 and 10 nM RA and transfected with pGL4-5′-Itg-α4. The cells were reactivated with anti-CD3/28 + IL2 in the presence of 20 nM RA for 16 h, and luciferase activity was normalized to control Renilla luciferase activity. (E) Naive WT or BATF KO CD4⁺ T cells were activated with anti-CD3/CD28 or concanavalin A for 4–5 d in the presence of IL-2 and 10 nM RA and the indicated HDAC inhibitors (TSA, BML-210, or EX-527), and the expression of CCR9 and α4β7 was measured by flow cytometry. Graphs show the percentage of cells expressing CCR9 or α4β7. All of the experiments were performed at least three times, and pooled (A, D, and E) or representative (B and C) data are shown. Error bars are SEM obtained from pooled data (A, D, and E) or differences between duplicated measurements (B and C). Significant differences from the KO control group are shown (*, P < 0.05).