Figure 7. XBP1 binds to the promoter of the Nfatc1 gene and promotes its transcription.

(A) A schematic of the promoter region of the Nfatc1 gene. The binding sites for NF-κB (gray circle), NFATc1 (black circles), and 2 putative XBP1s-binding sites (white rectangles) are shown. The left-right arrow indicates the expected PCR products for the ChIP assays in Figure 5B. (B) ChIP assays using control IgG, antibodies against XBP1 (αXBP1), or NFATc1 (αNFATc1). Cell extracts were collected from WT BMMs treated with (upper and middle panel) or without (lower panel) sRANKL. PCR of the cell extracts was performed using a set of primers either inside (–764 ~ 551) or outside (–942 ~ 811) of the promoter region containing the NFATc1-binding sites. Relative amount of the immunoprecipitated DNA fragment assessed by quantitative PCR (right panel). The amount of immunoprecipitated DNA of control IgG is set to 1. **P < 0.005. (C) Luciferase reporter assays using the WT construct (left, Nfatc1-800) or deletion mutants (middle, Nfatc1-800Δ-697-693; right, Nfatc1-800Δ-667-663) and the XPB1s and NFATc1 expression vectors. Fold induction of luciferase activity of each construct is presented. Luciferase activity of the cells transfected with reporter vector alone is set to 1. n = 3 replicates. Values represent mean ± SD. **P < 0.005. (D) Relative transcript expression levels of Nfatc1, Ctsk, and Acp5 in WT and Ern1^Mx1 BMMs transfected with GFP or NFATc1 expression vector. BMMs transduced with the indicated vectors were incubated in the presence of CSF1 and sRANKL for 3 to 4 days to induce differentiation prior to analysis. n = 4 replicates. Values represent mean ± SD. **P < 0.005; ***P < 0.0005. Statistical analysis was performed using Student’s t test.