FIGURE 8.

NFI-X3 induces the recruitment of RNA PolII to the gfap promoter. A, DNA was isolated from U373-Vec, U373-NFI-X1, and U373-NFI-X3 cells and bisulfite-converted, and methylation of suitable CpG was analyzed as described under “Experimental Procedures.” Empty circles indicate demethylated CpGs. B, lysates from U373-Vec, U373-NFI-X1-FLAG, and U373-NFI-X3-FLAG cells were immunoprecipitated with anti-FLAG antibodies, and precipitates were analyzed by Western blotting using anti-CBP and anti-NFI antibodies (inset). ChIP was performed using chromatin prepared from U373-Vec, U373-NFI-X1, and U373-NFI-X3 cells or from U373-Vec, U373-NFI-X1-FLAG, and U373-NFI-X3-FLAG cells as indicated. Both the gfap enhancer and core promoter were analyzed for the presence of acetylated histone H3, trimethylated histone H3 Lys⁴, RNA PolII, and NFI-X1 or NFI-X3 (anti-FLAG) using the antibodies described under “Experimental Procedures.” NRS, normal rabbit serum used for immunoprecipitation. Results are shown as a percentage of input. Experiments were performed three times. Error bars, S.D.