Figure 6.

KLF4, Tip60 and HDAC7 bind to HDC promoter by chromatin immunoprecipitation assay. (A) Overnight starved AGSE cells were treated with 10⁻⁸ M gastrin for 2 h. Cells were then fixed with formaldehyde followed by protein extraction as described in Materials and Methods section. Antibodies against KLF4, Tip60, HDAC7, acetyl-H4 plus control IgG were used to precipitate DNA–protein complexes. Purified DNA samples from precipitated DNA–protein complexes were used as template for PCR to amplify a fragment of HDC promoter, intron 1 and intron 2. (B) Gastrin treatment increased 107HDC promoter activity. Vector (pGL2), 107HDC reporter and 107HDCM reporter were transfected into AGSE cells. Before harvesting, cells were treated with 10⁻⁸ M gastrin from 2 h. Then, luciferase assays were performed as described. (C) mRNA levels of KLF4, HDC, Tip60 and HDAC7 after 10⁻⁸ M gastrin treatment were shown. Total RNA was extracted after gastrin treatment at different time points. Reverse transcription and follow-up quantitative RT-PCR was performed. Relative mRNA level was calculated as described in Materials and Methods section. Means ± SD for three independent experiments were shown, and statistical difference (P < 0.05) of the relative mRNA levels was indicated by a star (*).