Figure 6

MLH3 is a p73-specific target induced by hydroxyurea. (a) Western blotting analysis of lysates from untreated or hydroxyurea (HU)-treated HCT116-3(6) cells (1 mM, 16 hours) stably expressing lacZ, p53, or p73 small hairpin RNA (shRNA). (b) Effect of p53 or p73 knockdown on p21cip1 gene expression. Real-time RT-PCR analysis of the steady-state p21cip1 mRNA level in HCT116-3(6) cells stably expressing p53 or p73 shRNA *P < 0.05, n = 3 (c) Chromatin from untreated or HU-treated HCT116-3(6) cells (1 mM, 16 hours) were immunoprecipitated with the indicated antibodies and followed by PCR analysis using the primers flanking the ETF1 promoter region defined by the model-based algorithm for promoter array (MAP) promoter-score. Data are represented as log2 occupancy units from two independent ChIP experiments +/- standard deviation. Enrichment was confirmed by agarose gel electrophoresis analysis (bottom). (d) MAP window sequence. Primers used for qChIP analysis in (c) are indicated in bold. The core consensus sequence of p53 is underlined. (e) ETF1 mRNA level in the stable cell lines described in (a) with or without HU treatment was examined by RT-PCR analysis. (f) qChIP analysis of the MLH3 promoter was performed as in (c). (g) MAP window sequence. Primers used for qChIP analysis in (f) are shown in bold. The core consensus sequence of p53 is underlined. The p53 half site is highlighted in red and underline indicates mismatches. (h) Effect of p53 or p73 knockdown on MLH3 gene expression. Same analysis was carried out as in (e). *P < 0.05, n = 3.