A p53-Pax2 Pathway in Kidney Development: Implications for Nephrogenesis
Figure 5
Differential p53 binding at −844 in Pax2 promoter in mK4 (Pax2-expressing) versus mK3 (Pax2-non-expressing) cells. A
) (a) ChIP-Seq track showing p53 occupancy at Pax2 proximal promoter (Region 1). (b) That this enrichment is not an artifact of amplification and sequencing is shown by lack of enrichment in the Input sample. Presence of any spurious false peaks in the Input sample eliminates that region from an interval, reflecting high stringency set for the MACS program. (c) Grey bar shows region of p53 enrichment designated as an interval. The dotted green arrow denotes RefSeq TSS, solid green arrow shows TSS described by [51]. (d) p53 motifs (blue bars) identified by Genomatix and manually. (e) Position of amplicons to validate ChIP-Seq data and show differential p53 occupancy between mK3 and mK4 cells by ChIP-PCR. (f) Co-ordinates on mus chromosome 19. B) Chromatin was immunoprecipitated from both cell lines with anti-p53 antibody and amplified by PCR. PCR band intensities were quantified using the Alphaimager software as described in Methods, and band intensity of immunoprecipitated fragments was normalized to Input band intensity for each cell line. Normalized values for each amplicon were plotted as mK4/mK3 ratios. Values greater than 1.0 indicate fragment enrichment in mK4 relative to mK3, as a result of increased p53 binding and immunoprecipitation as seen for amplicons P3 and P7 (blue line). Values are a mean of three independent ChIP experiments.