Wild type and tumor-derived mutant p53 share a non-canonical mode of gene occupancy [ChIP-seq]

To directly examine the interplay between mutant p53 or Mdm2 and wild type p53 in gene occupancy and expression, an integrated RNA-seq and ChIP-seq analysis was performed in vivo using isogenically matched mouse strains. Response to radiation was used as an endpoint to place findings in a biologically relevant context. Unexpectedly, mutant p53 and Mdm2 only inhibit a subset of wild type p53 gene expression. In contrast to a dominant-negative or inhibitory role, the presence of either mutant p53 or Mdm2 actually enhances the occupancy of wild type p53 on many canonical targets. The C-terminal 19 amino acids of wild type p53 suppresses the p53 response allowing for survival at sublethal doses of radiation. Further, the mutant 172H p53 is shown to occupy genes and regulate their expression via a non-canonical means that is shared with wild type p53. This results in the heterozygous 172H/+ genotype having an expanded transcriptome compared to wild type p53+/+. Overall design: Briefly, approximately cells were crosslinked with 1% formaldehyde solution, quenched with glycine (0.125 M) and harvested in cold PBS. Sonication of crosslinked chromatin was performed using a Bioruptor® UCD-200TM Sonicator (Diagenode) with optimized cycles for each cell type. Immunoprecipitated DNA fragments (using CM5 antibody, or IgG (Rabbit IgG D1 antibody – Santa Cruz)) were purified the following day using the Qiagen Qiaquick PCR purification Kit Protocol and antibody/bead complexes isolated with Magna ChIPTM Protein A+G magnetic beads (Millipore). Complexes were washed, reverse crosslinked, and treated sequentially with RNase and proteinase K prior to DNA isolation. The sample and input DNA can be used for PCR or measured using the Qubit reader and used for ChIP-seq.