Targeted citrullination enables p53 binding to non-canonical sites [ChIP-seq]

TP53 encodes for the transcription factor p53, which binds to a diverse set of target genes in response to stress. Activation of p53 results in highly specific transcriptional responses, but the regulation of promoter selectivity by p53 is poorly understood. Here we report that sequence-specific binding of p53 is regulated by its target gene and binding partner PADI4. PADI4 enzymatically converts peptidyl-arginine to peptidyl-citrulline in a process known as citrullination. We show p53 is citrullinated in vitro at the C-terminus by PADI4, and we confirm two citrullination events in vivo (R306, R363). ChIP-seq reveals that PADI4 expression causes a redirection of p53 away from canonical binding sites to target genes associated with the ETS transcription factor and the interferon response. Chromatin profiling using citrullination-specific p53 antibodies supports this conclusion. These findings link citrullination to p53 function and illustrate how chromatin modifiers like PADI4 can redirect the p53 transcriptional response. Overall design: Chromatin immunoprecipitation followed by DNA-sequencing (ChIP-seq) of H3K27ac and p53 in HCT116 cells transfected with a tetracycline-inducible system to induce PADI4 or it's catalytically dead mutant (D350A) expression. Cells were either treated with vehicle (untreated), nutlin, or the combination of nutlin and doxycyline for 24 hours.