The complex architecture of p53 binding sites

Abstract

Sequence-specific protein-DNA interactions are at the heart of the response of the tumor-suppressor p53 to numerous physiological and stress-related signals. Large variability has been previously reported in p53 binding to and transactivating from p53 response elements (REs) due, at least in part, to changes in direct (base) and indirect (shape) readouts of p53 REs. Here, we dissect p53 REs to decipher the mechanism by which p53 optimizes this highly regulated variable level of interaction with its DNA binding sites. We show that hemi-specific binding is more prevalent in p53 REs than previously envisioned. We reveal that sequences flanking the REs modulate p53 binding and activity and show that these effects extend to 4–5 bp from the REs. Moreover, we show here that the arrangement of p53 half-sites within its REs, relative to transcription direction, has been fine-tuned by selection pressure to optimize and regulate the response levels from p53 REs. This directionality in the REs arrangement is at least partly encoded in the structural properties of the REs. Furthermore, we show here that in the p21-5′ RE the orientation of the half-sites is such that the effect of the flanking sequences is minimized and we discuss its advantages.

Dynamics of p53 DNA binding sites contributes to functional selectivity of p53-driven gene expression

“…We have previously compiled a list of 250 p53 REs, validated for binding in cellular context and for p53-dependent gene expression (27). These REs belong to 235 genes that are solely activating, do not contain spacer sequences between the two half sites, and are found within the promoter or the first intron/exon region (27). To classify these p53 genes by their cellular function, we looked for their specific function, the biological pathway (or process) initialized by them, and the functional outcome at the end of their biological pathway (Table S1).…”

“…p53 clusters (i.e., p53 REs that contain more than two half sites) are known to confer additional binding affinity and increase transactivation levels from the nearby gene (29,38). We chose to include only the most flexible full-site RE of any given cluster, based on our previous study, where we showed that there is a weak but significant correlation between RE conservation and deformability (27). We therefore hypothesize that the more conserved full-site RE (i.e., RE with sequence that is closer to the consensus sequence) is the primary RE of a cluster of REs, and hence will be bound first.…”

“…Here again, we analyzed the data by taking one RE per gene, except for the three sites containing three abutting half sites, mentioned above, where we included both sites in the analysis (i.e., once where the common middle half site is the 5’ half-site of RE1, and once where it is the 3’ half-site of RE2). Figure 2 shows representative patterns for selected functional groups, created by Weblogo (34,35), displaying dramatic changes between them, in all positions (see Figure S1 for the pattern of all other groups), and especially relative to the degenerate pattern of all 250 REs together, taken from our previous study (27). To quantitate the patterns observed in the sequence logos, we calculated Rseq for each pattern (Table S4).…”

“…We previously assembled a set of 250 p53 REs without spacers that contain sites validated for binding in cellular context and for p53-dependent gene expression (27) that belong to 235 genes (some genes contain multiple REs). We located each gene in the Genecards suite (https://www.genecards.org/) by their Entrez ID, gene symbol and gene name.…”

Evaluating the Influence of a G-Quadruplex Prone Sequence on the Transactivation Potential by Wild-Type and/or Mutant P53 Family Proteins through a Yeast-Based Functional Assay

“…Many studies have highlighted that RE recognition by P53 is regulated by the direct contacts with target DNA sequence; however, the so-called indirect readout provided by nucleotides within the RE that are not directly contacted by the P53 protein is also important [49,50]. This indirect readout, also referred to as "shape readout" since it appears to be related to structural properties at the P53 DNA target sites, has been recently extended to sequences surrounding the P53 RE [51].…”