Noncanonical DNA Motifs as Transactivation Targets by Wild Type and Mutant p53

Abstract

Sequence-specific binding by the human p53 master regulator is critical to its tumor suppressor activity in response to environmental stresses. p53 binds as a tetramer to two decameric half-sites separated by 0–13 nucleotides (nt), originally defined by the consensus RRRCWWGYYY (n = 0–13) RRRCWWGYYY. To better understand the role of sequence, organization, and level of p53 on transactivation at target response elements (REs) by wild type (WT) and mutant p53, we deconstructed the functional p53 canonical consensus sequence using budding yeast and human cell systems. Contrary to early reports on binding in vitro, small increases in distance between decamer half-sites greatly reduces p53 transactivation, as demonstrated for the natural TIGER RE. This was confirmed with human cell extracts using a newly developed, semi–in vitro microsphere binding assay. These results contrast with the synergistic increase in transactivation from a pair of weak, full-site REs in the MDM2 promoter that are separated by an evolutionary conserved 17 bp spacer. Surprisingly, there can be substantial transactivation at noncanonical ½-(a single decamer) and ¾-sites, some of which were originally classified as biologically relevant canonical consensus sequences including PIDD and Apaf-1. p53 family members p63 and p73 yielded similar results. Efficient transactivation from noncanonical elements requires tetrameric p53, and the presence of the carboxy terminal, non-specific DNA binding domain enhanced transactivation from noncanonical sequences. Our findings demonstrate that RE sequence, organization, and level of p53 can strongly impact p53-mediated transactivation, thereby changing the view of what constitutes a functional p53 target. Importantly, inclusion of ½- and ¾-site REs greatly expands the p53 master regulatory network.

A Regulatory Loop Composed of RAP80-HDM2-p53 Provides RAP80-enhanced p53 Degradation by HDM2 in Response to DNA Damage

“…A second p53-RE decamer is located 4 nt away and contains one mismatch in the CWWG core, making it at most a very weak contributor to p53 transactivation. Most functional p53 target genes have 0 -2-nt spacer (23,27,(43)(44)(45), and we recently demonstrated that a spacer of Ն3 nt greatly impairs p53 transactivation (26). Thus, although RE-5 fits the generally acknowledged consensus, our results demonstrate that the half-decamer is likely responsible for most of the p53-mediated transactivation at p53RE-5.…”

“…A 4-kb region surrounding the transcription start site of the human RAP80 promoter was scanned for the presence of p53 response element sequences (p53-REs). Although the commonly accepted consensus p53-RE consists of (RRRCWWGYYY followed by a 0 -13-nt spacer followed by RRRCWWGYYY) with up to approximately five mismatches, we recently established that a fully functional RE has a spacer of Յ3 nt and that a single decamer, a "noncanonical half-site" (23,25,26), could mediate transcriptional activation by p53. The CWWG core is important for p53 responsiveness, where C and G are essential and CATG is the strongest responder.…”

Understanding wild-type and mutant p53 activities in human cancer: new landmarks on the way to targeted therapies

“…Resnick (L9) made the same observation using a diploid yeast system, expressing p53 under the control of a galactose-dependent promoter and a reporter gene driven by p53RE. 3 In this system, increasing the number of spacer nucleotides resulted in a drastic decrease of both p53 transcriptional and DNA binding activities. He also showed that conservation of the p53RE core motif (CWWG) is the main requirement for the definition of a half site, the CATG motif appearing to be the most efficient one.…”

Transcriptional Regulation by P53

“…In fact, recent work has shown that tetrameric p53 is capable of binding to half-and even three quarterconsensus sites, such as those comprising the PIDD and Apaf-1 response elements (Jordan et al 2008). Using a Saccharomyces cerevisiae model system, one study found as much as a 1000-fold difference in transactivation by p53 for its weaker versus its stronger sites, with this difference being largely dependent on the central sequence element in the p53 RE (Inga et al 2002).…”