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.
Highlights
The tumor suppressor p53 (OMIM no. 191170) is a sequencespecific master regulatory gene that controls an extensive transcriptional network providing for genome integrity in response to cellular and environmental stresses or damage [1,2,3]. p53 differentially regulates the expression of target genes, as well as microRNAs associated with cell cycle control, apoptosis, DNA repair, angiogenesis, senescence and carbon metabolism [4,5]
A consensus p53 DNA binding sequence has been derived comprising a motif of two decamers RRRCWWGYYY (n) RRRCWWGYYY, where each decamer is composed of two adjacent p53 monomer binding sites in inverted orientation [16,17,18]. p53 binds cooperatively to the consensus response elements (REs) as a dimer of dimers, where a tetramer is the accepted functional unit required for full transcriptional activity [19,20,21,22,23,24,25,26]
While many factors may determine p53 transactivation of individual genes in human cells including stress stimuli, post-translational modifications, and transcriptional cofactors, the yeast system addresses the potential for wild type and mutant p53 to bind and transactivate from various REs derived from human genes when placed in a constant chromatin environment
Summary
The tumor suppressor p53 (OMIM no. 191170) is a sequencespecific master regulatory gene that controls an extensive transcriptional network providing for genome integrity in response to cellular and environmental stresses or damage [1,2,3]. p53 differentially regulates the expression of target genes, as well as microRNAs associated with cell cycle control, apoptosis, DNA repair, angiogenesis, senescence and carbon metabolism [4,5]. Paramount to p53 transcriptional function is the direct interaction between p53 and its targeted DNA sequence The nature of this interaction could per se determine transactivation capacity, as well as influence p53-mediated biological processes [9]. Such activities are often altered during human cancer development as highlighted by the frequent appearance of p53 missense mutations in its sequencespecific DNA binding domain [10,11] which can abrogate or alter p53 transactivational activity that result in changes in biological responses, such as the balance between apoptosis and survival in response to DNA damage. Most functional response elements (REs) identified in association with p53 target genes depart from this consensus, where base changes are tolerated at each position with the exception of the C and G at positions 4 and 7 in each half-site [27,28]
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