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. 2003 Aug 19;100(17):9934-9.
doi: 10.1073/pnas.1633803100. Epub 2003 Aug 8.

Functional mutants of the sequence-specific transcription factor p53 and implications for master genes of diversity

Michael A Resnick  1 , Alberto Inga
Affiliations

Functional mutants of the sequence-specific transcription factor p53 and implications for master genes of diversity

Michael A Resnick et al. Proc Natl Acad Sci U S A. .

Abstract

There are many sources of genetic diversity, ranging from programmed mutagenesis in antibody genes to random mutagenesis during species evolution or development of cancer. We propose that mutations in DNA sequence-specific transcription factors that _target response elements (REs) in many genes can also provide for rapid and broad phenotypic diversity, if the mutations lead to altered binding affinities at individual REs. To test this concept, we examined the in vivo transactivation capacity of wild-type human and murine p53 and 25 partial function mutants. The p53s were expressed in yeast from a rheostatable promoter, and the transactivation capacities toward >15 promoter REs upstream of a reporter gene were measured. Surprisingly, there was wide variation in transactivation by the mutant p53s toward the various REs. This is the first study to address directly the impact of mutations in a sequence-specific transcription factor on transactivation from a wide array of REs. We propose a master gene hypothesis for phenotypic diversity where the master gene is a single transcriptional activator (or repressor) that regulates many genes through different REs. Mutations of the master gene can lead to a variety of simultaneous changes in both the selection of _targets and the extent of transcriptional modulation at the individual _targets, resulting in a vast number of potential phenotypes that can be created with minimal mutational changes without altering existing protein-protein interactions.

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Figures

Fig. 1.
Fig. 1.
System for examining the transactivation capacity of wild-type and mutant p53s toward various response elements (REs). The p53 cDNA is expressed in yeast under the control of the GAL1,10 promoter, whose induction is regulated by varying the amounts of galactose. The ADE2 gene is controlled by p53 because a p53 RE (P21–3′ in the example) was inserted within a modified minimal promoter. Transactivation is assessed by colony color and depends on the amount of galactose in the synthetic medium (cells grown at five different concentrations are shown); the synthetic medium also contains 2% raffinose, which results in a basal level of p53 expression (18). A Western blot showing the amount of p53 protein is included.
Fig. 2.
Fig. 2.
Examples of p53-induced transactivation by using the ADE2 reporter gene assay and different levels of p53 expression. Wild-type human p53 and three functional mutants were examined at different levels of induction for their ability to induce transcription at four REs, as determined by change in colony color. The relative induction of p53 protein is shown. The supertrans T123A mutant showed enhanced activity with all four REs, whereas S215C, a mutant associated with familial breast cancer, exhibited subtle defects and the tumor hotspot mutant R282Q had no activity with the weak P21–3′ and BAX-B REs.
Fig. 3.
Fig. 3.
Relative transactivation capacity of p53 alleles with mutations in the DNA-BD. (A) Twenty-four human p53 mutations were examined in 15 isogenic yeast strains, each containing a different p53 RE that regulates expression of the ADE2 reporter gene. The REs are ranked from left to right according to their decreasing transactivation capacity with wild-type p53 (indicated at the bottom; also see Fig. 5). The p53 mutants are ordered according to their position in the primary sequence. The topological domains are shown. Red and black arrows identify mutations associated with familial breast cancer and sporadic cancers, respectively. The transactivation capacity of each allele toward each RE was determined by using variable expression of p53 under the GAL1 promoter and compared with the activity of wild-type p53. The relative transactivation capacities of mutants with respect to wild type is presented in a form similar to that for expression microarrays, with red indicating greatly increased, green indicating greatly decreased, blue indicating loss of function, and black equal to wild type. The quantification is based on the amounts of p53 protein required for transactivation with wild type or with a mutant allele. This was derived from the minimal amount of galactose required for transactivation to occur. p53 mutations with enhanced transactivation toward most REs are classified as supertrans (asterisks). Alleles with greatly altered patterns of specificity, including increase, decrease, and loss-of-function, are designated with squares. Alleles associated with sporadic and familial breast cancer are designated with black and red arrows, respectively. The transactivation capacity of murine wild-type p53 and of the T122L mutant (corresponding to T125L in the human protein) is also shown. The human and mouse wild-type p53s were not distinguishable in this assay. (B) In addition to the 15 REs examined above, seven supertrans and three altered specificity p53 mutants were characterized with six additional REs (Left). Although four REs (1× RGC through IGFBP3-A) are weak with wild-type p53 and one (IGFBP3-B) is not functional (see Fig. 5), enhanced transactivation was observed with most elements. Interestingly, the altered specificity mutants 125R and 125K showed reduced capacity with the strong RE of m-FAS but greatly enhanced activity with the weak PIG3 and IGFBP3-A REs. Three additional apoptotic REs were also investigated with nine more p53 alleles associated with cancer (Right). Mutant G279R showed enhanced activity with PUMA RE, reduced activity with BAX A+B, and lack of function with NOXA. The BAX A+B contains two adjacent p53 REs (see Table 1).
Fig. 4.
Fig. 4.
The piano analogy for the master gene of diversity hypothesis based on p53 responses. A master gene such as p53 controls the expression of many genes, similar to hands playing notes on a piano. The p(53) piano has many keys corresponding to direct _target genes (>50), and it is played by the “fingers” of the p53 “hand” via interaction with promoter REs. This creates a “chord” where each note (key) may have a different intensity (i.e., level of transactivation). In the case of p53 mutants that are completely nonfunctional, none of the _target genes are activated and “no sound” will emanate. However, for p53 mutants retaining partial function or exhibiting altered DNA-binding specificity, the p53 hand can have some inactive “fingers” that do not strike keys, and some fingers that are stronger (bold type) or weaker (smaller and lighter type). It is even possible that additional new keys (denoted by “x”) will be struck as would be the case for recognition of related REs. These changes can result in new chords and sounds with novel biological consequences.
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