Development of a panel of isogenic strains containing p53-responsive promoters.
We sought to develop an in vivo system to systematically evaluate the contributions of RE sequence and p53 protein level to differential transactivation. This required the ability to assess the capacities for transactivation by various amounts of p53 protein towards many REs inserted at the same position within a reporter gene. Yeast-based p53 functional assays have provided valuable tools for screening tumor samples, classifying mutant p53 alleles, and structure-function studies (8
). High-level, constitutive expression of wild-type p53, but not tumor mutants, could activate a promoter containing a p53 RE in yeast (66
). Using an inducible p53 expression system, we had previously identified various functional classes of p53 mutant alleles (36
). Moreover, at low expression levels, wild-type p53 was more active with RGC and P21 REs than with a BAX RE (37
). However, because of differences in strain backgrounds and structural arrangements of the REs, these comparisons were qualitative and an evaluation of differential transactivation could not be addressed.
To explore differential transactivation by p53, we developed a new set of isogenic haploid yeast strains based upon the ADE2
red-white p53 reporter system initially described by Flaman et al. (24
). Presented in Fig. is a general system for incorporating REs into a promoter. We utilized our recently developed two-step in vivo mutagenesis system, termed delitto perfetto (68
), to rapidly generate a panel of yeast strains with modified p53-responsive CYC1
promoters. Briefly, the wild-type ADE2
open reading frame was integrated into the chromosome, replacing the ade2
allele in the yIG397 background (see Materials and Methods). The ADE2
promoter was also replaced with a minimal CYC1
promoter that lacked upstream activating sequences. Hence, ADE2
transcription is inactive, and the strain, named yAFM, is red in medium containing low levels of adenine due to the interruption of adenine biosynthesis at the step prior to the one catalyzed by Ade2. Applying the delitto perfetto approach, a CORE cassette (CO, counterselectable marker; RE, reporter gene) was inserted by homologous recombination upstream of the minimal CYC1
promoter, and then the cassette was replaced by using double-stranded oligonucleotides containing a specific p53 RE. Using this approach, we created 22 p53 REs from mammalian p53-regulated genes plus four artificial sequences (Table ). The correct RE insertions were confirmed by DNA sequencing. At least two independent isolates for each RE, named yAFM-RE, were tested in the functional assay. With the exception of the c-fos
RE (i.e., yAFM-cFOS), all of the RE sequences cloned upstream of the CYC1
promoter contained a single 20- to 22-bp p53 tetramer binding site. Most of the REs have no spacer between the dimer binding sites. Table describes the actual sequence of each element along with the mammalian gene name from which it was derived.
Rheostatable expression of p53 protein controlled by the GAL1-10 promoter.
A single-copy centromere plasmid, pLS89, containing the human wild-type p53 cDNA under control of the GAL1
promoter was transformed into the yAFM-RE strains. The GAL
promoter is repressed on glucose, while on raffinose a derepressed, higher basal transcriptional state is achieved (34
). The p53 level on glucose is about 15 times lower than that on raffinose (data not shown). When galactose is added to the raffinose medium, the promoter can be induced to a high level. In order to establish that the system was rheostatable for p53 protein expression, we determined p53 levels after growth in medium containing raffinose plus low levels of galactose (Fig. ). The amount of p53 was quantified by using densitometric scans of Western blots (Fig. ). The levels of p53 protein were examined in five isogenic yAFM-RE strains, providing a mean relative p53 expression as a function of galactose concentration (Fig. ). The induction of p53 expression was approximately linear over the range of galactose concentration from 0 to 0.12%. The p53 level after induction with 2% galactose (see also Fig. ) increased only two to three times relative to that with 0.12% galactose.
FIG. 5. Quantitative assessment of ADE2 transcription. (A) The p53 REs do not differentially affect ADE2 expression in the absence of p53. The ratio of ADE2 to ACT1 (actin) mRNA levels was determined for yAFM strains lacking the p53 expression vector as well (more ...) Rheostatable expression of p53 reveals a broad range of transactivation capacities.
The relative abilities of p53 to transactivate the reporter gene at various REs were examined by using different levels of galactose inducer and ranking the amount of galactose inducer required for colonies to become pink or white. The various yAFM-RE transformants containing the p53 expression vector were streaked out for single colonies onto a series of 12 plates (which select for the vector) containing either glucose, raffinose, or raffinose plus different amounts of galactose (increasing by factors of 2, i.e., 0.001, 0.002, 0.004, 0.008, 0.016, 0.032, 0.12, 0.25, 0.5, and 2%). A small amount of adenine (5 mg/liter) was included to enable growth of the ade2 mutant cells to small red colonies if there was insufficient transactivation. Transactivation of ADE2 by p53 results in pink and white colonies, depending on the extent of induction. If the transactivation capacity with all p53 REs is the same, colony colors would be expected to change from red to pink to white at comparable levels of galactose. Instead, the responses often differed between REs based on level of galactose required to produce pink or white colonies. For example, the yAFM-P21-5′ strain was nearly white on glucose plates, as seen in Fig. , indicating high activity by basal levels of p53 toward this element. Strains such as those containing m-cFOS or IGF-BP3 B remained pink or red at maximum p53 expression (2% galactose), as shown in Fig. .
The 26 yAFM-REs could be ranked for ability to be induced by different amounts of p53 by using this phenotypic color assay. Since the relative p53 expression was measured under the same culture conditions and appeared to be linear over a broad range (r2 = 0.987) (Fig. ), the relative amount of p53 protein required for a phenotypic change (pink or white) could be determined. This allowed us to rank p53 transactivation capacity towards the 26 REs as the minimum level of p53 required to produce changes in colony color. (As described below, color correlates well with level of ADE2 transcription.) Furthermore, since all strains were isogenic, the transactivation capacities directly reflect the combination of the DNA binding affinity and the ability to transactivate from individual REs by p53.
The functional ranking of p53 transactivation at the various REs of p53-regulated genes is presented in Fig. ; P21-5′is the strongest, and the IGF-BP3 box B is the weakest (the Con and Mut-RGC response elements are described below). In addition to P21-5′, five yAFM-REs (p53R2 to hFAS) showed some transactivation at the basal levels of expression on glucose-only plates. All other strains were red on both glucose and raffinose. For 15 of the yAFM-REs (p53R2 to MDM2/RE1), white colonies could be detected at some level of p53 induction. For 12 of the latter (p53R2 to cyclin G), colonies appeared white (or nearly white as in the case of hFAS, PUMA, PCNA, and cyclin G) when there was less than a 20-fold induction of p53 protein relative to the level on glucose. The yAFM-NOXA, -P21-3′ and -MDM2/RE1 elements enabled some p53 transactivation with a 20-fold relative increase in induction of p53, based on the appearance of pink colonies; much more induction was required to produce white colonies. The remaining nine elements (PA26 to IGF-BP3 box B) did not enable high levels of transactivation, since the colonies did not turn white even under conditions of maximum p53 expression. Except for the IGF-BP3 box B RE, which was completely inactive, the colonies exhibited a light to dark pink color on 2% galactose.
Transactivation capacity measured by color assay corresponds to ADE2 mRNA levels.
The red-pink-white phenotypic assay for assessing p53 gene-specific transactivation capacity and the impact of different levels of expression is simple and highly reproducible. However, the mechanism of red pigment accumulation and metabolism by the ADE2 gene product to result in white colonies is unclear and might be affected by processes that are independent of p53. We therefore addressed the relationship between colony color and ADE2 transcription. The ADE2 mRNA levels were measured relative to actin mRNA (ACT1) by using a real-time RT-PCR approach (see Materials and Methods). First, we checked that there were comparable levels of ADE2 expression from various REs in the absence of p53 protein. Two of the strongest (yAFM-P21-5′ and -p53R2) and the two weakest (BAX-A and IGF-BP3 box B) p53-responsive strains were compared. The red yIG397 strain with a wild-type ADE2 promoter in front of the ade2,1 allele was also included (Fig. ). Total RNA was prepared from cultures grown in 2% raffinose medium for 24 h. Sufficient adenine was added to allow normal growth and prevent a selection for enhanced ADE2 expression. The uninduced ADE2 mRNA levels were comparable and reduced about 10 times for the p53-responsive promoters compared to the natural ADE2 promoter. The actin mRNA levels were similar for all cultures.
The levels of mRNA induced by p53 at various REs reflected colony color. p53-dependent ADE2 transactivation was measured with low (raffinose) and high (galactose) p53 expression. Total RNA was prepared from liquid raffinose cultures (i.e., basal level of induction) of yAFM-RE strains that produced red (RGC, PCNA, MDM2-RE1, BAX-A, p21-3′ cyclin G, and AIP1), pink (GADD45), and white (P21-5′ and p53-R2) colonies on this medium (Fig. ). Consistent with the phenotypic assay, the ADE2/ACT1 ratio was about five times greater for the two white strains than for the red strains. However, the pink yAFM-GADD45 strain was not distinguishable from the red strains.
RNA was also examined following growth in 2% galactose for all but the RGC and p53R2 strains. As shown in Fig. , the BAX-A strain, which is pink on 2% galactose plates (Fig. ), showed the lowest ADE2 induction. All of the other strains were white on 2% galactose. However, there were differences in mRNA induction that ranged from 1.5 times higher for the yAFM-P21-3′ strain to 60 times higher for the P21-5′ strain compared to levels in the BAX-A strain. The other strains showed between 10 and 20 times higher levels of ADE2 mRNA. Since the p53 protein levels of cells grown on 2% galactose were comparable, the differences in expression must be due to differences in transactivation capacity (Fig. ).
Finally, we examined the relative ADE2 transcription from the P21-5′, GADD45, PCNA, MDM2-RE1, and BAX-A REs at different levels of p53 induction. Total RNA was prepared from the same cultures used for the Western blots presented in Fig. , and the ADE2/ACT1 ratios are presented in Fig. . The yAFM-BAX-A strain exhibited poor induction at all levels of p53. The P21-5′, GADD45, and PCNA strains had various degrees of strong induction of ADE2 at relatively low levels of p53 protein. Except for P21-5′, the induction of transactivation reached a plateau at high levels of p53 protein expression. The yAFM-MDM2-RE1 strain showed a modest stimulation of ADE2 transactivation up to a 100-fold increase in p53. This RE becomes highly responsive only with large amounts of p53 (i.e., 2% galactose). We conclude that there is a good correlation between the phenotypic assay and transactivation of the ADE2 gene.
The CATG sequence at the center of a p53 dimer binding site greatly affects RE activity.
We noticed that the 5′-CATG sequence at the junction between p53 monomer binding sites is found in both dimer sequences of the four strongest REs described in Table : P21-5′, p53R2, m-FAS, and GADD45. This motif is present in only one dimer sequence in five other REs in Table . To evaluate the contribution of this sequence element to p53 transactivation capacity, we examined 4 additional artificial sequences, described in Table as Con-A, Con-B, Con-C and Mut-RGC. The Con sequences correspond to the consensus RRRCWWGYYY. Interestingly, Con-A, which contains the CATG sequence in both dimer elements, showed the strongest activity of all of the REs, as shown in Fig. (the colonies were white on glucose plates). Con-B, which is identical to Con-A but has CTAG central elements, ranked sixth, with an approximate 20-fold reduction in transactivation capacity. There was less transactivation from Con-C (it ranked eighth), possibly because it lacks any CATG element. We also constructed a modified RGC RE (Mut-RGC), replacing the CTTG sequence element with CATG in both dimers. This RE showed a dramatic increase in activity.
Subtle changes in transactivation capacity are revealed in p53 mutations associated with familial breast cancer.
The incidence of p53 mutations in familial breast cancer associated with germ line BRCA1/2 mutations is nearly 70%, compared to around 30% for sporadic breast cancer. Moreover, the spectrum of mutations appears to be different (30
). Interestingly, a subset of BRCA1-associated p53 mutant alleles appeared wild type in mammalian functional assays (67
), and four of the mutants, T150I, G199R, R202S, and S215C, were also wild type in a yeast transactivation assay at high p53 expression (11
). We tested these mutants for the ability to transactivate from 10 of the REs in this study at various levels of p53 expression. All of the mutants were comparable to the wild type at high levels of p53 expression (data not shown); however, the matrix of responses differed from wild type at low levels. The difference in the minimal galactose amount needed to obtain white colonies (or light pink colonies with BAX-B) with mutant and wild-type alleles was used to estimate the relative transactivation capacity. Subtle differences in the transactivation pattern were detected for all four alleles (Table ). Interestingly, the changes comprised both reduced and enhanced activity with specific REs and no effect with other REs relative to the wild type, suggesting that the amino acid changes do not simply affect protein stability.
Relative transactivation capacity at low levels of expression of p53 mutant alleles associated with familial breast cancera