The tumor suppressor p53 activates the transcription of human PIG3 through direct interaction with a polymorphic microsatellite sequence, (TGYCC)n. Here, the evolution of this p53-responsive element was recapitulated. Comparison between primate species revealed that the PIG3 promoter acquired this sequence element in its full length only in Hominoidea (apes and humans), whereas the number of TGYCC repeats is far lower in monkeys. Accordingly, only the PIG3 promoters from Hominoidea respond efficiently to p53, whereas those from monkeys respond poorly or not at all. In parallel, the PIG3 gene was strongly induced by p53 in human and chimpanzee cells but was unaffected by p53 in cells derived from a common marmoset monkey. Thus, a novel p53 target gene appeared as recently as during the evolution of primates. This suggests that mechanisms of tumor suppression are subject to ongoing evolution in humans and their closest relatives.
p53 suppresses the formation of malignant tumors through its ability to induce programmed cell death. At least to a large extent, this biological activity is accomplished by the activation of target genes. p53 interacts with the DNA of its target promoters and enhances the transcription of the corresponding genes (1, 2). Human PIG3 is activated by p53 and was suggested to function in p53-induced apoptosis through accumulation of reactive oxygen species (3). In accordance, p53 mutants that selectively fail to induce apoptosis but not cell cycle arrest also fail to activate PIG3 but not other p53-responsive genes (4, 5, 6).
We have shown previously that a microsatellite within the PIG3 promoter, (TGYCC)n, with Y = C or T, mediates the induction of PIG3 by p53 (7). p53 interacts directly and specifically with this sequence element in vitro and in vivo. This p53-responsive element is unusual not only for its relatively loose correspondence to the canonical p53-responsive consensus RRRCWWGYYYN(0–13)RRRCWWGYYY, with r = A or G, W = A or T, Y = C or T, and N = any nucleotide. Curiously, the PIG3 microsatellite is polymorphic in humans, the number of pentanucleotide repeats being 10, 15, 16, and 17. The size of the microsatellite correlates directly with the extent of promoter activation by p53 (7).
Microsatellites are prone to instability in the course of evolution (8). Therefore, we hypothesized that the p53-responsive element within the PIG3 promoter may differ from the human sequence even in closely related species. To test this, we analyzed the sequence and function of PIG3 promoters in apes and monkeys. It was found that PIG3 evolved its p53 responsiveness at the transition from monkeys to apes. These analyses allowed, for the first time, to recapitulate the occurrence of a p53-responsive promoter element during evolution.
Materials and Methods
Plasmids and Adenovirus Vectors.
We obtained reporter constructs containing the PIG3 promoter of different primate species by PCR amplification of the promoter region, using the primers CTC AGA TCT CAC GGA CAA GTG GGA ATG TAT AGC and CTC TAA GCT TTG CAC GGC TAA CAT ATT GTC TG, from the genomic DNA of different animals, followed by treatment with BglII and HindIII and ligation into pGL3-Basic (Promega).
Cell Culture and Transfections.
H1299 (a human TP53−/− cell line derived from an adenocarcinoma of the lung), CP132 cells (derived from chimpanzee fibroblasts), and G3SV1 cells (derived from ovarian granulosa cells of Callithrix jacchus; Ref. 12) were maintained in DMEM (Life Technologies, Inc.), supplemented with 10% FCS. For transfections, 2.5 × 105 H1299 cells were seeded on each well of a six-well dish (Greiner) and transfected with Lipofectamine 2000 (Life Technologies). Cells were harvested 24 h after transfection, followed by luciferase assays (Promega).
Semiquantitative Reverse Transcription-PCR.
To analyze the induction of the endogenous PIG3 by p53, G3SV1 cells were transduced with adenovirus vectors to express either p53 or a control vector expressing the green fluorescent protein. Similar experiments were done with H1299 and CP132 cells. The amount of virus and time of incubation were adjusted in each case to assure that 100% of the cells were transduced, as determined by green fluorescent protein expression. After 24 h (G3SV1 and H1299 cells) or 48 h (CP132 cells), total RNA was prepared (TRIzol Reagent; Life Technologies), followed by reverse transcription with Superscript II polymerase (Life Technologies), using the “RT” primers, and PCR amplification with Expand HiFi DNA polymerase (Roche), using the “forward” and “reverse” primers indicated below. We stopped the reaction after different numbers of temperature cycles and visualized the PCR product by ethidium bromide on an agarose gel. For the analysis of the different transcripts, the following primers were used:
RT MDM2: AAC ATC TGT TGC AAT GTG ATG G
MDM2 forward: TCA GGA TTC AGT TTC AGA TCA G
MDM2 reverse: CAT TTC CAA TAG TCA GCT AAG G
RT p73ΔN: CAG GTG GCT GAC TTG GCC GTG CTG,
p73ΔN forward: CGC CTA CCA TGC TGT ACG TCG GTG
p73ΔN reverse: TGC TGG AAA GTG ACC TCA AAG TGG
RT Apaf-1: AGA TCT GAT GTC TTC TCT GAG C
Apaf-1 forward: GTG TTA CAG ATT CAG TAA TGG G
Apaf-1 reverse: CTG AAG CTT CCC AGC GAT TGG G
RT GAPDH:3 GGT TCA CAC CCA TGA CGA ACA TG
GAPDH forward: TGA AGG TCG GAG TCA ACG GAT TTG GT
GAPDH reverse: GCA GAG ATG ATG ACC CTT TTG GCT C
For the analysis of the PIG3 expression on C. jacchus, the primer GCG CCT TGC TGT GCT GGT CTC C was used for reverse transcription, and the primers TGC TAC TGG GAC CCG CAA GAG C and CGT GCT CCT GCC TGG GAG TTC C were used for PCR amplification. For the amplification of the PIG3 transcript from H1299 and CP132 cells, we used the primer CGG TGA GCA GGC CTC TGG GAT GGC for reverse transcription and the primers GTG CAC TTT GAC AAG CCG GGA GGA and CAG CCT GGG TCA GGG TCA ATC CCT for PCR amplification.
Results and Discussion
The PIG3 Microsatellite Sequence in Apes and Monkeys.
As the number of TGYCC repeats varies even within one single species (human), we investigated the diversity of this number in related species. We amplified and sequenced the PIG3 promoter region of several primate species (Fig. 1). Great apes (Pan troglodytes, Pan paniscus, Gorilla gorilla, and Pongo pygmaeus) and the small ape Hylobates lar showed 14–19 TGYCC repeats, a number comparable with the one present in humans. In contrast, only 6 repeats were found in Old World monkeys (Macaca mulatta, and Colobus guereza) and 5 repeats in New World monkeys (Callicebus cupreus, Cebus apella, Callimico goeldii, and C. jacchus). Furthermore, the number of mismatches (pentanucleotide elements containing one nucleotide that does not correspond to the TGYCC consensus) was higher in Old and New World monkeys. These results indicate that the size of the PIG3 microsatellite decreases rapidly with the phylogenetic distance to humans.
Response of PIG3 Promoters from Monkeys and Apes to p53.
A reduced number of TGYCC repeats results in a weaker interaction with p53 (7). Therefore, we addressed the question whether the shorter PIG3 microsatellite observed in Old and New World monkeys also corresponds to a weaker promoter response to p53. The induction of the PIG3 promoter by p53 was quantified by reporter assays. As shown in Fig. 2, transactivation by p53 was strong when the PIG3 promoters of human, great ape, and gibbon species were analyzed. In contrast, the promoters from both Old World monkeys were only weakly inducible, and those from New World monkeys were not responsive at all. Thus, the response of the PIG3 promoter to p53 was acquired during the evolution of Hominoidea.
Response of PIG3 to p53 in Monkey Cells.
To test if p53 also fails to induce the endogenous PIG3 of a New World monkey, we used a granulosa cell line derived from the marmoset C. jacchus (12). These cells were transduced to overexpress p53, and we then analyzed the levels of several transcripts by reverse transcription-PCR. As shown in Fig. 3, the p53-responsive genes p73ΔN and MDM2 were induced in the presence of p53, and the same was found for the p53-responsive and apoptosis-related gene Apaf-1. In contrast, no induction was observed for PIG3, as in the GAPDH control. When using human H1299 cells, or CP132 cells from the chimpanzee P. troglodytes, PIG3 was clearly activated by p53. We conclude that the endogenous PIG3 in marmoset cells is not p53 responsive.
Concluding Remarks and Perspectives.
p53 and its functions as a regulator of transcription and inducer of programmed cell death are conserved from Caenorhabditis elegans to humans (13). Many target genes, including CDKN1A (p21), MDM2, TNFRSF6 (Fas), bax, Apaf-1, and PIG8, respond to p53 in murine as well as in human cells (14, 15, 16). In contrast, PIG3 evolved its p53 responsiveness during the most recent stages of evolution and, to our current knowledge, represents the phylogenetically youngest p53 target gene. Its regulation by p53 is unique to humans and apes, raising some caution about the analogies drawn between most animal models and human cancer. Differences in gene expression levels, rather than the structure of gene products, have long been proposed to be largely responsible for the different biological properties of distinct primate species (17, 18). However, examples of genes that are differentially regulated among primates are rare (19, 20), and the mechanistic basis of the differences in regulation is poorly understood. To our knowledge, PIG3 represents the clearest available example of a mechanistically explained difference in promoter regulation between primate species. Mechanisms of growth regulation have been established since the earliest stages of life, and molecular functions of tumor suppression are well established throughout mammalian species. Nonetheless, it appears that such mechanisms are still subject to ongoing evolution in humans and their closest relatives.
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Supported by the Wilhelm Sander-Stiftung and SET foundation. A. C. received a fellowship from PRAXIS XXI, FCT, Portugal.
The abbreviation used is: GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Internet address: http://warprc.org/psic/taxonomy.asp.
We thank H-D. Klenk for his continuous support, C. Lenz-Bauer for excellent technical assistance, N. Schuhmann for help with plasmid cloning, and J. Roth for helpful discussion. We also thank the important contribution of B. Husen and K. Lieder to the establishment of the marmoset granulosa cell line. Finally, we thank W. Enard and S. Pääbo for CP132 cells.