Diverse p63 and p73 isoforms regulate Δ133p53 expression through modulation of the internal TP53 promoter activity

Abstract

In response to stress, p53 binds and transactivates the internal TP53 promoter, thus regulating the expression of its own isoform, Δ133p53α. Here, we report that, in addition to p53, at least four p63/p73 isoforms regulate Δ133p53 expression at transcriptional level: p63β, ΔNp63α, ΔNp63β and ΔNp73γ. This regulation occurs through direct DNA-binding to the internal TP53 promoter as demonstrated by chromatin immunoprecipitation and the use of DNA-binding mutant p63. The promoter regions involved in the p63/p73-mediated transactivation were identified using deleted, mutant and polymorphic luciferase reporter constructs. In addition, we observed that transient expression of p53 family members modulates endogenous Δ133p53α expression at both mRNA and protein levels. We also report concomitant variation of p63 and Δ133p53 expression during keratinocyte differentiation of HaCat cells and induced pluripotent stem cells derived from mutated p63 ectodermal dysplasia patients. Finally, proliferation assays indicated that Δ133p53α isoform regulates the anti-proliferative activities of p63β, ΔNp63α, ΔNp63β and ΔNp73γ. Overall, this study shows a strong interplay between p53, p63 and p73 isoforms to orchestrate cell fate outcome.

Figure 1

Regulation of the internal TP53 promoter activity by p63 isoforms. (a) Schematic representation of the human TP53 gene. In addition to the proximal promoter (P1) located upstream exon 1, regulating p53 and Δ40 forms, a second promoter (P2) has been described from the end of intron 1 to the beginning of exon 5 that regulate Δ133 and Δ160 form expression. The main p53 response elements (p53REs) have been described at the exon 4/intron 4 junction. Dotted box: internal TP53 promoter introduced in pi3i4-Luc construct; P^*: promoter in TP53 intron 1 identified by Reisman et al.^, that regulates the expression of an unrelated p53 transcript encoded by TP53 intron 1. (b and c) Impact of p63 isoforms on the internal TP53 promoter activity in H1299 (b) and in MCF7 cells (c). Luciferase assays were performed in the presence of p63 isoforms using pi3i4-Luc construct. Among p63 isoforms, p63β, ΔNp63α and ΔNp63β strongly increased luciferase activity. ^*P<0.05; ^**P<0.005. (d) Expression levels of ectopic p63 isoforms in H1299 cells analyzed by western blot. 4A4: antibody specific for all p63 isoforms; Actin: loading control

Figure 2

Regulation of the internal TP53 promoter activity by p73 isoforms. (a and b) Impact of p73 isoforms on the internal TP53 promoter activity in H1299 (a) and in MCF7 cells (b). Luciferase assays were performed in presence of p73 isoforms using pi3i4-Luc construct. Among p73 isoforms, only ΔNp73γ strongly induced the activity of the internal TP53 promoter. ^*P<0.05; ^**P<0.005. (c) Expression levels of ectopic p73 isoforms in H1299 cells analyzed by western blot. 259A: antibody specific for all p73 isoforms; Actin: loading control. (d) Comparison of the transcriptional activities of p53 family members with the internal TP53 and the p21 promoters. The p53 family members showing the strongest transactivation activity on the internal TP53 promoter were used. The same range of activation was observed for p53α, ΔNp63α, ΔNp63β and ΔNp73γ on the two different promoters in H1299 cells. ^**P<0.005; ^***P<0.0005

Figure 3

Identification of internal TP53 promoter regions involved in the transactivation mediated by p53 family members. (a) Schematic representation of the sequential deletions introduced in the full-length pi3i4-Luc(A) construct to generate three deleted pi3i4-Luc vectors (C, G, and D). Only pi3i4-Luc(A) and pi3i4-Luc(C) retain the main p53REs. Arrow: initiation site of transcription; nucleotide number 1: corresponds to nucleotide +11523 – accession no. X54156, NCBI. (b) Basal luciferase activity of the deleted pi3i4-Luc constructs in MCF7 cells. As compared with the entire pi3i4-Luc(A) construct, the pi3i4-Luc(C) and pi3i4-Luc(D) showed a significant increase in basal luciferase activity, suggesting the presence of different regulatory elements on the internal TP53 promoter (silencers and enhancers). (c–f) Regions of the internal TP53 promoter involved in the transactivation mediated by p63β (c), ΔNp63β (d), ΔNp63α (e) and ΔNp73γ (f). The differences in basal activities have been normalized to evaluate the activation induced by p53 family members in MCF7 cells. Nucleotides 753–1042 are involved in the transactivation mediated by p63β, 953–1042 in the one mediated by ΔNp63α and ΔNp63β, 723–953 in the one mediated by ΔNp73γ. ^*P<0.05; ^**P<0.005; ^***P<0.0005

Figure 4

Role of cis-elements in the transactivation mediated by p53 family members. (a) Five p53 response elements (p53RE-A1 to -A5) have been identified at exon 4/intron 4 junction. In pi3i4-Luc construct, point mutations have been introduced in p53RE-A1 and -A2 to abolish their usage. WTp53REs: WT sequence of p53REs; MTp53RE-A1/2: mutant sequence of p53REs; bold: p53REs; underlined: mismatch between consensus p53RE and p53REs of the internal TP53 promoter; star: point mutations introduced by site-directed mutagenesis. (b and c) Impact of mutations within p53REs on the transactivation mediated by p53 family members on the internal TP53 promoter activity in H1299 (b) and in MCF7 cells (c). Luciferase assays were performed in the presence of p53 family members using pi3i4-Luc constructs carrying WTp53REs or MTp53RE-A1/2. Only p53α and ΔNp73γ were affected by mutations introduced in p53RE-A1 and p53RE-A2. ^*P<0.05; ^**P<0.005; ^***P<0.0005. (d) Impact of TP53 polymorphisms on the basal promoter activity. Luciferase assays were performed in MCF7 cells using pi3i4-Luc construct carrying four different haplotypes mimicking haplotypes derived from TP53 PIN3 (rs17878362 in intron 3: A1: non-duplicated; A2: 16-bp duplication) and from TP53 PEX4 (rs1042522, G>C in exon 4: R: arginine at codon 72; P: proline at codon 72). A1-R and A2-P presented the strongest intrinsic luciferase activity compared with A2-R and A1-P. ^**P<0.005. (e) Impact of TP53 polymorphisms on the transactivation of the internal TP53 promoter mediated by p53 family members. Luciferase assays were performed in MCF7 cells using the four polymorphic pi3i4-Luc constructs in the presence of p53 family isoforms. Differences in basal activities have been normalized to evaluate the activation induced by p53 family isoforms. TP53 PIN3 and PEX4 significantly affect ΔNp73γ-mediated transactivation. ^*P<0.05

Figure 5

Direct DNA-binding of p63 and p73 isoforms to the internal TP53 promoter. (a) Impact of WT and mutant ΔNp63α isoforms on the internal TP53 promoter. Luciferase assays were performed in H1299 cells using the pi3i4-Luc construct in the presence of WT or mutant ΔNp63α (contact mutation R279H and conformational mutation C306R). Presence of a single mutation in the DNA-binding domain of ΔNp63α alters the ΔNp63α-mediated transactivation on the internal TP53 promoter. ^**P<0.005; ^***P<0.0005. (b and c) Direct binding of p63 and p73 isoforms to the internal TP53 promoter. CHiPs were performed in HaCat cells using the 4A4 (p63 isoforms, B) or IMG-259 (p73 isoforms, C) antibodies. Two set of primers/probe were used: one hybridizing the exon 4/intron 4 junctions specific for the internal TP53 promoter and one hybridizing the TP53 intron 8 used as negative control. A representative experiment was illustrated

Figure 6

Modulation of Δ133p53α isoform expression and biological functions in response to ectopic expression of p63/p73 isoforms. (a and b) Variation of endogenous Δ133p53 expression in response to ectopic expression of p63/p73 isoforms. In mutant p53 MDA-MB-231 cells, introduction of p63/p73 isoforms resulted in a weak expression of Δ133p53 protein level (a) and in a significant increase of Δ133p53 mRNA level (b). Ku80: loading control; ^*P<0.05. (c) Proliferation assays in the presence of p53 family members. Cell numbers have been determined on three independent experiments by measuring sulforhodamine B staining using spectroscopy. Statistical analyses showed that p63β, ΔNp63β and ΔNp73γ had growth suppressive activities, which can be modulated by introduction of Δ133p53α. ^*P<0.05; ^**P<0.005 and ^***P<0.0005

Figure 7

Concomitant variation of p63, p73 and Δ133p53α expression during keratinocyte differentiation. (a and b) Differentiation of HaCat cells by increased concentration of calcium and analysis of the expression of p53 family members at both protein (a) and mRNA (b) levels. Forced differentiation of mutant p53 HaCat cells by addition of calcium decreased ΔNp63α and increased p73β expression. In parallel, an increased expression of Δ133p53α was observed at both mRNA and protein levels. d: days; ^*nonspecific band; Actin: loading control; ^*P<0.05; ^**P<0.005. (c) Epidermal differentiation of six iPSC lines expressing a WT or a mutant TP63 gene. Because of variability of epidermal fate efficiency between experiments, we illustrated here two representative experiments for control and mutated iPS individuals. In WT cells, differentiation was associated with an increased expression of p63 and a decreased expression of Δ133p53 at mRNA levels, whereas in mutant cells, only the increase of p63 was observed. WT: cells expressing WT TP63 gene; MT, cells expressing mutant TP63 gene (R304W or R204W)