The contribution of transactivation subdomains 1 and 2 to p53-induced gene expression is heterogeneous but not subdomain-specific

Abstract

Two adjacent regions within the transactivation domain of p53 are sufficient to support sequence-specific transactivation when fused to a heterologous DNA binding domain. It has been hypothesized that these two subdomains of p53 may contribute to the expression of distinct p53-responsive genes. Here we have used oligonucleotide microarrays to identify transcripts induced by variants of p53 with point mutations within subdomains 1, 2, or 1 and 2 (QS1, QS2, and QS1/QS2, respectively). The expression of 254 transcripts was increased in response to wild-type p53 expression but most of these transcripts were poorly induced by these variants of p53. Strikingly, a number of known p53-regulated transcripts including TNFRSF10B, BAX, BTG2, and POLH were increased to wild-type levels by p53(QS1) and p53(QS2) but not p53(QS1/QS2), indicating that either subdomain 1 or 2 is sufficient for p53-dependent expression of a small subset of p53-responsive genes. Unexpectedly, there was no evidence for p53(QS1)- or p53(QS2)-specific gene expression. Taken together, we found heterogeneity in the requirement for transactivation subdomains 1 and 2 of p53 without any subdomain-specific contribution to p53-induced gene expression.

Keywords: apoptosis; gene expression; microarray; p53; transcription factor.

Figure 1

Expression of transactivation subdomain variants of p53. (A) Schematic representation of epitopes recognized by the indicated monoclonal antibodies (DO-1, Pab1801, and Pab421), peptides (P) identified by mass spectroscopy (see Table W1), and p53 functional domains. SD1 and SD2 denote subdomains 1 and 2 within the acidic AD. PRD, proline-rich domain; DBD, DNA binding domain; TD, tetramerization domain; BD, basic domain. Numbers below indicate the amino acid position. (B) Immunoblot analysis of p53 expression 16 hours following infection of either HCT116p53^-/- or HeLa cells with the indicated recombinant adenovirus, using three different anti-p53 monoclonal antibodies. M and C represent mock- and control virus-infected samples whereas WT, QS1, QS2, and QS1/QS2 denote the wild-type and variant forms of p53. Similar blots were obtained with cell lysates derived from HCT116 and MDAH041 cells (data not shown). (C) Samples were collected at 8, 16, or 24 hours and subsequently analyzed by immunoblot analysis with the Pab421 monoclonal antibody.

Figure 2

Most p53 target genes are poorly induced by the QS variants. (A) Two hundred and fifty-four genes were induced by Adp53^wt. Of these, only 28, 23, and 1 were induced by QS1, QS2, and QS1/QS2, respectively. (B) The fold increase in expression of these 254 genes was determined following infection of cells with adenoviruses expressing wild-type, QS1, QS2, or QS1/QS2 variant of p53. The fold increase in the expression following infection with Adp53^QS1, Adp53^QS2, or Adp53^QS1/QS2 was less than the fold increase in response to Adp53^wt infection (one-way analysis of variance followed by a Tukey's Multiple Comparisons test, P ≤ .001). (C–E) The fold increase in expression due to Adp53^wt expression was compared to the fold increase in expression due to indicated transactivation subdomain variant of p53 for Adp53^wt-, Adp53^QS1-, and Adp53^QS2-induced genes (C, D, and E, respectively). The 254, 28, and 23 genes induced by Adp53^wt, Adp53^QS1, and Adp53^QS2 are listed in Tables W2 and W3.

Figure 3

Correlation between Adp53^QS1- and Adp53^QS2-induced genes. (A) A Venn diagram is used to represent the overlap between Adp53^QS1- and Adp53^QS2-induced genes, as defined in the Materials and Methods section. (B) The effect of Adp53^QS1 and Adp53^QS2 infection on the expression of the 254 Adp53^wt-, 28 Adp53^QS1-, and 23 Adp53^QS2-induced genes was determined. The genes induced by Adp53^wt, Adp53^QS1, and Adp53^QS2 are listed in Tables W2 and W3. A very tight correlation (R² values are inset) between Adp53^QS1- and Adp53^QS2-induced gene expression was observed within the subset of target genes.

Figure 4

Representative transcripts induced by wild-type p53. (A–C) Expression of the indicated transcript was determined by real-time RT-PCR using samples collected at the indicated time following virus infection (8, 16, or 24 hours). Expression of β-actin was used to normalize all RT-PCR results. Open, black, grey, hatched, and crosshatched bars represent control, Adp53^wt-, Adp53^QS1-, Adp53^QS2-, and Adp53^QS1/QS2-infected samples. Each value represents the mean fold increase in expression (± SEM) determined from a minimum of three independent experiments.

Figure 5

Effect of QS variants of p53 on cell viability and apoptosis. Apoptosis and cell viability were assessed 48 hours following infection with either control adenovirus or adenoviruses expressing the indicated variants of p53. Apoptosis was assessed by subdiploid DNA content (A) and viability was assessed by Trypan blue exclusion (B). Each point represents the mean (±SEM) determined from three independent experiments. Adp53wt induced more apoptosis than the variants (one-way analysis of variance followed by Tukey's Multiple Comparisons test, P ≤ .01 for apoptosis and P ≤ .05 for viability). No significant difference in viability or apoptosis was observed when comparing QS1 and QS2 variants.