Regulation of the p53 transcriptional response by structurally diverse core promoters

Abstract

p53 target promoters are structurally diverse and display pronounced differences in RNA polymerase II (RNAP II) occupancy even in unstressed cells, with higher levels observed on cell cycle arrest genes (p21) compared with apoptotic genes (Fas/APO1). This occupancy correlates well with their ability to undergo rapid or delayed stress induction. To understand the basis for such distinct temporal assembly of transcription complexes, we examined the role of core promoter structures in this process. We find that the p21 core promoter directs rapid, TATA box-dependent assembly of RNAP II preinitiation complexes (PICs), but permits few rounds of RNAP II reinitiation. In contrast, PIC formation at the Fas/APO1 core promoter is very inefficient but supports multiple rounds of transcription. We define a downstream element within the Fas/APO1 core promoter that is essential for its activation, and identify nuclear transcription factor Y (NF-Y) as its binding partner. NF-Y acts as a bifunctional transcription factor that regulates basal expression of Fas/APO1 in vivo. Thus, two critical parameters of the stress-induced p53 transcriptional response are the kinetics of gene induction and duration of expression through frequent reinitiation. These features are intrinsic, DNA-encoded properties of diverse core promoters that may be fundamental to anticipatory programming of p53 response genes upon stress.

Figure 1.

Functional characterization of the p21 and Fas/APO1 core promoters. (A) Schematic diagram of p21 and Fas/APO1 promoters, including their respective p53-binding sites. (B) In vitro transcription of p21 or Fas/APO1 plasmids was performed and analyzed by primer extension. (C) Schematic diagram of single- and multiple-round transcription in the presence of sarkosyl. Addition of sarkosyl to form PICs prevents new PIC formation. Templates without sarkosyl undergo multiple rounds of transcription. (D) Rates of PIC formation were analyzed by adding sarkosyl just before (time 0), or various times after, combining DNA templates with HeLa nuclear extracts (HNEs). Transcription was initiated by adding NTPs. (E) Quantification and graphical representation of D. (F) Rounds of transcription were analyzed by allowing PICs to form for 1 h before initiating transcription (time 0). The initiated reactions then underwent multiple rounds of transcription before sarkosyl addition at intervals from 15 sec to 30 min. The first lane shows that sarkosyl addition to DNA templates before exposure to HeLa extract prevents formation of functional PICs. (G) Rounds of transcription were also measured using DNA templates immobilized on magnetic beads. Extract and immobilized DNA were mixed for 1 h to allow PIC formation similar to C. For single-round transcription, the DNA template was washed with buffer followed by addition of NTPs to allow elongation, whereas this wash step was not included for multiple rounds of transcription. (H) Rounds of transcription were quantified, and the signal at each time point was compared with time 0 to calculate the rounds of reinitiation.

Figure 2.

Analysis of PIC assembly kinetics on full-length or core p21 and Fas/APO1 promoters. (A) Diagram of the full-length promoters containing p53 response elements or core promoters. (B) In vitro transcription reactions using HeLa extracts to compare promoter strength between full-length and core promoter templates. (C) Rates of PIC formation on p21 and Fas/APO1 full-length or core promoters were measured by in vitro transcription under conditions similar to Figure 1D.

Figure 3.

Functional core promoter elements mapped by scanning mutagenesis. (A,B) Diagram of p21 (A) and Fas/APO1 (B) promoter sequences indicating the location of scanning mutations. Progressive 10-bp transversion mutations were generated in the context of the full-length promoters. (C) In vitro transcription reactions of p21 scanning mutations. The two left lanes labeled “G” and “A” contain DNA sequencing reactions used to map the transcription start site(s). (D) Same as C but using Fas/APO1 scanning mutations. (E) Transcriptional analysis of the scanning mutations “F” and “G” of Fas/APO1 that were further defined by creating four 5-bp block mutations between Scans F and G to create Fas scan mutants F.1, F.2, G.1, and G.2. (F) Luciferase expression analysis in HCT116 cells using transiently transfected p21 or Fas/APO-1 core promoters within a pGL3 luciferase plasmid.

Figure 4.

Analysis of critical core promoter elements using chimeric templates. (A) Diagram of the Fas/APO1 promoter sequence indicating the location in which the p21 ATATCAG sequence was inserted to replace −23 to −29 and create the Fas-TATA promoter. (B) In vitro transcription to examine the activity of Fas-TATA compared with the Fas/APO1 promoter. (C) In vitro transcription analyzing the rate of PIC formation of p21, Fas/APO1, and Fas-TATA. HeLa nuclear extract was allowed to bind to template for 0–2 h before addition of sarkosyl, similar to Figure 2D. To generate multiple rounds of transcription, sarkosyl was not added to the last lane (2 h*). (D) Transcriptional analysis of Fas-TATA compared with F-TATA (Fas-TATA with the Scan F mutation). (E) Analysis of the Fas downstream element within the p21 wild-type or mutated full-length promoter.

Figure 5.

The NF-Y complex can bind to the critical Fas downstream element. (A) Sequences of the wild-type or mutant probes used for EMSA. (B) EMSA analysis of Fas downstream element (Fas “F–G”)-binding protein(s) from HeLa nuclear extracts. (C) EMSA competition assay using cold wild-type or mutated competitor F–G oligonucleotides. (First lane) Wild-type Fas “F–G” oligos were shifted with 4 μg of HNE in the absence or presence of cold wild-type or mutated competitor DNA. (D) Diagram indicating the steps to capture and characterize the complex binding the Fas downstream element. The DNA affinity pull-down was performed using immobilized multimers of the DNA sequence used for EMSA. (E) Proteins that bound to the wild-type Fas downstream element or the mutated sequence were step-eluted with buffer containing 0.1 M, 0.25 M, 0.5 M, or 1 M NaCl and tested for binding activity by EMSA. (F) Supershift analysis to test the specificity of NF-Y binding. In lanes 1–7, EMSA reactions were incubated for 30 min followed by addition of the specified antibody and incubated for 15 min. For lanes 8 and 9, antibodies were incubated with HNE for 10 min before mixing with DNA probe.

Figure 6.

NF-Y binds to and regulates the FAS/APO-1 promoter in MCF7 cells. (A) mRNA analysis of Fas/APO1 activation upon 5-FU (50 ng/mL) treatment for 14 h. (B) ChIPs of the FAS/APO-1, p21, and CCNB1 genes were assayed for the presence of NF-Y in MCF7 cells. (C) Western blot analysis of NF-Y subunits overexpressed in MCF7 cells. CMV-driven NF-YA, NF-YB, and NF-YC plasmids were used for transfection and assayed with subunit-specific antibodies. (D) mRNA from MCF7 cells overexpressing NF-Y were analyzed by qPCR.

Figure 7.

Schematic model of p21 and Fas/APO1 PIC formation and reinitiation kinetics. The p21 core promoter supports efficient PIC assembly through the TATA box for rapid transcriptional activation, but only poor reinitiation capability. In contrast to p21, the Fas/APO1 core promoter has low affinity for PIC recruitment but supports multiple reinitiation events. The downstream NF-Y-binding element is required for core promoter activity and may facilitate nucleation of the PIC.