Figure 1.
p53 Binds to Peptides that Mimic the Phosphorylated Pol II CTD.
Biotinylated CTD peptides bearing four heptad repeats (28-mers) that were unphosphorylated (CTD), Ser5-phosphorylated (Ser5-P-CTD), or Ser2-phosphorylated (Ser2-P-CTD), were adsorbed to streptavidin-coated magnetic beads. Recombinant HA-p53, bearing an HA tag on its N-terminus, was incubated with beads alone or beads bearing peptides. Bound proteins (B) and those remaining in the supernatant (S) were subjected to SDS-PAGE and the presence of p53 was detected using the 1801 mAb. (B) Graphical summary of peptide-bound p53 relative to unbound p53. Depicted are means ± S.E.M. (N = 4). A two-sample t-test (two-tailed, equal variance) comparing p53 binding to the Ser5-phosphorylated CTD peptide vs. the unphosphorylated CTD peptide results in a P value of 0.058. A corresponding analysis of p53 binding to the Ser2-phosphorylated CTD peptide results in a P value of 0.11.
Figure 2.
The Core Domain of p53 Mediates the p53-Pol II Interaction in Whole Cell Extracts.
(A) Domain structures of p53+ and p53coreΔ. TA1 and TA2, transactivation domains 1 and 2; Tetra., tetramerization domain; Reg., regulatory domain. (B) p53 immunoblot analysis of cells expressing p53+ (strain SY1000) and p53coreΔ (strain SY1004) regulated by the GAL1 promoter. Cells were grown in rich medium containing 1.8% raffinose and 0.2% galactose. p53 was detected through use of the DO-1 mAb; expression levels were internally normalized to those of Pgk1. (C) The Pol II large subunit co-immunoprecipitates with p53+, but not with p53coreΔ, in yeast WCEs. Immunoprecipitates were obtained from WCEs isolated from strains SY1000 and SY1004 cultivated as above using Myc- or p53-specific antibodies (9E10 or DO-1, respectively) (lanes 1–4). Rpb1 was detected using anti-CTD antiserum. Lanes 1 and 3 serve as negative controls as cells do not express a Myc-tagged protein. (D) p53+, but not p53coreΔ, co-IPs with Pol II. Immunoprecipitates were generated using either pre-immune or anti-Pol II (CTD) antiserum (lane 1–4) from extracts as above. p53 and p53coreΔ were detected using DO-1.
Figure 3.
Core domain Mutations Abolish Synthetic Growth Phenotypes Observed in p53-Expressing Cells.
(A) Fivefold serial dilutions of yeast cells (BY4741 background) expressing p53+, p53R273H or p53V143A behind the GAL1 promoter or expressing no ectopic protein (p53−). Cells were spotted onto synthetic rich medium containing either 2% glucose (SDC) or 1.6% raffinose/0.4% galactose; 6-azauracil (6-AU) was added where indicated. Cells were incubated at 30°C for 3 to 6 days. (B) Immunoblot analysis of p53+ and p53 core domain mutant-expressing strains grown in liquid synthetic medium containing 1.6% raffinose/0.4% galactose. p53+ and its mutant derivatives were detected using the 1801 mAb. (C) As in A, except p53 expression was regulated by the ADH1 promoter and BY4741 cells were grown on synthetic rich medium containing 2% glucose in the absence or presence of mycophenolic acid (MPA). (D) Spot dilution analysis of p53+ and p53 mutant-expressing cells as in A except grown on synthetic rich medium containing either 2% glucose or 1.5% raffinose/0.5% galactose in the absence or presence of MPA. (E) Fivefold serial dilutions of strain BY4741 (rows 1–3) or its isogenic dst1Δ counterpart (rows 4–6) transformed with p53+- or p53R175H-expressing plasmids (or empty vector; p53−) as in C and spotted onto synthetic rich medium. The SDC panel, while non-selective, shows that roughly equivalent numbers of cells were spotted. Note that in replicate experiments, p53+ BY4741 cells exhibited a slow growth phenotype on SDC -Leu, -His similar to that seen in C. (F) Immunoblot analysis of p53+ and p53R175H expression in the dst1Δ mutant grown in synthetic rich medium containing 2% glucose. (G) Spot dilution analysis of p53+ and p53V143A-expressing BY4741 ipk1Δ cells grown on synthetic medium containing either 2% glucose (SDC) or 1.6% raffinose/0.4% galactose as indicated.
Figure 4.
p53 Increases Pol II Density at Constitutively Transcribed Genes.
(A) Pol II ChIP analysis of the PMA1 gene in p53+ and p53− cells. Depicted is a multiplex PCR analysis (resolved on an 8% polyacrylamide gel) of DNA purified from immunoprecipitated chromatin samples isolated from SY1000 (PGAL1- p53) and SLY101 (p53−) cells subjected to a 2% galactose induction for 0, 60, 120 and 180 min. Immunoprecipitations were conducted using pre-immune (lanes 1, 6) or anti-CTD antiserum (lane 2–5, 7–10). Lane 11, DNA isolated from chromatin used in the ChIPs of lanes 1 and 2. An ORF-free region on chromosome V (ARS504) was co-amplified with the PMA1 loci, and serves as a non-specific IP control. (B) Summary of Pol II ChIP assays of the PMA1 5′-UTR, ORF, and 3′-UTR in p53+ and p53− cells conducted as in panel A. Depicted is Pol II abundance at each locus (net signal, immune minus pre-immune) relative to ARS504 (net signal, immune minus pre-immune). Shown are means ± S.D; N = 2. (C) As in panel B, except Pol II ChIP analysis of the ACT1 gene. (D) Northern analysis of PMA1 and ACT1 in p53+ and p53− cells following addition of galactose for the indicated times. Transcript levels were normalized to those of SCR1 and are presented in arbitrary units (shown are means ±S.D; N = 2).
Figure 5.
p53+, but not the Oncogenic Mutant p53V143A , Impairs Pol II Processivity on the Episomal PHO5-lacZ Gene.
(A) Physical maps of PHO5 reporter genes under GAL1 regulation used in the processivity assay. (B) Isogenic DST1+ and dst1Δ strains (SLY101 background) expressing the indicated p53 derivatives were transformed with either PGAL-PHO5 or PGAL-PHO5::lacZ reporter plasmids, and grown in synthetic selectable medium containing 1% raffinose and 1% galactose to early log phase. Cells were harvested and PHO5 expression levels were inferred by an acid phosphatase assay. GLAM ratio refers to the acid phosphatase activity of cells expressing the long transcript (PHO5-lacZ) relative to those expressing the short transcript (PHO5). Depicted are mean values ± S.E.M. of six independent transformants of each indicated strain/plasmid combination. Single asterisk signifies a significant difference between the indicated values (P<0.01; two-tailed t test); double asterisk signifies P<0.0005. (C) Western blot analysis of p53+ and p53V143A levels in DST1+ and dst1Δ strains employed in the GLAM assay (panel B). p53 derivatives were detected using the 1801 mAb; their relative levels, normalized to those of Pgk1, are provided below each lane. S, short transcript expressing cells; L, long transcript plasmid transformed cells; E, cells transformed with empty vector; T.U., transcription unit.
Table 1.
Yeast Strains.