Abstract
The tumor suppressor p53 principally functions as a gene-specific transcription factor. p53 triggers a variety of anti-proliferative programs by activating or repressing the transcription of effector genes in response to genotoxic stress. To date, much effort has been placed on understanding p53's ability to affect transcription in the context of its DNA-binding activity. How p53 regulates transcriptional output independent of DNA binding is less well understood. Here we provide evidence that human p53 can physically interact with the large subunit of RNA polymerase II (Pol II) both in in vitro interaction assays and in whole cell extracts, and that this interaction is mediated (at least in part) through p53's core DNA-binding domain and the Ser5-phosphorylated CTD of Pol II. Ectopic expression of p53, combined with mutations in transcription elongation factors or exposure to drugs that inhibit Pol II elongation, elicit sickness or lethality in yeast cells. These phenotypes are suppressed by oncogenic point mutations within p53's core domain. The growth phenotypes raise the possibility that p53 impairs Pol II elongation. Consistent with this, a p53-dependent increase in Pol II density is seen at constitutively expressed genes without a concomitant increase in transcript accumulation. Additionally, p53-expressing yeast strains exhibit reduced transcriptional processivity at an episomal reporter gene; this inhibitory activity is abolished by a core domain point mutation. Our results suggest a novel mechanism by which p53 can regulate gene transcription, and a new biological function for its core domain that is susceptible to inactivation by oncogenic point mutations.
Highlights
Immunoblot analysis shows that p53coreD is expressed at levels similar to that of full-length p53 in cells grown in the presence of 0.2% galactose/1.8% raffinose (Figure 2B)
We demonstrate in this study that the p53 tumor suppressor can physically and functionally interact with the large subunit of RNA polymerase II
Depicted is a multiplex PCR analysis of DNA purified from immunoprecipitated chromatin samples isolated from SY1000 (PGAL1- p53) and SLY101 (p532) cells subjected to a 2% galactose induction for 0, 60, 120 and 180 min
Summary
P53 is a tumor suppressor that transcriptionally regulates upwards of 2500 genes in response to genotoxic stress. Through its sequence-specific binding, p53 can promote transcription of genes involved in cell cycle arrest, apoptosis, and DNA repair. Six ‘‘hot spot’’ mutations map to the core domain and these mutations are divided into two classes depending on their effect on protein folding and DNA-binding ability [18] Contact mutations such as R248W (L3) and R273H (ß-sheet 10 [S10] in LSH) abolish p53’s ability to bind specific DNA sequences and activate expression of its target genes. Mutations in the core domain of p53 can affect cell fate by regulating DNA binding activity, and by interfering with p53’s protein interactions. Oncogenic mutations within the p53 core domain obviate this effect These and other results suggest a novel mechanism by which p53 can regulate gene transcription in eukaryotic cells
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