Heterochromatin silencing of p53 target genes by a small viral protein

Abstract

The transcription factor p53 (also known as TP53) guards against tumour and virus replication and is inactivated in almost all cancers. p53-activated transcription of target genes is thought to be synonymous with the stabilization of p53 in response to oncogenes and DNA damage. During adenovirus replication, the degradation of p53 by E1B-55k is considered essential for p53 inactivation, and is the basis for p53-selective viral cancer therapies. Here we reveal a dominant epigenetic mechanism that silences p53-activated transcription, irrespective of p53 phosphorylation and stabilization. We show that another adenoviral protein, E4-ORF3, inactivates p53 independently of E1B-55k by forming a nuclear structure that induces de novo H3K9me3 heterochromatin formation at p53 target promoters, preventing p53-DNA binding. This suppressive nuclear web is highly selective in silencing p53 promoters and operates in the backdrop of global transcriptional changes that drive oncogenic replication. These findings are important for understanding how high levels of wild-type p53 might also be inactivated in cancer as well as the mechanisms that induce aberrant epigenetic silencing of tumour-suppressor loci. Our study changes the longstanding definition of how p53 is inactivated in adenovirus infection and provides key insights that could enable the development of true p53-selective oncolytic viral therapies.

Fig 1. p53 is induced and phosphorylated in ΔE1B-55k infection but p53 activity is dominantly suppressed

a. SAECs were infected and protein lysates analyzed by immunoblotting. b. U2OS cells with inducible ARF were infected as indicated and analyzed for p53 levels and activation by immunoblotting. c. RT-QPCR of p53 transcriptional targets in infected SAECs (36 h.p.i.) plus/minus 10 Gy γ irradiation (IR). Error bars represent s.d. (n=3). d. Immunoblot of p53 protein phosphorylation in infected or doxorubicin (dox) treated SAECs (36 h.p.i.). e. Immunoblot of SAECs (36 h.p.i.) infected as indicated and treated with either control (−), dox, nutlin, or TSA at 24 h.p.i.

Fig 2. E4-ORF3 inactivates p53 independently of E1B-55k and p53 degradation

a. SAECs were infected with the indicated viruses (detailed description in Supp. Fig 8) and protein lysates (36 h.p.i.) analyzed for p53 activation by immunoblotting. b. SAECs were co-infected as indicated with either a GFP control virus (Ad-GFP, +) or a virus expressing E4-ORF3 (Ad-ORF3, +). Protein lysates (36 h.p.i.) were analyzed for p53 activation by immunoblotting. c. SAECs were infected and harvested over a 48hr time course as indicated and analyzed for p53 activation by immunoblotting. d. RT-QPCR of p53 transcriptional targets in infected SAECs at 36 h.p.i. Error bars represent s.d. (n=3).

Fig 3. E4-ORF3 induces heterochromatin formation and prevents p53-DNA binding at endogenous promoters

a. U2OS cells were transfected with p53-luc (solid line) or p53-mutant (dashed line) luciferase plasmids and infected with indicated viruses. Luminescence is plotted against time. b. and c. U2OS cells were infected as indicated or treated with doxorubicin. b. p53 induction was analyzed by immunoblotting and p53 transcriptional targets quantified by RT-QPCR (36 h.p.i.). Error bars represent s.d. (n=3) c. p53 ChIPs were analyzed by semi-quantitative PCR for p21 and MDM2 promoter sequences. d. p53 (green) and H3K9me3 (red) immunofluorescence of infected U2OS cells (36 h.p.i.). e. Co-localization of SUV39H1, SUV39H2, SETDB1 and G9a (green) with H3K9me3 (red) in Δ55k infected U2OS cells (36 h.p.i.).

Fig 4. E4-ORF3 forms a nuclear scaffold that specifies heterochromatin assembly and H3K9 trimethylation at p53 target promoters

a. Protein lysates from infected U2OS cells (36 h.p.i.) were analyzed for total Histone H3 or H3K9me3 levels by immunoblotting. H3K9me3 and p53 ChIPs were quantified by RT-QPCR, normalized relative to input DNA and plotted as fold change relative to mock. Error bars represent s.d. (n=2) b. H3K9me3 (green) and E4-ORF3 (red) localization in Δ55k infected SAECs (36 h.p.i.) was visualized by immunofluorescence. A high resolution confocal slice (0.3μm) through the nucleus is shown with a magnified section of E4-ORF3 and associated heterochromatin domains on the far right.

Fig 5. p53 transcriptional targets are silenced selectively in the backdrop of global transcriptional changes that drive oncogenic cellular and viral replication

Affymetrix global gene expression analyses of SAECsa. Heat map of the 1,730 overlapping differentially regulated genes (log FC>2 or <−2 with a false discovery rate (FDR) of 0.05) between Δ55k/ΔORF3 andΔ55k versus mock infected SAECs (36 h.p.i.). b. Unsupervised hierarchical clustering of 46 top differentially upregulated transcripts in both Δ55k/ΔORF3 infection and nutlin treatment. c. Pie-chart depicting the percentage of upregulated transcripts (log FC>2 and FDR of 0.05) in Δ55k/ΔORF3 versusΔ55k that have predicted p53 transcription factor binding sites and/or induced by a log FC>1.5 in response to nutlin. d. Summary and model.