E6 Oncoprotein Represses p53-Dependent Gene Activation via Inhibition of Protein Acetylation Independently of Inducing p53 Degradation  Mary C. Thomas, Cheng-Ming Chiang  Molecular Cell  Volume 17, Issue 2, Pages 251-264 (January 2005) DOI: 10.1016/j.molcel.2004.12.016

Figure 1 Acetyl-CoA and p300 Are Required for p53-Dependent Chromatin Transcription (A) Coomassie blue staining of proteins used for chromatin assembly. Molecular size markers are indicated in kDa. (B) G-less templates used for transcription assays. The p53 binding site (p53BS) containing four pentanucleotides is underlined. (C) Outline of the in vitro chromatin assembly reaction used for in vitro transcription and micrococcal nuclease (MNase) analysis. (D) MNase digestion of pWAFMLT chromatin. A 123 bp DNA ladder was used as size marker (M) in lane 1. (E) Transcription was suppressed from pWAFMLT chromatin template, but not from pWAFMLT DNA and pΔMLP internal control templates. (F) Coomassie blue staining of purified FLAG-tagged human p53 and various HATs. (G) Stimulation of p53-dependent chromatin transcription by p300 and acetyl-CoA. In vitro transcription was performed as outlined. Relative transcription (Rel Txn) in each set of reactions is defined as the signal intensity from the pWAFMLT chromatin or DNA template relative to that performed in the presence of p53, acetyl-CoA, and p300. Molecular Cell 2005 17, 251-264DOI: (10.1016/j.molcel.2004.12.016)

Figure 2 Acetylation of p53 and Nucleosomal Core Histones Correlates with p53-Dependent Chromatin Transcription (A) Schematic drawing and Coomassie blue staining of p300 proteins. (B) Desulfo-CoA inhibits transcription from pWAFMLT chromatin, but not from pΔMLP DNA. (C) Desulfo-CoA inhibits protein acetylation as evidenced by in vitro HAT assay detected by incorporation of [3H]acetyl-CoA. (D) p300-mediated acetylation of nucleosomal core histones is p53-dependent as demonstrated by in vitro HAT assays performed with free core histones (C.H.) or pWAFMLT chromatin in the absence or presence of p53, p300, and HeLa nuclear extract (NE) as indicated. (E) p53 activates transcription at a step prior to p300 entry and correlates with acetylation on p53 and nucleosomal core histones. p53 was added at different stages as outlined for both in vitro transcription and HAT assays. (F) p300 and CBP, but not PCAF and GCN5, enhance p53-dependent chromatin transcription. p53 was added at stage 2 or 3 as outlined in (E). Molecular Cell 2005 17, 251-264DOI: (10.1016/j.molcel.2004.12.016)

Figure 3 HPV E6 Oncoprotein Represses p53-Dependent Chromatin Transcription (A) Coomassie blue staining of purified E6 proteins encoded by HPV-18, HPV-16, and HPV-11. (B) Schematic of the chromatin transcription reaction performed in the absence (−) or presence (+) of HPV-18 E6 (18E6), p53, p300, or CBP as depicted. (C) E6 inhibits p300-mediated p53 transactivation from pWAFMLT chromatin in a dose-dependent manner. An increasing amount of 18E6 (5 ng, 15 ng, and 45 ng) was used in the transcription assays as indicated. Molecular Cell 2005 17, 251-264DOI: (10.1016/j.molcel.2004.12.016)

Figure 4 Recruitment of E6 and p300 by p53 Leads to Suppression of p53-Dependent Chromatin Transcription and Also Inhibition of Acetylation on p53 and Nucleosomal Core Histones (A) Outline of order-of-addition experiments with 18E6 added at different stages of the transcription reaction. (B) 18E6 efficiently inhibits p53-dependent chromatin transcription when added prior to the entry of p300. (C) Schematic of in vitro HAT assays performed with 18E6 included at different stages of the reaction. (D) 18E6 inhibits acetylation on p53 and nucleosomal core histones when added prior to p300 entry as evidenced by in vitro HAT assay. (E) 18E6 inhibits p300-mediated acetylation on p53 and free core histones, but not p300 autoacetylation, as examined by in vitro HAT assays performed in the absence or presence of p300, p53, free core histones (C.H.), and an increasing amount of 18E6 (5 ng, 15 ng, and 45 ng) as indicated. (F) Diagram of the in vitro ChIP assay. (G) Location of PCR primers used for in vitro ChIP assays. (H) Detection of protein binding to pWAFMLT chromatin with promoter-proximal or -distal primer pairs. Molecular Cell 2005 17, 251-264DOI: (10.1016/j.molcel.2004.12.016)

Figure 5 Interactions with p300 and p53 Are Necessary for E6-Mediated Repression of p53-Dependent Transcription In Vitro and In Vivo (A) Schematic of the E6-interacting regions spanning amino acids 340–413 and 1970–2220 of the full-length (FL) p300 protein. The truncated p300 protein (HAT) lacks the E6-interacting domains. (B) The E6-interacting regions on p300 are required for transcriptional inhibition by the E6 proteins encoded by HPV-18, HPV-16, and HPV-11. (C) E6 inhibits acetylation on p53 mediated by the full-length, but not truncated, p300 protein as shown by in vitro HAT assay conducted with FL or HAT p300 protein in the absence (−) or presence (+) of 18E6 and p53 as indicated. (D) E6 does not inhibit Gal4-VP16-mediated chromatin transcription. pG5MLT contains five copies of the Gal4 binding site linked to the adenovirus major late core promoter preceding a 380-nucleotide G-less cassette. (E) Amino acid sequence alignment of 11E6, 16E6, and 18E6 with ClustalW analysis (Chenna et al., 2003). Identical and similar amino acid residues are highlighted in yellow and gray, respectively. The p300/CBP-interacting region conserved among the E6 proteins is indicated in the red box. (F) E6 mutant defective in inducing p53 degradation, but not interaction with p300 and p53, retains the ability to suppress p53-dependent chromatin transcription. The first amino acid of the short (S) form of 16E6 corresponds to the second methionine found in the eighth amino acid residue of the long (L) form of 16E6 (see schematic). Lanes 1–4 show a Coomassie blue-stained gel of wild-type and mutant 16E6 used for in vitro transcription assay (lanes 5–12). (G) RT-PCR analysis of 16E6-mediated repression of endogenous p21 gene expression. HT1080 cells were transfected with pcDNA3 or expression plasmids for FLAG-tagged wild-type or mutant 16E6 with (+) or without (−) UV irradiation followed by MG132 treatment as indicated. (H) Detection of E6-p53-p300 trimeric complex formation by in vitro assembly assay. Immobilized GST-16E6(S) or GST alone was incubated with FLAG-tagged p300 with or without a prior incubation with FLAG-tagged p53 as outlined. The assembled complex was analyzed by Western blotting. Molecular Cell 2005 17, 251-264DOI: (10.1016/j.molcel.2004.12.016)

Figure 6 E6AP Is Not Involved in the Recruitment of p53-E6-p300 Complex to the Endogenous p21 Gene (A) E6AP is present in the immunoprecipitate containing 16E6 and 18E6, but not YYH and 11E6. Western blot of proteins coimmunoprecipitated with FLAG-tagged 16E6 or YYH transiently expressed in HT1080 (lanes 1–3) or mouse E6AP-knockout cells (lanes 4–6) or with FLAG-tagged 11E6 or 18E6 stably expressed in HT1080-derived cell lines (lanes 7–9). The level of induced p53 prior to immunoprecipitation (lysate) is shown. (B) The induced p21 gene expression is suppressed in HT1080 cells stably expressing 11E6 or 18E6 as demonstrated by RT-PCR. (C) 16E6, YYH, and 11E6, but not L37S and G134V, inhibit p21 gene transcription in E6AP-KO cells. Transfection and RT-PCR were performed as outlined in Figure 5G except MG132 was omitted. (D) E6-mediated inhibition of p21 gene transcription correlates with reduction of p21 protein as shown by RT-PCR and Western blotting. (E) 11E6 inhibits p53-mediated activation from a reporter construct containing the native human p21 promoter (pWWP-Luc), but not from the reporter harboring multimerized p53 binding sites (PG13-Luc). Transfection and luciferase assay were performed in Saos-2 cells without MG132 treatment. Error bars represent the average of two independent experiments ± SD. (F) Recruitment of E6 to the human p21 gene in UV-irradiated HT1080 cells correlates with reduced expression of p21 and also inhibition of acetylation on p53 and histone H4. Western blotting, RT-PCR, and in vivo ChIP assays were performed with MG132-treated HT1080 cells harboring pcDNA3 (−) or expressing FLAG-tagged HPV-18 E6 (f:18E6) with (+) or without (−) UV irradiation. (G) An E6AP-deficient complex containing E6-p300-p53 is recruited to the endogenous p21 gene. Re-ChIP assay of the E6 immunoprecipitates pulled down from HT1080-derived stable cell lines was carried out with or without UV irradiation. Molecular Cell 2005 17, 251-264DOI: (10.1016/j.molcel.2004.12.016)

Figure 7 Model for E6-Mediated Repression of p53-Dependent Chromatin Transcription In normal cells, p53-dependent recruitment of p300 leads to a conformational change of p300, acetylation of p53 and nucleosomal core histones, and eventually to transcriptional activation of p53-target genes. In the presence of HPV E6 protein, p53-dependent recruitment of E6 and p300 leads to a different conformational change on p300 and masks the ability of p300 to acetylate p53 and nucleosomal core histones without affecting p300 autoacetylation. In addition, E6 may convert chromatin bound p53-p300 activating complex to a repressing entity (dashed arrow), leading to inhibition of p53-target gene transcription. Molecular Cell 2005 17, 251-264DOI: (10.1016/j.molcel.2004.12.016)