The high-risk (HR) human papillomaviruses (HPV) are causative agents of anogenital tract dysplasia and cancers and a fraction of head and neck cancers. The HR HPV E6 oncoprotein possesses canonical oncogenic functions, such as p53 degradation and telomerase activation. It is also capable of stimulating expression of several oncogenes, but the molecular mechanism underlying these events is poorly understood. Here, we provide evidence that HPV16 E6 physically interacts with histone H3K4 demethylase KDM5C, resulting in its degradation in an E3 ligase E6AP- and proteasome-dependent manner. Moreover, we found that HPV16-positive cancer cell lines exhibited lower KDM5C protein levels than HPV-negative cancer cell lines. Restoration of KDM5C significantly suppressed the tumorigenicity of CaSki cells, an HPV16-positive cervical cancer cell line. Whole genome ChIP-seq and RNA-seq results revealed that CaSki cells contained super-enhancers in the proto-oncogenes and Ectopic KDM5C dampened these super-enhancers and reduced the expression of proto-oncogenes. This effect was likely mediated by modulating H3K4me3/H3K4me1 dynamics and decreasing bidirectional enhancer RNA transcription. Depletion of KDM5C or HPV16 E6 expression activated these two super-enhancers. These results illuminate a pivotal relationship between the oncogenic E6 proteins expressed by HR HPV isotypes and epigenetic activation of super-enhancers in the genome that drive expression of key oncogenes like and This study suggests a novel explanation for why infections with certain HPV isotypes are associated with elevated cancer risk by identifying an epigenetic mechanism through which E6 proteins expressed by those isotypes can drive expression of key oncogenes. .

<div>Abstract<p>The high-risk (HR) human papillomaviruses (HPV) are causative agents of anogenital tract dysplasia and cancers and a fraction of head and neck cancers. The HR HPV E6 oncoprotein possesses canonical oncogenic functions, such as p53 degradation and telomerase activation. It is also capable of stimulating expression of several oncogenes, but the molecular mechanism underlying these events is poorly understood. Here, we provide evidence that HPV16 E6 physically interacts with histone H3K4 demethylase KDM5C, resulting in its degradation in an E3 ligase E6AP- and proteasome-dependent manner. Moreover, we found that HPV16-positive cancer cell lines exhibited lower KDM5C protein levels than HPV-negative cancer cell lines. Restoration of KDM5C significantly suppressed the tumorigenicity of CaSki cells, an HPV16-positive cervical cancer cell line. Whole genome ChIP-seq and RNA-seq results revealed that CaSki cells contained super-enhancers in the proto-oncogenes <i>EGFR</i> and <i>c-MET</i>. Ectopic KDM5C dampened these super-enhancers and reduced the expression of proto-oncogenes. This effect was likely mediated by modulating H3K4me3/H3K4me1 dynamics and decreasing bidirectional enhancer RNA transcription. Depletion of KDM5C or HPV16 E6 expression activated these two super-enhancers. These results illuminate a pivotal relationship between the oncogenic E6 proteins expressed by HR HPV isotypes and epigenetic activation of super-enhancers in the genome that drive expression of key oncogenes like <i>EGFR</i> and <i>c-MET</i>.</p><p><b>Significance:</b> This study suggests a novel explanation for why infections with certain HPV isotypes are associated with elevated cancer risk by identifying an epigenetic mechanism through which E6 proteins expressed by those isotypes can drive expression of key oncogenes. <i>Cancer Res; 78(6); 1418–30. ©2018 AACR</i>.</p></div>

<p>Supplementary Figures - Supplementary Figures S1-9. Supplementary Figure S1. Mutual co-immunoprecipitation of HPV16 E6 and KDM5C; Supplementary Figure S2. HPV16 E6 attenuates the KDM5C protein level and KDM5C mRNA quantification; Supplementary Figure S3. HPV16 E6 destabilizes KDM5C and alters the global H3K4 methylation; Supplementary Figure S4. KDM5C does not visibly alters HPV 16E6, HPV 16E7 mRNA and protein levels; Supplementary Figure S5. KDM5C knock out increases tumorigenicity in mice xenograft assay; Supplementary Figure S6. H3K27Ac recruitment to chromatin in the parental cells was examined by the genomic analysis of ChIP-seq signals. ; Supplementary Figure S7. Normalized ChIP-seq signal for H3K27Ac across the EGFR gene and c-MET gene. Supplementary Figure S8. Normalized H3K4me1 levels at KDM5C-bound super enhancers in CaSki-vector and CaSki-KDM5C cells. Supplementary Figure S9. HPV 16E6 depletion leads to EGFR and c-MET super-enhancer inhibition. Supplementary Methods - Description of additional experimental methods including Transfection, siRNA and MG132 treatment, Antibodies, Co-immunoprecipitation and ubiquitination assays, Immunofluorescence Microscopy, Maltose Binding Protein (MBP) Pull-down Assay, Chromatin immunoprecipitation (ChIP)-PCR and ChIP-Seq, Cell Invasion Assay, Real Time Cell Proliferation Assay, Wound healing Assay, Trypsin Digestion and Mass spectrometry analysis Supplementary Tables - Supplementary Tables S1-2. Supplementary Table S1. List of qPCR primers used in this study. Table S2. List of DNA oligonucleotides used in this study.</p>

<p>Supplementary Figures - Supplementary Figures S1-9. Supplementary Figure S1. Mutual co-immunoprecipitation of HPV16 E6 and KDM5C; Supplementary Figure S2. HPV16 E6 attenuates the KDM5C protein level and KDM5C mRNA quantification; Supplementary Figure S3. HPV16 E6 destabilizes KDM5C and alters the global H3K4 methylation; Supplementary Figure S4. KDM5C does not visibly alters HPV 16E6, HPV 16E7 mRNA and protein levels; Supplementary Figure S5. KDM5C knock out increases tumorigenicity in mice xenograft assay; Supplementary Figure S6. H3K27Ac recruitment to chromatin in the parental cells was examined by the genomic analysis of ChIP-seq signals. ; Supplementary Figure S7. Normalized ChIP-seq signal for H3K27Ac across the EGFR gene and c-MET gene. Supplementary Figure S8. Normalized H3K4me1 levels at KDM5C-bound super enhancers in CaSki-vector and CaSki-KDM5C cells. Supplementary Figure S9. HPV 16E6 depletion leads to EGFR and c-MET super-enhancer inhibition. Supplementary Methods - Description of additional experimental methods including Transfection, siRNA and MG132 treatment, Antibodies, Co-immunoprecipitation and ubiquitination assays, Immunofluorescence Microscopy, Maltose Binding Protein (MBP) Pull-down Assay, Chromatin immunoprecipitation (ChIP)-PCR and ChIP-Seq, Cell Invasion Assay, Real Time Cell Proliferation Assay, Wound healing Assay, Trypsin Digestion and Mass spectrometry analysis Supplementary Tables - Supplementary Tables S1-2. Supplementary Table S1. List of qPCR primers used in this study. Table S2. List of DNA oligonucleotides used in this study.</p>

<div>Abstract<p>The high-risk (HR) human papillomaviruses (HPV) are causative agents of anogenital tract dysplasia and cancers and a fraction of head and neck cancers. The HR HPV E6 oncoprotein possesses canonical oncogenic functions, such as p53 degradation and telomerase activation. It is also capable of stimulating expression of several oncogenes, but the molecular mechanism underlying these events is poorly understood. Here, we provide evidence that HPV16 E6 physically interacts with histone H3K4 demethylase KDM5C, resulting in its degradation in an E3 ligase E6AP- and proteasome-dependent manner. Moreover, we found that HPV16-positive cancer cell lines exhibited lower KDM5C protein levels than HPV-negative cancer cell lines. Restoration of KDM5C significantly suppressed the tumorigenicity of CaSki cells, an HPV16-positive cervical cancer cell line. Whole genome ChIP-seq and RNA-seq results revealed that CaSki cells contained super-enhancers in the proto-oncogenes <i>EGFR</i> and <i>c-MET</i>. Ectopic KDM5C dampened these super-enhancers and reduced the expression of proto-oncogenes. This effect was likely mediated by modulating H3K4me3/H3K4me1 dynamics and decreasing bidirectional enhancer RNA transcription. Depletion of KDM5C or HPV16 E6 expression activated these two super-enhancers. These results illuminate a pivotal relationship between the oncogenic E6 proteins expressed by HR HPV isotypes and epigenetic activation of super-enhancers in the genome that drive expression of key oncogenes like <i>EGFR</i> and <i>c-MET</i>.</p><p><b>Significance:</b> This study suggests a novel explanation for why infections with certain HPV isotypes are associated with elevated cancer risk by identifying an epigenetic mechanism through which E6 proteins expressed by those isotypes can drive expression of key oncogenes. <i>Cancer Res; 78(6); 1418–30. ©2018 AACR</i>.</p></div>