Although the role of high-risk human papillomaviruses (hrHPVs) as etiological agents in cancer development has been intensively studied during the last decades, there is still the necessity of understanding the impact of the HPVE6andE7oncogenes on host cells, ultimately leading to malignant transformation. Here, we used newly established immortalized human keratinocytes with a well-defined HPV16E6E7expression cassette to get a more complete and less biased overview of global changes induced by HPV16 by employing transcriptome sequencing (RNA-Seq) and stable isotope labeling by amino acids in cell culture (SILAC). This is the first study combining transcriptome and proteome data to characterize the impact of HPV oncogenes in human keratinocytes in comparison with their virus-negative counterparts. To enhance the informative value and accuracy of the RNA-Seq data, four different bioinformatic workflows were used. We identified potential novel upstream regulators (e.g., CNOT7, SPDEF, MITF, and PAX5) controlling distinct clusters of genes within the HPV-host cell network as well as distinct factors (e.g., CPPED1, LCP1, and TAGLN) with essential functions in cancer. Validated results in this study were compared to data sets from The Cancer Genome Atlas (TCGA), demonstrating that several identified factors were also differentially expressed in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) and HPV-positive head and neck squamous cell carcinomas (HNSCs). This highly integrative approach allows the identification of novel HPV-induced cellular changes that are also reflected in cancer patients, providing a promising omics data set for future studies in both basic and translational research.IMPORTANCEHuman papillomavirus (HPV)-associated cancers still remain a big health problem, especially in developing countries, despite the availability of prophylactic vaccines. Although HPV oncogenes have been intensively investigated for decades, a study applying recent advances in RNA-Seq and quantitative proteomic approaches to a precancerous model system with well-defined HPV oncogene expression alongside HPV-negative parental cells has been missing until now. Here, combined omics analyses reveal global changes caused by the viral oncogenes in a less biased way and allow the identification of novel factors and key cellular networks potentially promoting malignant transformation. In addition, this system also provides a basis for mechanistic research on novel key factors regulated by HPV oncogenes, especially those that are confirmedin vivoin cervical cancer as well as in head and neck cancer patient samples from TCGA data sets.

Retinoic acid receptor ␤2 (RAR␤2) is often down-regulated during the multistep process to cervical cancer. In that way, its inhibitory function on the transcription factor AP-1, indispensable to maintain human papillomavirus (HPV) gene expression is relieved. Using HPV-18 positive HeLa cells as a model system, we show that ectopic expression of RAR␤2 is able to down-regulate HPV-18 transcription by selectively abrogating the binding of AP-1 to the viral regulatory region in a ligandindependent manner. This resulted in down-regulation of the viral mRNAs at the level of initiation of transcription. Decreased oncogene expression was accompanied by a re-induction of cell cycle inhibitory proteins such as p53, p21 CIP1 , and p27 KIP as well as by a cessation of cellular growth. Reduced transcriptional activity as a consequence of AP-1 reduction by selective c-Jun degradation apparently targets the HPV-18 regulatory region for epigenetic modification such as de novo methylation and nucleosomal condensation. This mechanism is otherwise counterbalanced by active and abundant viral transcription in malignant cells, because RAR␤2 itself becomes inactivated during cervical carcinogenesis. Hence, our study shows that the temporal co-existence of a potential repressor and viral oncoproteins is mutually exclusive and provides evidence of a cross-talk between a nuclear receptor, AP-1, and the epigenetic machinery.Cancer is a multistep process that is characterized by accumulation of various cell-damaging events such as chromosomal instabilities, inactivation of cell cycle regulatory proteins, and epigenetic modifications (1, 2). In the case of cervical cancer, the second leading malignancy in women worldwide, certain types of human papillomaviruses (HPV) 2 have been identified as etiological agents being involved both in initiation and maintenance of the transformed phenotype (3). The transcription of the viral oncogenes E6 and E7 is controlled by the viral upstream regulatory region (URR), harboring the tissue-specific enhancer and promoter elements (4). Here, beside other cellular transcription factors, activator protein-1 (AP-1) is the main regulator determining the efficiency of expression and in turn the intracellular net amount of the viral oncoproteins E6/E7 (5).As shown for many malignant cells, including HPV-positive cervical cancer cells, the retinoic acid receptor (RAR) ␤2 gene (RAR␤2), the most potent RAR involved in suppression of tumor-related phenotypes (6, 7), is epigenetically silenced via de novo methylation and chromatin remodeling (8). In other words, the absence of RAR␤ provides a selective advantage during multistep progression to cervical cancer, because the gene is obviously not compatible with unscheduled cell proliferation (9, 10). Indeed, re-introduction of RAR␤ into malignant cells interferes with anchorage-independent growth (6, 11) and diminishes tumor formation upon heterotransplantation into immunocompromised animals (12). Together with other isotypes of retinoid acid receptors (␣ and ␥) or reti...

In the present study, we used the human papillomavirus type 16 (HPV16)-positive cervical carcinoma cell line CaSki as a paradigmatic model to understand epigenetic silencing of viral multi-copy genomes. We show that most of the hypermethylated HPV16 copies are kept as 'occluded' chromatin that defines a condition where genes were refractory in their response to trans-acting transcription factors and to external reactivation efforts. This provides the first example that viral genomes are silenced by such a host cell mechanism, hitherto only known for endogenous genes to preserve a stable and robust phenotype. Moreover, considering an adaptive cross-talk between viral proteins and the epigenetic modification machinery, we demonstrate that particularly E2-but also ectopically delivered E6/E7-can induce significant de novo methylation within the enhancer and, to a less extent, within the promoter region. These data suggest that under certain physiological conditions, HPV can down-regulate its own gene expression, regardless of the presence of transcriptional activators. We propose that self-methylation of multi-copy HPV could be the first event prior to heterochromatin formation. These processes favour an 'occluded' chromatin conformation, finally being unresponsive to transcriptional activation. The shift from potentially competent heterochromatin towards an occluded state is basically irreversible, possibly using the same mechanism described for lineage differentiation. Along this line, it is tempting to speculate that virus-cell interaction is able to 'sense' viral copy number and down-regulates excess of gene activity in order to guarantee cell viability.