Papillomavirus-Associated Tumor Formation Critically Depends on c-Fos Expression Induced by Viral Protein E2 and Bromodomain Protein Brd4

Abstract

We investigated the mechanism of how the papillomavirus E2 transcription factor can activate promoters through activator protein (AP)1 binding sites. Using an unbiased approach with an inducible cell line expressing the viral transcription factor E2 and transcriptome analysis, we found that E2 induces the expression of the two AP1 components c-Fos and FosB in a Brd4-dependent manner. In vitro RNA interference confirmed that c-Fos is one of the AP1 members driving the expression of viral oncogenes E6/E7. Mutation analysis and in vivo RNA interference identified an essential role for c-Fos/AP1 and also for the bromodomain protein Brd4 for papillomavirus-induced tumorigenesis. Lastly, chromatin immunoprecipitation analysis demonstrated that E2 binds together with Brd4 to a canonical E2 binding site (E2BS) in the promoter of c-Fos, thus activating c-Fos expression. Thus, we identified a novel way how E2 activates the viral oncogene promoter and show that E2 may act as a viral oncogene by direct activation of c-Fos involved in skin tumorigenesis.

Involvement of Brd4 in different steps of the papillomavirus life cycle

“…The AP-1 member c-Fos has recently been demonstrated to be transcriptionally upregulated by a concerted action of E2 and Brd4. This activation has further been shown to be important for papillomavirus-induced tumor formation (Delcuratolo et al, 2016), which is also supported by previous findings showing that c-Fos is overexpressed in HPV-positive lesions (Nurnberg et al, 1995) and that a shift from c-Jun/Fra-1 to c-Jun/c-Fos dimers occurs during HPV-induced carcinogenesis (de Wilde et al, 2008). An increase of c-Fos expression via E2 and Brd4 also results in the activation of the viral promotor, which leads to an increase in oncogene expression and rendering all components of this regulatory cascade essential for tumorigenesis (Behren et al, 2005).…”

“…It is possible that other regions of Brd4, such as BID and phospho-NPS, might interact with E2 to stabilize the complex in nuclear foci. A model can be delineated suggesting that in the early small E1–E2-Brd4 foci, E2 predominantly acts as a transcription factor regulating expression of not only early viral genes (Sakakibara et al, 2013a), but also cellular genes including the immediate early gene c-Fos (Delcuratolo et al, 2016). At later time points, when the foci begin to amplify viral genomes the acetylated chromatin marks are dispersed to the periphery of the foci together with Brd4 and transcription may continue.…”

“…However, to date only a limited number of genes are validated to be regulated by E2. These include MMP-9 (Behren et al, 2005), hTERT (Lee et al, 2002), SF2/ASF (Klymenko et al, 2016; Mole et al, 2009), IL-10 (Bermudez-Morales et al, 2011), p21 (Steger et al, 2002), involucrin (Hadaschik et al, 2003), β 4-integrin (Oldak et al, 2010) and most recently c-Fos (Delcuratolo et al, 2016). Brd4 is necessary for E2’s function as a transcriptional activator when E2BS is positioned away from the promoter-proximal region in an enhancer-like sequence context (Lee and Chiang, 2009) as found in CRPV (see Fig.…”

“…With the help of two different shRNAs directed against rabbit Brd4 in the context of all early viral genes expressed at physiological levels, an important role of Brd4 for tumor formation and endogenous enhanced c-Fos expression in the wt CRPV-induced tumors was shown (Delcuratolo et al, 2016). Mutant genomes with an E2 defective for Brd4 binding (Jeckel et al, 2003) caused only very few tumors, which revealed severely retarded growth and were defective in enhanced c-Fos expression.…”

The Interaction Between Human Papillomaviruses and the Stromal Microenvironment

“…Finally, other transcription factors regulated by HPV can impact HIF-1 activity or independently respond to hypoxia, including E2F1, NFκB, AP1 and Myc 225,437,512–521 . IFNs can also decrease expression of angiogenic factors in part by inhibiting bFGF and VEGF production 235,522 , but the impact of HPV-induced suppression of IFN on angiogenesis specifically is not known.…”