Tandemly Integrated HPV16 Can Form a Brd4-Dependent Super-Enhancer-Like Element That Drives Transcription of Viral Oncogenes

2016-09-13 14:14:12

Journal American Society for Microbiology; 13 September 2016: DOI:10.1128/mBio.01446-16

Katharine E. Dooley, Alix Warburton, Alison A. McBride


In cancer cells associated with human papillomavirus (HPV) infections, the viral genome is very often found integrated into the cellular genome. The viral oncogenes E6 and E7 are transcribed from the viral promoter, and integration events that alter transcriptional regulation of this promoter contribute to carcinogenic progression. In this study, we detected highly enriched binding of the super-enhancer markers Brd4, MED1, and H3K27ac, visible as a prominent nuclear focus by immunofluorescence, at the tandemly integrated copies of HPV16 in cells of the cervical neoplasia cell line W12 subclone 20861. Tumor cells are often addicted to super-enhancer-driven oncogenes and are particularly sensitive to disruption of transcription factor binding to the enhancers. Treatment of 20861 cells with bromodomain inhibitors displaced Brd4 from the HPV integration site, greatly decreased E6/E7 transcription, and inhibited cellular proliferation. Thus, Brd4 activates viral transcription at this integration site, and strong selection for E6/E7 expression can drive the formation of a super-enhancer-like element to promote oncogenesis


Oncogenic human papillomaviruses play an essential role in the development of cervical cancer, and growth of these cancer cells requires continued expression of the viral E6 and E7 oncogenes. Integration of the virus into the host genome often results in deregulation of E6 and E7 expression, which provides a selective growth advantage and increases genetic instability of infected cells. We show here that tandemly integrated copies of the viral genome can form a super-enhancer-like element that drives E6/E7 transcription. Targeted disruption of factors binding to this element decreases viral transcription and causes cell death. Thus, cancer cells that harbor integrated HPV could be targeted by therapeutics that disrupt super-enhancer function.


Oncogenic human papillomaviruses (HPVs) are the cause of cervical cancer, and HPV genomes, which normally replicate extrachromosomally, are often found integrated into the host genome of these cancer cells. Commonly, either a single viral genome or multiple tandemly repeated viral genomes are integrated into the host DNA. The E6 and E7 oncogenes are expressed from the integrated genomes, most often as a fusion transcript expressed from the 3? junctional copy of HPV and the adjacent cellular DNA. Viral genome integration promotes carcinogenesis in a number of ways, but in almost all cases, the cancer-derived cells are dependent on expression of the E6 and E7 oncogenes for continued proliferation. Integration often occurs in the HPV E2 open reading frame, which disrupts the ability of E2 to repress E6 and E7 gene expression. The resulting dysregulation of E6 and E7 causes disruption of cell cycle control, leading to genetic instability and carcinogenesis. Even when the E2 gene remains intact, methylation of E2 binding sites inhibits binding and renders the viral promoter resistant to E2 regulation. Integration events also occur in the E1 gene: this not only removes the downstream E2 gene, but also eliminates the growth-suppressive properties of the E1 protein. In many cases, the E6-E7 virus-cell fusion transcript expressed from integrated DNA is more stable than the viral message, again increasing E6 and E7 levels. Only rarely does insertional mutagenesis result in modified expression of cellular oncogenes or tumor suppressors. In this study, we identify an additional mechanism of E6 and E7 oncogene upregulation. We show that multiple tandem copies of integrated HPV16 can act as a Brd4-dependent super-enhancer-like element that drives transcription of the E6 and E7 oncogenes.

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Key Words

CMA clinical testing | intragenic deletion | single-gene deletion |small CNV