In this study, we have shown that the E2 proteins from several papillomaviruses associate with transcriptionally active regions of cellular chromatin. Using a ChIP-chip approach, we have demonstrated that the BPV-1 E2 protein binds to a subset of human promoters in concert with the bromodomain protein Brd4. This binding correlates with signatures of active transcription, such as binding of RNA pol II, acetylation of histones, and methylation of histone H3 K4. Therefore, the E2 proteins associate with regions of active chromatin.
Notably, binding of the E2 proteins to cellular chromatin does not correlate with the presence of consensus E2 binding motifs in the cellular DNA. This is consistent with previous findings showing that the DNA binding function of E2 was not required for the association of E2 with cellular chromatin and that the chromatin binding determinants resided in the E2 transactivation domain (7
). Furthermore, it is unlikely that the binding sites for a viral protein would be conserved in host genes unless, by chance, they overlapped the binding site for an essential cellular factor or provided some other advantage to the host organism. Therefore, these interactions are most likely mediated by interaction of E2 with cellular host factors.
Although the E2 proteins bind to active cellular genes, there is no change in the occupancy of transcription factors bound to these sites and no change in the activity of these promoters, as measured by mRNA levels. This finding is actually not surprising. Papillomaviruses are persistent viruses that develop a long-term relationship with the host cell, and it is unlikely that they would cause global changes in cellular gene expression in the persistent phase of their life cycle. This is completely consistent with a recent microarray study that showed that when expressed in HeLa cells, BPV-1 E2 had no effect on the expression of cellular genes (6
). Rather than cause global changes in cellular gene expression, persistent viruses are much more likely to encode functions that specifically evade the host defense mechanisms and allow the virus to escape detection. For example, the papillomavirus E6 and E7 proteins can abrogate the cellular interferon response (reviewed in reference 20
We find that the cellular bromodomain protein, Brd4, also binds to all active cellular promoters. Brd4 is a ubiquitous protein that is a component of both the transcriptional elongation complex pTEFb (23
) and the Mediator complex (10
) and functions as a chromatin adaptor (21
). Brd4 is an essential gene (2
) that is important for G1
transcription and progression into S phase (13
). Based on the results presented here, it is likely that Brd4 is involved in the transcription of most, if not all, cellular genes. Binding of BPV-1 E2 to these promoters does not change the occupancy of Brd4 or other cellular factors. The effect of E2 and Brd4 on viral promoters, which are regulated by E2-specific binding sites, is very different. Under these circumstances, E2 can bind tightly to the promoters through its cognate DNA binding site and recruit Brd4 and other transcription factors. Notably, Brd4 seems to be important for the E2-dependent transcriptional regulation of all papillomavirus E2 proteins tested to date (11
Host cells have innate antiviral defenses to limit the expression of incoming foreign viral DNA, and in many cases, viral DNA is transcriptionally repressed and heterochromatinized (reviewed in reference 9
). However, viruses are equipped to counteract this response and maintain their genomes in a functional state. Furthermore, small DNA viruses, such as the papillomaviruses, must use all of the host transcriptional and replication machinery to express and replicate their own genome. Thus, we propose that the interaction of the E2 proteins with transcriptionally active promoters is an additional mechanism to localize E2, and the associated viral genomes, in subregions of the nucleus that are permissive for transcription and replication. Our results are very consistent with those of Kurg et al., who demonstrated biochemically that a fraction of E2, and associated viral genomes, was associated with fractions of transcriptionally active cellular chromatin (7
Studies are in progress to characterize the E2-Brd4 complexes that bind to cellular chromatin in mitosis, when cellular transcription is turned off. E2 tethers the viral genome to the host chromosomes in mitosis, and it has been proposed that this is important to partition the viral genome in approximately equal numbers to daughter cells and also to ensure that the viral genome is retained in the nucleus after cell division. We can now add a third reason for E2 to tether the viral genomes to cellular chromatin: to provide epigenetic memory and ensure that the genomes are retained in euchromatic regions of the nucleus to allow continued expression of the viral proteins important for its survival.