The E2 protein encoded by human papillomaviruses (HPVs) inhibits expression of the viral E6 oncoprotein, which, in turn, regulates p53 target gene transcription. To identify cellular proteins involved in E2-mediated transcriptional repression, we isolated an E2 complex from human cells conditionally expressing HPV-11 E2. Surprisingly, the double bromodomain-containing protein Brd4, which is implicated in cell cycle control and viral genome segregation, was found associated with E2 and conferred on E2 the ability to inhibit AP-1-dependent HPV chromatin transcription in an E2-binding site- [Keywords: HPV; E2; AP-1; Brd4; chromatin transcription; gene silencing] Supplemental material is available at http://www.genesdev.org.

Transcription in human papillomaviruses (HPVs) is mainly regulated by cellular transcription factors and virus-encoded E2 proteins that act as sequence-specific DNA-binding proteins. Although the functions of E2 as a transcriptional activator and a repressor have been well documented, the role of cellular factors involved in E2-mediated regulation of the HPV promoters and the mechanism by which E2 modulates viral gene expression remain unclear. Using reconstituted cell-free transcription systems, we found that cellular enhancerbinding factors and general cofactors, such as TAF II s, TFIIA, Mediator, and PC4, are not required for E2-mediated repression. Unlike other transcriptional repressors that function through recruitment of histone deacetylase or corepressor complexes, HPV E2 is able to directly target components of the general transcription machinery to exert its repressor activity on the natural HPV E6 promoter. Interestingly, preincubation of TATA binding protein (TBP) or TFIID with HPV template is not sufficient to overcome E2-mediated repression, which can be alleviated only via formation of a minimal TBP (or TFIID)-TFIIB-RNA polymerase II-TFIIF preinitiation complex. Our data therefore indicate that E2 does not simply work by displacing TBP or TFIID from binding to the adjacent TATA box. Instead, E2 appears to function as an active repressor that directly inhibits HPV transcription at steps after TATA recognition by TBP or TFIID.Transcription in eukaryotes is often regulated by extracellular molecules that act through distinct signal transduction pathways to modulate specific gene expression via controlling the activity of gene-specific transcription factors. These genespecific transcription factors then work in conjunction with general transcription factors (GTFs) and cofactors to enhance or inhibit the level of transcription. Although many studies have been conducted to elucidate the mechanisms of transcriptional activation in eukaryotes, relatively little is known about the mechanisms of repression. In general, transcriptional repressors can work either passively to antagonize the activator function or actively to inhibit the activity of the general transcription machinery (30). Counteraction of the activator function by passive repressors can be achieved by direct competition of the same DNA-binding sites (36,37,41,54,55), interference of overlapping or neighboring activator-binding sites (21,24,38,58), modification of the DNA-binding property of the activators (60), titrating away limiting protein factors required for activator function (15, 31), or masking and/or altering the function of the activation domain or blocking the DNA-binding activity of the activators through protein-protein interactions (3,24,46,61). In contrast, active repressors are able to directly inhibit the activity or the assembly of the general transcription machinery, with or without the help of corepressors (2,23,27,29,40,43,45,51). The recruitment of histone deacetylase complexes by some repressors or corepressors represen...

The full-length E2 protein, encoded by human papillomaviruses (HPVs), is a sequence-specific transcription factor found in all HPVs, including cancer-causing high risk HPV types 16 and 18 and wart-inducing low risk HPV types 6 and 11. To investigate whether E2 proteins encoded by high risk HPVs may function differentially from E2 proteins encoded by low risk HPVs and animal papillomaviruses, we conducted comparative DNAbinding and transcription studies using electrophoretic mobility shift assays and cell-free transcription systems reconstituted with purified general transcription factors, cofactor, RNA polymerase II, and with E2 proteins encoded by HPV-16, HPV-18, HPV-11, and bovine papillomavirus type 1 (BPV-1). We found that although different types of E2 proteins all exhibited transactivation and repression activities, depending on the sequence context of the E2-binding sites, HPV-16 E2 shows stronger transcription activity and greater DNA-binding affinity than those displayed by the other E2 proteins. Surprisingly, HPV-18 E2 behaves more similarly to BPV-1 E2 than HPV-16 E2 in its functional properties. Our studies thus categorize HPV-18 E2 and BPV-1 E2 in the same protein family, a finding consistent with the available E2 structural data that separate the closely related HPV-16 and HPV-18 E2 proteins but classify together the more divergent BPV-1 and HPV-18 E2 proteins.Human papillomaviruses (HPVs) 1 are a family of small DNA viruses that cause a wide variety of human diseases ranging from benign epithelial lesions, such as warts, to invasive cancers, such as cervical carcinoma. So far, more than 100 HPV types have been identified and fully sequenced, whereas more than 120 putative novel types have been partially characterized (1, 2). HPV types frequently found in invasive cancers include These are classified as high risk HPVs. In contrast, HPV types that are rarely found in cancers but are associated with genital warts, such as HPV-6 and HPV-11, are considered low risk HPVs (1-3). Because the genomic structures of HPVs are highly conserved, it is important to determine the functional differences among individual HPV gene products which lead to etiologically high and low risk phenotypes. Previous comparative studies have primarily focused on HPV-encoded E6-and E7-transforming proteins. These studies found that E6 and E7, from high risk HPVs, lead to cellular transformation much more readily than low risk E6 and E7 proteins (for review, see Refs. 4 and 5). In both high and low risk HPVs, expression of E6 and E7 is transcriptionally regulated via the E6 promoter by many cellular and viral proteins. The full-length viral E2 protein is a sequence-specific transcription factor that functions as an activator or repressor to regulate tightly the E6 promoter through four consensus E2-binding sites (E2-BSs), ACCGN 4 CGGT (6, 7), whose locations within the upstream regulatory region (URR) are highly conserved among genital HPVs. Efficient activation of the E6 promoter requires binding of E2 protein to the promoter...

TFIID is a general transcription factor required for the assembly of the transcription machinery on most eukaryotic promoters transcribed by RNA polymerase II. Although the TATA-binding subunit (TBP) of TFIID is able to support core promoter and activator-dependent transcription under some circumstances, the roles of TBP-associated factors (TAF II s) in TFIID-mediated activation remain unclear. To define the evolutionarily conserved function of TFIID and to elucidate the roles of TAF II s in gene activation, we have cloned the mouse TAF II 55 subunit of TFIID and further isolated mouse TFIID from a murine FM3A-derived cell line that constitutively expresses FLAG-tagged mouse TAF II 55. Both mouse and human TFIIDs are capable of mediating transcriptional activation by Gal4 fusions containing different activation domains in a highly purified human cellfree transcription system devoid of TFIIA and Mediator. Although TAF II -independent activation by Gal4-VP16 can also be observed in this highly purified human transcription system with either mouse or yeast TBP, TAF II s are strictly required for estrogen receptor-mediated activation independently of the core promoter sequence. In addition, TAF II s are necessary for transcription from a preassembled chromatin template. These findings clearly demonstrate an essential role of TAF II s as a transcriptional coactivator for estrogen receptor and in chromatin transcription.Regulation of eukaryotic transcription by gene-specific transcription factors often requires protein cofactors, in addition to the general transcription machinery. Currently, there are three classes of general cofactors commonly thought to be essential for activator-dependent transcription. The first class is RNA polymerase II-specific TBP-associated factors (TAF II s) 1 initially defined as components of TFIID (1-6). TAF II s are highly conserved through evolution and exhibit many properties accounting for the functional activities of TFIID. In general, TFIID is a core promoter-binding factor that has intrinsic activity to recognize the TATA box, initiator and downstream promoter elements, and initiates preinitiation complex assembly on both TATA-containing and TATA-less promoters (1,7,8). The nucleation pathway for preinitiation complex formation usually begins with TFIID binding to the core promoter region, followed either by sequential assembly of other general transcription factors (GTFs) and RNA polymerase II (pol II) or by recruitment of a preassembled pol II holoenzyme complex (9 -11). In addition to the core promoter-binding activity, TFIID has also been implicated as a general coactivator or corepressor in transducing the regulatory signals to the general transcription machinery, as exemplified by many protein-protein interactions occurring between gene-specific regulatory factors and components of TFIID (1)(2)(3)(12)(13)(14). A universal coactivator function of TFIID has recently been challenged by both in vivo yeast studies (15)(16)(17) and in vitro mammalian cell-free transcription assays ...