Alleviation of Human Papillomavirus E2-Mediated Transcriptional Repression via Formation of a TATA Binding Protein (or TFIID)-TFIIB-RNA Polymerase II-TFIIF Preinitiation Complex

Abstract

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...

Brd4 links chromatin targeting to HPV transcriptional silencing

“…4D, lanes 1-12), a conclusion further supported by the pairwise mutational studies (Fig. 4D, lanes 13-24) and also by previous in vivo and in vitro assays (Hou et al 2000 and references therein).…”

“…2A). We have previously shown that transcription in vitro from this HPV-11 URR-driven DNA template initiates at the correct start site and faithfully recapitulates E2-mediated regulation of E6 promoter activity as observed in vivo (Hou et al 2000). This HPV-11 DNA template was assembled into chromatin by incubating with purified HeLa core histones, Drosophila nucleosome assembly factor ACF (a protein dimer reconstituted from recombinant Acf1 and FLAG-tagged ISWI), and recombinant human NAP-1 histone chaperone (Fig.…”

“…To identify cellular proteins mediating the nuclear activity of E2 and to bypass potential cytotoxicity triggered by constitutive E2 production in mammalian cells, we established several tetracycline-regulated E2 cell lines conditionally expressing either dual hexahistidine/FLAGtagged HPV-11 E2 (367 residues) or dual hexahistidine/ FLAG-tagged dsE2C that contains 11 amino acids derived from the E1 ORF linked to the C-terminal 149 residues of E2 (Hou et al 2000). These proteins were efficiently induced, following the removal of tetracycline from cultured medium, in human embryonic kidney-derived 293 cells (Fig.…”

“…This configuration allows E2 to recognize distinct E2-binding sites (ACCN 6 GGT) with variable internal sequences (Hegde 2002). Although HPV E2, indeed, possesses weak transactivating activity (Hou et al 2000), it normally functions as a transcriptional repressor in its natural host to inhibit the expression of viral E6 and E7 oncoproteins (Bernard et al 1989). These virus-encoded oncoproteins, especially from high-risk genital HPVs that induce cervical cancers, are able to functionally inactivate p53 and pRb tumor suppressor proteins generally by targeting these cellular proteins for degradation (Münger et al 2004).…”

“…These E2 variants, including the double-spliced E2C (dsE2C) protein encoded by low-risk HPV type 11 (HPV-11), typically act as transcriptional repressors that inhibit HPV E6 promoter activity via specific E2-binding sites (Hou et al 2000). Surprisingly, while both full-length E2 (hereon referred to as E2) and dsE2C are able to suppress E6 promoter activity with HPV DNA templates in a cell-free transcription system reconstituted with recombinant TFIIB, TFIIE, TFIIF, and epitope-tagged RNA polymerase II (pol II), TFIID, and TFIIH (Hou et al 2000), inhibition of HPV transcription in cultured cells appears to additionally require the N-terminal region of E2 (Dowhanick et al 1995;Demeret et al 1997;Goodwin et al 1998). Presumably the N-terminal domain is necessary for E2 association with undefined cellular proteins to inhibit E6 promoter activity from HPV chromatin that is assembled in the cell.…”