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. 2001 Mar;75(6):2829-38.
doi: 10.1128/JVI.75.6.2829-2838.2001.

A novel silencer element in the bovine papillomavirus type 4 promoter represses the transcriptional response to papillomavirus E2 protein

K W Vance  1 , M S Campo, I M Morgan
Affiliations

A novel silencer element in the bovine papillomavirus type 4 promoter represses the transcriptional response to papillomavirus E2 protein

K W Vance et al. J Virol. 2001 Mar.

Abstract

The long control regions (LCRs) of mucosal epitheliotropic papillomaviruses have similar organizations: a promoter region, an enhancer region, and a highly conserved distribution of E2 DNA binding sites (C. Desaintes and C. Demeret, Semin. Cancer Biol. 7:339--347, 1996). The enhancer of these viruses is epithelial cell specific, as it fails to activate transcription from heterologous promoters in nonepithelial cell types (B. Gloss, H. U. Bernard, K. Seedorf, and G. Klock, EMBO J. 6:3735--3743, 1987). Using the bovine papillomavirus type 4 (BPV-4) LCR and a bovine primary cell system, we have shown previously that a level of epithelial specificity resides in a papillomavirus promoter region. The BPV-4 promoter shows an enhanced response to transcriptional activators in epithelial cells compared with that of fibroblasts (K. W. Vance, M. S. Campo, and I. M. Morgan, J. Biol. Chem. 274:27839--27844, 1999). A chimeric lcr/tk promoter suggests that the upstream BPV-4 promoter region determines the cell-type-selective response of this promoter in fibroblasts and keratinocytes. Promoter deletion analysis identified two novel repressor elements that are, at least in part, responsible for mediating the differential response of this promoter to upstream activators in fibroblasts and keratinocytes. One of these elements, promoter repressor element 2 (PRE-2), is conserved in position and sequence in the related mucosal epitheliotropic papillomaviruses, BPV-3 and BPV-6. PRE-2 functions in cis to repress the basal activity of the simian virus 40 promoter and binds a specific protein complex. We identify the exact nucleotides necessary for binding and correlate loss of binding with loss of transcriptional repression. We also incorporate these mutations into the BPV-4 promoter and demonstrate an enhanced response of the mutated promoter to E2 in fibroblasts. The DNA binding protein in the detected complex is shown to have a molecular mass of approximately 50 kDa. The PRE-2 binding protein represents a novel transcriptional repressor and regulator of papillomavirus transcription.

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Figures

FIG. 1
FIG. 1
E2-responsive promoter constructs. Promoter regions were PCR amplified as BglII-HindIII fragments and cloned into the pGL3 luciferase vector. Six E2 DNA binding sites were inserted into the BglII site immediately upstream of these promoters. The PV2 6E2 and tk 6E2 constructs have been described previously (39). The lcr/tk hybrid promoter, generated by splicing-by-overlap-extension PCR, contains the BPV-4 upstream promoter region from nucleotides 184 to 279 fused to the tk promoter region from nucleotides 120 to 199. This region of the tk promoter contains the TATA box and initiator element.
FIG. 2
FIG. 2
The lcr/tk hybrid promoter retains the enhanced epithelial response of the BPV-4 promoter to upstream activators. PalK and PalF cells were cotransfected with the indicated amounts of pCMV HPV16-E2 and pCGVP16-E2 expression vectors and 1 μg of either the PV2 6E2 (A), tk 6E2 (B), or lcr/tk 6E2 (C) reporter construct. The total amount of DNA transfected was made equal in each case with pCMV or pCG containing no insert. Results are expressed as fold transactivation relative to the luciferase activity in the absence of E2 or VP16-E2 expression vector.
FIG. 3
FIG. 3
(A) Sequence of the BPV-4 promoter from nucleotides 184 to 310. The TATA box and potential binding sites for transcription factors as shown by footprinting studies are shown in boldface (28). The TATA box proximal E2 binding sites are underlined. Mutations preventing E2 binding have been introduced into these sites, as these have been shown to mediate downregulation of transcription at high levels of E2. Promoter deletions are indicated by arrows. (B) E2-responsive promoter deletion constructs. A series of 5′ deletions of the BPV-4 promoter were PCR amplified as BglII-HindIII fragments. These fragments were cloned into pGL36E2, which contains six E2 DNA binding sites inserted into the BglII site of pGL3.
FIG. 4
FIG. 4
(A) Transcriptional activation of the BPV-4 promoter deletions by HPV-16 E2. PalK and PalF cells were cotransfected with 1 μg of reporter plasmid and 0.1 μg of pCMV HPV-16 E2 expression vector. This ratio has previously been shown to be optimal for maximal activation of the LCR promoter by E2. Results are expressed as fold activation relative to the luciferase activity of each reporter in the absence of E2. (B) Transcriptional activation of the minimal BPV-4 TATA-containing promoter. PalK and PalF cells were cotransfected with 1 μg of the 3bpTATA construct, which contains neither an initiator element nor a binding site for an upstream factor, and the indicated amount of either the pCMVHPV16E2 or the pCGVP16-E2 expression vector. pCMV or pCG was used to make the total amount of DNA transfected equal in all cases. Results are expressed as fold transactivation over the luciferase activity in the absence of activator. pGL3CONT, which contains the SV40 promoter and enhancer driving expression of the luciferase gene, was transfected in parallel in all cases to confirm efficient transfection. This construct demonstrates high levels of transcriptional activity in both keratinocytes and fibroblasts.
FIG. 5
FIG. 5
(A) PRE-2 SV40 constructs. Four copies of PRE-2 were inserted into the BglII site, upstream of the SV40 promoter in the pGL3 luciferase vector, in both a positive and a negative orientation. The position and sequence of PRE-2 are conserved among the mucosal epitheliotropic papillomaviruses BPV-4, BPV-3, and BPV-6. (B) PRE-2 strongly represses SV40 promoter activity in an orientation-independent manner in both PalK and PalF cells. PalK and PalF cells were transfected with the indicated amounts of pGL3PRO, PRO4XPRE-2, and PRO4XPRE-2(−). Results are expressed relative to the luciferase activity of pGL3PRO (set arbitrarily at 100%), which contains only the SV40 promoter.
FIG. 6
FIG. 6
PRE-2 binds a specific protein complex in both PalK and PalF cells. A single radiolabeled PRE-2 motif was used to probe PalK and PalF nuclear extracts in a band shift assay. One-hundredfold excesses of unlabeled PRE-2 and AP-1 oligonucleotides were used as indicated to assess the specificity of binding.
FIG. 7
FIG. 7
(A) Sequences of PRE-2 mutants. Footprinting studies demonstrate that PRE-2 contains a potential binding site for a transcription factor (underlined). Two double-stranded oligonucleotides, each containing a single PRE-2 motif with a 3-bp substitution in this footprint, were generated and tested for competition in the band shift assay. (B) PRE-2mt1 and PRE-2mt2 do not compete for binding to the detected protein complex. A single radiolabeled PRE-2 motif was used in a band shift assay to probe PalK and PalF nuclear extracts. Cold competitors were added as indicated at either a 100-fold (+) or a 500-fold (∗) molar excess.
FIG. 8
FIG. 8
Loss of complex binding to PRE-2 correlates with loss of transcriptional repression. Four copies of mt1 were inserted into the BglII site, upstream of the SV40 promoter in the pGL3PRO luciferase vector, generating PRO4Xmt1. PalK and PalF cells were transfected with the indicated amounts of pGL3PRO, PRO4XPRE-2, and PRO4Xmt1. Results are expressed relative to the luciferase activity of pGL3PRO (set arbitrarily at 100%), which contains only the SV40 promoter.
FIG. 9
FIG. 9
Mutation of PRE-2 results in an elevated transcriptional response of the BPV-4 promoter. PRE-2mt1 was introduced into the 80bpTATA promoter construct, generating 80bpmt1. One microgram of 80bpTATA and 80bpmt1 reporter constructs was cotransfected into PalK and PalF cells with 0.1 μg of pCMVHPV16-E2 expression vector. pCMV vector was added so that an equivalent amount of DNA was used in each transfection. Results are expressed as fold transactivation relative to the luciferase activity of each reporter in the absence of E2.
FIG. 10
FIG. 10
Molecular weight determination of the PRE-2 binding factor. PalK and PalF nuclear extracts were probed with a radiolabeled BrdU-substituted PRE-2 motif in a band shift assay. After UV exposure, the PRE-2 cross-linked protein complex was electrophoresed on an SDS–10% polyacrylamide gel. Competition band shift reactions with either 100-fold nonlabeled BrdU or 100-fold nonlabeled AP-1 were performed as indicated to assess the specificity of binding. Numbers at left are molecular masses in kilodaltons.
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