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represses human papillomavirus 11 upstream regulatory region expression through a promoter-proximal YY1-binding site
1 Northshore-Long Island Jewish Research Institute, 350 Community Drive, Manhasset, NY 11030, USA
2 Department of Otolaryngology, Long Island Jewish Medical Center, Long Island Campus of Albert Einstein College of Medicine, New Hyde Park, NY 11040, USA
Correspondence
Walter M. Ralph, Jr
wralphmd{at}optonline.net
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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(C/EBP) can function as a repressor or as an activator of human papillomavirus (HPV) gene expression, depending on which cell type the experiments are conducted. In this report, it was shown that within primary human foreskin keratinocyte cells (HFK) the activity of C/EBP can be switched from that of a repressor of HPV11 expression to an activator by mutating a single promoter-proximal consensus YY1-binding site within the HPV11 upstream regulatory region (URR). It was shown that in HFK cells, exogenous expression of C/EBP significantly activates the expression of mutant HPV11 URR reporter plasmids that contain deletions which overlap a 127 bp region (–269 to –142). Inclusive in this region are binding sites for multiple transcription factors, including AP1, YY1 and C/EBP
. Only mutation of the YY1 site resulted in the switch in phenotype, indicating that C/EBP represses HPV11 expression in these cells via YY1 binding. The level of YY1 activity was also measured in HFK cells transfected with a C/EBP expression plasmid and a significant increase in YY1 activity as compared with mock-transfected cells was found. C33A cells, which exhibit activation of wild-type HPV11 gene expression with exogenous C/EBP co-expression, failed to demonstrate C/EBP-induced YY1 activation. It was concluded that in HFK cells, exogenous C/EBP induces the activity of YY1, which, in turn, can repress HPV11 URR expression through the promoter-proximal YY1-binding site. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Sibbet & Campo, 1990; Bedell et al., 1991; Kyo et al., 1993; McCaffery & Jackson, 1994; Bauknecht et al., 1996; Maytin & Habener, 1998; Zhu et al., 1999). Briefly, the C/EBPs form a subfamily of proteins that belong to the super family of bZIP proteins. The subfamily consists of seven members: C/EBP, C/EBP, C/EBP
, C/EBP
, C/EBP
, CAA60698
[GenBank]
and CHOP (a.k.a. Gadd153). C/EBPs tend to form homodimers and heterodimers within the bZIP family and bind as a dimer to the palindromic DNA consensus sequence 5'-ATTGCGCAAT-3' as well as to the related CRE and PAR sites (Cao et al., 1991; Williams et al., 1991; Falvey et al., 1996; Vinson et al., 2002). Alternatively, C/EBPs have also been shown to be associated with non-leucine zipper containing proteins (LeClair et al., 1992; Nishio et al., 1993; Lee et al., 1994; Charles et al., 2001; Hadaschik et al., 2003; Schwartz et al., 2003). Actively dividing keratinocytes in culture as well as normal and laryngeal papilloma tissue mainly express C/EBP, C/EBP and CHOP (Jin et al., 1988; Maytin & Habener, 1998). Earlier studies from our laboratory have shown that altering the endogenous levels of C/EBP in human foreskin keratinocytes (HFK) does not affect the replication of HPV11 in these cells. However, reducing the endogenous levels of C/EBP greatly increases HPV11 replication, lending support to the observation that C/EBP functions in part to repress expression of HPV (Wang et al., 1996; Bauknecht & Shi, 1998).
Although several authors have shown that C/EBP (alias NF-IL-6) is a potent transcriptional activator of many genes bearing its consensus sequence, Kyo et al. (1993) first showed that C/EBP functioned uniquely as a potent repressor of HPV16 URR expression, and that mutation of the DNA-binding domain of C/EBP was sufficient to disrupt its repressive ability (Akira et al., 1990; Kyo et al., 1993; Agarwal et al., 1999; Robinson et al., 1998).
The ability of C/EBP to function as an activator or as a repressor has been attributed in part to the presence of multiple isoforms of C/EBP that are thought to be generated from leaky ribosomal scanning of the primary mRNA. Three isoforms have been characterized to date: the full-length protein (39 kDa), liver-enriched transcriptional activator protein or LAP (36 kDa), and liver-enriched inhibitory protein or LIP (20 kDa). LIP is missing the transactivating domain, but it possesses the basic DNA-binding motif as well as the leucine-zipper dimerization motif. It is this isoform that is thought to mediate C/EBP's repressive abilities (Descombes & Schibler, 1991).
Although consensus binding sites for C/EBPs do exist in the distal part of the HPV11 URR [of which C/EBP has been shown to bind (Auborn & Steinberg, 1991; Wang et al., 1996)], we currently report that a mutant reporter plasmid containing the HPV11 URR with all three C/EBP-binding sites mutated is still efficiently repressed by C/EBP in transient expression assays. This result would suggest that repression of the HPV11 URR with co-expression of C/EBP may not involve the binding of C/EBP to its consensus binding sites, but may involve another site in the URR. In addition, we show that while ectopic expression of C/EBP results in repression of the HPV11 URR in primary HFK, it results in activation of the HPV11 URR in the cervical carcinoma cell line C33A.
We tested a series of HPV11 URR expression plasmids containing deletions downstream of the C/EBP-binding site, and isolated a 127 bp region in the proximal promoter that is critical for C/EBP's repressive effects. This region contains a YY1-binding site that, when mutated, reverses the effect of co-expression of C/EBP from repression to activation. We also show that C/EBP can induce the activity of YY1 in a non-HPV system.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Plasmid construction.
The C/EBP expression vector pNF-IL-6 was a gift from S. Akira (Osaka University, Japan). It contains the full-length human C/EBP coding sequence driven by the cytomegalovirus (CMV) promoter. The empty CMV vector (pCMV) was the same plasmid with the C/EBP sequence removed.
The HPV11 URR-luciferase reporter plasmid pURR was a gift from T. DiLorenzo and B. Steinberg (Long Island Jewish Medical Center, New Hyde Park, NY, USA). It contains the full-length HPV11 URR from –833 (nt 7178) to +19 (nt 100) in the luciferase plasmid pGL3-basic (Promega). The mutant reporter plasmids p30O, p30C, p30F, pn11, p
BsrG1 and p127 were created as follows: p30O, p30C and p30F were created by digesting pURR with NdeI and performing exonuclease III digestions followed by mung bean nuclease digestion to blunt end the DNA. The three plasmids were three separate isolates. p30O has sequences from –286 to –480 deleted, p30C has sequences from –227 to –503 deleted and p30F has sequences from –115 to –414 deleted. pn11 was created by a double digestion of pURR with NdeI (–356) and Bsu361 (–142) followed by mung bean nuclease digestion to blunt end the DNA and T4 ligase blunt-end ligation. pBsrG1 was created by isolating the XbaI–HindIII fragment from pURR (which contains the entire URR) and subcloning it into pGEM7zf(+) (Promega) to create pX1. This was done in order to make the two BsrG1 sites (–269 and –248) within the URR unique. After digestion of pX1 with BsrG1 and religation, the URR was removed as a PvuII–HindIII fragment and inserted into the PvuII/HindIII site of pURR to create pBsrG1. p127 was created by a double digestion of pX1 with BsrG1(–269) and Bsu361 (–142) followed by mung bean nuclease digestion to blunt end the DNA and T4 ligase blunt-end ligation. The PvuII–HindIII fragment from this subclone was removed and inserted into the PvuII/HindIII site of pURR to create p127. All plasmids were confirmed by sequence analysis.
Plasmids pM3 and pM1 contain site-specific mutations in all three C/EBP-binding sites (pM3) or only the downstream site (pM1). They were generated from the wild-type plasmid by site-specific PCR mutagenesis using procedures described by Higuchi (1990). The plasmids pYY1 and pAP1 contain site-specific mutations in the YY1- (–215) and AP1-binding sites (–148), respectively. They were generated from the wild-type plasmid using procedures described in the GeneEditor in vitro site-directed mutagenesis system from Promega. All point mutations were confirmed by sequence analysis.
Plasmids pRLTK, the HSV tk renilla reporter plasmid (Promega), and pYY1-luc, the luciferase reporter plasmid designed to measure transcriptional activity of YY1 (Panomics), were obtained commercially.
Dual luciferase assays.
Dual luciferase assays were performed as described by Promega. Cells were plated at subconfluent levels and transfected with 2 µg reporter plasmid and 0·5 µg pRLTK. Cells were co-transfected with either pNF-IL-6 or pCMV (empty vector) at various concentrations. Transfections were performed using lipofectin reagent as described by Invitrogen. Transfection efficiencies were determined by co-transfection of cells in parallel experiments with 1 µg of the plasmid pIRES2-EGFP, which encodes the green fluorescent protein from Aequorea victoria (Clonetech), and the subsequent use of fluorescent microscopy to determine the percentage of fluorescent cells.
Western blot analysis.
Electrophoresis of cellular proteins (50 µg) was performed using 10 % SDS-PAGE pre-cast gels (Invitrogen) and transferred to Immobilon PVDF membranes (Millipore) by electroblotting. Membranes were blocked with 5 % non-fat dried milk with 0·1 % Tween 20. Blots were then incubated with anti-YY1 antibody H10 (Santa Cruz Biotechnology) at a 1 : 1000 dilution for 1 h, then incubated with the secondary antibody, donkey anti-rabbit HRP-labelled antibody (Santa Cruz). Appropriate bands were detected by chemiluminescence according to the ECL protocol (Amersham Pharmacia) and normalized with total protein levels determined by Coomassie staining (Sigma).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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has opposite effects on the HPV11 URR compared with the HSV tk promoter in HFK cells but similar effects in C33A cells can either repress or activate expression of various HPV genes, studies performed specifically with HPV11 have shown that expression of C/EBP results in the repression of HPV11 URR activity in many keratinocyte cell types. Given that Struyk et al. (2000) reported that the HPV16 long control region was activated in C33A cells by C/EBP, we also wanted to examine the effects of C/EBP on HPV11 URR in C33A cells compared to HFK cells. In addition, we wanted to simultaneously determine C/EBP's effect on a non-HPV promoter within the same cell context. Thus, both HFK and C33A cells were co-transfected with the full-length HPV11 URR firefly luciferase reporter (pURR) and the HSV tk promoter renilla luciferase reporter (pRLTK) in the presence or absence of the C/EBP expression vector, pNF-IL-6. The dual use of the firefly and renilla luciferase reporter plasmids made it possible to simultaneously assay C/EBP's effects on two different promoters within the same cell population, given that both the HPV11 URR and the HSV tk promoter have consensus C/EBP-binding sites.
As expected, firefly luciferase activity measured from pURR decreased in a dose-dependent manner with co-transfection of the C/EBP expression plasmid pNF-IL-6 in HFK cells (Fig. 1a). Within these same cells, however, the renilla luciferase activity measured from the HSV tk reporter pRLTK increased significantly in a dose-dependent manner (Fig. 1a). These results suggested that in a given cell at a given point in time, C/EBP may function simultaneously as a repressor and as an activator depending on the genomic context.
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activity, the results above were compared to those obtained in C33A cells. In C33A cells, the luciferase activity measured from both pURR and pRLTK increased significantly in a dose-dependent manner with co-transfection of the C/EBP expression vector (Fig. 1b
). Thus, despite the similar effects of pRLTK in HFK and C33A cells, the effect of C/EBP on the HPV11 URR was completely opposite in C33A cells compared with HFK cells. Taken together, these results suggested that cell type and genome context are independent determinants of whether C/EBP functions as a repressor or an activator.
C/EBP represses an HPV11 URR mutant lacking the C/EBP-binding sites
To analyse the importance of genome context on the activity of C/EBP, a mutant HPV11 URR reporter plasmid was created with the three consensus C/EBP-bindings sites mutated via site-directed mutagenesis (Fig. 2a). All three sites were mutated to divergent sequences that did not correspond to the binding sequence of any known factors. The resulting reporter plasmid was termed pM3. The basal level of luciferase activity for pM3 was repeatedly observed to be significantly lower than that of the wild-type plasmid pURR in HFK cells, suggesting that these sites serve to positively enhance HPV11 URR driven transcription in HFK cells. Co-transfection of pNF-IL-6 with pM3 resulted in a sevenfold dose-dependent decrease in luciferase activity, while a fivefold decrease was observed with co-transfection of pNF-IL-6 with pURR (Fig. 2b). These observations suggested that the ability of C/EBP to repress expression of HPV11 URR in HFK cells is not dependent on intact C/EBP-binding sites within the HPV11 genome.
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-binding sites might have an inhibitory influence on expression from HPV11 URR. However, co-transfection of pNF-IL-6 was not observed to significantly affect the expression of pM3 in C33A cells, while it significantly increased the activity of the wild-type plasmid pURR in similar experiments (Fig. 2c). Of note, a plasmid that contains a small deletion in the HSV tk reporter plasmid pRLTK that removes the lone consensus C/EBP-binding site was also not responsive to C/EBP in C33A or HFK cells when compared with the wild-type pRLTK plasmid (data not shown). Taken together, these results suggested that intact C/EBP-binding sites are important for the activation of HPV11 URR in C33A cells but are not required for its repression in HFK cells.
The C/EBP repressive element is localized to a YY1-binding site in HPV11 URR
Given that the C/EBP-binding sites in HPV11 URR did not seem to be required for repression by C/EBP in HFK cells, we wanted to identify cis-acting sequences that were specifically required for this phenotype. We constructed a series of reporter plasmids that contain deletion mutations in the HPV11 URR located downstream of the consensus C/EBP-binding site (Fig. 3). Using the results from the above experiments, we started with the assumption that the consensus C/EBP-binding site primarily mediates activation of the HPV11 URR and that there exist another region(s) that is responsible for the C/EBP-induced repression. Given that one of our deletion mutations might remove the repressive locus while keeping the C/EBP site intact, we would potentially create a mutant HPV11 URR reporter plasmid that would now be activated by exogenous C/EBP in HFK cells, instead of being repressed by it. Of the six deletion plasmids created, three of them showed a significant increase in luciferase activity as a response to exogenous C/EBP in HFK cells (Fig. 3). These three mutants all overlap a 127 bp region defined by the limits of the smallest deletion plasmid, p127. This plasmid was activated sixfold by C/EBP in HFK cells.
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, YY1, Oct-1, NF1, SEF4 and AP1. Several authors have suggested the involvement of either YY1 or AP1 as factors that may interact with C/EBP to influence its activities in other HPV types (Kyo et al., 1993; Bauknecht et al., 1995, 1996; Bauknecht & Shi, 1998). With this in mind, we engineered mutant plasmids with point mutations in the consensus sites for YY1, C/EBP or AP1 and tested them for their ability to be activated by exogenous C/EBP in HFK cells (Fig. 4). Only the plasmid pYY1, which contains the mutant YY1-binding site, was activated by C/EBP (Fig. 4). This result implicated the YY1 site as being necessary for C/EBP's repressive effects on HPV11 URR.
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affects the level of activity of endogenous YY1 in HFK cells but not in C33A cells; Riggs et al., 1993; Galvin & Shi, 1997; Shi et al., 1997; Thomas & Seto, 1999). Several authors have also established that YY1 acts as a repressive factor specifically with regards to HPV gene expression (May et al., 1994; Bauknecht et al., 1996; Tan et al., 2003). It is less well understood, however, how C/EBP affects YY1's repressive activity or vice versa. The above results strongly suggest that in HFK cells exogenous C/EBP exerts its repressive activity on HPV11 URR through the YY1-binding site and not through its own consensus binding site. Given that C/EBP by itself has never been shown to have an affinity for the YY1-binding site, this implied that C/EBP influences the activity of YY1 either directly or indirectly. To test this hypothesis, we assessed the ability of endogenous YY1 to activate a test plasmid (pYY1-luc) that is designed to detect increases in the activity of YY1. This commercially available luciferase reporter plasmid contains two tandem YY1-binding sites upstream of a single TATA box promoter. By co-transfecting the test plasmid in the presence or absence of an expression vector for C/EBP, we could potentially determine whether exogenous expression of C/EBP alters the activity of endogenous YY1 in both HFK and C33A cells. By itself, it is not assumed that C/EBP would have a direct effect on the test plasmid, given that it contains only a basal promoter and two tandem consensus YY1-binding sites (Panomics). When this experiment was performed in HFK cells, the YY1 test plasmid was induced fivefold with co-transfection of the C/EBP expression vector compared with co-transfection with the empty vector (Fig. 5a). This implied that in HFK cells, either the functional activity of YY1 or its protein level is increased in the presence of C/EBP. When this experiment was repeated in C33A cells (a cell line that not only fails to repress HPV11 URR activity in the presence of exogenous C/EBP but shows significant activation with it), the test plasmid failed to show any significant increase in activity in the presence of C/EBP (Fig. 5b). Given that pRLTK, the HSV tk renilla luciferase reporter plasmid that contains a consensus C/EBP-binding site, was simultaneously induced in these cells by C/EBP, it can be implied that although C/EBP maintains the ability to activate, it does not influence the level of activity of YY1 in C33A cells. Western blot analysis of either HFK or C33A total cell extracts failed to detect differences in the protein level of YY1 between cells co-transfected with C/EBP and those co-transfected with the empty vector (Fig. 6). Given our experimental conditions, transfection efficiencies were determined to be as low as 40 % for HFK cells and 38 % for C33A cells (data not shown). Since less than half of the cell population overexpressed C/EBP in these experiments, we could not make any definitive conclusions as to whether C/EBP significantly alters the protein level of endogenous YY1 in either cell type. Taken together, we concluded that in HFK cells, exogenous C/EBP induced the activity of endogenous YY1, which in turn functioned in part to repress expression of HPV11 URR. We also concluded that in C33A cells, exogenous C/EBP failed to activate endogenous YY1 activity and thus, solely functioned to activate HPV11 URR expression through direct interaction with its consensus C/EBP-binding site. At first glance, the activation of YY1 by C/EBP in HFK cells did not appear to be due to an increase in protein synthesis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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can act as a repressor or as an activator of gene expression in the same cell type depending on genomic context have shown that the repressive ability of C/EBP for HPV18 URR is promoter-specific, in that the albumin promoter is activated by C/EBP in HeLa cells in separate experiments. In this report, we show a similar phenomenon with a low risk HPV by examining the effect of overexpression of C/EBP on HPV11 URR and comparing it to the promoter expression from a non-HPV gene (HSV tk) in the context of primary HFK and the cervical carcinoma cell line C33A. C33A cells were chosen because they are HPV-negative and were shown by Struyk et al. (2000) to be one of the few cell lines in which another high risk HPV is activated by C/EBP overexpression. To take this one step further, we compared the effect of overexpression of C/EBP on these two promoters within cells simultaneously by using a dual reporter system.
We were able to show that while overexpression of C/EBP in HFK cells resulted in repression of HPV11 URR, it also produced activation of the HSV tk promoter within the same cellular milieu (Fig. 1a). This result would argue against the multiple isoform theory of C/EBP as being the principal reason for the different cellular phenotypes of C/EBP with regard to expression of HPV. Instead, this result suggests that the genomic context in which C/EBP is associated determines in part whether it activates or represses gene expression. Although the primary sequence of the C/EBP-binding sites are similar between HPV11 URR and the HSV tk promoter, the genomic context of the binding sites are very different.
This dual effect of C/EBP was not observed in C33A cells, however, suggesting that the machinery responsible for repression was defective in these cells. It is interesting to note that of all cell types tested to date, only two (HepG and C33A) fail to demonstrate C/EBP-induced repression of a wild-type HPV URR, suggesting that these cells are defective in this manner and that C/EBP-induced repression is the rule (Bauknecht & Shi, 1998; Struyk et al., 2000).
C/EBP-induced repression is independent of an intact binding site in HPV11 URR, but activation is binding-site dependent
Current models of the ability of C/EBP to repress and/or activate HPV expression in certain cells have attributed both functions to the direct interaction of C/EBP, or its isoforms, with its consensus binding site in the upstream region of the URR (LeClair et al., 1992; Struyk et al., 2000). In this report, we showed that mutation of all of the known C/EBP-binding sites (two and one ) did not diminish the ability of exogenous C/EBP to repress expression of HPV11 URR in HFK cells (Fig. 2b). In C33A cells, however, mutating the C/EBP-binding sites clearly abolished the ability of C/EBP to activate mutant URR (Fig. 2c). The activating function of C/EBP was dependent on the presence of an intact C/EBP-binding site in the reporter plasmid in both C33A (Fig. 2c) and HFK cells, given that the HSV tk promoter, which is activated in both cell types, looses its responsiveness to C/EBP in both cell types when its binding site is deleted (data not shown). The repressive function of C/EBP, on the other hand, proved to be completely independent of the presence of intact C/EBP-binding sites, supporting the findings of Bauknecht & Shi (1998), who reports that the disruption of the switch region in HPV18 URR (which contains the C/EBP consensus binding site) does not abrogate URR repression by exogenous C/EBP in HeLa cells. Taken together, these results support the presence of an activating and repressive activity of C/EBP, both operating by disparate mechanisms. These processes likely take place on spatially different loci, given that disruption of the activating locus did not affect the repressive locus. Clearly, a more involved mechanism of C/EBP repression/activation exists than would be suggested simply as being the result of ratio differences between isoforms. By deleting a 127 bp region of HPV11 URR DNA approximately 290 bp downstream of the intact C/EBP site and showing that this mutant is now able to be activated by overexpression of C/EBP in HFK cells (Fig. 3), we were able to show that (i) indeed two separate loci for C/EBP activities existed, (ii) that repression and activation probably occurred simultaneously on the same genome and if so (iii) that C/EBP-induced activation was phenotypically recessive to C/EBP-induced repression, given that C/EBP-induced activation of HPV11 URR is only observed in situations where the repressive machinery is defective (as in C33A cells) or the genomic locus is mutated (as with plasmid p127).
YY1 binding mediates C/EBP-induced repression
Since the repressive effects of C/EBP were not disrupted by mutation of the consensus C/EBP-binding site, we surmised that C/EBP might be interacting with another factor on the URR downstream of the C/EBP-binding site. Site-directed mutagenesis of the consensus binding sites for several known transcriptional factors within the 127 bp region of the downstream locus identified YY1 as a key factor responsible for C/EBP-induced repression of HPV11 URR expression (Fig. 4). Although Bauknecht et al. (1992) first identified YY1 as a key factor in HPV18 URR repression, significant differences exist between their results and ours that may shed more light on this topic. Whereas they show that overexpression of YY1 in HeLa cells causes activation of HPV18 URR expression and that mutation of the sole C/EBP site results in switching the phenotype to YY1-induced repression, we show that overexpression of C/EBP resulted in repression of the wild-type HPV11 URR expression in the more biologically relevant cells HFK, and that mutation of the promoter-proximal YY1-binding site resulted in switching the phenotype to C/EBP-induced activation. Of note, mutation of the sole C/EBP site in either HPV11 (our results) or HPV18 (Bauknecht & Shi, 1998) does not affect C/EBP-induced repression. Thus, we clearly show here that C/EBP-induced repression was mediated via YY1 binding to its promoter-proximal site, and that disruption of YY1 binding to this site was sufficient to switch C/EBP from being a repressor of HPV11 URR expression to being an activator in primary keratinocytes. Our results identified the region of –208 to –215 (nt 7804–7797) as being the ‘switch’ region of HPV11 URR, analogous to the ‘switch’ region (–243 to –251) of HPV18 (Bauknecht et al., 1995). Although the two switch regions contain different elements (a YY1 site for HPV11 and a C/EBP site for HPV18) and are located at different distances from their corresponding cis-element (130 bp between the C/EBP site and YY1 site in HPV18 and 340 bp between the C/EBP site and YY1 site in HPV11), we believe that they function similarly in the two viruses to mediate C/EBP-induced repression, given that both switch regions lie at somewhat similar distances from the start site of transcription (a 35 bp difference between the two), both involve elements that when mutated can switch the direction of induction, and both have been shown to involve YY1 binding. Although the term ‘switch’ is used to describe the observed phenotype change, it is not clear whether this effect represents a true change in activity of C/EBP/YY1 or just the uncovering of a less dominant process by a mutation in a more dominant process. Bauknecht & Shi (1998) hypothesized a model of synergistic interaction among C/EBP, YY1 and the TATA-binding protein (TBP) in which C/EBP interferes with the binding of TBP (Bauknecht & Shi, 1988; Bauknecht et al., 1995). In both viruses, this interference could be mediated via the promoter-proximal YY1-binding site, and disruption of YY1 binding to this site could potentially break the link between C/EBP and TBP. In future studies, we plan to use chromosome immunoprecipitation analysis to determine whether TBP binding is disrupted in the presence of C/EBP on HPV11 URR promoter plasmids with or without the switch region mutations.
C/EBP induces YY1 activity
In addition to the association seen in the context of the HPV genome, a functional link between C/EBP and YY1 was demonstrated by the fact that co-transfected C/EBP could alter the activity of a non-HPV test plasmid designed to detect changes in YY1 activity (Fig. 5a and b). The C/EBP-induced increase in YY1 activity was only observed in HFK cells, not in C33A cells, which was consistent with the finding that C/EBP only induces YY1 repression in HFK cells, not in C33A cells (Fig. 1a and b). Consistent with the fact that YY1 has been shown to physically interact with C/EBP (Bauknecht & Shi, 1988) is the observation that C/EBP-induced increases in YY1 activity appeared at first glance not to be at the level of protein synthesis (Fig. 6), but may be the result of direct protein–protein interaction between C/EBP and YY1. It must be stated that this observation is only a first approximation since repeated attempts at transfecting these cells have resulted in less than 50 % transfection efficiency. Nonetheless, this finding would be consistent with the findings of Dong et al. (1998) who noted no significant difference in the protein level of YY1 between C33A cells and other human epithelial cell lines (Dong et al., 1998). We are currently exploring the details of this C/EBP-induced increase in YY1 activity and the failure of this induction in C33A cells. Of particular note is the fact that C33A cells lack the protein BRG1 (a component of the mammalian SWI–SNF complex that regulates transcription through active modification of chromatin structure) and expresses a non-functional Rb protein (Muchardt & Yaniv, 1993; Murphy et al., 1999). Several lines of evidence strongly suggest that interactions among the SWI–SNF complex, Rb related proteins, YY1 and HDACs are important for modifying chromatin structure to allow for regulated control of repression or activation of gene expression (Murphy et al., 1999; Osborne et al., 2001; Siddiqui et al., 2003; Marenda et al., 2004).
It cannot be overemphasized that the results in this study are obtained from cells in which C/EBP has been exogenously introduced and overexpressed. Nevertheless, a significant number of important relationships between proteins and DNA sequences have been observed by this method in the literature and it continues to be an important tool in understanding gene regulation.
In summary, we report that we were able to switch the ability of exogenously expressed C/EBP from acting as a repressor of HPV11 URR expression to acting as an activator by mutating the promoter-proximal YY1-binding site in the HPV11 URR. Our report demonstrates that the interaction between YY1 and C/EBP and their influence on modulating HPV expression is not unique to the high-risk HPV types, but also exists for the low risk HPV type 11 as well and may represent a general mechanism of regulatory control. In addition, we show that in a non-HPV system, C/EBP can induce the activity of YY1 and enhance its function as either an activator or as a repressor.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 25 May 2005;
accepted 31 August 2005.
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