Institute of Virology, University of Cologne, Fürst-Pückler-Strasse 56, 50935 Cologne, Germany1
Author for correspondence: Gertrud Steger. Fax +49 221 478 3902. e-mail Gertrud.Steger{at}uni-koeln.de
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Abstract |
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We have observed that the E2 protein of human PV type 8 (HPV8), which is associated with the rare disease epidermodysplasia verruciformis (ev), is able to enhance the protein level of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) in the HPV-negative and p53-negative skin keratinocyte cell line RTS3b (Purdie et al., 1993 ) (data not shown). P21 plays important roles in regulation of cell growth, arrest or progression, DNA methylation, cell senescence, apoptosis and differentiation (reviewed in Dotto, 2000
). Its expression is regulated by many inducers, among them p53 and factors that control differentiation of diverse cell types including skin cells (Prowse et al., 1997
). Some of these factors mediate their effects on p21 gene expression via the promoter-proximal 210 nucleotides (Kardassis et al., 1999
and references therein). This region is GC-rich and contains five sequences that resemble Sp1 binding sites and there is no E2 consensus sequence present within this promoter fragment (Fig. 1A
In order to test whether this promoter-proximal region might be sufficient for HPV8 E2 to stimulate p21 activity, we transiently transfected a CMV promoter-driven HPV8 E2 expression vector (Stubenrauch & Pfister, 1994
) together with a luciferase reporter construct comprising the promoter-proximal region from +15 to -210 of p21 (Prowse et al., 1997
) (Fig. 1A
) in RTS3b cells. HPV8 E2 led to a low but distinct 2-fold activation. Surprisingly, when we expressed the HPV8 E2
N, lacking the AD, the promoter was stimulated 10-fold, suggesting that the AD of HPV8 E2 is not necessary for activation of the p21 promoter. Since the promoter fragment contains a cluster of Sp1 binding sites we wondered whether HPV8 E2 functionally interacts with Sp1 in activation and co-transfected an expression vector for Sp1. Sp1 on its own stimulated promoter activity 2·2-fold. HPV8 E2 and co-expressed Sp1 led to a 5·3-fold activation, indicating weak or no cooperativity. In contrast, co-expression of HPV8 E2
N and Sp1 resulted in a 24-fold activation. The enhancement of the activation mediated by HPV8 E2
N due to overexpression of Sp1 is significant (P=0·012), suggesting that activation by HPV8 E2
N involves functional interaction with Sp1. Since E2 proteins are rather conserved within their structure and function, we used the E2 protein of HPV18 to test whether this is true for another E2 protein. HPV18 E2 on its own also weakly stimulated the promoter (2-fold) as well as the mutant lacking the AD (HPV18 E2
N). These activations increased up to 4- and 5·5-fold, respectively, when Sp1 was co-expressed (Fig. 1A
). Thus, HPV18 E2
N does not cooperate with Sp1 in activation of the p21 promoter, in contrast to HPV8 E2
N. This difference is not related to differential expression of both proteins. Gel-shift assays using nuclear extracts from RTS3b cells transiently transfected with the corresponding expression vectors revealed that HPV18 E2
N was even present in higher amounts than HPV8 E2
N (Fig. 1D
). The lack of cooperativity between HPV18 E2
N and Sp1 does not result from squelching due to unphysiologically high amounts, since we tested a broad range of concentrations of expression vectors (data not shown). The E2 proteins are conserved within their DBDs and not within their internal hinge region (Giri & Yaniv, 1988
). Therefore, we wondered whether the hinge region of HPV8 E2 might be responsible for this cooperation with Sp1. Transfecting an expression vector encoding solely the hinge region of HPV8 E2 (HPV8 E2H) revealed that it was able to activate and to cooperate with co-expressed Sp1 to the same extend as HPV8 E2
N (Fig. 1B
). As shown by immunofluorescence tests of RTS3b cells transiently transfected with the plasmids expressing Flag-tagged HPV8 E2 proteins, HPV8 E2
N and HPV8 E2H were localized in the nucleus and expressed to comparable levels (Fig. 1D
).
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As in the case of HPV18 E2 (Fig. 2) and BPV1 E2 (Steger et al., 1995
), the AD of HPV8 E2 is required for cooperation with Sp1 in activation of the promoter containing high affinity Sp1 and E2 binding sites (Fig. 2
). This activation may involve the binding of the N terminus to components of the preinitiation complex or to co-factors, as identified for other E2 proteins (Miller Rank & Lambert, 1995
; Benson et al., 1997
; Yao et al., 1998
). These contacts seem to be absolutely required also for HPV8 E2, since HPV8 E2
N does not activate the synthetic promoter. Thus, the role of the direct interaction of the AD of BPV1 E2 and that of HPV18 E2 with Sp1 remains unclear for this kind of activation. In addition to Sp1, E2 shows cooperative activation with a variety of sequence-specific DNA binding factors such as AP1, USF, TEF-1, NF1/CTF when the corresponding binding sites as well as the E2 binding sites have been cloned upstream of the promoter (Ham et al., 1991
; Ushikai et al., 1994
). A direct interaction between E2 and these cooperation partners has not yet been shown, implying that this kind of cooperativity (also) may occur without direct binding. The role of the binding of the DBD to Sp1 in activation of transcription remains unclear as well. It is unlikely that this interaction is mediated by contaminating DNA since it occurs also in the presence of ethidium bromide (data not shown). Furthermore, we used purified proteins for our proteinprotein interaction studies. Interactions between transcription factors without any relevance for transactivation have been observed previously. For example, the binding of the DNA binding domain of Oct1 to TBP does not play any role in activation (Arnosti et al., 1993
). However, it might still be possible that these interactions are involved in other not yet characterized processes.
The mechanism of activation of the p21 promoter by the hinge region of HPV8 remains unclear. Our data presented here suggest that the direct interaction of this hinge region with Sp1 may be involved. It might be possible that the hinge mediates oligomerization of Sp1, which increases its affinity to the p21 promoter. Furthermore, the HPV8 E2H bound on the promoter via Sp1 may promote the recruitment of other cooperating factors such as p300. P300 has been shown to be required for p21 induction in differentiating keratinocytes (Xiao et al., 2000 ). In line with this model, we could show that p300 also interacts with the hinge of HPV8 E2 (A. Müller & G. Steger, unpublished results). A similar mechanism in stimulation of p21 gene expression has been described for c-Jun and the Smads protein (Kardassis et al., 1999
; Pardali et al., 2000
). Also in the case of c-Jun, which binds via its leucine zipper to Sp1, the AD is not necessary for activation of the p21 promoter.
The stronger activation of the p21 promoter by HPV8 E2N and E2H compared to the wild-type HPV8 E2 protein (Fig. 1A
, B
) might be due to the fact that E2
N and E2H are expressed to higher levels than the full-length protein (Fig. 1D
). Thus, small amounts of HPV8 E2 may not be sufficient for efficient cooperation with Sp1. Furthermore, the N-terminal AD may somehow interfere with Sp1 function, thereby inhibiting cooperativity mediated by the hinge. Moreover, regions required for functional interaction might only be accessible in the N-terminally truncated version. It is not known whether these forms of E2 are expressed in the case of HPV8, as has been shown for other E2 proteins (Lambert et al., 1987
; Liu et al., 1995
; Stubenrauch et al., 2000
). However, in an ev lesion induced by HPV5, which is closely related to HPV8, a series of spliced transcripts, with the potential to encode an E2 protein starting at amino acid 202, have been identified (Haller et al., 1995
).
Initially, the hinge region was suggested to function as a flexible linker to connect the AD and the DBD. Meanwhile, discrete functions for the hinge region of various E2 proteins have been described. For example, signals for phosphorylation and nuclear localization have been mapped to the hinge of BPV1 E2 and HPV11 E2, respectively (McBride et al., 1989a ; Lehman et al., 1997
; Pernose & McBride, 2000
; Zou et al., 2000
). Previously, we could demonstrate an involvement of the hinge of BPV1 E2 in regulation of gene expression (Steger et al., 1995
). In contrast to other E2 proteins, the E2 proteins of PV associated with ev display a very long hinge region, rich in arginine, serine and glycine residues, indicating specialized function. In correlation with this, the hinge of HPV5 E2 was shown to interact with splicing factors and to enhance splicing (Lai et al., 2000
). Our data presented here suggest a novel mechanism of transactivation by the hinge region of HPV8 E2, probably mediated through proteinprotein interaction with the important cellular regulator Sp1. By inducing the expression of p21 in infected keratinocytes, the hinge of HPV8 E2 might affect keratinocyte differentiation and thus contribute to HPV8-induced pathogenesis.
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Acknowledgments |
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References |
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Received 29 June 2001;
accepted 25 October 2001.