Institute of Virology, Universität zu Köln, Fürst-Pückler Str. 56, 50935 Köln, Germany1
Author for correspondence: Herbert Pfister.Fax +49 221 4783902. e-mail Herbert.Pfister{at}medizin.uni-koeln.de
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Abstract |
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Several mechanisms have been described to explain the different regulatory effects of YY1. In some cases, YY1 competes for DNA binding with an activator protein, such as nuclear serum response factor in muscle -actin (Lee et al., 1992
) and c-fos (Natesan & Gilman, 1995
) promoters and with NF-
B in the rat serum amyloid A1 gene promoter (Lu et al., 1994
). Another possibility is heterodimerization of YY1 with transcription regulators such as c-Myc (Shrivastava et al., 1993
), E1A (Lee et al., 1995
) or B23 (Inouye & Seto, 1994
). In addition, YY1 may act as a DNA-bending protein and regulate promoters through an effect on DNA structure (Natesan & Gilman, 1993
; Klug & Beato, 1996
). Recent findings indicate that YY1 interacts with the non-DNA-binding transcription factor RPD3 and negatively regulates transcription through tethering RPD3 to the promoter region (Yang et al., 1996
; Pazin & Kadonaga, 1997
).
Several YY1-binding sites have been reported in the HPV-16 LCR (May et al., 1994 ; O'Connor et al., 1996
). Single nucleotide exchanges observed in the promoter-distal (nt 77917799) and -proximal (nt 78407848) YY1-binding sites in HPV-16 DNA from cervical carcinomas abolished the binding capacity for YY1 protein in vitro and, in the context of the whole LCR, the point mutation in the promoter-distal YY1 site induced a roughly 4-fold increase in P97 activity in transient transfection assays (May et al., 1994
; Dong et al., 1994
). The mechanism of YY1 repressive activity in HPV-16 remains unclear. More recently, O'Connor et al. (1996)
concluded that YY1 repressed HPV-16 transcription by quenching AP1 activity. In this report, we provide evidence that YY1 represses promoter activity by competing with SP1 for DNA binding at the promoter-proximal YY1-binding motif.
The nucleotide sequence within the promoter-proximal YY1-binding site is CATGGG (nt 78427847), which could represent an SP1-binding motif. To address this possibility, we carried out electrophoretic mobility shift assays (EMSA) with double-stranded oligonucleotides (nt 78367875) that cover the promoter-proximal YY1-binding site, as described previously (Dong et al., 1994 ). Purified SP1 protein from nuclear extracts of HeLa cells was obtained from a commercial supplier (Promega). The HisYY1 fusion protein was expressed in E. coli RR cells grown at 30 °C to an OD600 of 0·7 (Shi et al., 1991
). The expression plasmid was induced by addition of IPTG (final concentration 1 mM). After a further 4 h incubation, bacteria were pelleted and lysed in 6 M guanidineHCl, pH 8·0. The HisYY1 protein was purified by affinity chromatography on NiNTA agarose (Qiagen) and renatured as described previously (May et al., 1994
). Different amounts of protein were mixed with 2000040000 Cerenkov c.p.m. of end-32P-labelled, double-stranded oligonucleotide probe in 20 µl and the DNAprotein complexes were separated from unbound probe in a 4% non-denaturing polyacrylamide gel (Dong et al., 1994
). The oligonucleotide WT, containing the wild-type HPV-16 sequence, formed complexes not only with YY1 but also with SP1 protein (Fig. 1
a, lanes 1 and 2). This demonstrates that, besides the SP1-binding site (nt 2833) upstream of P97 (Gloss & Bernard, 1990
), there was another SP1-binding motif within the 40 bp HPV-16 subfragment tested.
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Since the newly identified SP1 motif overlaps the promoter-proximal YY1-binding site completely, it is reasonable to think that SP1 will compete with YY1 for binding at this position. When YY1 and SP1 were mixed for EMSA with 32P-labelled oligonucleotide WT at the concentrations used for YY1- and SP1-specific shifts (Fig. 1a, lanes 1 and 2), there was no additional complex detectable, indicating that the proteins are not able to bind to the same DNA molecule and that no proteinprotein interaction occurs between DNA-bound YY1 and SP1 (Fig. 1a
, lane 3). The SP1-specific complex appeared clearly less prominent than in lane 2, suggesting a greater ability of YY1 to bind this site. With increasing amounts of SP1 protein, the SP1DNA complex became more apparent at the expense of the YY1DNA complex (Fig. 1a
, lane 4).
To evaluate the relative occupation of nucleotides 78407848 by YY1 and SP1 at the concentrations that occur in cervical carcinoma cells, we performed EMSA with nuclear extracts of HT3, HeLa and C33a cells. The cells were grown in DMEM supplemented with 10% foetal calf serum and harvested by trypsinization. After washing twice with PBS (Gibco), the cells were lysed on ice for 15 min with 1 ml lysis buffer (0·6% NP-40, 300 mM sucrose, 10 mM HEPES, pH 7·9, 1 mM EDTA, 1·5 mM MgCl2, 50 mM PMSF, 0·5 mM DTT). The nuclei were pelleted by centrifugation and incubated on ice for 1 h in 0·1 ml elution buffer (25%, v/v, glycerol, 10 mM HEPES, pH 7·9, 0·1 mM EDTA, 0·1 mM EGTA, 1·5 mM MgCl2, 525 mM PMSF, 0·5 mM DTT). The supernatants were collected and stored in aliquots at -80 °C after determining the protein concentration. After mixing the nuclear extracts with 32P-labelled, double-stranded oligonucleotide WT, several complexes were detected by EMSA, two of which (A and D) migrated to similar positions to those of the SP1- and YY1-specific complexes obtained with purified proteins (Fig. 2, lanes 15). The identity of these complexes was confirmed by competition experiments with 50- or 400-fold excesses of different unlabelled oligonucleotides. Complex A was competed for by oligonucleotide SP1 and more efficiently by oligonucleotide PM (Fig. 2
, lanes 912), which both contain SP1-binding sites. Complex D was not detectable in the presence of oligonucleotide AAV (Fig. 2
, lanes 7 and 8), containing a genuine YY1-binding site. Both complexes were not affected by 50-fold excesses of the complementary competitors or of oligonucleotide C (Fig. 2
, lane 14), binding neither SP1 nor YY1. No specificity could be assigned to complexes B, C and E, which were removed by a 400-fold excess of any unlabelled competitor. Both YY1- and SP1-specific complexes were clearly visible with all three nuclear extracts tested, with the SP1-specific band appearing considerably stronger.
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This analysis thus provides evidence for another mechanism of YY1 repression. SP1-binding sites are usually located and clustered in front of TATA boxes or TATA-less promoters. SP1 acts as a transcription activator or initiator through binding to its cognate sequences. In the HPV-16 LCR, one SP1-binding site (nt 2833) has been mapped in front of promoter P97 close to an E2-binding site (Gloss & Bernard, 1990 ). Although SP1 is generally considered to be a protein that interacts with promoter-proximal elements, it also mediates long-range activation of transcription via proteinprotein interactions between DNA-bound SP1 molecules or SP1 and other transcription factors. Two SP1-recognition sites within the HPV-16 LCR offer the possibility to form a loop (Su et al., 1991
), which could contribute to the activation of P97 because the enhancer element would move closer to the transcription initiation complex. Competition between YY1 and SP1 for binding to nt 78407848 could theoretically hinder SP1-mediated activation of P97.
As expected in view of the completely overlapping YY1 and SP1 recognition sites, binding of both proteins to the oligonucleotide 78367875 in vitro was indeed mutually exclusive. At the same protein concentration, YY1 bound more efficiently than SP1. However, the SP1-specific complex turned out to be more prominent than the YY1-specific complex when using nuclear extracts of cervical carcinoma cell lines for EMSA. This suggests a predominance of SP1, which is in line with the presence of high levels of SP1 in C33a cells (Apt et al., 1996 ). One would therefore expect that repression by YY1 through competition with SP1 is more relevant in normal keratinocytes, with lower SP1 concentrations compared with tumour cells (Apt et al., 1996
). In spite of the rather unfavourable ratio of YY1 and SP1 in C33a cells, the PM mutation led to an approximately 5-fold activation. This will be the result of both removal of competitive YY1 binding and the higher affinity of SP1 further increasing the amount of SP1 binding to this site.
An increased concentration of SP1 was not only observed in transformed keratinocytes but also in differentiated ones (Apt et al., 1996 ). The newly established competition between the activator SP1 and repressor YY1 could therefore contribute to the increased activity of the HPV-16 oncogene promoter in the course of epithelial differentiation and tumour progression.
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Acknowledgments |
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Footnotes |
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References |
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Received 11 February 1999;
accepted 12 April 1999.