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Sp1 binding is inhibited by mCpmCpG methylation
Gene 195 (1997) 67–71
Sp1 binding is inhibited by mCpmCpG methylation Susan J. Clark a,b,*, Janet Harrison a, Peter L. Molloy b a Kanematsu Laboratories, Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050, Australia b CSIRO, Division of Molecular Science, P.O. Box 184, North Ryde, NSW 2113, Australia Received 8 December 1996; accepted 9 February 1997; Received by H. Cedar
Abstract Previously it has been found that binding of the Sp1 transcription factor is not significantly affected by methylation of the CpG dinucleotide within its binding site, 5∞-GGGCGG ( lower strand, 5∞-CCGCCC ). Since it has been established that mammalian cells also have the capacity to methylate cytosines (C ) at CpNpG sites we examined the effect of methylation of the outer C of the CpCpG on Sp1 binding. We find that methylation of the outer C is inhibitory and in particular methylation of both cytosines mCpmCpG inhibits binding by 95%. Furthermore, we have identified endogenous mCpmCpG methylation of an Sp1 site in the CpG island promoter of the retinoblastoma (Rb) gene by genomic sequencing. This occurs in a proportion of retinoblastoma tumors which are extensively CpG methylated in the Rb promoter. The results raise the possibility that mCpmCpG methylation could have a biological function in preventing Sp1 binding, thereby contributing to the subsequent abnormal methylation of CpG islands often observed in tumor cells. © 1997 Elsevier Science B.V. Keywords: Transcription factor binding; CNG Methylation; Retinoblastoma; Gene regulation
1. Introduction In vertebrate genomes almost all of the identified post-replicative modification of DNA is in the form of methylation of cytosine at symmetric CpG dinucleotides. Changes in methylation of specific genes are seen during development and during neoplastic transformation and progression (Holliday, 1990; Jones and Buckley, 1990; Herman et al., 1995). Substantial data have accumulated which demonstrate that methylation of CpG dinucleotides in gene-regulatory regions of both introduced and endogenous genes can block their expression (Razin and Cedar, 1991). Whether methylation acts as a primary trigger or to reinforce other cellular events during development has not been established, but aberrant methylation clearly has the capacity to act as an epigenetic mutation. It has been demonstrated that DNA methylation can act both directly and indirectly to inhibit gene expression. Methylated DNA is localized to inactive chromatin and it has been suggested that through bind* Corresponding author at address b. Tel. +61 2 98864948; Fax +61 2 94905805; e-mail: [email protected] Abbreviations: DMS, dimethyl sulfate; mC, 5-methyl cytosine; PCR, polymerase chain reaction; Rb, Retinoblastoma tumor suppressor gene. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 1 64 - 9
ing to specific methylated DNA binding proteins clusters of methylated CpGs may promote the formation of inactive chromatin and the exclusion of the transcription machinery (Antequerra et al., 1989; Meehan et al., 1989; Graessmann and Graessmann, 1993). For some transcription factors, (e.g., E2F, CREB and USF ) methylation at specific CpGs has been shown to directly inhibit protein binding and thus inhibit transcription ( Kovesdi et al., 1987; Watt and Molloy, 1988; Iguchi-Ariga and Schaffner, 1989). For other factors such as Sp1, methylation of a CpG site within the recognition sequence does not interfere with protein binding (Harrington et al., 1988; Holler et al., 1988; Ben-Hattar et al., 1989). In fact there is accumulating evidence that Sp1 sites are important in maintaining CpG-rich regions in an unmethylated state (Brandeis et al., 1994; Macleod et al., 1994). We have recently shown that mammalian cells also have the capacity to maintain methylation of cytosine at CpNpG sites of introduced plasmid DNA (CpApG and CpTpG) and also to de novo methylate these and CpCpG sites to a low level (Clark et al., 1995). This raises the possibility that such sites of methylation are present in endogenous genes and may have a role either in regulation of gene expression or another biological
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function. The core binding site for Sp1, 5∞-GGGCGG ( lower strand, 5∞-CCGCCC ), contains a potentially methylatable CpCpG site. Here we examine the effect of methylation of the outer C of the CpCpG present in the Sp1 recognition site on its binding and also present evidence that such methylation can occur in genomic DNA.
2. Experimental A series of oligonucleotides corresponding to both upper and lower strands of a consensus Sp1 site ( Thiesen and Bach, 1990) were synthesized, differing only in which cytosines were methylated (see Fig. 1). Methylation was introduced at the central CpG dinucleotide (bases C5 and C6∞), the outer cytosine of the CpCpG triplet (C7∞) or in cytosines at a CpNpG site flanking the core Sp1 binding region (C-1, C2∞). Binding of Sp1 to gel-purified double-stranded oligonucleotides, methylated in various configurations, was studied under standard conditions as shown in Fig. 1. Consistent with previous reports (Harrington et al., 1988; Holler et al., 1988; Ben-Hattar et al., 1989), methylation of the central CpG dinucleotide on either or both strands, had little or no effect on the efficiency of Sp1 binding (tracks B, F and G). Methylation of the flanking CpNpG site on either or both strands (tracks E, J and K ) also had no effect on binding. However, methylation of the single outer (C7∞) cytosine adjacent to the central CpG on the lower strand reduced Sp1 binding by nearly 50% (track D). This inhibition was strongly enhanced when both cytosines on the lower strand were methylated (track C ) and was of similar extent whether or not the upper strand cytosine (C5) was methylated (tracks C and H ). Equivalent results have been obtained when either the upper or lower strands were labeled (data not shown). These results are consistent with detailed mutational and methylation interference studies of Sp1 binding sites (Letovsky and Dynan, 1989; Thiesen and Bach, 1990; Kuwahara et al., 1993). Previous studies have shown that Sp1 can bind to sites containing alternate bases at position C5 of the recognition sequence (Letovsky and Dynan, 1989; Kudo and Fukuda, 1994). This is consistent with the lack of effect of methylation at C5 (Fig. 1, Track F ). Also, G5∞ on the lower strand is not protected from methylation by dimethyl sulfate (DMS ) when Sp1 is bound, though prior methylation does inhibit binding ( Kuwahara et al., 1993). Both methylation protection and interference assays indicate close protein contact with G6. By contrast, methylation of G7 does not interfere with Sp1 binding and this base shows enhanced reactivity with DMS when Sp1 is bound. Considering that this base pair is strictly conserved in Sp1 binding sites, it is possible that critical contact is made with the
lower strand C7∞. Consideration of the mode of contact of zinc finger domains with their binding sites (Nardelli et al., 1991; Fairall et al., 1993) has raised the possibility that there could be a direct H-bond to C7∞, particularly if the helix is distorted from ideal B-form. as suggested by the enhanced DMS reactivity of G7 when Sp1 is bound ( Kuwahara et al., 1993). The presence of methyl cytosines in successive base pair steps is also likely to constrain the DNA structure ( Fratini et al., 1982) and this could limit the capacity for Sp1 binding. Either such a structural effect or direct interference with hydrogen bonding would be consistent with the strong inhibition of Sp1 binding observed. It has been established by restriction enzyme studies that the promoter region of the Retinoblastoma tumor suppressor gene (Rb) is methylated in a proportion of retinoblastoma tumors (Sakai et al., 1991). Using samples previously characterized by restriction enzyme analysis we have been studying the specific patterns and extent of DNA methylation across the Rb promoter using bisulfite genomic sequencing (Stirzaker et al., 1997). In this work we have identified a number of cases where the outer cytosine, C7∞, of the CpCpG on the lower strand of the Sp1 site is methylated. Fig. 2 shows the methylation state of the cytosines in the CpCpG trinucleotide within the Sp1 binding site of the Rb promoter from a retinoblastoma patient (patient 252). An example of sequence from an individual clone shows methylation of both cytosines in the CpCpG Sp1 binding site ( Fig. 2A). Direct PCR sequencing indicates that about 28% of the outer C∞s are methylated in the DNA of this patient ( Fig. 2B, C ). Altogether three of eight patients with a hypermethylated Rb gene showed 20–30% methylation of the outer cytosine (C7∞). In all cases, methylation of the outer C occurred only where the inner cytosine (C6∞, i.e. the CpG site) was methylated. A significant occurrence of methylation of other CpNpG cytosines (mainly at CpCpG sites) was also seen in these patients. It is possible either that the aberrant de novo methylation has occurred only in a subset of the tumor cell population and is then faithfully maintained, or conversely, that CpCpG maintenance methylation is inefficient in each generation. The presence of methylation of the outer cytosine of a CpCpG sequence present in the core Sp1 binding site in genomic DNA and the capacity of this modification to block Sp1 binding raises the question of whether this may be of biological significance. CpG islands are rich in Sp1 sites and Sp1 binding has been proposed to play a critical role in maintaining CpG islands in an unmethylated state since mutation of the Sp1 sites in CpG islands allows the island to become methylated (Brandeis et al., 1994; Macleod et al., 1994). While CpG methylation of the Sp1 binding site is permissive for Sp1 binding, aberrant methylation of the outer cytosine in the core
S.J. Clark et al. / Gene 195 (1997) 67–71
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Fig. 1. Inhibition of Sp1 binding by DNA methylation. The left panel shows the sequence of the double-stranded oligonucleotides, with the core Sp1 binding site shown in upper case. The reference nucleotides are numbered −1 to 9 for the upper oligonucleotide and −1∞ to 9∞ for the lower oligonucleotide according to Kuwahara et al. (1993). (A)–( K ) show the methylation pattern for each double-stranded DNA set. Methylated and unmethylated cytosines are represented by filled and open circles, respectively. An electrophoretic mobility shift assay (EMSA) showing free oligonucleotide DNA and DNA complexed with Sp1 is shown to the right. The addition or absence of Sp1 in the binding reaction is indicated by + or −, respectively. The level of Sp1 binding relative to the unmethylated DNA is shown in the central column. Methods: oligonucleotides, containing 5-methyl cytosine (mC ) as indicated, were synthesized on an Applied Biosystems 370A synthesizer and purified according to manufacturer’s instructions. Double-stranded DNAs were prepared by annealing one oligonucleotide, radiolabeled with c32P-ATP and T4-polynucleotide kinase, with a complementary unlabeled oligonucleotide. They were purified by elecrophoresis through 12% non-denaturing polyacrylamide gels. DNA probes, 5 fmol, were incubated in 20 ml reactions containing 10 mM N-2-Hydroxyethyl piperazine-N∞-2-ethanesulfonic acid, 10% glycerol, 50 mM KCl, 6 mM MgCl , 25 mM ZnCl , 0.5 mM ethylenediaminetetraacetic acid, 5 mM dithiothreitol, 100 mM Pefabloc (Boehringer), 0.1% 2 2 Nonidet P40, containing 100 ng poly dI.dC, 50 mg bovine serum albumin, with or without 0.5 footprint units of Sp1 (Promega). Sp1-bound and free probe were separated by electrophoresis through a 6% polyacrylamide (40:1) in 90 mM Tris base, 90 mM boric acid, 1 mM ethylenediaminetetraacetic acid, pH 8.3. Phosphorimaging of the dried gel was done using a Molecular Dynamics Model No 400E and quantitation of binding was done using ImageQuant software version 3.3 (Molecular Dynamics).
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Fig. 2. Cytosine methylation of the CpCpG triplet in an Sp1 binding site of the Rb promoter. (A) shows the sequence surrounding the Sp1 binding site within the Rb promoter from an individual clone isolated from polymerase chain reaction (PCR)-amplified bisulfite-treated retinoblastoma tumor DNA. Arrows denote unconverted cytosines, corresponding to cytosines methylated in the parent DNA strand. The outer C of the CpCpG is indicated by *. The converted sequence as read in the clone and the original unconverted sequence are shown to the right; the position of the Sp1 binding site is indicated. (B) and (C ) show the cytosine (C ) and thymine (T ) Genescan profiles which were derived from quantitative genomic sequence analysis of PCR-amplified bisulfite-treated retinoblastoma tumor DNA, as described in Paul and Clark (1996). The original sequence (bisulfite unconverted) and the bisulfite converted methylated sequence are shown above the scans. The sequence resulting from conversion of unmethylated DNA is shown beneath the T profile. Unconverted C∞s corresponding to methylated cytosines in the sequence are indicated with arrows; the unmethylated cytosines are denoted with open triangles under the T profile. The methylated outer C of the CpCpG triplet is indicated by *. Percentage methylation for any cytosine is measured as the peak height of C over the peak heights of C+T. Methylation of the outer C of the CpCpG triplet is measured as 28% and the inner C as 92%. Methods: DNA was subjected to bisulfite modification and the promoter region of the Rb gene amplified by PCR as described (Clark et al., 1994). The specific nested primers used for amplification of the bottom strand of the Rb gene were: Outer Set, Rb15 (1662–1695): 5∞-ACCCCAGCCTAAAAAAAATAATTCTAAATAAAA and Rb16 (1714–1743): 5∞-GATTTTTTTGGATTTTGGTTATAAAAATAA; Inner Set, Rb17 (1926–1948): 5∞-AATACCTCCTAAAAAACCCCTAAACCCAC and Rb18 (1949–1978): 5∞-TTTTAAGGTTTTTTGAGAAAAAT. The coordinates of the primers indicate their location on the Rb sequence (Accession number: L11910). Direct sequence analysis was done as described (Paul and Clark, 1996). PCR products were cloned and the individual clones sequenced as described (Clark et al., 1994).
binding site would prevent Sp1 from binding, thus acting like a mutation at the Sp1 site. As seen for Sp1 site mutations, this could trigger CpG methylation of the rest of the island. For an island such as that of the Rb tumor suppressor gene, the resulting gene silencing could contribute to clonal expansion and tumor development. Though CpG methylation would be faithfully maintained, the lower efficiency of maintenance of CpNpG methylation may mean that its presence in the population is transient. In this context CpNpG methylation may be thought of as an epigenetic mutation which could initiate the silencing of tumor suppressor genes by CpG methylation.
3. Conclusions (1) Binding of the transcription factor Sp1 is inhibited by mCpmCpG methylation. (2) Endogenous mCpmCpG methylation of a critical Sp1 site was identified in the hypermethylated promoter of the Rb tumor suppressor gene in three of eight retinoblastoma tumors analysed. (3) We propose that by blocking Sp1 binding, mCpmCpG methylation could act to initiate de novo CpG methylation of the CpG island, leading to promoter silencing.
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Acknowledgement We thank Ted Dryja for providing the retinoblastoma tumor DNAs, Cheryl Paul, Clare Stirzaker and Doug Millar for genomic sequencing, Marianne Frommer for helpful discussions and Robin Holliday and Horace Drew for critical reading of the manuscript. This work was partially supported by an Anthony Rothe Memorial Grant.
References Antequerra, F., MacLeod, D., Bird, A.P., 1989. Specific protection of methylated CpGs in mammalian nuclei. Cell 58, 509–517. Ben-Hattar, J., Beard, P., Jiricny, J., 1989. Cytosine methylation in CTF and Sp1 recognition sites of an HSV tk promoter: effects on transcription in vivo and on factor binding in vitro. Nucleic Acids Res. 17, 10179–10190. Brandeis, M., Frank, D., Keshet, I., Siegfried, Z., Mendelsohn, M., Nemes, A., Temper, V., Razin, A., Cedar, H., 1994. Sp1 elements protect a CpG island from de novo methylation. Nature 371, 435–438. Clark, S.J., Harrison, J., Frommer, M., 1995. CpNpG methylation in mammalian cells. Nature Genet. 10, 20–27. Clark, S.J., Harrison, J., Paul, C.L., Frommer, M., 1994. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 22, 2990–2997. Fairall, L., Schwabe, J.W.R., Chapman, L., Finch, J.T., Rhodes, D., 1993. The crystal structure of a two zinc-finger peptide reveals an extension of the rules for zinc-finger/DNA recognition. Nature 366, 483–487. Fratini, A.V., Kopka, M.L., Drew, H.R., Dickerson, R.E., 1982. Reversible bending and helix geometry in a B-DNA dodecamer: CGCGAATTBrCGCG. J. Biol. Chem. 257, 14686–14707. Graessmann, M., Graessmann, A., 1993. DNA methylation, chromatin structure and the regulation of gene expression. In: Jost, J.P., Saluz, H.P. ( Eds), DNA Methylation: Molecular Biology and Biological Significance. Birkhauser, Basel, pp 404–424. Harrington, M.A., Jones, P.A., Imagawa, M., Karin, M., 1988. Cytosine methylation does not affect binding of transcription factor Sp1. Proc. Natl. Acad. Sci. USA 85, 2066–2070. Herman, J.G., Merlo, A., Mao, L., Lapidus, R.G., Issa, J.-P.J., Davidson, N.E., Sidransky, D., Baylin, S.B., 1995. Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res. 55, 4525–4530. Holler, M., Westin, G., Jiricny, J., Schaffner, W., 1988. Sp1 transcrip-
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tion factor binds DNA and activates transcription even when the binding site is CpG methylated. Genes Dev. 2, 1127–1135. Holliday, R., 1990. Mechanisms for the control of gene activity during development. Biol. Rev. 65, 431–471. Iguchi-Ariga, S.M.M., Schaffner, W., 1989. CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation. Genes Dev. 3, 612–619. Jones, P.A., Buckley, J.D., 1990. The role of DNA methylation in cancer. Adv. Cancer Res. 54, 1–23. Kovesdi, I., Reichel, R., Nevins, J.R., 1987. Role of adenovirus E2 promoter binding factor in E1A-mediated coordinate gene control. Proc. Natl. Acad. Sci. USA 84, 2180–2184. Kudo, S., Fukuda, M., 1994. Transcriptional activation of human leukosialin (CD43) gene by Sp1 through binding to a GGGTGG motif. Eur. J. Biochem. 223, 319–327. Kuwahara, J., Yonezawa, A., Futamura, M., Sugiura, Y., 1993. Binding of transcription factor Sp1 to GC box DNA revealed by footprinting analysis: different contact of three zinc fingers and sequence recognition mode. Biochemistry 32, 5994–6001. Letovsky, J., Dynan, W.S., 1989. Measurement of the binding of transcription factor Sp1 to a single GC box recognition sequence. Nucleic Acids Res. 17, 2639–2653. Macleod, D., Charlton, J., Mullins, J., Bird, A.P., 1994. Sp1 sites in the mouse aprt gene promoter are required to prevent methylation of the CpG island. Genes Dev. 8, 2282–2292. Meehan, R.R., Lewis, J.D., McKay, S., Kleiner, E.L., Bird, A.P., 1989. Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs. Cell 58, 499–507. Nardelli, J., Gibson, T.J., Vesque, C., Charnay, P., 1991. Base sequence discrimination by zinc-finger DNA-binding domains. Nature 349, 175–178. Paul, C.L., Clark, S.J., 1996. Cytosine methylation: quantitation by automated genomic sequencing and GENESCAN@ analysis. BioTechniques 21, 126–133. Razin, A., Cedar, H., 1991. DNA methylation and gene expression. Microbiol. Rev. 55, 451–458. Sakai, T., Toguchida, J., Ohtani, N., Yandell, D.W., Rapaport, J.M., Dryja, T., 1991. Allele-specific hypermethylation of the retinoblastoma tumor-suppressor gene. Am. J. Hum. Genet. 48, 880–888. Stirzaker, S.C., Millar, D.S., Paul, C.L., Warnecke, P.M., Harrison, J., Vincent, P.C., Frommer, M., Clark, S.J., 1997. Extensive DNA methylation spanning the Rb promoter in retinoblastoma tumors. Cancer Res. 57 Thiesen, H.J., Bach, C., 1990. Target Detection Assay ( TDA): a versatile procedure to determine DNA binding sites as demonstrated on SP1 protein. Nucleic Acids Res. 18, 3203–3209. Watt, F., Molloy, P.L., 1988. Cytosine methylation prevents binding to DNA of a HeLa cell transcription factor required for optimal expression of the adenovirus major late promoter. Genes Dev. 2, 1136–1143.
Sp1 binding is inhibited by mCpmCpG methylation Susan J. Clark a,b,*, Janet Harrison a, Peter L. Molloy b a Kanematsu Laboratories, Royal Prince Alfred Hospital, Missenden Road, Camperdown, NSW 2050, Australia b CSIRO, Division of Molecular Science, P.O. Box 184, North Ryde, NSW 2113, Australia Received 8 December 1996; accepted 9 February 1997; Received by H. Cedar
Abstract Previously it has been found that binding of the Sp1 transcription factor is not significantly affected by methylation of the CpG dinucleotide within its binding site, 5∞-GGGCGG ( lower strand, 5∞-CCGCCC ). Since it has been established that mammalian cells also have the capacity to methylate cytosines (C ) at CpNpG sites we examined the effect of methylation of the outer C of the CpCpG on Sp1 binding. We find that methylation of the outer C is inhibitory and in particular methylation of both cytosines mCpmCpG inhibits binding by 95%. Furthermore, we have identified endogenous mCpmCpG methylation of an Sp1 site in the CpG island promoter of the retinoblastoma (Rb) gene by genomic sequencing. This occurs in a proportion of retinoblastoma tumors which are extensively CpG methylated in the Rb promoter. The results raise the possibility that mCpmCpG methylation could have a biological function in preventing Sp1 binding, thereby contributing to the subsequent abnormal methylation of CpG islands often observed in tumor cells. © 1997 Elsevier Science B.V. Keywords: Transcription factor binding; CNG Methylation; Retinoblastoma; Gene regulation
1. Introduction In vertebrate genomes almost all of the identified post-replicative modification of DNA is in the form of methylation of cytosine at symmetric CpG dinucleotides. Changes in methylation of specific genes are seen during development and during neoplastic transformation and progression (Holliday, 1990; Jones and Buckley, 1990; Herman et al., 1995). Substantial data have accumulated which demonstrate that methylation of CpG dinucleotides in gene-regulatory regions of both introduced and endogenous genes can block their expression (Razin and Cedar, 1991). Whether methylation acts as a primary trigger or to reinforce other cellular events during development has not been established, but aberrant methylation clearly has the capacity to act as an epigenetic mutation. It has been demonstrated that DNA methylation can act both directly and indirectly to inhibit gene expression. Methylated DNA is localized to inactive chromatin and it has been suggested that through bind* Corresponding author at address b. Tel. +61 2 98864948; Fax +61 2 94905805; e-mail: [email protected] Abbreviations: DMS, dimethyl sulfate; mC, 5-methyl cytosine; PCR, polymerase chain reaction; Rb, Retinoblastoma tumor suppressor gene. 0378-1119/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S 03 7 8 -1 1 1 9 ( 9 7 ) 0 0 1 64 - 9
ing to specific methylated DNA binding proteins clusters of methylated CpGs may promote the formation of inactive chromatin and the exclusion of the transcription machinery (Antequerra et al., 1989; Meehan et al., 1989; Graessmann and Graessmann, 1993). For some transcription factors, (e.g., E2F, CREB and USF ) methylation at specific CpGs has been shown to directly inhibit protein binding and thus inhibit transcription ( Kovesdi et al., 1987; Watt and Molloy, 1988; Iguchi-Ariga and Schaffner, 1989). For other factors such as Sp1, methylation of a CpG site within the recognition sequence does not interfere with protein binding (Harrington et al., 1988; Holler et al., 1988; Ben-Hattar et al., 1989). In fact there is accumulating evidence that Sp1 sites are important in maintaining CpG-rich regions in an unmethylated state (Brandeis et al., 1994; Macleod et al., 1994). We have recently shown that mammalian cells also have the capacity to maintain methylation of cytosine at CpNpG sites of introduced plasmid DNA (CpApG and CpTpG) and also to de novo methylate these and CpCpG sites to a low level (Clark et al., 1995). This raises the possibility that such sites of methylation are present in endogenous genes and may have a role either in regulation of gene expression or another biological
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function. The core binding site for Sp1, 5∞-GGGCGG ( lower strand, 5∞-CCGCCC ), contains a potentially methylatable CpCpG site. Here we examine the effect of methylation of the outer C of the CpCpG present in the Sp1 recognition site on its binding and also present evidence that such methylation can occur in genomic DNA.
2. Experimental A series of oligonucleotides corresponding to both upper and lower strands of a consensus Sp1 site ( Thiesen and Bach, 1990) were synthesized, differing only in which cytosines were methylated (see Fig. 1). Methylation was introduced at the central CpG dinucleotide (bases C5 and C6∞), the outer cytosine of the CpCpG triplet (C7∞) or in cytosines at a CpNpG site flanking the core Sp1 binding region (C-1, C2∞). Binding of Sp1 to gel-purified double-stranded oligonucleotides, methylated in various configurations, was studied under standard conditions as shown in Fig. 1. Consistent with previous reports (Harrington et al., 1988; Holler et al., 1988; Ben-Hattar et al., 1989), methylation of the central CpG dinucleotide on either or both strands, had little or no effect on the efficiency of Sp1 binding (tracks B, F and G). Methylation of the flanking CpNpG site on either or both strands (tracks E, J and K ) also had no effect on binding. However, methylation of the single outer (C7∞) cytosine adjacent to the central CpG on the lower strand reduced Sp1 binding by nearly 50% (track D). This inhibition was strongly enhanced when both cytosines on the lower strand were methylated (track C ) and was of similar extent whether or not the upper strand cytosine (C5) was methylated (tracks C and H ). Equivalent results have been obtained when either the upper or lower strands were labeled (data not shown). These results are consistent with detailed mutational and methylation interference studies of Sp1 binding sites (Letovsky and Dynan, 1989; Thiesen and Bach, 1990; Kuwahara et al., 1993). Previous studies have shown that Sp1 can bind to sites containing alternate bases at position C5 of the recognition sequence (Letovsky and Dynan, 1989; Kudo and Fukuda, 1994). This is consistent with the lack of effect of methylation at C5 (Fig. 1, Track F ). Also, G5∞ on the lower strand is not protected from methylation by dimethyl sulfate (DMS ) when Sp1 is bound, though prior methylation does inhibit binding ( Kuwahara et al., 1993). Both methylation protection and interference assays indicate close protein contact with G6. By contrast, methylation of G7 does not interfere with Sp1 binding and this base shows enhanced reactivity with DMS when Sp1 is bound. Considering that this base pair is strictly conserved in Sp1 binding sites, it is possible that critical contact is made with the
lower strand C7∞. Consideration of the mode of contact of zinc finger domains with their binding sites (Nardelli et al., 1991; Fairall et al., 1993) has raised the possibility that there could be a direct H-bond to C7∞, particularly if the helix is distorted from ideal B-form. as suggested by the enhanced DMS reactivity of G7 when Sp1 is bound ( Kuwahara et al., 1993). The presence of methyl cytosines in successive base pair steps is also likely to constrain the DNA structure ( Fratini et al., 1982) and this could limit the capacity for Sp1 binding. Either such a structural effect or direct interference with hydrogen bonding would be consistent with the strong inhibition of Sp1 binding observed. It has been established by restriction enzyme studies that the promoter region of the Retinoblastoma tumor suppressor gene (Rb) is methylated in a proportion of retinoblastoma tumors (Sakai et al., 1991). Using samples previously characterized by restriction enzyme analysis we have been studying the specific patterns and extent of DNA methylation across the Rb promoter using bisulfite genomic sequencing (Stirzaker et al., 1997). In this work we have identified a number of cases where the outer cytosine, C7∞, of the CpCpG on the lower strand of the Sp1 site is methylated. Fig. 2 shows the methylation state of the cytosines in the CpCpG trinucleotide within the Sp1 binding site of the Rb promoter from a retinoblastoma patient (patient 252). An example of sequence from an individual clone shows methylation of both cytosines in the CpCpG Sp1 binding site ( Fig. 2A). Direct PCR sequencing indicates that about 28% of the outer C∞s are methylated in the DNA of this patient ( Fig. 2B, C ). Altogether three of eight patients with a hypermethylated Rb gene showed 20–30% methylation of the outer cytosine (C7∞). In all cases, methylation of the outer C occurred only where the inner cytosine (C6∞, i.e. the CpG site) was methylated. A significant occurrence of methylation of other CpNpG cytosines (mainly at CpCpG sites) was also seen in these patients. It is possible either that the aberrant de novo methylation has occurred only in a subset of the tumor cell population and is then faithfully maintained, or conversely, that CpCpG maintenance methylation is inefficient in each generation. The presence of methylation of the outer cytosine of a CpCpG sequence present in the core Sp1 binding site in genomic DNA and the capacity of this modification to block Sp1 binding raises the question of whether this may be of biological significance. CpG islands are rich in Sp1 sites and Sp1 binding has been proposed to play a critical role in maintaining CpG islands in an unmethylated state since mutation of the Sp1 sites in CpG islands allows the island to become methylated (Brandeis et al., 1994; Macleod et al., 1994). While CpG methylation of the Sp1 binding site is permissive for Sp1 binding, aberrant methylation of the outer cytosine in the core
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Fig. 1. Inhibition of Sp1 binding by DNA methylation. The left panel shows the sequence of the double-stranded oligonucleotides, with the core Sp1 binding site shown in upper case. The reference nucleotides are numbered −1 to 9 for the upper oligonucleotide and −1∞ to 9∞ for the lower oligonucleotide according to Kuwahara et al. (1993). (A)–( K ) show the methylation pattern for each double-stranded DNA set. Methylated and unmethylated cytosines are represented by filled and open circles, respectively. An electrophoretic mobility shift assay (EMSA) showing free oligonucleotide DNA and DNA complexed with Sp1 is shown to the right. The addition or absence of Sp1 in the binding reaction is indicated by + or −, respectively. The level of Sp1 binding relative to the unmethylated DNA is shown in the central column. Methods: oligonucleotides, containing 5-methyl cytosine (mC ) as indicated, were synthesized on an Applied Biosystems 370A synthesizer and purified according to manufacturer’s instructions. Double-stranded DNAs were prepared by annealing one oligonucleotide, radiolabeled with c32P-ATP and T4-polynucleotide kinase, with a complementary unlabeled oligonucleotide. They were purified by elecrophoresis through 12% non-denaturing polyacrylamide gels. DNA probes, 5 fmol, were incubated in 20 ml reactions containing 10 mM N-2-Hydroxyethyl piperazine-N∞-2-ethanesulfonic acid, 10% glycerol, 50 mM KCl, 6 mM MgCl , 25 mM ZnCl , 0.5 mM ethylenediaminetetraacetic acid, 5 mM dithiothreitol, 100 mM Pefabloc (Boehringer), 0.1% 2 2 Nonidet P40, containing 100 ng poly dI.dC, 50 mg bovine serum albumin, with or without 0.5 footprint units of Sp1 (Promega). Sp1-bound and free probe were separated by electrophoresis through a 6% polyacrylamide (40:1) in 90 mM Tris base, 90 mM boric acid, 1 mM ethylenediaminetetraacetic acid, pH 8.3. Phosphorimaging of the dried gel was done using a Molecular Dynamics Model No 400E and quantitation of binding was done using ImageQuant software version 3.3 (Molecular Dynamics).
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Fig. 2. Cytosine methylation of the CpCpG triplet in an Sp1 binding site of the Rb promoter. (A) shows the sequence surrounding the Sp1 binding site within the Rb promoter from an individual clone isolated from polymerase chain reaction (PCR)-amplified bisulfite-treated retinoblastoma tumor DNA. Arrows denote unconverted cytosines, corresponding to cytosines methylated in the parent DNA strand. The outer C of the CpCpG is indicated by *. The converted sequence as read in the clone and the original unconverted sequence are shown to the right; the position of the Sp1 binding site is indicated. (B) and (C ) show the cytosine (C ) and thymine (T ) Genescan profiles which were derived from quantitative genomic sequence analysis of PCR-amplified bisulfite-treated retinoblastoma tumor DNA, as described in Paul and Clark (1996). The original sequence (bisulfite unconverted) and the bisulfite converted methylated sequence are shown above the scans. The sequence resulting from conversion of unmethylated DNA is shown beneath the T profile. Unconverted C∞s corresponding to methylated cytosines in the sequence are indicated with arrows; the unmethylated cytosines are denoted with open triangles under the T profile. The methylated outer C of the CpCpG triplet is indicated by *. Percentage methylation for any cytosine is measured as the peak height of C over the peak heights of C+T. Methylation of the outer C of the CpCpG triplet is measured as 28% and the inner C as 92%. Methods: DNA was subjected to bisulfite modification and the promoter region of the Rb gene amplified by PCR as described (Clark et al., 1994). The specific nested primers used for amplification of the bottom strand of the Rb gene were: Outer Set, Rb15 (1662–1695): 5∞-ACCCCAGCCTAAAAAAAATAATTCTAAATAAAA and Rb16 (1714–1743): 5∞-GATTTTTTTGGATTTTGGTTATAAAAATAA; Inner Set, Rb17 (1926–1948): 5∞-AATACCTCCTAAAAAACCCCTAAACCCAC and Rb18 (1949–1978): 5∞-TTTTAAGGTTTTTTGAGAAAAAT. The coordinates of the primers indicate their location on the Rb sequence (Accession number: L11910). Direct sequence analysis was done as described (Paul and Clark, 1996). PCR products were cloned and the individual clones sequenced as described (Clark et al., 1994).
binding site would prevent Sp1 from binding, thus acting like a mutation at the Sp1 site. As seen for Sp1 site mutations, this could trigger CpG methylation of the rest of the island. For an island such as that of the Rb tumor suppressor gene, the resulting gene silencing could contribute to clonal expansion and tumor development. Though CpG methylation would be faithfully maintained, the lower efficiency of maintenance of CpNpG methylation may mean that its presence in the population is transient. In this context CpNpG methylation may be thought of as an epigenetic mutation which could initiate the silencing of tumor suppressor genes by CpG methylation.
3. Conclusions (1) Binding of the transcription factor Sp1 is inhibited by mCpmCpG methylation. (2) Endogenous mCpmCpG methylation of a critical Sp1 site was identified in the hypermethylated promoter of the Rb tumor suppressor gene in three of eight retinoblastoma tumors analysed. (3) We propose that by blocking Sp1 binding, mCpmCpG methylation could act to initiate de novo CpG methylation of the CpG island, leading to promoter silencing.
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Acknowledgement We thank Ted Dryja for providing the retinoblastoma tumor DNAs, Cheryl Paul, Clare Stirzaker and Doug Millar for genomic sequencing, Marianne Frommer for helpful discussions and Robin Holliday and Horace Drew for critical reading of the manuscript. This work was partially supported by an Anthony Rothe Memorial Grant.
References Antequerra, F., MacLeod, D., Bird, A.P., 1989. Specific protection of methylated CpGs in mammalian nuclei. Cell 58, 509–517. Ben-Hattar, J., Beard, P., Jiricny, J., 1989. Cytosine methylation in CTF and Sp1 recognition sites of an HSV tk promoter: effects on transcription in vivo and on factor binding in vitro. Nucleic Acids Res. 17, 10179–10190. Brandeis, M., Frank, D., Keshet, I., Siegfried, Z., Mendelsohn, M., Nemes, A., Temper, V., Razin, A., Cedar, H., 1994. Sp1 elements protect a CpG island from de novo methylation. Nature 371, 435–438. Clark, S.J., Harrison, J., Frommer, M., 1995. CpNpG methylation in mammalian cells. Nature Genet. 10, 20–27. Clark, S.J., Harrison, J., Paul, C.L., Frommer, M., 1994. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 22, 2990–2997. Fairall, L., Schwabe, J.W.R., Chapman, L., Finch, J.T., Rhodes, D., 1993. The crystal structure of a two zinc-finger peptide reveals an extension of the rules for zinc-finger/DNA recognition. Nature 366, 483–487. Fratini, A.V., Kopka, M.L., Drew, H.R., Dickerson, R.E., 1982. Reversible bending and helix geometry in a B-DNA dodecamer: CGCGAATTBrCGCG. J. Biol. Chem. 257, 14686–14707. Graessmann, M., Graessmann, A., 1993. DNA methylation, chromatin structure and the regulation of gene expression. In: Jost, J.P., Saluz, H.P. ( Eds), DNA Methylation: Molecular Biology and Biological Significance. Birkhauser, Basel, pp 404–424. Harrington, M.A., Jones, P.A., Imagawa, M., Karin, M., 1988. Cytosine methylation does not affect binding of transcription factor Sp1. Proc. Natl. Acad. Sci. USA 85, 2066–2070. Herman, J.G., Merlo, A., Mao, L., Lapidus, R.G., Issa, J.-P.J., Davidson, N.E., Sidransky, D., Baylin, S.B., 1995. Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res. 55, 4525–4530. Holler, M., Westin, G., Jiricny, J., Schaffner, W., 1988. Sp1 transcrip-
71
tion factor binds DNA and activates transcription even when the binding site is CpG methylated. Genes Dev. 2, 1127–1135. Holliday, R., 1990. Mechanisms for the control of gene activity during development. Biol. Rev. 65, 431–471. Iguchi-Ariga, S.M.M., Schaffner, W., 1989. CpG methylation of the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishes specific factor binding as well as transcriptional activation. Genes Dev. 3, 612–619. Jones, P.A., Buckley, J.D., 1990. The role of DNA methylation in cancer. Adv. Cancer Res. 54, 1–23. Kovesdi, I., Reichel, R., Nevins, J.R., 1987. Role of adenovirus E2 promoter binding factor in E1A-mediated coordinate gene control. Proc. Natl. Acad. Sci. USA 84, 2180–2184. Kudo, S., Fukuda, M., 1994. Transcriptional activation of human leukosialin (CD43) gene by Sp1 through binding to a GGGTGG motif. Eur. J. Biochem. 223, 319–327. Kuwahara, J., Yonezawa, A., Futamura, M., Sugiura, Y., 1993. Binding of transcription factor Sp1 to GC box DNA revealed by footprinting analysis: different contact of three zinc fingers and sequence recognition mode. Biochemistry 32, 5994–6001. Letovsky, J., Dynan, W.S., 1989. Measurement of the binding of transcription factor Sp1 to a single GC box recognition sequence. Nucleic Acids Res. 17, 2639–2653. Macleod, D., Charlton, J., Mullins, J., Bird, A.P., 1994. Sp1 sites in the mouse aprt gene promoter are required to prevent methylation of the CpG island. Genes Dev. 8, 2282–2292. Meehan, R.R., Lewis, J.D., McKay, S., Kleiner, E.L., Bird, A.P., 1989. Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs. Cell 58, 499–507. Nardelli, J., Gibson, T.J., Vesque, C., Charnay, P., 1991. Base sequence discrimination by zinc-finger DNA-binding domains. Nature 349, 175–178. Paul, C.L., Clark, S.J., 1996. Cytosine methylation: quantitation by automated genomic sequencing and GENESCAN@ analysis. BioTechniques 21, 126–133. Razin, A., Cedar, H., 1991. DNA methylation and gene expression. Microbiol. Rev. 55, 451–458. Sakai, T., Toguchida, J., Ohtani, N., Yandell, D.W., Rapaport, J.M., Dryja, T., 1991. Allele-specific hypermethylation of the retinoblastoma tumor-suppressor gene. Am. J. Hum. Genet. 48, 880–888. Stirzaker, S.C., Millar, D.S., Paul, C.L., Warnecke, P.M., Harrison, J., Vincent, P.C., Frommer, M., Clark, S.J., 1997. Extensive DNA methylation spanning the Rb promoter in retinoblastoma tumors. Cancer Res. 57 Thiesen, H.J., Bach, C., 1990. Target Detection Assay ( TDA): a versatile procedure to determine DNA binding sites as demonstrated on SP1 protein. Nucleic Acids Res. 18, 3203–3209. Watt, F., Molloy, P.L., 1988. Cytosine methylation prevents binding to DNA of a HeLa cell transcription factor required for optimal expression of the adenovirus major late promoter. Genes Dev. 2, 1136–1143.