Sp1 binding is inhibited by mCpmCpG methylation
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 binding 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 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.
Section snippets
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
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.
Acknowledgements
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.
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