Elsevier

Gene

Volume 195, Issue 1, 11 August 1997, Pages 67-71
Gene

Sp1 binding is inhibited by mCpmCpG methylation

https://doi.org/10.1016/S0378-1119(97)00164-9Get rights and content

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.

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.

References (27)

  • A.V. Fratini et al.

    Reversible bending and helix geometry in a B-DNA dodecamer: CGCGAATTBrCGCG

    J. Biol. Chem.

    (1982)
  • P.A. Jones et al.

    The role of DNA methylation in cancer

    Adv. Cancer Res.

    (1990)
  • R.R. Meehan et al.

    Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs

    Cell

    (1989)
  • F. Antequerra et al.

    Specific protection of methylated CpGs in mammalian nuclei

    Cell

    (1989)
  • J. Ben-Hattar et al.

    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.

    (1989)
  • M. Brandeis et al.

    Sp1 elements protect a CpG island from de novo methylation

    Nature

    (1994)
  • S.J. Clark et al.

    CpNpG methylation in mammalian cells

    Nature Genet.

    (1995)
  • S.J. Clark et al.

    High sensitivity mapping of methylated cytosines

    Nucleic Acids Res.

    (1994)
  • L. Fairall et al.

    The crystal structure of a two zinc-finger peptide reveals an extension of the rules for zinc-finger/DNA recognition

    Nature

    (1993)
  • Graessmann, M., Graessmann, A., 1993. DNA methylation, chromatin structure and the regulation of gene expression. In:...
  • M.A. Harrington et al.

    Cytosine methylation does not affect binding of transcription factor Sp1

    Proc. Natl. Acad. Sci. USA

    (1988)
  • J.G. Herman et al.

    Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers

    Cancer Res.

    (1995)
  • M. Holler et al.

    Sp1 transcription factor binds DNA and activates transcription even when the binding site is CpG methylated

    Genes Dev.

    (1988)
  • Cited by (0)

    View full text