Cytosine methylation does not affect binding of transcription factor Sp1

Maureen Harrington, P. A. Jones, M. Imagawa, M. Karin

Research output: Contribution to journalArticle

133 Citations (Scopus)

Abstract

DNA methylation may be a component of a multilevel control mechanism that regulates eukaryotic gene expression. We used synthetic oligonucleotides to investigate the effect of cytosine methylation on the binding of the transcription factor Sp1 to its target sequence (a G + C-rich sequence known as a 'CC box'). Concatemers of double-stranded 14-mers containing a GC box successfully competed with the human metallothionein IIA promoter for binding to Sp1 in DNase 1 protection experiments. The presence of 5-methylcytosine in the CpG sequence of the GC box did not influence Sp1 binding. The result was confirmed using double-stranded 20-mers containing 16 base pairs of complementary sequence. Electrophoretic gel retardation analysis of annealed 28-mers containing a GC box incubated with an Sp1-containing HeLa cell nuclear extract demonstrated the formation of DNA-protein complexes; formation of these complexes was not inhibited when an oligomer without a GC box was used as a competitor. Once again, the presence of a 5-methylcytosine residue in the GC box did not influence the binding of the protein to DNA. The results therefore preclude a direct effect of cytosine methylation on Sp1-DNA interactions.

Original languageEnglish (US)
Pages (from-to)2066-2070
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume85
Issue number7
DOIs
StatePublished - 1988
Externally publishedYes

Fingerprint

Sp1 Transcription Factor
5-Methylcytosine
Cytosine
Methylation
Deoxyribonucleases
Metallothionein
DNA
DNA-Binding Proteins
DNA Methylation
Cell Extracts
HeLa Cells
Oligonucleotides
Base Pairing
Gels
Gene Expression
Proteins

Keywords

  • 'GC box'
  • DNA methylation
  • metallothionein gene expression

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Cytosine methylation does not affect binding of transcription factor Sp1. / Harrington, Maureen; Jones, P. A.; Imagawa, M.; Karin, M.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 85, No. 7, 1988, p. 2066-2070.

Research output: Contribution to journalArticle

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N2 - DNA methylation may be a component of a multilevel control mechanism that regulates eukaryotic gene expression. We used synthetic oligonucleotides to investigate the effect of cytosine methylation on the binding of the transcription factor Sp1 to its target sequence (a G + C-rich sequence known as a 'CC box'). Concatemers of double-stranded 14-mers containing a GC box successfully competed with the human metallothionein IIA promoter for binding to Sp1 in DNase 1 protection experiments. The presence of 5-methylcytosine in the CpG sequence of the GC box did not influence Sp1 binding. The result was confirmed using double-stranded 20-mers containing 16 base pairs of complementary sequence. Electrophoretic gel retardation analysis of annealed 28-mers containing a GC box incubated with an Sp1-containing HeLa cell nuclear extract demonstrated the formation of DNA-protein complexes; formation of these complexes was not inhibited when an oligomer without a GC box was used as a competitor. Once again, the presence of a 5-methylcytosine residue in the GC box did not influence the binding of the protein to DNA. The results therefore preclude a direct effect of cytosine methylation on Sp1-DNA interactions.

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