Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Feb 13;386(1):72-80.
doi: 10.1016/j.jmb.2008.11.054. Epub 2008 Dec 6.

Effects of CpG methylation on recognition of DNA by the tumour suppressor p53

Miriana Petrovich  1 , Dmitry B Veprintsev
Affiliations

Effects of CpG methylation on recognition of DNA by the tumour suppressor p53

Miriana Petrovich et al. J Mol Biol. .

Abstract

Methylation of DNA is one of the mechanisms controlling the expression landscape of the genome. Its pattern is altered in cancer and often results in the hypermethylation of the promoter regions and abnormal expression of tumour suppressor genes. Methylation of CpG dinucleotides located in the binding sites of transcription factors may contribute to the development of cancers by preventing their binding or altering their specificity. We studied the effects of CpG methylation on DNA recognition by the tumour suppressor p53, a transcription factor involved in the response to carcinogenic stress. p53 recognises a large number of DNA sequences, many of which contain CpG dinucleotides. We systematically substituted a CpG dinucleotide at each position in the consensus p53 DNA binding sequence and identified substitutions tolerated by p53. We compared the binding affinities of methylated versus non-methylated sequences by fluorescence anisotropy titration. We found that binding of p53 was not affected by cytosine methylation in a majority of cases. However, for a few sequences containing multiple CpG dinucleotides, such as sites in the RB and Met genes, methylation resulted in a four- to sixfold increase in binding of p53. This approach can be used to quantify the effects of CpG methylation on the DNA recognition by other DNA-binding proteins.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
To access the whole variety of DNA sequences recognised by p53, we systematically introduced CpG or 5-methyl-CpG at every possible position in the sequence.
Fig. 2
Fig. 2
Fluorescence anisotropy R reflects the tumbling rate of molecules in solution. It is ideal for studying strong protein–DNA interactions, as the complex formed is larger and tumbles more slowly than does the unbound oligonucleotide. A displacement of the Alexa488-labelled reporter oligonucleotide from the complex by the unlabelled competitor oligonucleotide allows accurate measurement of the difference in the Kd between two sequences. For clarity, the data shown represent a single titration using reference sequence number 300 from Table 1. Reporter DNA sequence (20 nM) was mixed with 125 nM full-length p53 protein. Competitor DNA was added to the sample in 25 steps. Such a titration was done for every DNA sequence studied. We multiplexed our measurements to perform 96 such titrations in parallel, allowing simultaneous analysis of a number of different sequences (see Materials and Methods for details).
Fig. 3
Fig. 3
(a) Effects of CpG methylation on affinity of p53 for DNA. CpG (black) or 5-methyl-CpG (red) dinucleotide was systematically substituted into the reference sequence at positions 1–10 of the first half-site. The CpG position refers to the position of the C in the first half of the p53 binding sequence GGACATGTCC. The difference in affinity relative to the unmethylated reference sequence is shown (Δlog Kd = log Kd(i) − log Kd(ref)). The log Kd of the reference sequence was − 7.55. (b) The difference in the affinity that can be attributed specifically to methylation is the difference in height of the bars at the same position, ΔΔlog Kd. = Δlog Kd(met) − Δlog Kd(non-met). Introduction of CpG dinucleotide at positions 4 and 6 has small overall impact on the affinity of p53 and results in the biggest methylation-specific response. Error bars represent one standard deviation based on at least three individual titrations.
Fig. 4
Fig. 4
Effects of single-strand methylation on binding of p53 to DNA. (a) Schematic diagram representing a consensus sequence of a p53 half-site. (b) A change in the affinity caused by a specific methylated cytosine or a combination of cytosines in a CpG dinucleotide. Methylation of CpG dinucleotides at positions 4 and 6 has the largest effect on binding of p53. These modifications affect invariant nucleotides at positions 4 and 6′, respectively. When methylated cytosine is present on only one strand, the effects of methylation at positions 5 and 6 had a similar effect as double methylation. 4, methylation on the top strand; 4′, methylation on the bottom strand; 4 + 4′, methylation on both strands. Likewise for position 6. The log Kd of the reference sequence was − 7.55.
Fig. 5
Fig. 5
Effects of multiple methylated CpG on the affinity of p53 for DNA. (a) Multiple CpG substitution results in progressively weaker binding because of the increasingly suboptimal DNA sequence. However, if the CpG dinucleotides are methylated, the overall increase in the log Kd is smaller. (b) The difference between the affinity of the methylated and non-methylated sequence increased proportionally to the number of methylated CpG dinucleotides. The log Kd of the reference sequence was − 7.55.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Robertson K.D. DNA methylation and human disease. Nat. Rev. Genet. 2005;6:597–610. - PubMed
    1. Brena R.M., Costello J.F. Genome–epigenome interactions in cancer. Hum. Mol. Genet. 2007;16(Spec. No. 1):R96–R105. - PubMed
    1. Bird A. DNA methylation patterns and epigenetic memory. Genes Dev. 2002;16:6–21. - PubMed
    1. Jones P.A., Baylin S.B. The epigenomics of cancer. Cell. 2007;128:683–692. - PMC - PubMed
    1. Watt F., Molloy P.L. Cytosine methylation prevents binding to DNA of a HeLa cell transcription factor required for optimal expression of the adenovirus major late promoter. Genes Dev. 1988;2:1136–1143. - PubMed

Publication types