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

Petrovich M, Veprintsev DB - J. Mol. Biol. (2008)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Protein Engineering, Cambridge CB2 0QH, UK.

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.

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(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.
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fig3: (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.

Mentions: We compared the affinity of oligonucleotides containing single methylated and unmethylated CpG dinucleotide to the affinity of unlabelled reference sequence. The changes in the affinity caused by all possible substitutions of CpG dinucleotide within the reference oligonucleotide (Table 1) are presented in Fig. 3. Because of the symmetry of the sequence used (it is the same sequence on the complementary strand in the 5′ to 3′ direction) only the first half-site was scanned, the sequence of the second half-site being unaltered. First, we investigated if p53 binds to sequences containing CpG dinucleotide by comparing the affinity of the CpG-containing sequence to the reference sequence (Fig. 3a, black series). The substitution of the CpG dinucleotide in the reference sequence makes it suboptimal in many cases. Substitutions at positions 3, 5 and 7 resulted in a particularly large reduction of the affinity (increase in the log Kd value). These substitutions replace the invariant C and G with G and C at positions 4 and 6, respectively (Fig. 4). These results are consistent with our previous data on the sequence dependence of the affinity using single-nucleotide substitutions, as well as the consensus sequence definition.15,16,20 The CpG at position 5 replaces the WW (W = A or T) motif at positions 5/6. This motif is important for recognition of DNA, with the TA dinucleotide being the best both for binding and for transactivation activity.15,16,20


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

Petrovich M, Veprintsev DB - J. Mol. Biol. (2008)

(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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2666794&req=5

fig3: (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.
Mentions: We compared the affinity of oligonucleotides containing single methylated and unmethylated CpG dinucleotide to the affinity of unlabelled reference sequence. The changes in the affinity caused by all possible substitutions of CpG dinucleotide within the reference oligonucleotide (Table 1) are presented in Fig. 3. Because of the symmetry of the sequence used (it is the same sequence on the complementary strand in the 5′ to 3′ direction) only the first half-site was scanned, the sequence of the second half-site being unaltered. First, we investigated if p53 binds to sequences containing CpG dinucleotide by comparing the affinity of the CpG-containing sequence to the reference sequence (Fig. 3a, black series). The substitution of the CpG dinucleotide in the reference sequence makes it suboptimal in many cases. Substitutions at positions 3, 5 and 7 resulted in a particularly large reduction of the affinity (increase in the log Kd value). These substitutions replace the invariant C and G with G and C at positions 4 and 6, respectively (Fig. 4). These results are consistent with our previous data on the sequence dependence of the affinity using single-nucleotide substitutions, as well as the consensus sequence definition.15,16,20 The CpG at position 5 replaces the WW (W = A or T) motif at positions 5/6. This motif is important for recognition of DNA, with the TA dinucleotide being the best both for binding and for transactivation activity.15,16,20

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: MRC Centre for Protein Engineering, Cambridge CB2 0QH, UK.

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.

Show MeSH
Related in: MedlinePlus