Petrovich Miriana, Veprintsev Dmitry B
MRC Centre for Protein Engineering, Cambridge CB2 0QH, UK.
J Mol Biol. 2009 Feb 13;386(1):72-80. doi: 10.1016/j.jmb.2008.11.054. Epub 2008 Dec 6.
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.
DNA甲基化是控制基因组表达格局的机制之一。其模式在癌症中会发生改变，常导致启动子区域的高甲基化以及肿瘤抑制基因的异常表达。位于转录因子结合位点的CpG二核苷酸甲基化可能通过阻止转录因子结合或改变其特异性而促进癌症的发展。我们研究了CpG甲基化对肿瘤抑制因子p53识别DNA的影响，p53是一种参与致癌应激反应的转录因子。p53可识别大量DNA序列，其中许多含有CpG二核苷酸。我们在p53 DNA结合共有序列的每个位置上系统地替换一个CpG二核苷酸，并确定p53能够耐受的替换情况。我们通过荧光各向异性滴定比较了甲基化和未甲基化序列的结合亲和力。我们发现，在大多数情况下，p53的结合不受胞嘧啶甲基化的影响。然而，对于一些含有多个CpG二核苷酸的序列，如RB和Met基因中的位点，甲基化导致p53的结合增加了4至6倍。这种方法可用于量化CpG甲基化对其他DNA结合蛋白识别DNA的影响。
