NORMAL GENOMIC

• Hendrich B, Hardeland U, Ng HH, Jiricny J, Bird A
• The thymine glycosylase MBD4 can bind to the product of deamination at methylated CpG sites.
• Nature. 1999; 401: 301-4
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• In addition to its well-documented effects on gene silencing, cytosine methylation is a prominent cause of mutations. In humans, the mutation rate from 5-methylcytosine (m5C) to thymine (T) is 10-50-fold higher than other transitions and the methylated sequence CpG is consequently under-represented. Over one-third of germline point mutations associated with human genetic disease and many somatic mutations leading to cancer involve loss of CpG. The primary cause of mutability appears to be hydrolytic deamination. Cytosine deamination produces mismatched uracil (U), which can be removed by uracil glycosylase, whereas m5C deamination generates a G x T mispair that cannot be processed by this enzyme. Correction of m5CpG x TpG mismatches may instead be initiated by the thymine DNA glycosylase, TDG. Here we show that MBD4, an unrelated mammalian protein that contains a methyl-CpG binding domain, can also efficiently remove thymine or uracil from a mismatches CpG site in vitro. Furthermore, the methyl-CpG binding domain of MBD4 binds preferentially to m5CpG x TpG mismatches-the primary product of deamination at methyl-CpG. The combined specificities of binding and catalysis indicate that this enzyme may function to minimize mutation at methyl-CpG.

• Cross SH, Meehan RR, Nan X, Bird A
• A component of the transcriptional repressor MeCP1 shares a motif with DNA methyltransferase and HRX proteins.
• Nat Genet. 1997; 16: 256-9
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• Methylation of cytosines within the sequence CpG is essential for mouse development and has been linked to transcriptional suppression in vertebrate systems. Methyl-CpG binding proteins (MeCPs) 1 and 2 bind preferentially to methylated DNA and can inhibit transcription. The gene for MeCP2 has been cloned and a methyl-CpG binding domain (MBD) within it has been defined. A search of DNA sequence databases with the MBD sequence identified a human cDNA with potential to encode an MBD-like region. Sequencing of the complete cDNA revealed that the open reading frame also encodes two cysteine-rich domains that are found in animal DNA methyltransferases (DNMTs) and in the mammalian HRX protein (also known as MLL and All-1). HRX is related to Drosophila trithorax. The protein, known as Protein Containing MBD (PCM1), was expressed in bacteria and shown to bind specifically to methylated DNA. PCM1 also repressed transcription in vitro in a methylation-dependent manner. A polyclonal antibody raised against the protein was able to 'supershift' the native MeCP11 complex from HeLa cells, indicating that PCM1 is a component of mammalian MeCP1.

• Boyes J, Bird A
• DNA methylation inhibits transcription indirectly via a methyl-CpG binding protein.
• Cell. 1991; 64: 1123-34
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• We have studied the mechanism by which DNA methylation inhibits transcription both in cell-free nuclear extracts and in the living cell. Repression of transcription in vitro for four different promoters was shown to be an indirect effect. The mediator of repression had properties indistinguishable from those of a methyl-CpG binding protein (MeCP-1) that has been previously identified. Use of differentially methylated promoters and methylated competitors in transient transfection assays suggested that indirect repression via MeCP-1 also occurs in the living cell. This was supported by the fact that MeCP-1-deficient cells showed much reduced repression of methylated genes.