The mammalian protein MBD4 contains a methyl-CpG binding domain and can enzymatically remove thymine (T) or uracil (U) from a mismatched CpG site in vitro. These properties suggest that MBD4 might function in vivo to minimize the mutability of 5-methylcytosine by removing its deamination product from DNA. We tested this hypothesis by analyzing Mbd4-/- mice and found that the frequency of of C --> T transitions at CpG sites was increased by a factor of three. On a cancer-susceptible Apc(Min/+) background, Mbd4-/- mice showed accelerated tumor formation with CpG --> TpG mutations in the Apc gene. Thus MBD4 suppresses CpG mutability and tumorigenesis in vivo.

Gene silencing through de novo methylation of CpG island promoters contributes to cancer. We find that Mbd2, which recruits co-repressor complexes to methylated DNA, is essential for efficient tumorigenesis in the mouse intestine. As Mbd2-deficient mice are viable and fertile, their resistance to intestinal cancer may be of therapeutic relevance.

MBD4 was originally identified through its methyl binding domain, but has more recently been characterized as a thymine DNA glycosylase that interacts with the mismatch repair (MMR) protein MLH1. In vivo, MBD4 functions to reduce the mutability of methyl-CpG sites in the genome and mice deticient in MBD4 show increased intestinal tumorigenesis on an Apc Min/ þ background. As MLH1 and other MMR proteins have been functionally linked to apoptosis, we asked whether MBD4 also plays a role in mediating the apoptotic response within the murine small intestine. Mice deficient for MBD4 showed significantly reduced apoptotic responses 6 h following treatment with a range of cytotoxic agents including cirradiation, cisplatin, temozolomide and 5-fluorouracil (5-FU). This leads to increased clonogenic survival in vivo in Mbd4 À/À mice following exposure to either 5-FU or cisplatin. We next analysed the apoptotic response to 5-FU and temozolomide in doubly mutant Mbd4À/À mice but observed no additive decrease. The results imply that MBD4 and MLH1 lie in the same pathway and therefore that MMR-dependent apoptosis is mediated through MBD4. MBD4 deficiency also reduced the normal apoptotic response to c-irradiation, which we show is independent of Mlh1 status (at least in the murine small intestine), so suggesting that the reliance upon MBD4 may extend beyond MMR-mediated apoptosis. Our results establish a novel functional role for MBD4 in the cellular response to DNA damage and may have implications for its role in suppressing neoplasia.

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