Deficiency of Mbd2 suppresses intestinal tumorigenesis.

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 deficiency does not increase mutation or accelerate tumorigenesis in mice lacking MMR.

Mbd4 (methyl-binding domain 4) has been shown to be mutated in a high percentage of mismatch repair (MMR)-deficient colorectal tumours that exhibit microsatellite instability (MSI). However, the significance of these mutations is still unclear as they are predominantly monoallelic and the majority occur at a poly-A tract. Apart from MMR-deficient tumours, no other reports of mutations of Mbd4 in human neoplasia are as yet published. To address the significance of loss of Mbd4 in the absence of MMR, we have crossed Mbd4-deficient mice to mice lacking DNA MMR. We show that, in the context of MMR deficiency, additional loss of Mbd4 does not alter spontaneous mutation frequency at the endogenous Dlb-1b locus, nor does it modify tumour onset, tumour spectrum or MSI compared to singly mutant Msh2 or Mlh1 mice. Taken together, these findings show that nullizygosity or heterozygosity for Mbd4 does not affect MMR-dependent tumorigenesis.

Apoptosis and mutation in the murine small intestine: loss of Mlh1- and Pms2-dependent apoptosis leads to increased mutation in vivo.

The mismatch repair (MMR) protein Msh2 has been shown to function in the apoptotic response to alkylating agents in vivo. Here, we extend these studies to the MutL homologues (MLH) Mlh1 and Pms2 by analysing the apoptotic response within the small intestine of gene targeted strains. We demonstrate significant differences between Msh2, Mlh1 and Pms2 mutations in influencing apoptotic signalling following 50mg/kg N-methyl-nitrosourea (NMNU), with no obvious reliance upon either Mlh1 or Pms2. However, following exposure to 100mg/kg temozolomide or lower levels of NMNU (10mg/kg) both Mlh1- and Pms2-dependent apoptosis was observed, indicating that the apoptotic response at these levels of DNA damage is dependent on the MutL homologues. Given our ability to observe a MutLalpha dependence of the apoptotic response, we tested whether perturbations of this response directly translate into increases in mutation frequency in vivo. We show that treatment with temozolomide or 10mg/kg NMNU significantly increases mutation in both the Mlh1 and Pms2 mutant mice. At higher levels of NMNU, where the apoptotic response is independent of Mlh1 and Pms2, no gene dependent increase in mutation frequency was observed. These results argue that the MutSalpha and MutLalpha are not equally important in their ability to signal apoptosis. However, when MMR does mediate apoptosis, perturbation of this response leads to long-term persistence of mutant cells in vivo.

Enhanced CpG mutability and tumorigenesis in MBD4-deficient mice.

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.