Functional consequences of Rett syndrome mutations on human MeCP2.

The neurodevelopmental disorder known as Rett syndrome has recently been linked to the methyl-CpG-binding transcriptional repressor, MeCP2. In this report we examine the consequences of these mutations on the function of MeCP2. The ability to bind specifically to methylated DNA and the transcription repression capabilities are tested, as well as the stability of proteins in vivo. We find that all missense mutations (R106W, R133C, F155S, T158M) within the methyl-binding domain impair selectivity for methylated DNA, and that all nonsense mutations (L138X, R168X, E235X, R255X, R270X, V288X, R294X) that truncate all or some of the transcriptional repression domain (TRD) affect the ability to repress transcription and have decreased levels of stability in vivo. Two missense mutations, one in the TRD (R306C) and one in the C-terminus (E397K), had no noticeable effects on MeCP2 function. Together, these results provide evidence of how Rett syndrome mutations can affect distinct functions of MeCP2 and give insight into these mutations that may contribute to the disease.

Effects of Rett syndrome mutations of the methyl-CpG binding domain of the transcriptional repressor MeCP2 on selectivity for association with methylated DNA.

We have investigated the properties of mutant forms of the methyl-CpG binding transcriptional repressor MeCP2 associated with Rett syndrome, a childhood neurodevelopmental disorder. We find that four Rett syndrome mutations at known sites within the methyl-CpG binding domain (MBD) impair binding to methylated DNA, but have little effect on nonspecific interactions with unmethylated DNA. Three of these mutations (R106W, R133C, and F155S) have their binding affinities for methylated DNA reduced more than 100-fold; this is consistent with the hypothesis that impaired selectivity for methylated DNA of mutant MeCP2 contributes to Rett syndrome. However, a fourth mutant, T158M, has its binding affinity for methylated DNA reduced only 2-fold, indicative either of additional distinct regulatory functions associated with the MBD or of an exquisite sensitivity of developing neurons to the selective association of MeCP2 with methylated DNA.

The p53 response to DNA damage in vivo is independent of DNA-dependent protein kinase.

Ionizing radiation (IR) exposure causes mammalian cells to undergo p53-dependent cell cycle arrest and/or apoptosis. The in vivo role of DNA-dependent protein kinase (DNA-PK) in the transduction of the DNA damage signal to p53 remains unresolved. To determine the relationship between DNA-PK and p53, we studied the cell cycle and apoptotic responses to IR in mice deficient in DNA-PK. Using the slip mouse, which harbors an inactivating mutation of the DNA-PK catalytic subunit (DNA-PKcs), we demonstrated not only that these DNA-PKcs null mutants were highly radiosensitive but also that upon IR treatment, p53 accumulated in their cultured cells and tissue. Induced p53 was transcriptionally active and mediated the induction of p21 and Bax in slip cells. Examination of the thymic cell cycle response to IR treatment indicated that the slip G(1)/S-phase cell cycle checkpoint function was intact. We further show that slip mice exhibited a higher level of spontaneous thymic apoptosis as well as a more robust apoptotic response to IR than wild-type mice. Together, these data demonstrate that the p53-mediated response to DNA damage is intact in cells devoid of DNA-PK activity and suggest that other kinases, such as the product of the gene (ATM) mutated in ataxia telangiectasia, are better candidates for regulating IR-induced phosphorylation and accumulation of p53.