Characterization of HIPK2 that, by Associating with MeCP2, Might Function as a Modifier Gene in Rett Syndrome.

Mutations in the methyl CpG-binding protein 2 (MECP2) gene, located on Xq28, are responsible for almost all cases of classic RTT. Conversely, less than half of the patients with one of the variant forms of RTT carry mutations in MECP2. It seems, thus, that other genes are involved in causing RTT; moreover the fact that there are patients with milder phenotypes in spite of severe mutations argues that modifier genes might restrict the clinical outcome by regulating MeCP2 functions. To search for MeCP2 interacting proteins possibly involved in RTT we performed a yeast two-hybrid screening and identified among the positive clones HIPK2 (homeodomain interacting protein kinase 2) that belongs to a family of Ser/Thr kinases originally identified as corepressors for homeodomain transcription factors. HIPK2 has a clear role in regulating cell growth and genotoxic stress-induced apoptosis. Furthermore, its involvement in the nervous system is indicated by the neuronal defects of null mice that partially overlap those observed in Mecp2 ko mice. Since important MeCP2 functions in the nervous system are regulated by its phosphorylation we found it interesting to analyze the functional role of its interaction with HIPK2. We have thus confirmed that the two proteins associate in vitro and in vivo and phosphorylation assays have shown that MeCP2 is significantly phosphorylated by HIPK2 in vitro. Importantly, these assays have also allowed us to establish that Ser80 within the MBD of MeCP2 is a specific target of HIPK2. Functional assays have shown that ectopic MeCP2 causes an increase in cell death and an additive effect of the two proteins in inducing apoptosis in cultured cells was observed. Importantly, the role of MeCP2 in inducing apoptosis together with HIPK2 is lost when Ser80 is mutated or a kinase dead derivative of HIPK2 is used. Presently we are analyzing whether MeCP2 is a target of the kinase also in vivo and the role of the interaction for the nervous system. In favor of the hypothesis that the two proteins work in a common molecular pathway we have shown by immunohistochemistry experiments that the expression pattern of MeCP2 and HIPK2 in the brain of adult mice is highly similar. We therefore believe that these studies are relevant for understanding whether this novel MeCP2 interactor acts as a modifier gene influencing disease severity in RTT patients with mutations in MeCP2.