Two highly related p66 proteins comprise a new family of potent transcriptional repressors interacting with MBD2 and MBD3

Abstract

Methyl-CpG-binding domain proteins (MBD) mediate functional responses of methylated DNA. MBD2 and MBD3 are components of the MeCP1 protein complex, which contains the Mi-2/NuRD complex and includes 66-and 68-kDa polypeptides. Here we identified two highly related 66-kDa proteins in a yeast two-hybrid screen with MBD2b. Based on the high degree of sequence conservation to the previously identified Xenopus p66 subunit of the Mi-2/NuRD complex, we termed these proteins hp66α and hp66β. hp66α is the human orthologue of Xenopus p66, whereas hp66β, previously identified as a component of the human MeCP1 complex, is a second member of a p66 gene family. Coprecipitation of hp66α and MBD2 demonstrates their in vivo association. Furthermore, confocal microscopy shows a nuclear colocalization of hp66α with hp66β and MBD2 in a speckled pattern. hp66α is a potent transcriptional repressor reducing gene activity about 100-fold and is ubiquitously coexpressed with hp66β in cell lines and in fetal and adult tissues. We demonstrate direct binding of both p66 family members to MBD2 as well as MBD3. Interestingly, hp66α, which binds with a higher affinity than hp66β, interacts via two interaction domains in contrast to a single interaction domain present in hp66β. These results demonstrate that two highly related mammalian p66 proteins display overlapping functions and are involved in methylation dependent transcriptional repression.