Multiple domains of the MBD2 protein are required for transcriptional repression in B cells. (P4382)

Abstract We demonstrated previously that the mb-1 (Igα; Cd79a) promoter is demethylated progressively during its activation in early B cell development. Demethylation of promoter CpGs is initiated by binding of the transcription factor, Early B cell Factor 1 (EBF1). EBF1 increases local accessibility and demethylation of mb-1 promoter DNA, which allows for binding by Pax5 prior to transcription. This process is inhibited by Mi-2/NuRD chromatin remodeling complexes. This was evidenced by knockdown of the ATPase subunit of Mi-2/NuRD, Mi-2β (CHD4). Recent work in the laboratory has focused on other components of the Mi-2/NuRD complex to assess their importance for association of Mi-2/NuRD with methylated DNA. A subset of Mi-2/NuRD complexes contains Methyl Binding Domain protein 2 (MBD2). MBD2 binds methylated, but not hydroxymethylated or unmethylated cytosines. We have demonstrated that knockdown of MBD2 using shRNA potentiated mb-1 transcriptional activation only in the presence of EBF1 and Pax5. MBD2 is important for recruiting Mi-2/NuRD complexes to gene targets and for maintenance of heterochromatin. MBD2 comprises arginine-glycine (RG)-rich, Methyl CpG binding (MBD) and C-terminal (CTD) domains. Through mutational analysis, we have demonstrated that multiple domains of MBD2 are important for transcriptional attenuation of the mb-1 gene in B cells. We are now investigating how MBD2 binding is inhibited to de-repress the B cell program in early B cell progenitors.