SUMO modification enhances p66-mediated transcriptional repression of the Mi-2/NuRD complex

Human p66 alpha and p66 beta are two potent transcriptional repressors that interact with the methyl-CpG-binding domain proteins MBD2 and MBD3. An analysis of the molecular mechanisms mediating repression resulted in the identification of two major repression domains in p66 alpha and one in p66 beta. Both p66 alpha and p66 beta are SUMO-modified in vivo: p66a at two sites (Lys-30 and Lys-487) and p66 beta at one site (Lys-33). Expression of SUMO1 enhanced the transcriptional repression activity of Gal-p66 alpha and Gal-p66 beta. Mutation of the SUMO modification sites or using a SUMO1 mutant or a dominant negative Ubc9 ligase resulted in a significant decrease of the transcriptional repression of p66a and p66 beta. The Mi-2/NuRD components MBD3, RbAp46, RbAp48, and HDAC1 were found to bind to both p66 alpha and p66 beta in vivo. Most of the interactions were not affected by the SUMO site mutations in p66 alpha or p66 beta, with two exceptions. HDAC1 binding to p66 alpha was lost in the case of a p66 alpha K30R mutant, and RbAp46 binding was reduced in the case of a p66 beta K33R mutant. These results suggest that interactions within the Mi-2/NuRD complex as well as optimal repression are mediated by SUMOylation.