Methylation of CpG dinucleotides in eukaryotes is an epigenetic mark that is implicated in transcriptional silencing. Methyl-CpG binding protein MBD2 serves to recruit the Mi-2/NuRD repressive complex to a methylated promoter. Human p66alpha and p66beta, which are components of Mi-2/NuRD complex, are two potent transcriptional repressors that interact with MBD2, but no details concerning the mechanism in hp66 proteins-mediated transcriptional repression have been described.
The current work showed that transcriptional repression mediated by hp66alpha and hpbeta is partially dependent on histone deacetylation. Two major repression domains in hpalpha, and one in hp66beta were characterized. In addition, the amino acid Lys-149 of hp66beta was identified to be essential for the interaction with MBD2 and the nuclear localization of hp66alpha. Emerging evidence indicated that SUMO (small ubiquitin-like modifier) modification negatively regulates the transcriptional activity of transcription factors. The study gave evidence that both hp66beta; and hpbeta proteins can be SUMOylated, and furthermore that SUMO modification enhances hp66-mediated transcriptional repression. Two major SUMO modification sites at Lys-30 and Lys-487 of hp66alpha, and one major SUMO modification site at Lys-33 of hpbeta were identified. Mutational analysis of the SUMO modification sites in hp66alpha or hp66beta revealed that there is no change in localization in comparison to wild type hp66. But interestingly, the Mi-2/NuRD complex component HDAC1 (histone deacetylase 1) is recruited to the SUMO modification site Lys-30 of hp66alpha which shows TSA (Trichostatin A) sensitivity, whereas mutation of the SUMO modification site Lys-33, which shows TSA insensitivity, abolishes the interaction between hp66beta and RbAp46 (Rb associated protein 46) in vivo. Taken together, these results suggest that both, interactions within the Mi-2/NuRD complex as well as optimal repression are mediated by SUMOylation.
Moreover, to gain further insights into protein complexes containing hp66 proteins, stable cell lines expressing individually both hp66 proteins were established. After a two-step chromatographic purification and a subsequent FLAG affinity purification protein complexes were isolated. Western blotting analysis revealed that several subunits of the Mi-2/NuRD complex as well as MBD2, and PRMT5 were found to be associated with FLAG-hp66 proteins.
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