Abstract
Histone acetylation and histone deacetylation are processes mediated by complexes containing histone acetyl transferases or histone deacetylases (HDAC) and play an important role in transcriptional regulation. Here, new molecular insights are presented for two class I HDACs containing complexes: Sin3 and Mi-2/NuRD. Chapter 1 introduces class I HDAC containing complexes. Chapter 2 presents a perspective on experimental approaches used in this thesis. In Chapter 3 we revealed a critical role for PAH2 residue Phe7 as well as a stabilizing role for PAH2 residues Val14 and Gln39 in the interaction between the PAH2 domain of the mSin3b co-repressor and the tumor suppressor Mad. In chapter 4 we identify peptides interacting with the PAH1 and PAH2 domain of mSin3b. Substitution in PAH2 of Phe7 and Val14 with their respective PAH1 residues affected PAH2 identified peptides. Two PAH1 identified peptides shared an LVXLL motif and were affected by substitution of Leu14 and Lys39 in PAH1 with their respective PAH2 residues. Our results suggest that amino acids 7, 14 and 39 define the specificity of the PAH1 and the PAH2 domain of Sin3 for recruitment of DNA binding repressors. In chapter 5 we revealed that MBD2 and MBD3 assemble into mutually distinct functional Mi-2/NuRD complexes. We observed co-purification of a novel putative tumor suppressor, DOC-1. We showed that the arginine methyl transferase PRMT5 associates and methylates the N-term region of MBD2. PRMT5 is recruited in vivo to methylated CpG islands, together with MBD2 in a DNA methylation dependent manner. Our data suggests that Mi-2/NuRD comprises a family of protein complexes with distinct biochemical and functional properties. This thesis should provide the necessary ground to understand how the PAH1 and PAH2 domain of Sin3 establish an association with their interacting partners and will help hopefully to elucidate the mechanism by which Mi-2/NuRD repress transcription.
