Molecular characterization of Sin3 PAH-domain interactor specificity and identification of PAH partners.
Sin3 is the central component of a multisubunit co-repressor complex. A number of DNA-binding proteins are targeted by the Sin3 complex to chromatin through association with its paired amphipathic helix (PAH) domains. Here, we performed a yeast two-hybrid screening using a peptide aptamer library and identified peptides that interact with either PAH1 or PAH2. Analysis of PAH2 interacting peptides uncovered motifs similar to previously characterized PAH2 interacting proteins, Mad, Ume6 and kruppel-like members, while analysis of PAH1 interacting peptides revealed an LXXLL motif. In addition, a tandem affinity purification (TAP)-tagging approach of Sin3b resulted in the isolation of known and novel interactors amongst which neural retina leucine (NRL) zipper. Strikingly, one of the identified PAH2 interacting peptide showed strong resemblance to the NRL region amino acids 125-150. Direct association between PAH2 and NRL was shown and NRL(125-150) mediated transcriptional repression in reporter assays. Finally, we reveal that PAH1 and PAH2 amino acids 7, 14 and 39 shown previously to be important for Mad-PAH2 interaction, also play an important role in the specificity of interaction between PAH1, PAH2 and identified aptamers. Our results provide novel insights into the molecular determinant of the specificity of PAH1 and PAH2 for their interacting partners.
Le Guezennec
Xavier
X
Department of Molecular Biology, NCMLS, Radboud University Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
Vermeulen
Michiel
M
Stunnenberg
Hendrik G
HG
D020816:Amino Acid Motifs; D000818:Animals; D052158:Aptamers, Peptide / Q000032:analysis; D050976:Basic-Leucine Zipper Transcription Factors / Q000378:metabolism; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism*; D006655:Histone Deacetylases / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D051379:Mice; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D056264:Sin3 Histone Deacetylase and Corepressor Complex; D014157:Transcription Factors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D052158:Aptamers, Peptide; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D005136:Eye Proteins; D005656:Fungal Proteins; C490037:NRL protein, human; D012097:Repressor Proteins; C115812:Sin3b protein, mouse; D014157:Transcription Factors; D006655:Histone Deacetylases; D056264:Sin3 Histone Deacetylase and Corepressor Complex