The organized chromatin domain of the repressed yeast a cell-specific geneSTE6 contains two molecules of the corepressor Tup1p per nucleosome

In yeast alpha cells the a cell-specific genes STE6 and BAR1 are packaged a s gene-sized chromatin domains of positioned nucleosomes. Organized chromat in depends on Tup1p, a corepressor that interacts with the N-terminal regio ns of H3 and H4. If Tup1p functions to organize or stabilize a chromatin do main, the protein might be expected to be present at a level stoichiometric with nucleosomes. Chromatin immunoprecipitation assays using Tup1p antibod ies showed Tup1p to be associated with the entire genomic STE6 coding regio n. To determine stoichiometry of Tup1p associated with the gene, a yeast pl asmid containing varying lengths of the STE6 gene including flanking contro l regions and an Escherichia coli lac operator sequence was constructed, Af ter assembly into chromatin in vivo in Saccharomyces cerevisiae, minichromo somes were isolated using an immobilized lac repressor. In these experiment s, Tup1p was found to be specifically associated with repressed STE6 chroma tin in vivo at a ratio of about two molecules of the corepressor per nucleo some. These observations strongly suggest a structural role for Tup1p in re pression and constrain models for organized chromatin in repressive domains .