Ducker C E, Simpson R T
Department of Biochemistry and Molecular Biology, 308 Althouse, Pennsylvania State University, University Park, PA 16802, USA.
EMBO J. 2000 Feb 1;19(3):400-9. doi: 10.1093/emboj/19.3.400.
In yeast alpha cells the a cell-specific genes STE6 and BAR1 are packaged as gene-sized chromatin domains of positioned nucleosomes. Organized chromatin depends on Tup1p, a corepressor that interacts with the N-terminal regions of H3 and H4. If Tup1p functions to organize or stabilize a chromatin domain, the protein might be expected to be present at a level stoichiometric with nucleosomes. Chromatin immunoprecipitation assays using Tup1p antibodies showed Tup1p to be associated with the entire genomic STE6 coding region. To determine stoichiometry of Tup1p associated with the gene, a yeast plasmid containing varying lengths of the STE6 gene including flanking control regions and an Escherichia coli lac operator sequence was constructed. After assembly into chromatin in vivo in Saccharomyces cerevisiae, minichromosomes were isolated using an immobilized lac repressor. In these experiments, Tup1p was found to be specifically associated with repressed STE6 chromatin in vivo at a ratio of about two molecules of the corepressor per nucleosome. These observations strongly suggest a structural role for Tup1p in repression and constrain models for organized chromatin in repressive domains.
在酵母α细胞中，a细胞特异性基因STE6和BAR1被包装成具有定位核小体的基因大小的染色质结构域。有组织的染色质依赖于Tup1p，它是一种与H3和H4的N端区域相互作用的共抑制因子。如果Tup1p起到组织或稳定染色质结构域的作用，那么预计该蛋白的存在水平与核小体呈化学计量关系。使用Tup1p抗体进行的染色质免疫沉淀分析表明，Tup1p与整个基因组STE6编码区域相关联。为了确定与该基因相关的Tup1p的化学计量，构建了一个酵母质粒，其中包含不同长度的STE6基因，包括侧翼控制区域和一个大肠杆菌乳糖操纵序列。在酿酒酵母体内组装成染色质后，使用固定化的乳糖阻遏物分离微型染色体。在这些实验中，发现Tup1p在体内与被抑制的STE6染色质特异性相关，其比例约为每个核小体有两个共抑制因子分子。这些观察结果强烈表明Tup1p在抑制中具有结构作用，并限制了抑制结构域中有组织染色质的模型。
