Miller Joanna A, Widom Jonathan
Department of Biochemistry, Molecular Biology and Cellular Biology, Northwestern University, Evanston, Illinois 60208-3500, USA.
Mol Cell Biol. 2003 Mar;23(5):1623-32. doi: 10.1128/MCB.23.5.1623-1632.2003.
The mechanism by which gene regulatory proteins gain access to their DNA target sites is not known. In vitro, binding is inherently cooperative between arbitrary DNA binding proteins whose target sites are located within the same nucleosome. We refer to such competition-based cooperativity as collaborative competition. Here we show that arbitrarily chosen foreign DNA binding proteins, LexA and Tet repressor, cooperate with an adjacently binding endogenous activator protein, Gcn4, to coactivate expression of chromosomal reporter genes in Saccharomyces cerevisiae. Coactivation requires that the cooperating target sites be within a nucleosome-length distance; it leads to increased occupancy by Gcn4 at its binding site; and it requires both Gcn5 and Swi/Snf which, at an endogenous Gcn4-dependent promoter, act subsequent to Gcn4 binding. These results imply that collaborative competition contributes to gene regulation in vivo. They further imply that, even in the presence of the cell's full wild-type complement of chromatin remodeling factors, competition of regulatory proteins with histone octamer for access to regulatory target sites remains a quantitative determinant of gene expression levels. We speculate that initial target site recognition and binding may occur via spontaneous nucleosomal site exposure, with remodeling factor action required downstream to lock in higher levels of regulatory protein occupancy.
基因调控蛋白获取其DNA靶位点的机制尚不清楚。在体外,对于靶位点位于同一个核小体内的任意DNA结合蛋白而言,结合本质上具有协同性。我们将这种基于竞争的协同性称为协作竞争。在此我们表明,任意选择的外源DNA结合蛋白LexA和四环素阻遏物,可与相邻结合的内源性激活蛋白Gcn4协同作用,共同激活酿酒酵母中染色体报告基因的表达。协同激活要求协同的靶位点处于一个核小体长度的距离之内;它会导致Gcn4在其结合位点的占有率增加;并且它需要Gcn5和Swi/Snf,在内源性Gcn4依赖性启动子处,它们在Gcn4结合之后发挥作用。这些结果表明,协作竞争在体内对基因调控有贡献。它们进一步表明,即使存在细胞中完整的野生型染色质重塑因子互补物,调控蛋白与组蛋白八聚体竞争获取调控靶位点仍然是基因表达水平的一个定量决定因素。我们推测,最初的靶位点识别和结合可能通过自发的核小体位点暴露发生,下游需要重塑因子的作用来锁定更高水平的调控蛋白占有率。