Adolf-Butenandt-Institut, University of Munich, Schillerstr. 44, 80336 Munich, Germany.
Mol Cell Biol. 2010 Aug;30(16):4060-76. doi: 10.1128/MCB.01399-09. Epub 2010 Jun 21.
The yeast PHO5 promoter is a classical model for studying the role of chromatin in gene regulation. To enable biochemical dissection of the mechanism leading to PHO5 activation, we reconstituted the process in vitro. Positioned nucleosomes corresponding to the repressed PHO5 promoter state were assembled using a yeast extract-based in vitro system. Addition of the transactivator Pho4 yielded an extensive DNase I-hypersensitive site resembling induced PHO5 promoter chromatin. Importantly, this remodeling was energy dependent. In contrast, little or no chromatin remodeling was detected at the PHO8 or PHO84 promoter in this in vitro system. Only the PHO5 promoter harbors a high-affinity intranucleosomal Pho4 binding site (UASp) where Pho4 binding can compete with nucleosome formation, prompting us to test the importance of such competition for chromatin remodeling by analysis of UASp mutants in vivo. Indeed, the intranucleosomal location of the UASp element was critical, but not essential, for complete remodeling at the PHO5 promoter in vivo. Further, binding of just the Gal4 DNA binding domain to an intranucleosomal site could increase PHO5 promoter opening. These data establish an auxiliary role for DNA binding competition between Pho4 and histones in PHO5 promoter chromatin remodeling in vivo.
酵母 PHO5 启动子是研究染色质在基因调控中作用的经典模型。为了能够对导致 PHO5 激活的机制进行生化剖析，我们在体外重新构建了该过程。使用基于酵母提取物的体外系统，组装了与受抑制的 PHO5 启动子状态相对应的定位核小体。添加转录激活因子 Pho4 后，会产生类似于诱导的 PHO5 启动子染色质的广泛的 DNase I 超敏位点。重要的是，这种重塑是能量依赖性的。相比之下，在该体外系统中，在 PHO8 或 PHO84 启动子上几乎检测不到或检测到很少的染色质重塑。只有 PHO5 启动子含有高亲和力的核小体内部 Pho4 结合位点（UASp），Pho4 结合可以与核小体形成竞争，促使我们通过体内分析 UASp 突变体来测试这种竞争对染色质重塑的重要性。事实上，UASp 元件的核小体内部位置对于 PHO5 启动子在体内的完全重塑是至关重要的，但不是必需的。此外，Gal4 DNA 结合域与核小体内部位点的结合可以增加 PHO5 启动子的开放性。这些数据确立了 Pho4 和组蛋白之间的 DNA 结合竞争在体内 PHO5 启动子染色质重塑中的辅助作用。
