Fascher K D, Schmitz J, Hörz W
Institut für Physiologische Chemie, Universität München, Germany.
J Mol Biol. 1993 Jun 5;231(3):658-67. doi: 10.1006/jmbi.1993.1317.
The PHO5 promoter from Saccharomyces cerevisiae can exist in two chromatin configurations depending on its state of activity. In the repressed promoter a short hypersensitive site containing a binding site for the transcription factor PHO4 is flanked by specifically positioned nucleosomes. After induction two nucleosomes upstream and two downstream of the hypersensitive site are disrupted, and the entire promoter becomes accessible. We have investigated mechanisms responsible for setting up the structure of the repressed state and for the transition. Episomal centromeric plasmids bearing the PHO5 promoter show the same chromatin structure as the endogenous chromosomal copy arguing that the chromosomal context is not essential and that the nucleosomal organization is not set up from a distance. Deleting most of the hypersensitive region including the PHO4 binding site also leaves the positioning of the adjacent nucleosomes in the repressed promoter unchanged indicating that histone-DNA interactions play an important role in setting up nucleosome positions. However, when half of the DNA of a nucleosome is deleted a new nucleosome forms at the same location with respect to the neighboring nucleosome indicating that boundary effects also contribute to nucleosome positioning in the native promoter. Disruption of the nucleosomes under activating conditions is shown to require interaction of PHO4 with its binding site located within the hypersensitive region. This disruption takes place also in two independent constructs in which the TATA box had been deleted and as a result the gene was not transcribed. This result shows for the first time that the generation of active chromatin at a regulated promoter is not the result of gene expression but occurs prior to transcription.
来自酿酒酵母的PHO5启动子可根据其活性状态以两种染色质构型存在。在受抑制的启动子中，一个包含转录因子PHO4结合位点的短超敏位点两侧是特定定位的核小体。诱导后，超敏位点上游和下游的两个核小体被破坏，整个启动子变得可及。我们研究了负责建立抑制状态结构和转变的机制。携带PHO5启动子的附加型着丝粒质粒显示出与内源性染色体拷贝相同的染色质结构，这表明染色体背景并非必需，并且核小体组织不是从远处建立的。删除包括PHO4结合位点在内的大部分超敏区域，也不会改变受抑制启动子中相邻核小体的定位，这表明组蛋白-DNA相互作用在建立核小体位置中起重要作用。然而，当一个核小体的一半DNA被删除时，相对于相邻核小体，会在同一位置形成一个新的核小体，这表明边界效应也有助于天然启动子中的核小体定位。在激活条件下核小体的破坏表明需要PHO4与其位于超敏区域内的结合位点相互作用。这种破坏也发生在两个独立的构建体中，其中TATA框已被删除，结果基因未被转录。这一结果首次表明，在受调控的启动子处活性染色质的产生不是基因表达的结果，而是发生在转录之前。
