Shei G J, Broach J R
Department of Molecular Biology, Princeton University, New Jersey 08544, USA.
Mol Cell Biol. 1995 Jul;15(7):3496-506. doi: 10.1128/MCB.15.7.3496.
The mating-type loci located at the ends of chromosome III in Saccharomyces cerevisiae are transcriptionally repressed by a region-specific but sequence-nonspecific silencing apparatus, mediated by cis-acting silencer sequences. Previous deletion analyses have defined the locations and organizations of the silencers in their normal context and have shown that they are composed of various combinations of replication origins and binding sites for specific DNA-binding proteins. We have evaluated what organization of silencer sequences is sufficient for establishing silencing at a novel location, by inserting individual silencers next to the MAT locus and then assessing expression of MAT. The results of this analysis indicate that efficient silencing can be achieved by inserting either a single copy of the E silencer from HMR or multiple, tandem copies of either the E or I silencer from HML. These results indicate that while all silencers are functionally equivalent, they have different efficiencies; HMR E is more active than HML E, which itself is more active than HML I. Both HMR E and HML E exhibit orientation-dependent silencing, and the particular organization of binding elements within the silencer domain is critical for function. In some situations, silencing of MAT is conditional: complete silencing is obtained when cells are grown on glucose, and complete derepression occurs when cells are shifted to a nonfermentable carbon source, a process mediated in part by the RAS/cyclic AMP signaling pathway. Finally, the E silencer from HMR is able to reestablish repression immediately upon a shift back to glucose, while the silencers from HML exhibit a long lag in reestablishing repression, thus indicating distinctions between the two silencers in their reestablishment capacities. These results demonstrate that silencers can serve as nonspecific gene inactivation centers and that the attendant silencing can be rendered responsive to potential developmental cues.
酿酒酵母中位于III号染色体末端的交配型基因座受到一种区域特异性但序列非特异性的沉默机制的转录抑制，该机制由顺式作用沉默子序列介导。先前的缺失分析已经确定了沉默子在其正常环境中的位置和组织，并表明它们由复制起点和特定DNA结合蛋白的结合位点的各种组合组成。我们通过在MAT基因座旁边插入单个沉默子，然后评估MAT的表达，来评估沉默子序列的何种组织足以在新位置建立沉默。该分析结果表明，通过插入来自HMR的单个E沉默子拷贝或来自HML的E或I沉默子的多个串联拷贝，可以实现有效的沉默。这些结果表明，虽然所有沉默子在功能上是等效的，但它们具有不同的效率；HMR E比HML E更活跃，而HML E本身比HML I更活跃。HMR E和HML E都表现出方向依赖性沉默，并且沉默子结构域内结合元件的特定组织对功能至关重要。在某些情况下，MAT的沉默是有条件的：当细胞在葡萄糖上生长时获得完全沉默，而当细胞转移到不可发酵碳源时发生完全去抑制，这一过程部分由RAS/环磷酸腺苷信号通路介导。最后，来自HMR的E沉默子在转回葡萄糖后能够立即重新建立抑制，而来自HML的沉默子在重新建立抑制方面表现出很长的延迟，因此表明这两个沉默子在重新建立能力上存在差异。这些结果表明，沉默子可以作为非特异性基因失活中心，并且伴随的沉默可以对潜在的发育线索作出反应。
