ACTION OF A RAP1 CARBOXY-TERMINAL SILENCING DOMAIN REVEALS AN UNDERLYING COMPETITION BETWEEN HMR AND TELOMERES IN YEAST
ACTION OF A RAP1 CARBOXY-TERMINAL SILENCING DOMAIN REVEALS AN UNDERLYING COMPETITION BETWEEN HMR AND TELOMERES IN YEAST
复制标题
DOI：
10.1101/gad.9.3.370
复制
发表时间：
1995-02-01
影响因子：
10.5
通讯作者：
SHORE, D
中科院分区：
文献类型：
--
作者：
BUCK, SW;SHORE, D
RAP1是酵母中的一种序列特异性DNA结合蛋白，可以抑制或激活转录。以前的研究已经证明RAP1在HM交配型基因座和端粒沉默中起直接作用。在这里，我们证明了当RAP1的一个小的羧基末端结构域与GAL4 DNA结合域(G(BD))融合并靶向含有GAL4 DNA结合位点的突变的HMR消音器时，足以建立抑制。G(BD)/RAP1杂交体的沉默，就像HMR的正常沉默一样，需要反式作用因子Sir2、SIR3和SIR4。然而，G(BD)/RAP1介导的沉默不依赖于SIR1，SIR1的产物通常是建立HMR抑制所必需的。靶向沉默也对沉默缺陷RAP1(S)突变显示出不寻常的反应。在G(BD)/RAP1杂交种中引入RAP1(S)错义突变可以改善靶向性沉默，但野生型G(BD)/RAP1杂交种不能在内源RAP1基因座携带RAP1(S)突变的菌株中建立抑制。此外，我们还发现在RAP1(S)菌株中端粒沉默增加。我们认为，RAP1(S)突变通过改变HMR和端粒沉默之间的平衡，有利于端粒沉默，创造了HMR特异性沉默缺陷。这种平衡受端粒长度和RAP1羧基末端与Rif1和SIR4蛋白之间的相互作用的调节。为了支持这一模型，我们证明了异常长的端粒可以对抗HMR的沉默，并且在双杂交实验中，RAP1(S)杂交蛋白与SIR4显示了更强的相互作用。
RAP1 is a sequence-specific DNA-binding protein in yeast that can either repress or activate transcription. Previous studies have demonstrated a direct role for RAP1 in silencing at HM mating type loci and telomeres. Here, we show that a small carboxy-terminal domain of RAP1 is sufficient to establish repression when fused to the GAL4 DNA-binding domain (G(BD)) and targeted to mutated HMR silencers containing GAL4 DNA-binding sites. Silencing by G(BD)/RAP1 hybrids, like normal silencing at HMR, requires the trans-acting factors SIR2, SIR3, and SIR4. However, G(BD)/RAP1-mediated silencing is independent of SIR1, whose product is normally required for the establishment of repression at HMR. Targeted silencing also displays an unusual response to silencing-defective rap1(s) mutations. The incorporation of a rap1(s) missense mutation into G(BD)/RAP1 hybrids can improve targeted silencing, yet wild-type G(BD)/RAP1 hybrids fail to establish repression in strains in which the endogenous RAP1 locus carries a rap1(s) mutation. In addition, we find that telomeric silencing is increased in rap1(s) strains. We propose that the rap1(s) mutation creates an HMR-specific silencing defect by shifting a balance between silencing at HMR and telomeres in favor of telomeric silencing. This balance is regulated by telomere length and by interactions between the RAP1 carboxyl terminus and both RIF1 and SIR4 proteins. In support of this model, we show that abnormally long telomeres antagonize silencing at HMR and a rap1(s) hybrid protein displays a strengthened interaction with SIR4 in a two-hybrid assay.