Promoter Specificity of Transcription Factors

描述（由申请人提供）：特异性基因表达由与启动子和增强子中存在的元件结合的转录因子控制。在许多情况下，多个转录因子可以识别相同的DNA序列，但每个因子可以激活不同的基因。因此，转录因子的DNA结合特征不足以确定启动子特异性。酵母转录因子Swi 5和Ace 2具有几乎相同的锌指DNA结合结构域，并且这两种因子显示出相似的细胞周期调控模式，在M和G1中存在于细胞核中有限的时间。虽然Swi 5和Ace 2在体外识别相同的DNA序列，但它们是体内激活不同细胞周期调控基因所必需的。Swi 5激活HO的转录，但不激活CTS 1，Ace 2激活CTS 1，但不激活HO。通常，Swi 5激活促进细胞周期进程的基因，而Ace 2激活细胞分离所需的基因。染色质免疫沉淀（ChIP）实验表明，有两种不同的机制，以防止这些因素激活基因。在一种情况下，因子与启动子结合，但不激活，而在另一种情况下，因子在体内不与其在体外识别的启动子位点结合。Swi 5与CTS 1结合但不激活转录。我们的数据表明，Fkh 1和Fkh 2蛋白结合在CTS 1启动子阻止Swi 5激活转录。因此，Fkh蛋白充当选择性阻遏物，阻断Swi 5而非Ace 2促进CTS 1表达。提出实验来了解这种选择性抑制机制是如何工作的。数据表明，Ace 2招募激酶的启动子，这种激酶克服了这种抑制。Ace 2在体外可以与HO启动子结合，但ChIP显示其在体内不与HO启动子结合。实验提出，以确定顺式作用位点和反式作用的监管机构，防止Ace 2在HO结合，并表征这种调节。Ace 2在体外与SIC 1启动子结合良好，但在体内结合较差。这些数据表明，Ace 2的不同部分形成结合抑制剂的结构单元。结构的研究提出了Ace 2蛋白质的这一区域的性质的特征，并提出了遗传筛选，以确定和表征抑制剂。激酶突变阻断Ace 2在体内结合DNA，尽管Ace 2仍然存在于细胞核中。这种DNA结合缺陷可以通过Ace 2的N-末端区域的突变来抑制。这些数据表明，N-末端区域作为一个自动抑制剂，阻断DNA结合的C-末端DNA结合域，与磷酸化的N-末端区域的Ace 2缓解这种抑制。生化和遗传实验提出了这种自抑制的特点。 项目叙述：基因表达的调节对于真核生物中不同细胞类型的正确分化和维持是必不可少的，并且基因表达的异常可导致包括癌症在内的特定疾病。酿酒酵母（Saccharomycescerevisiae）是研究基因调控的模式真核生物，由于转录调控机制在酵母和脊椎动物之间是保守的，因此在酵母中的研究具有普遍性。在这里，我们研究了两个转录因子具有相同的DNA结合结构域，识别相同的DNA序列在体内，以确定什么机制限制他们的能力，在体内激活特定基因的表达。

DESCRIPTION (provided by applicant): Specific gene expression is controlled by transcription factors binding to elements present in promoters and enhancers. In many instances multiple transcription factors can recognize the same DNA sequence, but each factor can activate different genes. Thus the DNA-binding characteristics of a transcription factor are not sufficient to determine promoter specificity. The yeast transcription factors Swi5 and Ace2 have nearly identical zinc finger DNA-binding domains, and both factors show similar pattern of cell cycle regulation, being present in the nucleus for a limited period in M and G1. Although Swi5 and Ace2 recognize the same DNA sequences in vitro, they are required for the activation of different cell cycle regulated genes in vivo. Swi5 activates transcription of HO, but not CTS1, and Ace2 activates CTS1, but not HO. Generally, Swi5 activates genes that promote cell cycle progression while Ace2 activates genes required for cell separation. Chromatin immunoprecipitation (ChIP) experiments show that there are two distinct mechanisms operative to prevent these factors from activating genes. In one case a factor binds to a promoter, but does not activate, and in the other case a factor does not bind in vivo to a promoter site that it recognizes in vitro. Swi5 binds to CTS1 but does not activate transcription. Our data suggests that the Fkh1 and Fkh2 proteins bound at the CTS1 promoter prevent Swi5 from activating transcription. Thus, the Fkh proteins act as a selective repressor, blocking Swi5 but not Ace2 from promoting CTS1 expression. Experiments are proposed to understand how this selective repression machinery works. Data suggest that Ace2 recruits a kinase to the promoter, and that this kinase overcomes this repression. Ace2 can bind to the HO promoter in vitro, but ChIPs show that it does not bind to the HO promoter in vivo. Experiments are proposed to identify both cis acting sites and trans acting regulators that prevent Ace2 from binding at HO, and to characterize this regulation. Ace2 binds well to the SIC1 promoter in vitro, but poorly in vivo. The data suggest that distinct portions of Ace2 form a structural unit that binds an inhibitor. Structural studies are proposed to characterize the nature of this region of the Ace2 protein, and a genetic screen is proposed to identify and characterize the inhibitor. A kinase mutation blocks Ace2 from binding DNA in vivo, although Ace2 is still in the present in the nucleus. This DNA-binding defect can be suppressed by mutations in the N-terminal region of Ace2. The data suggest that the N-terminal region acts as an autoinhibitor, blocking DNA-binding by the C-terminal DNA-binding domain, with phosphorylation in the N-terminal region of Ace2 relieving this inhibition. Biochemical and genetic experiments are proposed to characterize this autoinhibition. Project Narrative: Regulation of gene expression is essential for the proper differentiation and maintenance of distinct cell types in eukaryotes, and abnormalities in gene expression can cause specific diseases including cancer. The yeast Saccharomyces cerevisiae with its powerful genetic tools has proven to be the model eukaryotic organism for studies of gene regulation, and since the transcription regulatory machinery is conserved between yeast and vertebrates the insights gained from studies in yeast are generally universal. Here we study two transcription factors with identical DNA-binding domains that recognize the same DNA sequence in vivo, to determine what mechanisms restrict their ability to activate expression of specific genes in vivo.