Promoter Specificity of Transcription Factors

描述(申请人提供)：特定的基因表达由转录因子控制，转录因子与启动子和增强子中存在的元件结合。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结合特性不足以确定启动子的特异性。酵母转录因子Swi5和ACE2具有几乎相同的锌指DNA结合域，并且这两个因子表现出相似的细胞周期调节模式，在M和G1中仅在细胞核中存在一段时间。虽然Swi5和ACE2在体外识别相同的DNA序列，但它们在体内激活不同的细胞周期调控基因是必需的。Swi5激活HO的转录，但不激活CTS1；ACE2激活CTS1，但不激活HO。一般来说，Swi5激活促进细胞周期进程的基因，而ACE2激活细胞分离所需的基因。染色质免疫沉淀(CHIP)实验表明，有两种不同的机制可以阻止这些因子激活基因。在一种情况下，因子与启动子结合，但不激活，在另一种情况下，因子在体内不结合到它在体外识别的启动子位置。Swi5与CTS1结合，但不激活转录。我们的数据表明，结合在CTS1启动子上的Fkh1和Fkh2蛋白阻止了Swi5激活转录。因此，Fkh蛋白作为一个选择性抑制物，阻止Swi5而不是ACE2促进CTS1的表达。为了了解这种选择性压抑机制是如何工作的，我们提出了一些实验。数据表明，ACE2向启动子招募了一个激酶，而这个激酶克服了这种抑制。ACE2在体外可以与HO启动子结合，但在体内芯片显示它不与HO启动子结合。有人建议进行实验，以确定阻止ACE2与HO结合的顺式作用位点和反式作用调节器，并对这一调控进行表征。ACE2在体外与SIC1启动子结合良好，但在体内结合较差。数据表明，ACE2的不同部分形成了一个结合抑制剂的结构单元。建议进行结构研究来表征ACE2蛋白这一区域的性质，并建议通过遗传筛选来鉴定和表征该抑制物。虽然ACE2仍然存在于细胞核中，但一种激酶突变会阻止ACE2在体内与DNA结合。这种DNA结合缺陷可以通过ACE2的N-末端区域的突变来抑制。这些数据表明，N-末端区域作为一种自身抑制物，阻断了C-末端DNA结合域与DNA的结合，而ACE2的N-末端区域的磷酸化解除了这种抑制。生物化学和遗传学实验被用来描述这种自我抑制。 项目简介：基因表达调控对于真核生物中不同细胞类型的正确分化和维持至关重要，基因表达异常可导致包括癌症在内的特定疾病。酿酒酵母凭借其强大的遗传工具已被证明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，因此从酵母研究中获得的见解通常是普遍存在的。在这里，我们研究两个具有相同DNA结合域的转录因子，它们在体内识别相同的DNA序列，以确定是什么机制限制了它们在体内激活特定基因表达的能力。

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.