Promoter Specificity of Transcription Factors

描述（由申请人提供）：特定的基因表达是由转录因子结合到启动子和增强子中的元件来控制的。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，并且这两个因子具有相似的细胞周期调节模式，在M期和G1期在细胞核中存在有限的时间。虽然Swi5和Ace2在体外识别相同的DNA序列，但在体内，它们是激活不同细胞周期调节基因所必需的。Swi5激活HO的转录，但不能激活CTS1， Ace2激活CTS1，但不能激活HO。通常，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，一个因子与启动子结合，但不激活，在另一种情况下，一个因子在体内不与它在体外识别的启动子位点结合。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蛋白作为一种选择性抑制因子，阻断Swi5而非Ace2促进CTS1表达。提出了实验来了解这种选择性抑制机制是如何工作的。数据表明，Ace2向启动子招募一个激酶，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ChIPs显示它在体内不与HO启动子结合。我们建议通过实验来确定阻止Ace2在HO上结合的顺式作用位点和反式作用调控位点，并对这种调控进行表征。Ace2在体外与SIC1启动子结合良好，但在体内结合较差。这些数据表明，Ace2的不同部分形成一个结合抑制剂的结构单元。提出了结构研究来表征Ace2蛋白这一区域的性质，并提出了遗传筛选来鉴定和表征该抑制剂。尽管Ace2仍存在于细胞核中，但在体内，一个激酶突变阻止了Ace2与DNA的结合。这种dna结合缺陷可以通过Ace2 n端区域的突变来抑制。数据表明，n端区域作为一种自身抑制剂，通过c端dna结合域阻断dna结合，而在Ace2的n端区域磷酸化可以缓解这种抑制作用。生物化学和基因实验被提出来表征这种自抑制。

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.