Promoter Specificity of Transcription Factors

转录因子的启动子特异性

• 批准号: 6470277
• 负责人: David J Stillman
• 金额: $ 26.25万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6470277
• 关键词: Saccharomyces cerevisiae; binding sites; cell cycle; cell cycle proteins; functional /structural genomics; fungal genetics; fungal proteins; gene mutation; genetic promoter element; genetic regulation; molecular genetics; protein binding; transcription factor

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 the same pattern of cell cycle regulation, being present in the nucleus for a limited period in G1. Although Swi5 and Ace2 recognize the same DNA sequences in vitro, they are required for the activation of two different G1-specific genes in vivo. Swi5 activates transcription of HO, but not CTS1, and Ace2 activates CTS1, but not HO. The Swi5 and Ace2 transcription factors function at the M/G1part of the cell cycle. A combination of S1 nuclease protection and microarray assays will be used to characterize all of the genes activated by these two factors, providing tools for mechanistic studies of gene regulation. Our analysis thus far suggests that 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 can bind to the CTS1 promoter, but it does activate transcription. We have identified cis-acting sequences within the CTS1 promoter and trans-acting factors that prevent activation by Swi5. Experiments are proposed to investigate how promoter bound factors can block promoter bound Swi5 from activating transcription, while allowing Ace2 to function in transcriptional activation. Although Swi5 and Ace2 enter the nucleus at approximately the same time, several genetic experiments suggest that Ace2 accumulation in the nucleus may be slightly delayed at anaphase relative to Swi5. Recent work shows that Ace2 has a novel nuclear export sequence. Based on cis- and trans- mutations that affect Ace2 activity, we believe we have identified set of cell cycle genes that function to inhibit nuclear export of Ace2 and thus allow it to activate transcription. Experiments are proposed to dissect this nuclear export. Ace2 can bind to the HO promoter in vitro, but chromatin immunoprecipitation shows that it does not bind to the HO promoter in vivo. If Swi5 enters the nucleus slightly before Ace2 during mitosis, then it is possible that after Swi5 binds the promoter acquires a block to Ace2 binding. We propose to characterize the promoter sites, regulatory proteins, and cell cycle machinery that produce this block to transcription factor binding.

特异的基因表达受转录因子控制，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同的细胞周期调节模式，在G1期仅存在于细胞核中。虽然Swi5和ACE2在体外识别相同的DNA序列，但在体内激活两个不同的G1特异性基因是必需的。Swi5激活HO的转录，但不激活CTS1；ACE2激活CTS1，但不激活HO。Swi5和ACE2转录因子在细胞周期的M/G1部分起作用。结合S1核酸酶保护和微阵列分析将被用来表征这两个因素激活的所有基因，为基因调控的机制研究提供工具。到目前为止，我们的分析表明，Swi5激活了促进细胞周期进程的基因，而ACE2激活了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可以与CTS1启动子结合，但它确实激活了转录。我们已经确定了CTS1启动子中的顺式作用序列和阻止Swi5激活的反式作用因子。实验旨在研究启动子结合因子如何阻止启动子结合的Swi5激活转录，同时允许ACE2在转录激活中发挥作用。虽然Swi5和ACE2几乎同时进入细胞核，但一些遗传学实验表明，相对于Swi5，ACE2在细胞核中的积累在后期可能略有延迟。最近的工作表明，ACE2具有一种新的核输出序列。基于影响ACE2活性的顺式和反式突变，我们相信我们已经确定了一组细胞周期基因，它们的功能是抑制ACE2的核输出，从而允许它激活转录。有人提议进行实验来剖析这种核出口。ACE2在体外可以与HO启动子结合，但在体内染色质免疫沉淀显示它不与HO启动子结合。如果Swi5在有丝分裂过程中稍早于ACE2进入细胞核，则有可能在Swi5结合后，启动子获得了与ACE2结合的阻断。我们建议对产生这种转录因子结合阻断的启动子位点、调节蛋白和细胞周期机制进行表征。