Promoter Specificity of Transcription Factors

特定的基因表达是由转录因子结合的元件存在于启动子和增强子控制。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期的有限时间内存在于细胞核中。虽然Swi5和Ace2在体外识别相同的DNA序列，但它们是激活体内两种不同的g1特异性基因所必需的。Swi5激活HO的转录，但不能激活CTS1， Ace2激活CTS1，但不能激活HO。Swi5和Ace2转录因子在细胞周期的M/ g1部分起作用。S1核酸酶保护和微阵列检测的结合将用于表征所有由这两个因子激活的基因，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激活促进细胞周期进展的基因，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可以与CTS1启动子结合，但它确实激活转录。我们已经确定了CTS1启动子内的顺式作用序列和防止Swi5激活的反式作用因子。我们提出了实验来研究启动子结合因子如何阻断启动子结合的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，但一些遗传实验表明，相对于Swi5， Ace2在细胞核中的积累可能在后期略有延迟。最近的研究表明，Ace2具有一个新的核输出序列。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，这些基因可以抑制Ace2的核输出，从而使其激活转录。有人建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，但染色质免疫沉淀显示它在体内不与HO启动子结合。如果在有丝分裂过程中，Swi5比Ace2先进入细胞核，那么在Swi5结合后，启动子可能获得对Ace2结合的阻断。我们提出表征启动子位点，调控蛋白，和细胞周期机制产生这种转录因子结合阻滞。

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.