MAINTAINANCE OF HOMEOTIC GENE TRANSCRIPTION

同源基因转录的维持

• 批准号: 6138601
• 负责人: Mark D BIGGIN
• 金额: $ 9.07万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6138601
• 关键词: DNA binding protein; Drosophilidae; affinity chromatography; crosslink; developmental genetics; embryogenesis; gene induction /repression; gene interaction; gene mutation; genetic mapping; genetic transcription; homeobox genes; transcription factor; ultraviolet radiation

In the fruit fly, a temporally ordered regulatory cascade of developmental control genes has been discovered that acts to initiate and maintain highly precise patterns of gene expression. This involves the activation of genes in specific cells and repression in other cells. The long term goal of this proposal is to determine the mechanisms that maintain repression of transcription during development, using the Drosophila homeotic gene Ultrabithorax (Ubx) as a model. Ubx is expressed in the central portion of the embryo. In the region anterior of this, Ubx expression is repressed during early embryogenesis by a transiently expressed transcription factor called hunchback. Thereafter, this initial pattern of repression is maintained throughout development by a ubiquitously expressed group of proteins, the Polycomb group (PcG). This system is in effect a "molecular memory", since it must in some way continuously mark repressed genes, even during DNA replication. We have discovered the first example of a single factor binding site that acts in this system and have shown that the protein acting on this site is zeste. This breakthrough should greatly aid studies of how PcG repression works. Our current experiments establish that zeste is required for maintaining repression of Ubx promoter constructs which closely reproduce the pattern of Ubx expression. The data suggest that zeste's function in controlling the endogenous Ubx gene is shared by other proteins. To provide further evidence for this, we will determine if the derepression of the endogenous Ubx gene that results from mutation of PcG genes is enhanced by zeste mutations. We will also determine if zeste genetically interacts with only a specific subset of PcG genes. The functional domains of zeste protein required for repression of Ubx transgenic promoter constructs will be determined. Proteins that bind to minimal regions of zeste that mediate repression will be identified and purified from embryo nuclear extracts. We will test whether PcG repressor proteins or general transcription factors directly interact with minimal zeste repression domains. It has been suggested that PcG proteins may be targeted to promoters by other proteins. We will test if zeste is one of these proteins. Many of the PcG proteins are conserved in mammals, and these proteins have been shown to be involved in repression of the mammalian homeotic genes. Our experiments will significantly further our understanding of what appears to be a highly conserved developmental regulatory process.

在果蝇中，已经发现了发育控制基因的时序调控级联，其作用是启动和维持基因表达的高度精确模式。这涉及特定细胞中基因的激活和其他细胞中的抑制。这项建议的长期目标是确定在发育过程中维持转录抑制的机制，使用果蝇同源异型基因Ultrabithorax（Ubx）作为模型。Ubx在胚胎的中央部分表达。在此之前的区域，Ubx的表达在早期胚胎发生期间被一种称为hunchback的瞬时表达转录因子抑制。此后，这种初始的抑制模式在整个发育过程中由一组普遍表达的蛋白质（Polycomb组（PcG））维持。这个系统实际上是一个“分子记忆”，因为它必须以某种方式连续标记被抑制的基因，即使在DNA复制期间。我们已经发现了在该系统中起作用的单个因子结合位点的第一个例子，并表明作用于该位点的蛋白质是zeste。这一突破将极大地帮助研究PcG抑制是如何起作用的。我们目前的实验证实，zeste是维持Ubx启动子结构的抑制所必需的，该启动子结构紧密地再现了Ubx表达的模式。这些数据表明，zeste在控制内源性Ubx基因方面的功能是由其他蛋白质共享的。为了提供进一步的证据，我们将确定是否内源性Ubx基因的去阻遏，PcG基因突变的结果是增强zeste突变。我们还将确定zeste是否仅与PcG基因的特定子集发生遗传相互作用。将确定阻遏Ubx转基因启动子构建体所需的zeste蛋白的功能结构域。将从胚核提取物中鉴定并纯化与介导阻遏的zeste最小区域结合的蛋白质。我们将测试是否PcG阻遏蛋白或一般转录因子直接与最小zeste阻遏结构域相互作用。已经表明PcG蛋白可以被其他蛋白质靶向启动子。我们将测试zeste是否是这些蛋白质之一。许多PcG蛋白在哺乳动物中是保守的，并且这些蛋白已被证明参与哺乳动物同源异型基因的抑制。我们的实验将大大促进我们对高度保守的发育调控过程的理解。