MAINTAINANCE OF HOMEOTIC GENE TRANSCRIPTION

在果蝇中，发育控制基因的时序调控级联已被发现，其作用是启动和维持高度精确的基因表达模式。这涉及到特定细胞中基因的激活和其他细胞中的抑制。该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，使用果蝇同源基因超abithorax （Ubx）作为模型。Ubx在胚胎的中心部分表达。在此之前的区域，Ubx的表达在早期胚胎发生时被一种称为驼背的瞬时表达的转录因子抑制。此后，这种最初的抑制模式在整个发育过程中由一个无处不在的表达蛋白群Polycomb group （PcG）维持。这个系统实际上是一种“分子记忆”，因为它必须以某种方式持续标记被抑制的基因，即使在DNA复制过程中也是如此。我们已经发现了在这个系统中起作用的单因子结合位点的第一个例子，并表明作用于这个位点的蛋白质是zeste。这一突破将极大地帮助研究PcG抑制如何起作用。我们目前的实验表明，zeste是维持Ubx启动子结构的抑制所必需的，而Ubx启动子结构与Ubx的表达模式密切相关。这些数据表明，zeste控制内源性Ubx基因的功能是由其他蛋白质共享的。为了进一步证明这一点，我们将确定由PcG基因突变引起的内源性Ubx基因的抑制是否会被zeste突变增强。我们还将确定zeste基因是否仅与PcG基因的特定子集相互作用。zeste蛋白抑制Ubx转基因启动子结构所需的功能域将被确定。结合到介导抑制的zeste最小区域的蛋白质将被鉴定并从胚胎核提取物中纯化。我们将测试PcG抑制蛋白或一般转录因子是否直接与最小的zeste抑制域相互作用。有研究表明，PcG蛋白可能被其他蛋白靶向于启动子。我们将测试zeste是不是其中一种蛋白质。许多PcG蛋白在哺乳动物中是保守的，这些蛋白已被证明参与抑制哺乳动物同源基因。我们的实验将大大加深我们对高度保守的发育调节过程的理解。

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.