MAINTAINANCE OF HOMEOTIC GENE TRANSCRIPTION

在果蝇中，已经发现了发育控制基因的时间有序的调节级联，它启动和维持了基因表达的高度精确的模式。这涉及到特定细胞中基因的激活和其他细胞中的抑制。这项建议的长期目标是以果蝇同源基因超双胸(UBX)为模型，确定在发育过程中维持转录抑制的机制。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育过程中被一种名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都由一组普遍表达的蛋白质维持，即多梳群(PcG)。这个系统实际上是一个“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复制期间也是如此。我们已经发现了第一个在这个系统中作用的单因子结合位点的例子，并表明作用在这个位点上的蛋白质是ZEST。这一突破应该会极大地帮助研究PcG抑制是如何发挥作用的。我们目前的实验证明，ZEST是维持对Ubx启动子结构的抑制所必需的，该结构紧密地复制了Ubx的表达模式。这些数据表明，ZEST在控制内源Ubx基因方面的功能与其他蛋白质相同。为了进一步证明这一点，我们将确定由PcG基因突变导致的内源Ubx基因的去抑制是否会因ZEST突变而增强。我们还将确定ZEST是否只与特定的PcG基因子集相互作用。将确定抑制Ubx转基因启动子构建所需的ZEST蛋白的功能结构域。将从胚胎核提取物中鉴定和纯化与ZEST最小区域结合的介导抑制的蛋白质。我们将测试PcG抑制蛋白或一般转录因子是否直接与最小ZEST抑制域相互作用。已有研究表明，PcG蛋白可能被其他蛋白靶向于启动子。我们将测试Zust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，这些蛋白被证明参与了哺乳动物同源异型基因的抑制。我们的实验将大大加深我们对似乎高度保守的发育调控过程的理解。

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.