MAINTAINANCE OF HOMEOTIC GENE TRANSCRIPTION

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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.