MOLECULAR ANALYSIS OF SEGMENTATION IN DROSOPHILA

University Of California Berkeley, Berkeley CA

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Abstract

DESCRIPTION (adapted from application): This is an application to continue his investigations of complex loci of Drosophila to understand the regulation of promoter-enhancer interactions. He made seminal contributions in this area by elucidating how localized patterns of the gene even-skipped (eve) are established along the anteroposterior axis of the precellular Drosophila embryo. The regulation of the segmentation stripes is due to the combined action of broadly distributed activators and narrowly-expressed, short-range repressors. Short range repressors act at distance of less than 100 bp and repress the action of specific activators while long range repression regulates specific enhancers that lie >1000 bp away and are thought to act through repression of general machinery. The present application extends beyond this earlier work focusing on two general goals: 1) The mechanism of action of repressors, both short-range and long-range, and 2) The mechanism by which long-range enhancer-promoter interactions are modulated, in both a positive sense via facilitator proteins and in a negative direction via the use of insulator DNA or competition. There are 3 specific aims: (i) Identify repressors as either short-range or long-range and investigate the mechanism of repression by identifying co-repressors. (ii) Identify proteins required for the enhancer-blocking activity of the specific attenuator Fab-7 and the gypsy insulator. (iii) Investigate the role of promoter competition in the regulation of specific enhancer-promoter interactions in the ANT-C and BX-C gene complexes. The general approach in all of these aims is to use transgenic reporters whose expression is modulated by precisely engineered promoter and enhancers. For example, the initial experiments use a fly strain with a beta-galactosidase reporter transgene. The expression is controlled by enhancers from the eve loci, known as stripe 2 and stripe 3 enhancers since they are responsible for eve expression in these localized region of the precellular embryo. Repressor binding sites positioned close to stripe 2 enhancer and at a distance from stripe 3 can reconstruct the two modes of repression. Repression activity is monitored by the disappearance of blue stripes in the embryo. Other transgenes use Drosophila eye color gene white as the reporter.