The proposed study is a continuation of current efforts to analyze the regulation of the Drosophila segmentation gene, even-skipped (eve). It encodes a homeobox protein that is transcribed in a series of 7 stripes in the early embryo. These stripes foreshadow the subdivision of the embryo into segments, and it is hoped that a detailed understanding of how eve stripes are formed will provide general insights into the segmentation process. It is known that individual eve stripes are controlled by separate enhancers in the eve promoter. For example, the regulation of stripe #2 depends on a 500 bp enhancer that is located between -1.5 Kb and -1 Kb upstream of the transcription start site. The stripe #2 enhancer is activated by broadly distributed activators, including the bicoid morphogen; the stripe borders are formed by selective repression. Activation and repression is mediated by tightly linked binding sites within the stripe #2 enhancer. The overall goal of the proposed study is to determine how the eve stripe enhancers function as integrating pattern elements that direct sharp on/off stripes of gene expression in response to crude gradients of transcriptional activators and repressors. This information should provide general insights into how eukaryotic enhancers work. For example, the kappa light chain enhancer probably directs B-cell specific expression by discerning relatively small differences in the levels of activators and repressors that are common to many different hematopoietic lineages. The research plan includes 5 specific aims. first, in vitro binding assays will be done to determine whether stripe #2 activators bind DNA cooperatively, and whether repressors compete with activators for common sites. Second, in vivo assays will investigate the detailed regulation of eve stripe #2, with a particular emphasis on the mechanisms underlying repression and multiplicative activation. Third, the stripe #3 enhancer will be characterized to determine whether there are general rules governing stripe formation. Fourth, the organization of the eve promoter will be examined by altering the spacing between individual stripe enhancers. And finally, the origins of segment polarity will be investigated by performing genetic complementation assays with defective eve promoters.
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