Embryos regulate their growth and development in many ways, but control of gene transcription is particularly important for directing cells along particular developmental pathways. In Drosophila, a cascade of nuclear regulatory events establishes very early differences in cell fates by producing intricate patterns of gene expression. Many of these pattern-forming genes encode DNA binding proteins that regulate each others expression, and subsequently instruct the rest of the genome in a manner appropriate to each position in the organism. These regulatory proteins are conserved across the evolutionary distance separating flies and humans. This applies to both their primary structure, implying similarity in mechanism, and often their developmental function. That is, the regulatory scheme in which they function solves a common problem of developing multi-cellular organisms. Our studies drew a detailed picture of the basic cis-regulatory landscape of Drosophila even skipped (eve) gene during embryonic development. The eve locus is a textbook example of gene regulation by modular enhancers, each one driving expression in a distinct spatial and temporal subset of the overall pattern of expression throughout embryogenesis. Recently, our studies have evolved to explore chromatin-based gene regulation in the larger context of chromosomal architecture.
Even-skipped (Eve) is a transcription factor that regulates developmental processes in a highly conserved fashion. Eve uses both Groucho-dependent and -independent mechanisms to repress transcription. By our laboratory and others, all enhancers required for the complex eve expression pattern were identified. Therefore, we have the ability to completely rescue null mutants with a transgene, allowing us to functionally replace the endogenous gene. This facilitates our studies of its regulation in vivo. For example, Eve is expressed in ganglion mother cells (GMCs) in the developing Drosophila nervous system that develop into RP2 and a/pCC motoneurons. We showed that without Eve expression in these GMCs and neurons, their axonal projections are altered. Their axons normally project to dorsal muscles, but without Eve, they rarely exit from the central nervous system. By modifying the Eve coding region in the context of the eve rescue transgene, we showed that Eve function is to repress target genes in this context, and that this function is conserved.
Currently, we are analyzing how long-range repression and activation occur over an entire genetic locus, through the regulation of chromatin structure. We found two kinds of distinct regulatory elements, Polycomb response elements (PREs) and insulators, which exert their influence through the regulation of repressive chromatin and chromosomal architecture, respectively. We showed that eve is regulated by two PREs, located near the promoter and at the 3' end of the locus, which together facilitate packaging of the eve locus into repressive chromatin in the OFF state. These elements work epigenetically to keep eve from disrupting development of the CNS and other tissues through mis-expression. We further showed that these PREs have the ability to facilitate the maintenance of not only repression of transcription, but also activation of transcription, in cells where continued eve expression is required. In addition to the eve PREs, we identified two other novel elements that flank the eve locus (homie and nhomie), separating it from neighboring genes both functionally and structurally, in terms of chromosomal architecture. We showed that these insulators interact specifically and directionally with each other to form a “stem-loop”-type chromosomal domain, wherein eve enhancers and PRE functions are confined, and that enhancers and promoters located far apart on a chromosome can be connected functionally by interacting insulators, independent of the topology of the intervening DNA.
Both of these kinds of elements function in a variety of genes, and in mammals as well as in Drosophila, to regulate developmental processes like stem cell maintenance and differentiation. Understanding the underlying mechanisms will provide novel ways to attack problems such as cancer, which is caused in part by mis-regulation of gene expression and chromatin structure.