Mark D. Biggin
The regulation of morphogenesis and development is probably best understood in Drosophila melanogaster. Genetic experiments have uncovered many genes that lie within a temporally ordered regulatory cascade controlling Drosophila development. Many of these genes have been shown to be transcription factors. We are using molecular and genetic techniques to determine which genes are the direct target of these transcription factors. We are using classical biochemical approaches to uncover new classes of transcription factors that also regulate spatial and temporal patterns of transcription. We are also examining the mechanisms and underlying biochemical principles by which all of these factors act.
One group of developmental control genes we are studying share an homologous DNA binding domain termed the homeodomain. Understanding how these proteins act has proven difficult, partly because it has not been easy to determine how these proteins bind DNA in vivo. We have developed an improver! in vivo UV crosslinking assay and have usec! it to examine binding of two homeodomain proteins, even skipped (eve) and fushitarazu (ftz). Interestingly, these two proteins bind in a manner that differs significantly from that predicted by earlier models. For example, eve and ftz proteins bind in an unprecedentedly uniform manner throughout the length of expected target genes. They also bind at only slightly lower levels to a number of genes we did not initially expect them to regulate. We have now shown that some of this latter class of gene (including the heat shock 70 genes and the Actin SC gene) are spatially regulated in a manner suggesting they may be controlled by eve and ftz. We are now testing new models that may account for how these proteins act.
In other experiments: we are using in vitro transcription experiments to determine how eve protein represses transcription; we are using transgenic promoter analysis and classical genetics to determine the biological function of three biochemically identified transcription factors; and we have purified the RNA polymerase II general transcription factors from Drosophila embryos to study the detailed mechanisms by which transcriptional regulators interact with them.
Upper diagram: the binding sites of four activator proteins (Z. G. NT and A) on a target promoter repressed by eve protein. Lower Diagram: Cooperative associations between eve molecules bound at high and low affinity DNA sites form a stable structure that excludes activator binding and represses transcription.