It is widely assumed that the regulation of gene expression by sequence specific DNA-binding proteins involves interactions between these proteins and cognate cis-regulatory sequences of their downstream target genes. However, DNA binding-independent regulatory activities have now been described for a number of different transcription factors. This project uses a combination of molecular and genetic approaches to investigate a novel, DNA-binding independent pathway of repression by Runt, the founding member of the Runt domain family of transcriptional regulators. Quantitation of the in vivo potency of a DNA-binding defective form of Runt reveals differential requirements for DNA-binding in the regulation of different downstream target genes. DNA-binding is not required for repression of the odd-numbered stripes of the segment polarity gene engrailed, but does contribute to Runt's role as a regulator of sloppy-paired another downstream target gene in the pathway of segmentation. DNA elements that are responsible for generating the odd-numbered engrailed stripes will be identified by examining the in vivo expression of reporter genes containing different segments of engrailed cis-regulatory DNA. Reporter gene constructs that express the odd-numbered engrailed stripes will be examined for their ability to respond to Runt-dependent repression. Functional dissection of minimal elements that mediate the response to Runt will provide a platform for further studies at the molecular level.
Regions of the Runt protein that are specifically required for repression of engrailed will be identified and Runt deletion derivatives will be examined for their regulatory activities in transgenic Drosophila embryos. This approach is based on previous observations on the modular organization of the conserved regions of Runt. A comparison of the relative potency of different deletion derivatives on engrailed and other target genes will reveal if specific regulatory properties can be attributed to the different conserved regions of the Runt protein. Finally, genetic screens will be carried out to identify other factors that participate in the DNA binding-independent repression of engrailed by Runt. Preliminary genetic screens have identified a maternally-expressed DNA-binding protein encoded by the tramtrack locus that cooperates with runt to repress engrailed.. The extension of the genetic screens for runt-interacting factors will provide a rigorous framework for future studies on the molecular mechanism of DNA binding-independent regulation by the Runt protein. These studies will provide new information on the context-dependent activities of Runt domain proteins and may provide fundamental insights into the regulation of gene transcription in eukaryotic systems.
