It is widely assumed that the regulation of gene transcription during embryonic development involves controlling the assembly of the core transcriptional machinery at the promoter and the initiation of transcription. However, recent results indicate that the initial expression of several genes that are involved in cell fate specification in the Drosophila embryo is regulated downstream of transcription initiation and involves developmentally regulated transcriptional elongation. This project uses the well-defined developmental context of the Drosophila blastoderm stage embryo to investigate developmentally regulated transcriptional elongation. In particular, this project takes advantage of several unique attributes offered by the sloppy-paired-1 (slp1) gene for investigating the mechanism of regulation by Runt, the founding member of the Runx family of developmental regulators, and transcription factors encoded by other pair-rule segmentation genes. One key advantage is the simple combinatorial rules that are responsible for modulating Runt's activity as both an activator and a repressor of slp1 expression. Indeed, using straightforward genetic manipulations it is possible to achieve uniform, physiologically relevant Runt-dependent activation or repression of slp1 in all somatic cells of a late blastoderm stage embryo. These embryos provided a platform for biochemical studies on the in vivo protein-DNA interactions associated with slp1 repression that led to the finding that slp1 expression is regulated at the level of transcriptional elongation. This project uses a combination of biochemical and genetic approaches to clarify the specific step in the transcription cycle that is blocked in slp1-repressed embryos and to identify the factor(s) that mediate regulation of elongation in response to Runt and other transcription factors. The project also takes advantage of recent results on the structure of the slp1 cis-regulatory region to investigate the functional contribution of different cis-regulatory DNA elements to both the establishment as well as the developmentally regulated elongation of a paused Pol II complex on the slp1 promoter. These studies will provide new insights on transcriptional regulatory mechanisms in the Drosophila embryo that are certain to have significance extending beyond this model system.
The regulation of gene expression is crucial for development and aberrations in gene expression are associated with developmental defects and disease. Recent findings that developmentally regulated gene expression involves control of transcriptional elongation are surprising and open up new areas of research that are of potentially widespread relevance and importance. The project will have a broad positive impact in promoting research training and career development for students at several different educational levels, including graduate, undergraduate and high school students with active participation by students from under-represented minorities.
