Investigating mechanisms of gene regulation CALS Impact Statement

We are developing information and methodologies that can be broadly used to understand and manipulate gene expression in a broad array of organisms. This information is not only critical to understanding critical stages in cell and organismal regulation, but it is also of fundamental importance in understanding and treating disease, as well as in the control of genes that influence agricultural production and resistance to disease.

We have developed and are applying new strategies for investigating molecular interactions that modulate the level of eukaryotic gene expression in living cells and organisms. One novel--and we hope generally applicable--approach has been the selection of RNAs (RNA aptamers) that bind tightly to specific protein components of the regulated transcription and RNA-processing machinery of eukaryotic cells. These RNA aptamers are in effect drugs that can be selected to particular targets and then engineered and expressed as genetic units in a controlled manner in cells and organisms. The controlled expression of such high-affinity, inhibitory aptamer RNA provides a means of rapidly inactivating targeted domains of proteins, and thereby allowing an assessment of the primary function and mechanism of action of particular proteins in vivo.

We have used both well-established and new technologies to identify protein factors that participate in gene regulation and to define, with high temporal and spatial resolution, in vivo protein-nucleic acid and protein-protein interactions during the process of gene activation. The second stage of our analysis is to identify protein interactions that are critical to the process. We have succeeded in defining many of these protein factors and their mechanism of actions. In the process, we identified a new mode of regulation, called promoter-proximal pausing, that is now seen to play a broad role in gene regulation in animals. We have also succeeded in developing technologies for selecting RNAs that bind tightly to a specific protein in vitro, and apply them in vivo to whole animals. We are currently selecting RNAs designed to interfere with specific steps in gene transcription and its control.

These investigations should provide critical insights into the important mechanisms that dictate how genes are regulated. This information is providing insights into how genes are and can be regulated. Moreover, this research provides the tools to manipulate gene expression in ways that should prove useful in health sciences and agricultural production. Additionally, the RNA aptamer technology, and the aptamer (drugs) that we select and engineer, should prove to be powerful in the modulation or moderation of abnormal regulatory processes seen in disease states, as well as providing new tools in gene regulation that should find broad application in agricultural production.

Overview