The advent of microarray technology has allowed, for the first time, the ability to assess the genome-wide changes in transcription that occur in response to specific stress, drugs or toxicants. The waves of gene changes that occur in response to a specific stimulus are transmitted by the assembly of transcription factors at specific promoters. Thus a key component of the code which couples environmental stimuli to changes in gene expression is the synthesis, modification and aggregation of activating and inactivating transcription factors at the promoters which are induced or suppressed. Studies of this crucial component of information flow in genetic networks have been limited by the difficulty to identify and characterize the proteins that interact with intact regulatory regions of genes whose expression is modulated. While great advances have been made in identifying which transcription factors are capable of activating particular gene constructs, direct knowledge is limited about which factors activate the actual gene in an intact chromosome and the secondary modification of those factors when interacting with the intact gene. In order to facilitate studies into these questions, the investigators propose to develop a new technique that will allow mass spectrometric identification of the proteins associated with specific gene promoters. If successful, this new technique has the potential to vastly improve the researchers' capacity to identify and characterize the protein assembly that interacts with specific promoters following an external stimulus and thereby significantly contribute to unraveling the structure and organization of genetic networks. The investigators propose to develop and validate this approach with a prokaryotic model, and then to apply the technique to the modulation of a well characterized eukaryotic promoter that forms a central part of the environmentally sensitive reproductive system. Specifically, the technique will be applied to the identification and characterization of proteins that interact with the mouse LhB promoter when induced by pulsatile hormone administration. This gene has a well characterized tertiary gene promoter that requires induction and synthesis of other transcription factors for its induction. The researchers will utilize this newly developed technique to identify the transcription factors that bind the intact LhB promoter during pulsatile GnRH induction.
