The transcriptional corepressor CtBP participates in pathways important for development, cell cycle regulation, and transformation. One such pathway involves the oncoprotein Evi-1, which has been linked to human acute myelogenous leukemia. Recently, the principal investigator demonstrated that CtBP is regulated by the nicotinamide adenine dinucleotides, NAD and NADH, in a manner that regulates its ability to interact with a variety of transcriptional repressors. This work suggested a completely novel mechanism for transcriptional regulation, in which the formation of a repressor:corepressor complex is regulated by changes in the nuclear levels of free NAD/NADH, with CtBP serving as a redox sensor for transcription. The main thrust of the current project is to extend this model by measuring the levels of free nicotinamide adenine dinucleotides in the nucleus, establishing the impact of this regulatory mechanism on the properties of the human transforming protein Evi-1, and elucidating the mechanism of CtBP-mediated transcriptional repression. Three specific aims are proposed: 1) Determine whether the free nuclear concentrations of nicotinamide adenine dinucleotides are appropriate for their proposed role in CtBP regulation. The affinities of NAD and NADH for CtBP will be measured in vitro and compared to their free nuclear concentrations, determined in vivo using two-photon fluorescence microscopy, 2) Establish whether the ability of CtBP to sense the nuclear redox state regulates the function of human oncoprotein Evi-1 and test whether blockers of the NAD/NADH binding site in CtBP inhibit Evi-1 function, 3) Identify components of the human cellular CtBP complex and determine the contributions of these components to transcriptional repression. Regulation of Evi-1 function through the proposed NAD/NADH-induced allosteric effect on CtBP is likely to affect the course of cell transformation. Blockers of the NAD/NADH-induced change may therefore be therapeutically useful in certain types of leukemia. Through a mentored career development plan, the principal investigator will gain proficiency in the field of gene regulation as it relates to molecular oncology. The mentor's laboratory and institution are rich in intellectual and physical resources that provide an excellent environment for facilitating the principal investigator's transition to independent scientist.
NIH ExPorter
NIH ExPorter