Dr. Mackey plans to pursue a career as a physician-scientist in molecular endocrinology. His major area of interest is understanding the mechanism by which hormones regulate gene expression. His training goal over the next five years is to expand his knowledge base and technical expertise to become an independent investigator. He will work with Dr. David Talmadge in the Institute of Human Nutrition at Columbia University. Transcriptional repression by steroid hormones has been shown to involve multiple mechanisms. Among these are interactions of steroid receptors with cell-specific transcription factors and binding of steroid receptors to DNA sequences that differ from the upregulatory consensus sequences. The objective of this research proposal is to characterize the DNA-protein interactions which underlie transcriptional repression of the parathyroid hormone (PTH) gene in response to 1,25 (OH)2D3 as a model system of steroid mediated transcriptional repression.
Three specific aims have been outlined. The first will address the DNA sequences required for down regulation in parathyroid cell using native and mutant hPTH-luciferase fusion genes in transient infections in dispersed parathyroid cells.
The second aim will address whether the 1,25 (OH)2D3 receptor can bind to the hPTH DNA sequence alone, which DNA bases are required for binding, and whether the 1,25(OH)2D3 receptor binds as monomer or heterodimer.
The third aim will focus on identifying the accessory protein(s) involved in down regulation. This will involve the purification of the protein(s) and the cloning of the cDNA which encodes the protein(s). Toward this end, two strategies will be used: affinity chromatography and screening of a parathyroid cDNA expression library. Analyzing the DNA-protein interactions of the 1,23(OH)2D3 mediated downregulation of PTH gene transcription and gene regulation by steroid hormones will be of interest in general, but may also provide the basis for new approaches to the treatment and prevention of secondary hyperparathyroidism in chronic renal disease.
