Every year in the United States over 550,000 deaths occur in more than five million Americans affected with atherosclerosis. The cause of atherosclerosis is not clear, however several risk factors are strongly related to its development. Of all known risk factors promoting atherosclerosis and heart disease, a high serum LDL-cholesterol level is the most important, and its significance in the pathogenesis of atherosclerotic coronary heart disease is well established. LDL receptors bind to the most atherogenic plasma protein LDL , and interestingly, the LDL receptor gene is negatively regulated at the transcriptional level by both the intracellular cholesterol obtained from plasma LDL and the cholesterol synthesized de novo. The goal of our research is to unravel the molecular mechanism of transcriptional regulation of the LDL receptor gene by sterols in human cells. The proposed research addresses a specific question concerning the mechanism of sterol regulation of the LDL receptor gene, by blending the biochemical and molecular biology techniques with yeast genetics. The proposed approach has become available only recently. All attempts to identify a protein involved in the sterol regulation of this receptor gene using the conventional in vitro methods have failed. Earlier, we have provided evidence for the in vivo role of the sterol regulatory element in controlling the LDL receptor gene expression by sterols. Now, protein-DNA interactions that occur in vivo in the promoter region of the LDL receptor gene in response to sterols will be investigated. Initial results using the in vivo footprinting technique are very encouraging, and the significance of these footprints in the sterol regulation of the LDL receptor gene will be established in future experiments. Furthermore, our investigation will provide an opportunity to isolate the cDNA of physiologically relevant crucial factor(s) involved in the sterol regulation of the LDL receptor gene by developing novel in vivo genetic screening systems in yeast. We will use human LDL receptor gene promoter placed upstream of two different reporter genes to screen for SREBP cDNA clones using yeast. The first system, Sp1-interactive selection system will screen for cDNA clones that causes enhancement of the LacZ gene expression in an SRE-1 dependent manner, and the second system, growth selection system, utilizes HIS3 reporter gene. Both systems provides a tight """"""""ON-OFF"""""""" genetic selection and have proved useful in cloning the cDNAs of difficult transcription factors. Finally, experiments are also designed to understand the molecular mechanism of cytokines induction of the LDL receptor gene transcription, and its repression by sterols in human cells. These have been included as a result of new In vivo footprint observed in the human LDL receptor gene promoter. Interestingly, in vivo footprinting studies have identified a new region In the LDL receptor gene promoter which footprints only in the absence of sterols, and contain consensus sequences for two transcription factors binding sites (Ets and CREBP), whose activities are modulated by growth factors, cAMP and intracellular calcium levels. We are confident that this pioneering work will enable us to refine the techniques involved, and apply them successfully to other less well characterized but important sterol-responsive genes that have complex promoters.
