Project 3: MANIPULATION OF GENE EXPRESSION WITH SMALL MOLECULES

Duke University, Durham NC

Investigators

Linked publications & trials

Abstract

The goal of this project is to create cell permeable synthetic molecules capable of activating the[unreadable] expression of specific genes. The "synthetic transcription factor mimics" would be capable of localizing to a[unreadable] specific promoter region and recruiting the transcriptional machinery to a nearby gene, thus mimicking a[unreadable] basic function of native transactivator proteins. These molecules would be tools of outstanding utility in[unreadable] biomedical research and could potentially be elaborated into a new class of therapeutic agents.[unreadable] It is envisioned that a synthetic activator could be created by fusing together a DMA-binding molecule,[unreadable] specifically a hairpin polyamide with the appropriate DMA recognition characteristics, with a molecule[unreadable] capable of binding the RNA polymerase II holoenzyme, thus recruiting it to the target promoter. There is[unreadable] considerable evidence from our laboratory and others that this is a valid approach, but while synthetic[unreadable] activators capable of functioning in nuclear extracts have been reported, the goal of molecules that function[unreadable] in living cells remains elusive. We have recently made an exciting breakthrough with the discovery of a cell[unreadable] permeable peptoid that functions as an activation domain equivalent in living cells. This is the first[unreadable] observation of such activity. We plan to link this peptoid and improved derivatives to hairpin polyamides with[unreadable] appropriate sequence recognition properties to create cell permeable synthetic activators. These[unreadable] compounds will be employed to manipulate metabolism in cell lines and human islets. In particular, we will[unreadable] attempt to activate the Nkx6.1 gene and the cytosolic, NADPH-dependent isocitrate dehydrogenase gene in[unreadable] islets and determine the effect of this stimulation of the metabolism of the cell. These studies will be in[unreadable] collaboration with the Newgard laboratory. Following the lead of recent results in the Newgard laboratory, we[unreadable] also plan to use genome-wide chromatin immunoprecipitation assays to help to identify direct Nkx6.1 target[unreadable] genes and will also then design synthetic molecules to turn on these genes as well.[unreadable] Throughout the course of this project, consistent efforts will be made to develop ever more potent[unreadable] synthetic activators. To do so, we will take advantage of a novel cell-based screen that we have developed[unreadable] which allows synthetic combinatorial libraries to be screened for activation domain mimics directly.[unreadable] Furthermore, we will also set up cellular assays to optimize polyamides for binding to the desired promoters.