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