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MANIPULATION OF GENE EXPRESSION WITH SMALL MOLECULES

用小分子操纵基因表达

基本信息

批准号：
8147684
负责人：
Thomas J. Kodadek
金额：
$ 28.34万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-01 至 2012-05-09
项目状态：
已结题

项目摘要

The goal of this project is to create cell permeable synthetic molecules capable of activating the expression of specific genes. The "synthetic transcription factor mimics" would be capable of localizing to a specific promoter region and recruiting the transcriptional machinery to a nearby gene, thus mimicking a basic function of native transactivator proteins. These molecules would be tools of outstanding utility in biomedical 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 molecule capable of binding the RNA polymerase II holoenzyme, thus recruiting it to the target promoter. There is considerable evidence from our laboratory and others that this is a valid approach, but while synthetic activators capable of functioning in nuclear extracts have been reported, the goal of molecules that function in living cells remains elusive. We have recently made an exciting breakthrough with the discovery of a cell permeable peptoid that functions as an activation domain equivalent in living cells. This is the first observation of such activity. We plan to link this peptoid and improved derivatives to hairpin polyamides with appropriate sequence recognition properties to create cell permeable synthetic activators. These compounds will be employed to manipulate metabolism in cell lines and human islets. In particular, we will attempt to activate the Nkx6.1 gene and the cytosolic, NADPH-dependent isocitrate dehydrogenase gene in islets and determine the effect of this stimulation of the metabolism of the cell. These studies will be in collaboration with the Newgard laboratory. Following the lead of recent results in the Newgard laboratory, we also plan to use genome-wide chromatin immunoprecipitation assays to help to identify direct Nkx6.1 target genes 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 potent synthetic activators. To do so, we will take advantage of a novel cell-based screen that we have developed which 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.

该项目的目标是创造能够激活特定基因表达的细胞渗透性合成分子。“合成转录因子模拟物”能够定位于特定的启动子区域并将转录机制募集到附近的基因，从而模拟天然反式激活蛋白的基本功能。这些分子将是生物医学研究中具有突出实用性的工具，并可能被精心制作成一类新的治疗剂。可以设想，合成激活剂可以通过将DMA结合分子（特别是具有适当DMA识别特征的发夹聚酰胺）与能够结合RNA聚合酶II全酶的分子融合在一起，从而将其募集到靶启动子来产生。我们的实验室和其他实验室有相当多的证据表明这是一种有效的方法，但是尽管已经报道了能够在核提取物中发挥作用的合成活化剂，但发挥作用的分子的目标是活细胞中的基因仍然难以捉摸。我们最近取得了一个令人兴奋的突破，发现了一种细胞可渗透的类肽，在活细胞中起着激活结构域等同物的作用。这是对这种活动的第一次观察。我们计划将这种类肽和改进的衍生物与具有适当序列识别特性的发夹聚酰胺连接起来，以创造细胞可渗透的合成激活剂。这些化合物将用于操纵细胞系和人类胰岛中的代谢。特别是要试图激活胰岛中的Nkx6.1基因和胞质NADPH依赖性异柠檬酸脱氢酶基因，并确定这种刺激对细胞代谢的影响。这些研究将与Newgard实验室合作。根据Newgard实验室最近的结果，我们还计划使用全基因组染色质免疫沉淀分析来帮助识别直接的Nkx6.1靶点。然后设计合成分子来开启这些基因。在整个项目过程中，将不断努力开发更有效的合成活化剂。为此，我们将利用我们开发的一种新的基于细胞的筛选，该筛选允许直接筛选合成的组合文库的激活结构域模拟物。此外，我们还将建立细胞测定来优化聚酰胺与所需启动子的结合。