Project 3: MANIPULATION OF GENE EXPRESSION WITH SMALL MOLECULES

该项目的目标是创造能够激活细胞的细胞渗透性合成分子。 特定基因的表达。"合成的转录因子模拟物"将能够定位于一个转录因子。 特异性启动子区域，并将转录机器募集到附近的基因，从而模拟一个启动子区域。 天然反式激活蛋白的基本功能。这些分子将是非常有用的工具， 生物医学研究，并可能被精心制作成一类新的治疗剂。 设想合成活化剂可以通过将DMA结合分子融合在一起来产生， 特别是具有适当的DMA识别特性的发夹聚酰胺， 能够结合RNA聚合酶II全酶，从而将其募集到靶启动子。有 来自我们实验室和其他人的大量证据表明，这是一种有效的方法， 已经报道了能够在核提取物中起作用的活化剂， 活细胞中的基因仍然难以捉摸。我们最近取得了令人兴奋的突破， 在活细胞中作为激活结构域等价物起作用的可渗透类肽。这是第一 观察这种活动。我们计划将这种拟肽和改进的衍生物与发夹聚酰胺连接， 合适的序列识别特性以产生细胞可渗透的合成活化剂。这些 化合物将用于操纵细胞系和人胰岛中的代谢。特别是要 试图激活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。这些研究将在 与Newgard实验室合作。根据Newgard实验室最近的结果，我们 他们还计划使用全基因组染色质免疫沉淀分析来帮助识别Nkx6.1的直接靶点 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方法， 其允许直接筛选合成的组合文库的活化结构域模拟物。 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。

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.