Project 3: MANIPULATION OF GENE EXPRESSION WITH SMALL MOLECULES

该项目的目标是创造细胞可渗透的合成分子，能够激活 特定基因的表达。“合成转录因子模拟物”将能够定位到 特定的启动子区域，并将转录机制招募到附近的基因，从而模仿 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，并有可能被精心设计成一类新的治疗剂。 可以设想通过将DMA结合分子融合在一起来生成合成激活剂， 具体地说是一种具有适当的DMA识别特征的发夹聚酰胺， 能够结合RNA聚合酶II的全酶，从而将其招募到目标启动子上。的确有 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，但尽管是人工合成的 据报道，能够在核提取液中发挥作用的激活剂，是发挥作用的分子的目标 在活细胞中的作用仍然难以捉摸。我们最近取得了一项令人兴奋的突破，发现了一种细胞 在活细胞中作为激活域的等价物的渗透性类肽。这是第一次 对这种活动的观察。我们计划将这种类肽和改进的衍生品与发夹聚酰胺联系起来 适当的序列识别特性，以产生细胞渗透性合成激活剂。这些 化合物将被用于操纵细胞系和人类胰岛的新陈代谢。特别是，我们将 尝试激活Nkx6.1基因和胞质NADPH依赖的异柠檬酸脱氢酶基因 并确定这种刺激对细胞新陈代谢的影响。这些研究将在 与纽加德实验室的合作。根据纽加德实验室的最新结果，我们 我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靶标 然后还将设计合成分子来启动这些基因。 在这个项目的整个过程中，我们将始终如一地努力开发出更强大的 合成激活剂。为此，我们将利用我们开发的一种新的基于细胞的屏幕 这使得可以直接筛选合成组合文库中的激活结构域模拟物。 此外，我们还将建立细胞分析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.