GFP-Based Assays to Probe Transcriptional Controls(RMI)

描述(由申请人提供)：将开发两(2)个基于绿色荧光蛋白(GFP)的分析来探索转录和转录后调控的机制。第一个测试将利用含有GFP基因的细胞，该基因被表观遗传机制沉默。用已知的组蛋白脱乙酰酶(HDAC)抑制剂(HDI)小分子处理这些细胞，可以使GFP强劲地重新激活。这一发现表明沉默机制可能包括(但不限于)组蛋白去乙酰化。HDI已被公认为抗癌药物，使用这种基于GFP细胞的分析方法对化学文库进行高通量筛选(HTS)应该可以识别新的HDI，以及针对维持沉默所需的其他蛋白质的化合物。初步结果包括几个针对检测的特异性、敏感性和背景的原则性证明实验。拟议的实验将测试该检测方法对多井形式的适应性。将进行已知化合物(HDI)和化学库的试点筛选。GFP沉默的细胞也将受到siRNAs的挑战，siRNAs将下调HDAC的表达，预计这种治疗也将重新激活沉默的GFP基因。还将进行其他实验，以评估全基因组siRNA HTS的可行性，以确定与维持表观遗传沉默有关的其他基因。第二个基于GFP的检测将被用来探索通过组蛋白mRNA的稳定性来协调DNA复制和组蛋白生物合成的转录后控制途径。在本实验中，将产生编码GFP和组蛋白mRNA的3‘-非翻译区的融合转录本。据预测，抑制DNA合成将导致GFP融合转录本的特异性降解。中断这一调控途径的化学物质或siRNA预计将稳定融合转录本，并允许持续的GFP表达。将进行可行性研究，以验证这一分析。由于两种检测方法都是基于GFP的，因此HTS格式的GFP检测的优化条件将适用于两种系统。这两种分析方法都涉及细胞调控的基本方面(表观遗传沉默、细胞周期控制)，这些方面都与癌症的发展有关。这些分析有可能揭示新的先导化合物，以及治疗的新基因靶点。

DESCRIPTION (provided by applicant): Two (2) green fluorescence protein (GFP)-based assays will be developed to probe mechanisms of transcriptional and post-transcriptional control. The first assay will utilize cells that contain a GFP gene that is silenced by epigenetic mechanisms. Treatment of these cells with small molecules that are known histone deacetylase (HDAC) inhibitors (HDIs), results in robust reactivation of GFP. This finding indicates that the silencing mechanisms likely include (but are not limited to) histone deacetylation. HDIs have been recognized as anti-cancer drugs and high throughput screens (HTSs) of chemical libraries using this GFP cell-based assay should identify new HDIs, as well as compounds that target other proteins that are required for maintenance of silencing. Preliminary results include several proof of-principle experiments that address specificity, sensitivity and background of the assay. The proposed experiments will test the adaptability of the assay to a multi-well format. Pilot screens with known compounds (HDIs) and chemical libraries will be carried out. GFP-silent cells will also be challenged with siRNAs that will knockdown the expression of HDACs and this treatment is also predicted to reactivate the silent GFP gene. Additional experiments will also be carried out to assess the feasibility of a genome-wide siRNA HTS to identify other genes involved in the maintenance of epigenetic silencing. A second GFP based assay will be used to probe the pathway of posttranscriptional control that coordinates DNA replication and histone biosynthesis, through histone mRNA stability. In this assay, a fusion transcript will be generated encoding GFP and the 3'-untranslated region of histone mRNA. It is predicted that inhibition of DNA synthesis will result in specific degradation of the GFP fusion transcript. Chemicals or siRNAs that interrupt this regulatory pathway are predicted to stabilize the fusion transcript and allow continued GFP expression. Feasibility studies will be carried out to validate this assay. As both assays are GFP-based, optimized conditions for GFP detection in a HTS format will be applicable to both systems. Both of these assays address fundamental aspects of cell regulation (epigenetic silencing, cell cycle control) that have been implicated in cancer development. The assays have the potential to reveal new lead compounds, as well as new gene targets for therapeutics.