GFP-Based Assays to Probe Transcriptional Controls(RMI)

描述（由申请人提供）：将开发两(2)种基于绿色荧光蛋白（GFP）的检测方法来探测转录和转录后控制机制。第一个试验将利用含有GFP基因的细胞，该基因被表观遗传机制沉默。用已知的小分子组蛋白去乙酰化酶（HDAC）抑制剂（hdi）处理这些细胞，结果是GFP的强劲再激活。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已经被认为是抗癌药物，使用这种基于GFP细胞的检测方法的化学文库的高通量筛选（hts）应该能识别出新的hdi，以及靶向维持沉默所需的其他蛋白质的化合物。初步结果包括几个原理证明实验，解决特异性，敏感性和背景分析。提出的实验将测试该方法对多井格式的适应性。将对已知化合物（hdi）和化学文库进行试点筛选。GFP沉默的细胞也会受到sirna的挑战，sirna会降低hdac的表达，这种治疗也被预测会重新激活沉默的GFP基因。还将进行其他实验，以评估全基因组siRNA HTS的可行性，以确定参与维持表观遗传沉默的其他基因。第二个基于GFP的分析将用于通过组蛋白mRNA的稳定性来探索协调DNA复制和组蛋白生物合成的转录后控制途径。在这个实验中，将产生编码GFP和组蛋白mRNA的3'-未翻译区域的融合转录物。据预测，抑制DNA合成将导致GFP融合转录物的特异性降解。据预测，中断这一调控途径的化学物质或sirna可以稳定融合转录物并允许持续的GFP表达。将进行可行性研究以验证该分析。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。这两种检测方法都涉及与癌症发展有关的细胞调控的基本方面（表观遗传沉默、细胞周期控制）。这些分析有可能揭示新的先导化合物，以及新的治疗基因靶点。

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.