Coordinate Gene Regulation in Animal Cells

描述（由申请人提供）： 热休克（HS）基因是一组高度调控的基因，特别适合研究诱导型mRNA产生的基本特征。在这里，既建立和新的技术将被用来确定参与HS基因mRNA的生产的因素，并定义与高时间和空间分辨率时，在哪里，以及与谁这些因素在基因激活过程中相互作用。将在体内评价通过耗尽或失活特定转录因子或这些因子的特定表面对hsp 70启动子和基因造成的后果。目的1是一系列筛选，以确定参与HS诱导的转录和RNA加工的因素，使用Polytene染色体免疫荧光，细胞培养物的RNAi处理，以及现有的果蝇突变体的测定。目的2通过多线染色体免疫染色和实时PCR定量ChIP测定的互补方法，研究HS基因激活过程中启动子的结构和功能以及因子募集的动力学。这些策略将通过体内化学足迹分析、通过实时、逆转录PCR和北方分析定量特定转录物以及通过核运行进行的延伸接合RNA聚合酶II（Pol II）分析来增强。此外，将使用双光子和FRET显微镜检查CFP-和YFP-标记的转录因子对在多线染色体上的相互作用，提供了在真实的时间和活细胞中基因激活期间在特定位点的因子相互作用的独特视图。目的3测试特定因子在启动子结构和功能中的作用，使用一组互补的和逐渐更高分辨率的策略来耗尽或消除特定因子或这些因子的特定表面。策略包括RNAi治疗，使用现有的果蝇突变体，产生和使用快速作用的温度敏感等位基因，应用高度特异性的药物/抑制剂，以及产生和使用高亲和力RNA适体与关键转录因子的不同相互作用表面。在所有情况下，对启动子和基因结构和功能的影响将通过目标2中描述的体内策略进行检查。由此产生的信息将是关键，在建立分子模型的各个步骤中的转录激活的基因和耦合的RNA加工事件在体内，并在提供必要的正常和疾病状态的分子理解的背景。

DESCRIPTION (provided by applicant): The heat shock (HS) genes are a highly-regulated set of genes particularly well-suited to investigate fundamental features of inducible mRNA production. Here, both established and new technologies will be used to identify factors that participate in the HS gene mRNA production, and to define with high temporal and spatial resolution when, where, and with whom these factors interact during the process of gene activation. The consequences on the hsp70 promoter and gene caused by depleting or inactivating specific transcription factors or particular surfaces of these factors will be evaluated in vivo. Aim 1 is a series of screens to identify factors involved in HS-induced transcription and RNA processing using Polytene chromosome immunofluorescence, RNAi treatments of cell cultures, and assays of existing fly mutants. Aim 2 examines promoter architecture and function during HS gene activation and the kinetics of factor recruitment by complementary methods of immuno-staining of polytene chromosomes and real-time PCR-quantified ChIP assays. These strategies will be augmented by in vivo chemical footprinting assays, quantification of specific transcripts by real-time, reverse-transcription PCR and Northern assays, and assays of elongationally-engaged RNA Polymerase II (Pol II) by nuclear run-on. Additionally, pairs of CFP- and YFP-tagged transcription factors will be examined for their interactions on polytene chromosomes using two photon and FRET microscopy, providing a unique view of factor interactions at specific loci during gene activation in real time and in living cells. Aim 3 tests the role of specific factors in promoter architecture and function using a battery of complementary and progressively higher resolution strategies to deplete or inactivate particular factors or particular surfaces of these factors. Strategies include RNAi treatments, use of existing Drosophila mutants, generation and use of fast-acting temperature-sensitive alleles, application of highly-specific drugs/inhibitors, and generation and use of high affinity RNA aptamers to different interacting surfaces of key transcription factors. In all cases, the effects on promoter and gene structure and function will be examined by the in vivo strategies described in Aim 2. The resulting information will be key in establishing molecular models for the various steps in the transcriptional activation of genes and the coupled RNA processing events in vivo, and in providing the background necessary for a molecular understanding of normal and disease states.