Coordinate Gene Regulation in Animal Cells

点击翻译按钮获取中文摘要

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.