Physiological Model of Gene Regulation in Drosophila

果蝇基因调控的生理模型

• 批准号: 10415987
• 负责人: John B. Reinitz
• 金额: $ 65.92万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10415987
• 关键词: Alleles; Architecture; Atlases; Bacteria; Basic Science; Behavior; Binding; Binding Sites; Biological Assay; Biology; Chironomus Genus; Chromatin; Chromatin Loop; Chromosomes; Code; Codon Nucleotides; Complex; Congenital Abnormality; Coupling; DNA; DNA Sequence; Data; Development; Dissection; Distant; Drosophila genus; Drosophila melanogaster; Embryo; Enhancers; Frequencies; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Code; Genetic Transcription; Genome; Goals; Homologous Gene; Human; Individual; Investigation; Learning; Location; Malignant Neoplasms; Mammals; Medical; Modeling; Modernization; Monitor; Mutation; Organism; Output; Pattern; Positioning Attribute; Process; Proteins; RNA; RNA chemical synthesis; Resolution; Role; Series; Site; Specific qualifier value; Structure; System; Techniques; Testing; Time; Transcript; Transcription Initiation; Transcriptional Regulation; Variant; Work; cell fate specification; design and construction; developmental disease; experimental study; flexibility; genomic locus; in vivo; physiologic model; programs; promoter; support tools; transcription factor; translational medicine

Abstract/Project Summary The goal of this proposal is to discover and interpret the code by which cis-regulatory DNA controls gene expression. This regulatory DNA controls the specification of cell fates with exquisite precision in multicellular organisms, including humans, and its dysregulation underlies both developmental diseases and cancer. The manner in which this control is coded into the genome remains poorly understood. Moreover, the recent discovery that metazoan genes are transcribed in random bursts raises the problem of understanding how this random process is controlled to give rise to the highly precise distribution of mature transcripts observed. Both of these problems constitute a roadblock to further progress in basic science and translational medicine, and we propose to remove them by the work proposed here. The key supporting tool is an established model of transcriptional control that takes DNA sequence and the concentrations of transcription factors as inputs and gives RNA synthesis rate as output. This model is not limited to enhancers, but can also treat an entire genetic locus. We previously used this model to understand how conservation of enhancer function across phylogeneti- cally distant species occurred in the absence of conservation of DNA sequence. We found that the conserved entities were small clusters of binding sites in which the exact positions of binding sites and the identity of bound transcription factors can vary, but only within certain limits. These clusters, which we call “soft codons,” may have a role as essential as the structural genetic code. To test this, we propose to Aim 1: (a) Discover and model soft codons in the entire eve locus of D. melanogaster, D. virilis, and D. erecta in their native context, and selected enhancers from distant dipterans in the genuses Megaselia, Clogmia, and Chironomus expressed in D. melanogaster. The random bursts of transcription observed in vivo are also under the control of transcription factors. We propose to extend our transcription model to treat control of these bursts by a program of parallel experimen- tation and modeling. All experiments will be conducted in the context of a native intact locus, in which we will analyze the effects of a series of carefully selected perturbations. Specifically, we propose to Aim 2: Perform an in vivo regulatory dissection of the Drosophila eve locus in which we will monitor bursting in (a) The whole locus; (b) A series of key stripe two enhancer constructs designed to vary strength and variability of transcription; (c) Rearrangements of enhancers within the whole locus; and (d) Pure transvective constructs in which all interactions between the enhancer and basal promoter are in trans. We will use the resulting data, together with our preexisting quantitative atlas of gene expression at cellular resolution to Aim 3: Construct a new stochastic model of transcriptional control by coupling our current model of transcription to a simple model of stochastic transcription initiation.

摘要/项目摘要 该提案的目标是发现并解释顺式调控 DNA 控制基因的密码 这种调节性 DNA 在多细胞中以极其精确的方式控制着细胞命运的规范。 包括人类在内的生物体及其失调是发育性疾病和癌症的基础。 这种控制编码到基因组中的方式仍然知之甚少。 后生动物基因以随机爆发方式转录的发现提出了理解如何转录的问题 这个随机过程受到控制，以产生观察到的成熟转录本的高度精确的分布。 这两个问题都构成了基础科学和转化医学进一步进步的障碍， 我们建议通过这里提出的工作来消除它们，关键的支持工具是一个既定的模型。 以 DNA 序列和转录因子浓度作为输入的转录控制 并给出 RNA 合成率作为输出。该模型不仅限于增强子，还可以处理整个。 遗传位点。 我们之前使用这个模型来了解增强子功能的保守性如何在系统发育过程中发挥作用。 很远的物种发生在DNA序列不保守的情况下。 实体是一小群结合位点，其中结合位点的确切位置和身份 结合的转录因子可以变化，但仅在一定范围内，这些簇，我们称之为“软密码子”。 可能与结构遗传密码一样重要。为了测试这一点，我们提出目标 1：（a）发现。 以及 D. melanogaster、D. virilis 和 D.ectera 整个 eve 基因座中的模型软密码子 原生环境，以及来自 Megaselia、Clogmia 属的遥远双翅目的选定增强子， 摇蚊在黑腹果蝇中表达。 体内观察到的随机转录爆发也受到转录因子的控制。 建议扩展我们的转录模型，通过并行实验程序来处理这些爆发的控制- 所有实验都将在本地完整基因座的背景下进行，我们将在其中进行。 具体来说，我们建议目标 2：执行。 果蝇 eve 基因座的体内调节性解剖，我们将监测（a）中的爆发 (b) 一系列关键条带两个增强子构造，旨在改变强度和 转录的变异性；(c) 整个基因座内增强子的重排；以及 (d) 纯 平转构建体，其中增强子和基础启动子之间的所有相互作用都在 我们将使用所得数据以及我们预先存在的基因表达定量图谱。 目标 3 的细胞分辨率：通过耦合构建转录控制的新随机模型 我们当前的转录模型转变为随机转录起始的简单模型。