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Physiological Model of Gene Regulation in Drosophila

果蝇基因调控的生理模型

基本信息

批准号：
10633284
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
项目类别：
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/10633284
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 Phylogenetic Analysis 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 fly genomic locus in vivo physiologic model programs promoter support tools transcription factor translational medicine

项目摘要

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/项目摘要本研究的目的是发现并解释顺式调控DNA控制基因的密码子表情这种调控DNA在多细胞生物中以极其精确的方式控制着细胞命运的特异性。生物体，包括人类，它的失调是发育疾病和癌症的基础。的这种控制被编码到基因组中的方式仍然知之甚少。而且，最近的后生动物基因以随机爆发的方式转录的发现，提出了理解后生动物基因如何转录的问题。控制该随机过程以产生观察到的成熟转录物的高度精确的分布。这两个问题构成了基础科学和转化医学进一步发展的障碍，我们建议通过此处提出的工作来消除这些缺陷。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为输入的转录控制并给出RNA合成速率作为输出。该模型不仅限于增强剂，还可以治疗整个基因位点我们以前使用这个模型来了解增强子功能如何在基因组中保守，远缘种在DNA序列不保守的情况下出现。我们发现保守的实体是结合位点的小簇，其中结合位点的确切位置和结合的转录因子可以变化，但仅在一定限度内。这些簇，我们称之为“软密码子”，可能和结构遗传密码一样重要。为了验证这一点，我们提出目标1：（a）发现并在D. melanogaster、黑腹杜父鱼D. virilis和D.直立在他们的天然环境，以及来自Megaselia，Clogmia，和摇蚊在D.黑腹菌在体内观察到的转录的随机爆发也在转录因子的控制下。我们我建议扩展我们的转录模型，通过一个平行实验的程序来处理这些突发的控制- 定位和建模。所有实验将在天然完整基因座的背景下进行，其中我们将分析一系列精心挑选的扰动的影响。具体而言，我们建议目标2：执行果蝇eve基因座的体内调节解剖，我们将在（a）中监测爆发整个基因座;（B）一系列关键条带双增强子构建体，其被设计为改变强度和转录的可变性;（c）整个基因座内增强子的重排;和（d）纯其中增强子和基础启动子之间的所有相互作用都在译我们将使用由此产生的数据，连同我们先前存在的基因表达定量图谱，目标3的细胞解决方案：构建一个新的转录调控随机模型我们目前的转录模型变成了一个简单的随机转录起始模型。