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/项目摘要该提案的目的是发现和解释顺式调节DNA控制基因的代码表达。该调节性DNA控制多细胞中独有精度的细胞命运的规范包括人类在内的生物体及其失调均基于发育疾病和癌症。该控制被编码到基因组中的方式仍然很少理解。而且，最近发现后生基因被随机爆发转录的发现引发了理解如何该随机过程受到控制，以引起观察到的成熟转录本的高度精确分布。这两个问题都是在基础科学和翻译医学中进一步进步的障碍，我们建议通过此处提出的工作将其删除。关键支持工具是一个既定的模型采用DNA序列的转录控制和转录因子的浓度作为输入并给出RNA合成速率作为输出。该模型不仅限于增强剂，而且还可以对待整个遗传基因座。我们以前使用此模型来了解如何在系统生殖力的范围内保护增强子功能在缺乏DNA序列的情况下，发生远处的物种。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身份绑定的转录因子可能会有所不同，但仅在一定范围内。这些簇，我们称为“软密码子”，可能具有与结构遗传密码一样重要的作用。为了测试这一点，我们建议目标1：（a）发现并在整个前夕的软密码子，D。Melanogaster，D。Virilis和D. erecta中本地环境，以及来自远处的二角体的精选增强子，Clogmia，Clogmia， Chironomus在D. Melanogaster中表达。在体内观察到的转录的随机爆发也受转录因子的控制。我们提议扩展我们的转录模型以通过平行专家计划来治疗对这些爆发的控制 - 构建和建模。所有实验将在本地完整基因座的背景下进行，我们将在其中进行分析一系列精心选择的扰动的效果。具体说明，我们建议目标2：执行果蝇前夕基因座的体内调节解剖，我们将监测（a）中爆裂整个基因座；（b）一系列关键条纹两个增强子构建体，旨在改变强度和转录的可变性；（c）整个轨迹内增强子的重排；（d）纯净增强子和基本启动子之间所有相互作用的经过感染构造都在反式。我们将使用最终的数据，以及我们的先前存在的基因表达的定量地图集目标3：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对随机转录启动的简单模型。