Mechanisms controlling stochastic gene expression during eye development

眼睛发育过程中控制随机基因表达的机制

基本信息

批准号：
10652357
负责人：
Robert John Johnston
金额：
$ 40.17万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-01 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10652357
关键词：
ATAC-seq Affect Anosmia Automobile Driving Biological Process Cells ChIP-seq Chromatin Clustered Regularly Interspaced Short Palindromic Repeats Color Complement DNA Data Development Disease Drosophila genus Elements Enhancers Exhibits Eye Eye Development Fluorescent in Situ Hybridization Frequencies Gene Expression Genes Genetic Transcription Genomics Goals Human Image Immune Immunologic Deficiency Syndromes Individual Investigation Knock-out Link Lymphoma Modeling Molecular Monitor Motor Neurons Mutation Neurons Noise Pattern Photoreceptors Physiologic pulse RNA Regulation Regulator Genes Reporter Resolution Retina Rhodopsin Role Signal Transduction Source Specific qualifier value System Testing Time Transcript Transcriptional Regulation Undifferentiated Vision Disorders Visual impairment Visualization autism spectrum disorder cell fate specification cell type color detection experimental study fly histone modification imaging approach mosaic mutant olfactory receptor olfactory sensory neurons promoter retinal neuron stem cells transcription factor visual photoreceptor

项目摘要

Project Summary Cell fate specification is driven by lineage, signaling, and stochastic regulatory inputs. The mechanisms controlling stochastic fate specification, in which a cell randomly chooses between two or more fates, are poorly understood. Stochastic fate specification is critical for diversifying retinal neurons, olfactory sensory neurons, motor neurons, immune cells, and stem cells. Breakdowns in these mechanisms cause debilitating human disorders, including vision impairments, anosmia, autism, immunodeficiencies, and lymphoma. The main goal of this project is to determine how chromatin state and transcriptional variability control stochastic fate specification, using the random patterning of photoreceptor subtypes in the fly retina as a paradigm. The fly eye contains a random mosaic of two color-detecting R7 photoreceptor subtypes, defined by expression of Rhodopsin 4 (Rh4) or Rhodopsin 3 (Rh3). This fate decision is controlled by the transcription factor Spineless (Ss), which is expressed in a random subset of mature R7s. SsON R7s express Rh4, while SsOFF R7s express Rh3. Our data support a two-step mechanism regulating ssON/OFF expression in mature R7s. In step 1, the early enhancer drives an early pulse of ss transcription in R7 precursors that opens the chromatin at the ss locus. In step 2, the transcriptional pulse ceases and chromatin variably closes, defining the accessibility of the late enhancer. Depending on the degree of chromatin compaction, the late enhancer either turns on (open chromatin) or remains off (closed chromatin) for the lifetime of the mature R7. How regulation of transcription and chromatin compaction is integrated to turn genes randomly on or off during development is poorly understood. We will use DNA FISH, genomics, CRISPR, and lacO/LacI-based live imaging approaches to assess the role of chromatin dynamics and the temporality of regulatory inputs in the two-step mechanism (Aim 1). Identification of the source of variability driving stochastic fate specification in metazoans has not been achieved. Our data suggest that variability in transcription (initiation, elongation, frequency, and duration) in individual cells influences terminal R7 fate specification. To test this hypothesis, we will use nascent multi-color RNA FISH and MS2/MCP-based live imaging to assess transcriptional parameters and relate them to R7 subtype fates (Aim 2). Successful completion of these experiments will identify a source of variability that drives a cell fate decision and inform how molecular noise is utilized to diversify cell types during metazoan development.

项目概要细胞命运规范是由谱系、信号传导和随机调控输入驱动的。这控制随机命运规范的机制，其中细胞随机选择两个或更多的命运，人们知之甚少。随机命运规范对于多样化至关重要视网膜神经元、嗅觉感觉神经元、运动神经元、免疫细胞和干细胞。这些机制的故障会导致使人衰弱的疾病，包括视力损伤、嗅觉丧失、自闭症、免疫缺陷和淋巴瘤。该项目的主要目标是确定染色质状态和转录变异如何控制随机命运规范，使用果蝇视网膜中感光亚型的随机图案作为范例。果蝇眼睛包含两种颜色检测 R7 光感受器亚型的随机镶嵌，由视紫质 4 (Rh4) 或视紫红质 3 (Rh3) 的表达来定义。这个命运决定是被掌控的由转录因子 Spineless (Ss) 引起，该因子在成熟 R7 的随机子集中表达。 SsON R7s 表达 Rh4，而 SsOFF R7s 表达 Rh3。我们的数据支持两步机制调节成熟 R7 中的 ssON/OFF 表达。在步骤 1 中，早期增强器驱动早期脉冲 R7 前体中的 ss 转录在 ss 基因座处打开染色质。在步骤 2 中，转录脉冲停止，染色质不同程度地关闭，从而定义了晚期的可及性增强剂。根据染色质压缩程度，晚期增强子要么开启（开放染色质）或在成熟 R7 的一生中保持关闭（封闭染色质）。如何转录和染色质压缩的调节被整合以随机打开或关闭基因开发过程中关闭的情况知之甚少。我们将使用 DNA FISH、基因组学、CRISPR 和基于 lacO/LacI 的实时成像方法评估染色质动力学的作用和两步机制中监管投入的临时性（目标 1）。鉴定驱动后生动物随机命运规范的变异源尚未实现。我们的数据表明转录的变异性（起始、延伸、单个细胞中的频率和持续时间影响终端 R7 命运规范。为了测试这个假设，我们将使用新生的多色 RNA FISH 和基于 MS2/MCP 的实时成像评估转录参数并将其与 R7 亚型命运联系起来（目标 2）。成功的完成这些实验将确定驱动细胞命运决定的变异性来源并告知如何利用分子噪音在后生动物发育过程中使细胞类型多样化。

项目成果

期刊论文数量（12）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Thyroid hormone signaling specifies cone subtypes in human retinal organoids.

甲状腺激素信号传导指定人类视网膜类器官中的视锥细胞亚型。

DOI：
发表时间：
2018
期刊：
影响因子：
0
作者：
Eldred, Kiara C;Hadyniak, Sarah E;Hussey, Katarzyna A;Brenerman, Boris;Zhang, Ping;Chamling, Xitiz;Sluch, Valentin M;Welsbie, Derek S;Hattar, Samer;Taylor, James;Wahlin, Karl;Zack, Donald J;Johnston Jr, Robert J
通讯作者：
Johnston Jr, Robert J

Characterization of histone inheritance patterns in the Drosophila female germline.

果蝇雌性种系中组蛋白遗传模式的表征。

DOI：
发表时间：
2021
期刊：
影响因子：
7.7
作者：
Kahney, Elizabeth W;Zion, Emily H;Sohn, Lydia;Viets;Johnston, Robert;Chen, Xin
通讯作者：
Chen, Xin

The insulator protein BEAF-32 is required for Hippo pathway activity in the terminal differentiation of neuronal subtypes.

绝缘子蛋白 BEAF-32 是神经元亚型终末分化中 Hippo 通路活性所必需的。

DOI：
发表时间：
2016
期刊：
影响因子：
0
作者：
Jukam, David;Viets, Kayla;Anderson, Caitlin;Zhou, Cyrus;DeFord, Peter;Yan, Jenny;Cao, Jinshuai;Johnston Jr, Robert J
通讯作者：
Johnston Jr, Robert J

Buffering and Amplifying Transcriptional Noise During Cell Fate Specification.

在细胞命运规范过程中缓冲和放大转录噪音。