How histone modifications influence transcriptional bursting in a developing embryo

组蛋白修饰如何影响发育中胚胎的转录爆发

• 批准号: 9760849
• 负责人: Joseph M Zinski
• 金额: $ 6.16万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9760849
• 关键词: 3' Untranslated Regions; Acetylation; Acetyltransferase; Acylation; Affect; Binding; Biological Assay; CRISPR imaging; Cell Cycle; Cells; ChIP-seq; Characteristics; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; DNA-Directed RNA Polymerase; Data; Development; Disease; Drosophila genus; EP300 gene; Embryo; Enhancers; Epigenetic Process; Equilibrium; Etiology; Eukaryota; Exhibits; Frequencies; Gene Expression; Genes; Genetic Transcription; Goals; Heterogeneity; Histone Deacetylase; Histones; Image; Individual; Kinetics; Label; Link; Malignant Neoplasms; Measurement; Measures; Mediating; Messenger RNA; Methods; Methylation; Modeling; Modification; Molecular; Oocytes; Pattern; Play; Polycomb; Post-Translational Protein Processing; Prevalence; Production; RNA; RNA Interference; Regulation; Reporter; Role; Site; Stochastic Processes; Testing; Transcriptional Regulation; Transgenes; cell fate specification; effective therapy; efficacy testing; experimental study; fluorescence imaging; genome-wide; histone acetyltransferase; histone demethylase; histone methyltransferase; histone modification; in vivo; knock-down; mathematical model; nuclease; promoter; theories

Abstract The goal of this proposal is to link histone post-translational modifications (HPTMs) to transcription rates and bursting frequencies. In all eukaryotes studied, transcription at individual gene loci exhibits random oscillations between active and inactive states, a phenomenon known as transcription bursting. Cell fate specification in embryos is driven by genes whose bursting characteristics determine synchrony and robustness during development, but the molecular interactions which generate the bursting frequencies observed in development are unknown. To understand how transcription controls development, it is necessary to uncover the molecular determinants that control the duration of bursts and the rates at which bursts occur in vivo. Eukaryotic transcription is characterized by the enrichment of HPTMs at enhancers and promoters. Despite the strong correlation between HPTMs and gene activity, it is unclear how HPTMs determine transcription rates and set bursting frequencies. Moreover, HPTMs occur in many combinations at promoters and enhancers, generating in theory many possible promoter states. Here, I will determine whether HPTMs confer multiple transcriptional states during development and whether those states determine specific rates of transcription bursting. The most powerful methods currently available to measure transcription rates are single mRNA FISH and live nascent site imaging. To quantitatively describe state transitions using these assays, I will implement a mathematical model of transcription states. The “two-state” model has been widely used to quantitatively describe burst duration and frequency in terms of the average rates of switching between the active and inactive states. However, it is untested whether the simple two-state approach can accurately describe the transcription of a gene undergoing complex regulation during development, such as the gap gene hunchback. Here I will determine whether the two-state model is sufficient to describe bursting frequencies of endogenous hunchback. I will insert RNA loops into the endogenous hunchback locus to create an endogenous reporter. I will use single mRNA FISH and live-imaging of the endogenous hunchback reporter to measure transcriptional bursting kinetics. I will then determine how HPTMs affect the kinetics of hunchback transcription. I will maternally knockdown an array of histone methyltransferases, acetyltransferases, demethylases, and deacetylates and measure their effects on the transcriptional bursting of hunchback. For each HPTM knockdown, I will determine which mathematical model best describes the single mRNA FISH and live-imaging data. Together, these experiments will determine how changes in specific histone marks change hunchback bursting kinetics and promoter state.

抽象的 该建议的目的是将组蛋白翻译后修改（HPTMS）与转录率和 突发的频率。在所有真核生物研究中，单个基因基因座的转录均表现出随机振荡 在主动状态和非活性状态之间，一种被称为转录爆发的现象。细胞脂肪规格 胚胎是由爆发特征决定同步和鲁棒性的基因驱动的 开发，但是产生发育中观察到的突发频率的分子相互作用 是未知的。要了解转录如何控制发展，有必要发现分子 控制爆发持续时间的决定因素和体内发生爆发的速率。真核生物 转录的特征是HPTM在增强子和启动子上的富集。尽管很强 HPTM与基因活性之间的相关性，尚不清楚HPTM如何确定转录率并设置 突发的频率。此外，HPTM在发起人和增强剂的许多组合中发生，产生 从理论上讲，许多可能的启动子状态。在这里，我将确定HPTM是否授予多个转录 在开发过程中的状态以及这些状态是否确定转录爆发的特定速率。这 目前可用于测量转录率的最强大方法是单mrna鱼和活 新生站点成像。为了使用这些测定法进行定量描述状态转变，我将实施 转录状态的数学模型。 “两态”模型已被广泛用于定量 描述爆发持续时间和频率，以活动和活动之间的平均切换率 不活动状态。但是，未经测试的简单两国方法是否可以准确描述 在发育过程中接受复杂调节的基因的转录，例如GAP基因驼背。 在这里，我将确定两国模型是否足以描述内源性的爆发频率 偻。我将插入RNA环中的内源性驼背基因座以创建内源性记者。 将使用单个mRNA鱼和内源性驼背记者的现场成像来测量转录 爆发动力学。然后，我将确定HPTM如何影响驼背转录的动力学。我会 母体敲除一系列组蛋白甲基转移酶，乙酰转移酶，去甲基酶和 脱乙酰盐并测量其对驼背转录爆发的影响。对于每个HPTM 敲除，我将确定哪种数学模型最能描述单个mRNA鱼和现场模仿 数据。这些实验将共同确定特定组蛋白标记的变化如何改变驼背 破裂的动力学和启动子状态。