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 该提案的目标是将组蛋白翻译后修饰 (HPTM) 与转录速率和 在所有研究的真核生物中，单个基因位点的转录表现出随机振荡。 在活性和非活性状态之间，这种现象被称为细胞命运规范。 胚胎是由基因驱动的，其爆发特征决定了胚胎发育过程中的同步性和鲁棒性。 发育，但产生发育中观察到的爆发频率的分子相互作用 要了解转录如何控制发育，有必要揭示该分子。 控制爆发持续时间和体内爆发发生速率的决定因素。 转录的特点是 HPTM 在增强子和启动子处富集，尽管其强度很高。 HPTMs 和基因活性之间的相关性，目前尚不清楚 HPTMs 如何确定转录率并设置 此外，HPTM 在启动子和增强子处以多种组合出现，产生 理论上有许多可能的启动子状态。在这里，我将确定 HPTM 是否赋予多种转录。 发育过程中的状态以及这些状态是否决定转录爆发的具体速率。 目前可用于测量转录率的最强大方法是单 mRNA FISH 和活体 为了使用这些测定定量描述状态转变，我将实施一个 “双态”模型已广泛用于定量转录状态。 根据活动和活动之间切换的平均速率来描述突发持续时间和频率 然而，简单的两种状态方法是否可以准确地描述尚未测试。 发育过程中经历复杂调控的基因转录，例如间隙基因驼背。 这里我将确定二态模型是否足以描述内源性的爆发频率 我会将 RNA 环插入内源性驼背基因座中，以创建内源性报告基因 I。 将使用单 mRNA FISH 和内源驼背报告基因的实时成像来测量转录 然后我将确定 HPTM 如何影响驼背转录的动力学。 母体敲低一系列组蛋白甲基转移酶、乙酰转移酶、去甲基酶和 去乙酰化并测量它们对驼背转录爆发的影响。 敲除，我将确定哪种数学模型最能描述单个 mRNA FISH 和实时成像 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。 爆裂动力学和启动子状态。