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.

摘要 该提案的目标是将组蛋白翻译后修饰（HPTM）与转录速率联系起来， 爆发频率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 在活跃和不活跃状态之间，这种现象被称为转录爆发。细胞命运质量标准 胚胎是由基因驱动的，这些基因的爆发特征决定了胚胎发育过程中的同步性和健壮性。 发展，但分子间的相互作用，产生突发频率观察发展 是未知的。为了理解转录是如何控制发育的，有必要揭示转录的分子机制。 控制爆发持续时间和爆发在体内发生的速率的决定因素。真核 转录的特征在于HPTM在增强子和启动子处的富集。尽管大力 HPTM与基因活性之间的相关性，尚不清楚HPTM如何决定转录速率和基因活性。 爆发频率此外，HPTM以许多组合出现在启动子和增强子处，产生 理论上有许多可能的启动子状态。在这里，我将确定是否HPTM赋予多个转录 在发展过程中的状态，以及这些状态是否决定转录爆发的具体速率。的 目前可用于测量转录率的最有效的方法是单个mRNA FISH和活的mRNA FISH。 新生部位成像。为了使用这些分析来定量描述状态转换，我将实现一个 转录状态的数学模型“两态”模型已被广泛用于定量分析 描述突发持续时间和频率的平均切换率之间的活动和 不活跃的国家。然而，简单的两态方法是否能准确地描述 在发育过程中经历复杂调控的基因的转录，例如差距基因hunchback。 在这里，我将确定两态模型是否足以描述内源性的爆发频率。 驼背我会把RNA环插入内源性hunchback基因座来制造内源性报告基因。我 将使用单个mRNA FISH和内源性hunchback报告基因的实时成像来测量转录水平。 爆裂动力学然后，我将确定HPTM如何影响hunchback转录的动力学。我会 母源性敲低组蛋白甲基转移酶、乙酰转移酶、脱甲基酶和 去乙酰化并测量它们对hunchback转录爆发的影响。对于每个HPTM 敲除后，我将确定哪种数学模型最能描述单个mRNA FISH和实时成像 数据总之，这些实验将确定特定组蛋白标记的变化如何改变驼背 爆裂动力学和促进剂状态。