How histone modifications influence transcriptional bursting in a developing embryo

摘要 这项建议的目标是将组蛋白翻译后修饰(HPTM)与转录速率和 爆发频率。在所有被研究的真核生物中，单个基因位点的转录表现出随机振荡。 在活跃和非活跃状态之间，这一现象被称为转录爆发。中的细胞命运规范 胚胎是由基因驱动的，其破裂特征决定了胚胎在发育过程中的同步性和健壮性 发育，但产生发育中观察到的猝发频率的分子相互作用 都是未知的。为了了解转录是如何控制发育的，有必要揭示分子。 在活体内控制爆发持续时间和爆发速率的决定因素。真核生物 转录的特征是HPTM在增强子和启动子上的富集性。尽管强者 HPTM与基因活性之间的相关性，目前尚不清楚HPTM如何决定转录速率和SET 爆发频率。此外，HPTM在启动子和增强子中以多种组合出现，产生 从理论上讲，有很多种可能的启动状态。在这里，我将确定HPTM是否授予多个转录 在发育过程中的状态，以及这些状态是否决定特定的转录爆发速率。这个 目前可用于测量转录速率的最强大的方法是单个mRNAFISH和活的 初露端倪的现场成像。为了使用这些分析定量描述状态转换，我将实现一个 转录状态的数学模型。“两态”模型已被广泛应用于定量研究 描述脉冲串持续时间和频率，根据活动和 非活动状态。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，如GAP基因驼背。 在这里，我将确定两态模型是否足以描述内源性 驼背。我将在内源性驼背基因座中插入RNA环，以创建内源报告基因。我 将使用单个mRNAFISH和内源性驼背报告的实时成像来测量转录 爆破动力学。然后，我将确定HPTM如何影响驼背转录的动力学。这就做 母体基因敲除了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。对于每个HPTM 击倒后，我将确定哪个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 爆破动力学和启动子状态。

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.