Developmental regulation of histone genes by pioneer factors and three-dimensional architecture

项目概述：母体沉积因素控制着动物发育的最早阶段。什么时候 母性因子被耗尽，转录责任转移到受精卵身上。这一过程称为 合子基因组激活(ZGA)在动物中是保守的，对于准确的基因表达是必要的 和细胞分化。母体组蛋白在早期胚胎发育过程中尤其关键，因为它们的 水平调节细胞/核分裂的时间：组蛋白的耗尽导致细胞周期停滞，而 组蛋白的过度表达会导致细胞的非同步分裂。合子组蛋白基因的表达被激活 早，在ZGA之前。组蛋白基因是一套独特的调控因子的靶点，统称为 组蛋白位点小体(HLb)。然而，目前尚不清楚HLB组分如何直接将该基因座识别为无 与DNA相互作用。最近的数据表明，受精性组蛋白基因是专门的转录因子的靶标。 称为先锋因子，识别核小体受阻的结合部位。先锋因素促进整体 ZGA通过招募染色质重塑复合体来准备其他转录因子。先锋因素 因此，它们是很好的候选基因，可以针对沉默的合子组蛋白基因座，并为HLB做准备 形成和组蛋白基因表达。 我的初步数据显示，ZGA的先锋因子塞尔达--ZGA的“主调节器”--占据了 果蝇组蛋白基因先于合子组蛋白基因表达。一个缺乏Zelda结合的组蛋白基因阵列 与野生型阵列相比，SITS在招募组蛋白基因表达所需的HLB因子方面效率较低。 在ZGA期间，塞尔达与另一家先锋因素夹具合作。我们之前发现夹子 参与合子HLb的形成和组蛋白基因的表达。在这项提案中，我建议利用 在果蝇中研究先锋因子对组蛋白基因贡献的有力工具 ZGA期间的监管。我推测塞尔达和卡普合作准备受精卵的组蛋白基因 HLB的形成和组织。 在目标1中，我将通过利用转基因的组蛋白来确定Zelda在合子HLb形成中的作用 缺少塞尔达结合位点的阵列，在不同发育时间人工将塞尔达与转基因捆绑在一起 ZGA周围的点，显微镜下分析HLB的形成。在目标2a中，我将定义塞尔达的关系 并通过在存在的组蛋白基因座上执行先锋因子切割和运行来夹住合子组蛋白基因座 以及缺乏另一个先锋因素。在目标2B中，我将探索组蛋白基因座中先锋因子的作用 通过DAMID和长读排序进行组织。该项目的长期目标是定义 先锋因子对不可或缺的组蛋白基因调控的贡献。对精确受精卵的需求 组蛋白调控是广泛保守的，来自果蝇的见解将促进我们对组蛋白调控的理解 有助于人类早期发展的机制。

PROJECT SUMMARY: Maternally deposited factors control the earliest stages of animal development. When maternal factors are depleted, the transcriptional responsibility shifts to the zygote. This process, known as zygotic genome activation (ZGA), is conserved across animals and is necessary for accurate gene expression and cellular differentiation. Maternal histone proteins are especially critical during early embryogenesis as their levels regulate the timing of cellular/nuclear divisions: depletion of histones leads to cell cycle arrest, while histone overexpression causes asynchronous cell division. Expression of the zygotic histones genes is activated early, prior to ZGA. The histone genes are targeted by a suite of unique regulatory factors collectively called the Histone Locus Body (HLB). However, it is unclear how the HLB components identify the locus as none directly interact with DNA. Recent data suggest that zygotic histone genes are targeted by specialized transcription factor called pioneer factors, which recognize nucleosome-obstructed binding sites. Pioneer factors facilitate overall ZGA by recruiting chromatin remodeling complexes in preparation for other transcription factors. Pioneer factors are therefore excellent candidates that may target the silent zygotic histone locus and prepare it fore HLB formation and histone gene expression. My preliminary data suggest that the pioneer factor Zelda, the “master regulator” of ZGA, occupies the Drosophila histone locus prior to zygotic histone gene expression. A histone gene array lacking Zelda binding sites is less efficient at recruiting HLB factors needed for histone gene expression compared to a wild type array. During ZGA, Zelda cooperates with another pioneering factor CLAMP. We previously discovered that CLAMP participates in zygotic HLB formation and histone gene expression. In this proposal, I propose to leverage powerful tools in Drosophila melanogaster to investigate the contribution of pioneer factors to histone gene regulation during ZGA. I hypothesize that Zelda and CLAMP collaborate to prepare the zygotic histone locus for HLB formation and organization. In Aim 1, I will determine the role of Zelda in zygotic HLB formation by leveraging a transgenic histone array lacking Zelda binding sites, artificially tethering Zelda to the transgene at different developmental time points around ZGA, and assaying HLB formation by microscopy. In Aim 2A, I will define the relationship of Zelda and CLAMP at the zygotic histone locus by performing pioneer factor CUT&RUN at histone loci in the presence and absence of the other pioneer factor. In Aim 2B, I will probe the role of pioneer factors in histone locus organization through DamID and long-read sequencing. The long-term goal of this project is to define the contribution of pioneer factors to regulation of the indispensable histone genes. The need for precise zygotic histone regulation is broadly conserved, and insights from Drosophila will advance our understanding of mechanisms that contribute to early human development.