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

母体沉积因子控制动物发育的最早阶段。当 当母体因子耗尽时，转录责任转移到合子。这个过程，被称为 合子基因组激活（ZGA）在动物中是保守的，并且是精确基因表达所必需的 和细胞分化。母体组蛋白在早期胚胎发生过程中特别关键，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， 组蛋白过表达导致细胞分裂不同步。合子组蛋白基因的表达被激活 早在ZGA之前。组蛋白基因被一套独特的调控因子所靶向，这些调控因子统称为 组蛋白基因座体（HLB）。然而，目前还不清楚HLB组分如何直接将该位点鉴定为无 与DNA相互作用。最近的数据表明合子组蛋白基因是由专门的转录因子靶向的 称为先锋因子，它识别核小体阻碍的结合位点。先锋因素促进整体 ZGA通过募集染色质重塑复合物为其他转录因子做准备。先锋因素 因此，是很好的候选人，可以靶向沉默合子组蛋白基因座，并准备它的HLB 形成和组蛋白基因表达。 我的初步数据表明，先驱因子塞尔达，ZGA的“主调节器”，占据了 合子型组蛋白基因表达前的果蝇组蛋白基因座。一种缺乏Zelda结合的组蛋白基因阵列 与野生型阵列相比，位点在募集组蛋白基因表达所需的HLB因子方面效率较低。 在ZGA期间，塞尔达与另一个先锋因素CLAMP合作。我们之前发现CLAMP 参与合子HLB形成和组蛋白基因表达。在这份提案中，我提议利用 在果蝇中研究先驱因子对组蛋白基因贡献的有力工具 在ZGA期间进行调整。我假设塞尔达和CLAMP合作准备合子组蛋白基因座， HLB的形成和组织。 在目标1中，我将通过利用转基因组蛋白来确定Zelda在合子HLB形成中的作用 缺乏Zelda结合位点的阵列，在不同发育时间人工将Zelda与转基因连接 点，并通过显微镜测定HLB形成。在目标2A中，我将定义塞尔达的关系 和CLAMP在合子组蛋白基因座通过执行先锋因子CUT&RUN在组蛋白基因座存在 以及缺乏其他先驱因素。在目标2B中，我将探讨先驱因子在组蛋白基因座中的作用 组织通过DamID和长读序。该项目的长期目标是定义 先锋因子对调控不可或缺的组蛋白基因的贡献。需要精确的合子 组蛋白调控是广泛保守的，果蝇的见解将促进我们对组蛋白调控的理解。 有助于人类早期发育的机制。

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.