Mechanisms of Nuclear Body Formation in the Early Embryo

总结： 协调的基因调控在合子基因组起始的发育胚胎中尤其重要 第一次抄写。促进协调基因调控的保守策略是形成 保守的结构称为核体，亚核区域，在那里特定的因子积累， 执行调节和转录功能。组蛋白基因座体（HLB）是一种保守的核小体 我发现锌指蛋白，CLAMP（染色质连接蛋白） MSL蛋白的衔接子），是果蝇HLB调节所必需的。我现在的研究目标是 解决关键问题：CLAMP如何以特定于上下文的方式启动核主体 在合子基因组激活的关键发育阶段形成？ 在本项目的目标1中，我将定义CLAMP在染色质可及性和因子募集过程中的作用。 合子基因组激活我将从性别鉴定的胚胎池中进行ATAC-seq和转录因子ChIP-seq 在存在和不存在CLAMP的情况下，在合子基因组激活的时间点附近。我将收到 我的导师布朗大学的埃里卡·拉森博士给我的指导。这些实验将解决如何 染色质状态影响核体的形成，以及染色质的组成如何在ZGA时发生变化， 核体具有其独特的性质。在目标2中，我将使用遗传工具来确定 CLAMP在HLB的时间要求和允许HLB形成的顺式元件的特定质量， 在北卡罗来纳大学教堂山分校的罗伯特·杜罗尼奥博士的指导下，这 目的是比较整个开发过程中HLB启动和维持的机制。第3章我会 进行蛋白质组学分离染色质片段在早期胚胎中的存在和不存在 CLAMP，然后进行质谱分析。我将确定未知的HLB组件及其可靠性 在我的共同导师普林斯顿大学的Paul Schedl博士的指导下，这一目标 将阐明早期胚胎核体形成的机制，包括有序招募 已知和新的因素。 我将执行拟议的实验，同时也获得经验和关键的背景， 发育生物学和蛋白质组学。我还将通过实践来发展我的教学和指导技能。 经验和领导力/实验室管理课程。我提议的研究将产生重大影响 新的实验方向，我提出的职业发展将支持我，因为我开始我的 独立实验室

SUMMARY: Coordinated gene regulation is especially critical in the developing embryo where the zygotic genome initiates transcription for the first time. A conserved strategy to facilitate coordinated gene regulation is formation of conserved structures called nuclear bodies, sub-nuclear regions at which specific factors accumulate to perform regulatory and transcriptional functions. The histone locus body (HLB) is a conserved nuclear body that regulates the histone genes, and I discovered that the zinc finger protein, CLAMP (Chromatin Linked Adaptor for MSL Proteins), is essential to HLB regulation in Drosophila melanogaster. My research now aims to address the key question: How does CLAMP act in a context-specific manner to initiate nuclear body formation during the critical developmental stage of zygotic genome activation? In Aim 1 of this project I will define the role of CLAMP in chromatin accessibility and factor recruitment during zygotic genome activation. I will perform ATAC-seq and transcription factor ChIP-seq from sexed embryo pools in the presence and absence of CLAMP, around the time point of zygotic genome activation. I will receive mentorship from my mentor, Dr. Erica Larschan at Brown University. These experiments will address how chromatin state affects nuclear body formation and how the composition of chromatin changes at ZGA when nuclear bodies assume their unique properties. In Aim 2 I will use genetic tools to determine the developmental timing requirements of CLAMP at the HLB and the specific qualities of cis-elements that allow HLB formation, under mentorship from my co-mentor Dr. Robert Duronio at the University of North Carolina-Chapel Hill. This aim will compare the mechanism(s) of HLB initiation and maintenance throughout development. In Aim 3 I will perform Proteomics of Isolated Chromatin Segments in early embryos in the presence and absence of CLAMP, followed by mass spectrometry analysis. I will identify unknown HLB components and their reliance on CLAMP recruitment, under mentorship from my co-mentor Dr. Paul Schedl at Princeton University. This aim will illuminate the mechanism of early embryonic nuclear body formation, including the ordered recruitment of known and novel factors over developmental time. I will perform the proposed experiments while also gaining experience and critical background in developmental biology and proteomics. I will also develop my teaching and mentoring skills through hands on experience and leadership/laboratory management courses. My proposed research will generate significant new experimental directions, and my proposed career development will support me, as I initiate my independent laboratory.