Mechanisms of Nuclear Body Formation in the Early Embryo

摘要： 在合子基因组开始启动的发育中的胚胎中，协调的基因调控尤其关键。 这是第一次抄写。促进协调基因调控的一种保守策略是形成 被称为核体的保守结构，亚核区，在那里特定的因子积累到 执行调节和转录功能。组蛋白基因座小体是一种保守的核小体 这调节组蛋白基因，我发现锌指蛋白，CLAMP(染色质连接 MSL蛋白的接头)，对果蝇HLB的调节是必不可少的。我的研究现在的目标是 解决关键问题：夹具如何以特定于上下文的方式启动核机构 合子基因组激活关键发育阶段的形成？ 在这个项目的目标1中，我将确定钳子在染色质可及性和因子招募过程中的作用 合子基因组激活。我会从性别鉴定的胚胎池中进行atac-seq和转录因子芯片-seq 在有无钳制的情况下，合子基因组激活前后的时间点。我会收到 我的导师，布朗大学的Erica Larschan博士的指导。这些实验将解决如何 染色质状态影响核体的形成以及在ZGA时染色质的组成如何变化 核物体具有其独特的性质。在目标2中，我将使用基因工具来确定发育 HLB处的钳位的时序要求和允许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我的共同导师罗伯特·杜罗尼奥博士的指导下。这 AIM将对整个开发过程中HLB的启动和维护机制(S)进行比较。在《目标3》中我会 在存在和不存在的情况下对早期胚胎中分离的染色质片段进行蛋白质组学研究 钳制，然后进行质谱分析。我将确定未知的HLB组件及其依赖关系 在普林斯顿大学我的共同导师Paul Schedl博士的指导下，谈到了Clip招聘。这一目标 将阐明早期胚胎核体形成的机制，包括有序招募 随着发育时间的推移，已知的和新的因素。 我将执行拟议的实验，同时在以下方面获得经验和关键背景 发育生物学和蛋白质组学。我还将通过亲身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。我提出的研究将产生重要的 新的实验方向和我提议的职业发展将支持我，因为我开始 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.