Mechanisms Driving Meiotic Chromosome Morphogenesis

项目摘要 这项提议的总体目标是促进我成为一名主要的染色体研究者的愿望。 在一所研究密集型大学学习生物学。这个项目将建立在我在哺乳动物方面的研究生背景之上。 在减数分裂的同时扩大我的技术能力，以促进我在其他物种的染色体生物学研究 环境，如人类疾病。这项拟议的研究利用酿酒酵母研究染色体。 发生在第一次减数分裂之前的动力学。在这段时间内，核被重新组织以允许 同源染色体的排列。同源染色体的正确配对取决于 一系列分子通路，包括内源性DNA损伤和修复、检查点信号转导和 构建一种称为联会复合体(SC)的结构支架。SC由两个侧面组成 沿着每条染色体的长度形成的元件和连接侧生染色体的一个中心元件 同源染色体的元素。保守的轴蛋白Hop1(HORMAD1/2蛋白在小鼠和 人类)是侧向元件的重要组成部分，也是SC组装的中央调节因子。 尽管进行了广泛的研究，但Axis蛋白如何定位到染色质，然后招募中央 建造SC的要素仍不清楚。事实上，Hop1的很大一部分是从侧向分子中解离出来的 随着中心元素的沉积，提出了一个问题：Hop1是SC的结构组件还是 只需要调节中心元素的沉积。AIM 1利用条件蛋白诱导和 耗竭实验以测试SC地层对Hop1的要求。它还将决定Hop1的作用 在这个过程中的解离。目的2研究DNA与侧向元件之间的界面 表现出正常的DSB诱导和修复动力学但DNA改变的新的Hop1分离功能等位基因 断裂模式，这可能反映了改变的Hop1染色质关联。HOP1的这个等位基因移除了一个 Hop1中心的未特定化区域，与规范的PhD域共享特征 对于阅读和响应组蛋白标记很重要。AIM 2-1使用该Hop1突变体来确定影响 通过芯片序列对AXIS蛋白质的AXIS图案化并将这些效应与改变的断裂图案化相关联 通过全染色体Southern印迹分析观察。AIM 2-2利用靶向生物素化和质量 以确定Hop1结合位点周围的染色质环境。这项实验将是 用于确定候选蛋白，这些候选蛋白将使用酵母进行AXIS蛋白定位 删除库。这一建议的结果将为了解减数分裂的机制提供重要的新见解。 染色体形态发生，并将作为一个框架，以了解不孕症和出生缺陷 人类。这项提议有很强的训练成分，将把我的知识扩展到整个基因组 测序技术、计算生物学和蛋白质组学。总之，这次培训将为我提供 建立独立的染色体生物学研究项目所需的经验。

Project Summary The overall goal of this proposal is to facilitate my aspiration to become a primary investigator of chromosome biology at a research-intensive university. This project will build on my graduate background in mammalian meiosis while expanding my technical abilities to facilitate my investigation of chromosome biology in other contexts, such as human disease. The proposed research utilizes S. cerevisiae to investigate the chromosome dynamics that occur prior to the first meiotic division. During this time, the nucleus is reorganized to allow for the alignment of homologous chromosomes. The proper alignment of homologous chromosomes depends on a series of molecular pathways, including endogenous DNA damage and repair, checkpoint signaling, and the construction of a structural scaffold known as the synaptonemal complex (SC). The SC is made of two lateral elements that form along the lengths of each chromosome and one central element that connect the lateral elements of homologous chromosomes. The conserved axis proteins Hop1 (HORMAD1/2 proteins in mice and humans) are an essential component of the lateral element and a central regulator of SC assembly. Despite extensive research, how the axis proteins localize to chromatin and then recruit the central element to build the SC remains unclear. Indeed, a large fraction of Hop1 dissociate from the lateral elements as the central element is deposited, raising the question whether Hop1 is a structural component of the SC or only needed to mediate deposition of the central element. Aim 1 utilizes conditional protein induction and depletion experiments to test the requirements of Hop1 in SC formation. It will also determine the role of Hop1 dissociation in this process. Aim 2 investigates the interface between DNA and the lateral element by using a novel hop1 separation-of-function allele that exhibits normal DSB induction and repair kinetics but altered DNA breakage patterns, which likely reflect altered Hop1 chromatin association. This allele of HOP1 removes an uncharacterized region in the center of Hop1 that shares features with PHD domains, which are canonically important for reading and responding to histone marks. Aim 2-1 uses this hop1 mutant to determine the effects the axis patterning by ChIP-seq of axis proteins and to correlate these effects with altered break patterning observed by whole-chromosome Southern blot analysis. Aim 2-2 utilizes targeted biotinylation and mass spectrometry to define the chromatin environment surrounding Hop1 binding sites. This experiment will be used to identify candidate proteins that will be tested for a role in axis protein positioning using the yeast deletion library. The results of this proposal will provide important new insight into the mechanism of meiotic chromosome morphogenesis and will serve as a framework for understanding infertility and birth defects in humans. This proposal has a strong training component and will expand my knowledge to whole genome sequencing techniques, computational biology, and proteomics. Altogether, this training will provide me with the experience needed for establishing an independent research program in chromosome biology.