Mechanisms Driving Meiotic Chromosome Morphogenesis

项目摘要 这个建议的总体目标是促进我的愿望，成为一个主要的染色体研究者 在一所研究密集型大学学习生物学。这个项目将建立在我的研究生背景在哺乳动物 减数分裂，同时扩大我的技术能力，以促进我在其他染色体生物学的调查 例如人类疾病。本研究利用S.酿酒酵母研究染色体 发生在第一次减数分裂之前的动态。在此期间，细胞核进行重组， 同源染色体的排列同源染色体的正确排列取决于 一系列的分子途径，包括内源性DNA损伤和修复，检查点信号传导， 一种称为联会复合体（SC）的结构支架的构建。SC由两个侧面组成 每个染色体的沿着长度形成的元件和一个连接侧染色体的中心元件 同源染色体的成分。保守的轴蛋白Hop 1（HORMAD 1/2蛋白在小鼠和 人）是侧部元件的重要组成部分和SC组装的中心调节器。 尽管进行了广泛的研究，轴蛋白如何定位到染色质，然后招募中央 建立SC的要素仍不清楚。事实上，Hop 1的很大一部分从横向元件中分离出来 随着中心元素的沉积，提出了一个问题，Hop 1是SC的结构组成部分，还是 只需要介导中心元素的沉积。Aim 1利用条件蛋白诱导， 耗尽实验以测试SC形成中Hop 1的需求。它还将决定Hop 1的作用 在这个过程中的分裂。目的2：利用一种新的方法研究DNA与侧链元件之间的界面， 一种新的hop 1功能分离等位基因，表现出正常的DSB诱导和修复动力学，但改变了DNA 断裂模式，这可能反映了改变Hop 1染色质协会。HOP 1的这个等位基因去除了一个 Hop 1中心的未表征区域，与PHD结构域共享特征，这些结构域是典型的 对组蛋白标记的阅读和反应很重要。Aim 2-1使用这种hop 1突变体来确定 通过ChIP-seq的轴蛋白的轴模式，并将这些效应与改变的断裂模式相关联 通过全染色体Southern印迹分析观察。目标2-2利用靶向生物素化和质量 通过荧光光谱法确定Hop 1结合位点周围的染色质环境。这个实验将是 用于鉴定候选蛋白质，所述候选蛋白质将被测试在使用酵母的轴蛋白定位中的作用 删除库这一结果将为深入研究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，了解不孕症和出生缺陷， 人类这个建议有一个强大的培训组成部分，将扩大我的知识，以整个基因组 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 建立一个独立的染色体生物学研究计划所需的经验。

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.