Mechanisms of sperm chromatin remodeling in vivo and in vitro

精子染色质体内外重塑机制

基本信息

批准号：
10753662
负责人：
Saher Sue Hammoud
金额：
$ 53.96万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-15 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10753662
关键词：
ATP phosphohydrolase Affect Antibodies Appearance Back Binding Biochemical Biological Assay Cells ChIP-seq Chromatin Chromatin Remodeling Factor Complex Couples DNA DNA Packaging Data Deposition Development Diagnostic Epitopes Etiology Event Excision Fertility Generations Genetic Genome Genomic Segment Genotype Germ Cells Global Change Goals Haploidy Histones ISWI Immunoprecipitation In Vitro Infertility Investigation Kinetics Knock-out Knowledge Label Mass Spectrum Analysis Mediating Meiosis Methods Mitosis Modeling Molecular Molecular Chaperones Mus Nucleosomes Oocytes PRM2 gene Pattern Phenotype Physical condensation Population Positioning Attribute Process Production Protamines Protein Subunits Proteins Publishing Reagent Recombinant Proteins Role SMARCA5 gene Sexual Reproduction Slide Somatic Cell Spermatids Spermiogenesis Testing Testis Woman biochemical tools biophysical properties biophysical tools chromatin remodeling effective therapy egg genomic tools in vivo insight laser tweezer loss of function men mutant novel optic tweezer programs protamine 1 public health relevance reconstitution reproductive fitness single molecule sperm cell

项目摘要

Abstract Unlike oocytes (and all somatic cells), which package their DNA with histones, the DNA of mature sperm is bound by protamines, a highly basic and rapidly evolving protein that is essential for compaction of the paternal genome. This differential packaging of egg and sperm DNA traces back >500 million years, yet our understanding of the principles of genome organization remains poorly understood. To examine this fundamental yet poorly understood process we have developed novel genetic and biophysical tools to identify proteins implicated in the histone-to-protamine exchange and to dissect the molecular mechanisms of chromatin reorganization during spermiogenesis. In preliminary studies we developed epitope-tagged protamine 1 and 2 mice and relevant reagents such as specific antibodies. Immunoprecipitation followed by mass spectrometry has identified multiple potential chromatin remodelers/chaperones, which we will functionally characterize in this proposed study, both in vivo and in vitro. Since protamine packaging results in a super-condensed chromatin state, which does not easily lend itself to mechanistic investigations in vivo, we have developed novel methods to purify protamines from sperm to use in both in vitro DNA curtain assays and in vitro reconstitution assays of the histone-to-protamine exchange process. The overall goal of this proposal is to apply our diverse suite of genetic, molecular, and biophysical tools to gain mechanistic insights into normal sperm chromatin packaging, which is essential for genome fidelity and reproductive fitness of the species.

抽象的与用组蛋白包装 DNA 的卵母细胞（以及所有体细胞）不同，成熟精子的 DNA 是与鱼精蛋白结合，鱼精蛋白是一种高度碱性且快速进化的蛋白质，对于父体的压缩至关重要基因组。卵子和精子 DNA 的这种差异包装可以追溯到 5 亿年前，但我们的对基因组组织原理的理解仍然知之甚少。为了检验这个基本然而人们对这个过程知之甚少，我们开发了新的遗传和生物物理工具来识别蛋白质参与组蛋白与鱼精蛋白的交换并剖析染色质的分子机制精子发生过程中的重组。在初步研究中，我们开发了表位标记的鱼精蛋白 1 和 2 小鼠及特异性抗体等相关试剂。免疫沉淀随后进行质谱分析确定了多个潜在的染色质重塑剂/伴侣，我们将在本文中对其进行功能表征拟议的体内和体外研究。由于鱼精蛋白包装会产生超浓缩的染色质状态，这不容易适合体内机械研究，我们开发了新方法从精子中纯化鱼精蛋白，用于体外 DNA 帘式测定和体外重组测定组蛋白与鱼精蛋白的交换过程。该提案的总体目标是应用我们多样化的套件遗传、分子和生物物理工具，以获得正常精子染色质包装的机制见解，这对于物种的基因组保真度和繁殖适应性至关重要。