Mechanisms of sperm chromatin remodeling in vivo and in vitro

精子染色质体内外重塑机制

• 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
• 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

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.

摘要 与卵母细胞（和所有体细胞）不同，成熟精子的DNA是用组蛋白包装的。 由鱼精蛋白结合，鱼精蛋白是一种高度碱性和快速进化的蛋白质，对父亲的致密化至关重要 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，但我们的研究结果表明， 对基因组组织原理的理解仍然知之甚少。为了检验这个基本的 尽管我们对这一过程知之甚少，但我们已经开发出了新的遗传和生物物理工具来识别蛋白质。 涉及组蛋白-鱼精蛋白交换，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 小鼠和相关试剂如特异性抗体。免疫沉淀法和质谱法 确定了多种潜在的染色质重塑/分子伴侣，我们将在本研究中对其进行功能表征。 建议的研究，包括体内和体外研究。由于鱼精蛋白包装导致超浓缩的染色质 国家，这并不容易借给自己的机制调查在体内，我们已经开发了新的方法 从精子中纯化鱼精蛋白，以用于体外DNA帘幕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的多样化套件， 遗传、分子和生物物理学工具，以获得对正常精子染色质包装的机械见解， 这对于物种的基因组保真度和生殖适应性是必不可少的。