Mechanisms of sperm chromatin remodeling in vivo and in vitro

摘要 不像卵母细胞(和所有体细胞)用组蛋白包装它们的DNA，成熟精子的DNA是 鱼精蛋白是一种高度碱性和快速进化的蛋白质，对父系的紧凑是必不可少的 基因组。卵子和精子DNA的这种不同包装可以追溯到5亿年前，但我们的 对基因组组织原理的理解仍然很少。要考察这一基本原理 然而，对过程知之甚少，我们已经开发出新的遗传和生物物理工具来识别蛋白质 与组蛋白到鱼精蛋白的交换有关，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。免疫沉淀和质谱分析 确定了多个潜在的染色质重构体/伴侣，我们将在此对其进行功能性表征 建议进行体内和体外研究。由于鱼精蛋白包装会导致染色质超浓缩 状态，这不容易适合在活体内进行机械性研究，我们开发了新的方法 从精子中提纯精蛋白以用于体外DNA窗帘试验和体外重组试验 组蛋白到鱼精蛋白的交换过程。这项提案的总体目标是应用我们多样化的 遗传、分子和生物物理工具，以获得对正常精子染色质包装的机械洞察， 这对物种的基因组保真度和生殖适合性是必不可少的。

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.