Investigating the contribution of paternal nucleosomes using the Gcn5 knock-out mouse

摘要 在美国，大约15%的夫妇受到不孕不育的影响，其中近 这些案件中有50%。不孕症的一个潜在机制是父系表观基因组的改变。 哺乳动物精子表现出一种独特的、高度紧凑和浓缩的DNA结构，这种结构强烈地 依赖于表观遗传机制，包括组蛋白超乙酰化和随后的核小体驱逐。 具体地说，90%-99%的精子核小体被驱逐并被鱼精蛋白取代，从而实现了这一点 压实程度惊人。人类精子的精蛋白比例发生改变或组蛋白过多 保留与IVF/ICSI后的不孕症和胚胎发生改变有关。然而，准确的 由于核小体异常滞留而导致的胚胎发育改变的原因，最终是潜在的 父系贡献的组蛋白的调节功能和机制影响早期发育， 在很大程度上仍然不为人知。我们之前已经开发出一种有条件的小鼠突变体，其中组蛋白 乙酰基转移酶Gcn5在减数分裂前生殖细胞(Gcn5cKO)中被烧毁。该模型的一个相关特性是 成熟的精子增加了组蛋白的保留，降低了生育力。该模型提供了以下功能 研究父性核小体对胚胎的影响，并确定 父系核小体异常对胚胎发育的影响。我们建议这位父亲 表观基因组，特别是核小体，在调节早期胚胎染色质和 转录动力学，从而导致适当的胚胎发育。 我们将利用我们的Gcn5cKO小鼠模型来引入精子核小体的异常补充 并研究以下特定目的：(1)确定父系核小体是否过剩 改变植入前胚胎的动力学和成功发育以及(2)确定是否异常 保留的父系核小体改变着植入前胚胎的染色质动态和转录。 总之，拟议的研究将为早期胚胎的管理机制提供重要的见解。 发育，包括父系表观基因组对染色质动力学和合子基因组的影响 激活。它还将提供证据，说明为什么父系染色质异常会导致不育和 改变了人类的胚胎发生。这些研究的结果有可能最终影响临床 诊断为不孕不育的患者的处理。

ABSTRACT Infertility affects approximately 15% of couples in the United States, with males contributing in nearly 50% of these cases. One potential mechanism underlying infertility are alterations to the paternal epigenome. Mammalian spermatozoa exhibit a unique, highly compacted and condensed DNA structure that is strongly dependent on epigenetic mechanisms, including histone hyperacetylation followed by nucleosome eviction. Specifically, 90-99% of sperm nucleosomes are evicted and replaced with protamines, allowing for this remarkable degree of compaction. Human sperm exhibiting altered ratios of protamines or excess histone retention are associated with infertility and altered embryogenesis following IVF/ICSI. However, the exact cause of altered embryogenesis as a result of abnormal nucleosome retention, and ultimately, potential regulatory functions and mechanisms by which paternally contributed histones affect early development, remain largely unknown. We have previously developed a conditional mouse mutant where the histone acetyltransferase Gcn5 is ablated in pre-meiotic germ cells (Gcn5cKO). One relevant feature of this model is that mature sperm have increased histone retention and decreased fertility. This model provides the ability to study the effect of paternal nucleosome contributions to the embryo, and determine the consequences of abnormal paternal nucleosome contribution on embryonic development. We propose that the paternal epigenome, specifically nucleosomes, play a role in regulating early embryonic chromatin and transcriptional dynamics, thus leading to proper embryonic development. We will utilize our Gcn5cKO mouse model to introduce an abnormal complement of sperm nucleosomes into embryos and investigate the following Specific Aims: (1) To determine if excess paternal nucleosomes alter the kinetics and successful development of pre-implantation embryos and (2) determine if abnormally retained paternal nucleosomes alter chromatin dynamics and transcription in pre-implantation embryos. Together, the proposed research will provide important insight into the mechanisms governing early embryonic development, including the effects of the paternal epigenome on chromatin dynamics and zygotic genome activation. It will additionally provide evidence as to why abnormal paternal chromatin results in infertility and altered embryogenesis in humans. The results of these studies have the potential to ultimately impact clinical management of patients diagnosed with infertility.