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

摘要 不孕症影响着美国大约15%的夫妇，其中男性占近10%。 其中50%的案件。不育的一个潜在机制是父系表观基因组的改变。 哺乳动物精子具有独特的、高度致密和浓缩的DNA结构， 依赖于表观遗传机制，包括组蛋白超乙酰化，然后是核小体驱逐。 具体来说，90-99%的精子核小体被驱逐并被鱼精蛋白取代， 压实度非常高。显示鱼精蛋白或过量组蛋白比例改变的人精子 尿潴留与IVF/ICSI后的不育和胚胎发生改变有关。但具体 异常核小体滞留导致胚胎发生改变的原因，最终， 父亲贡献的组蛋白影响早期发育的调节功能和机制， 仍然在很大程度上未知。我们之前已经开发了一种条件性小鼠突变体， 乙酰转移酶Gcn 5在减数分裂前生殖细胞中被消除（Gcn 5cKO）。该模型的一个相关特征是 成熟的精子会增加组蛋白的保留并降低生育能力。该模型提供了以下能力： 研究父核小体对胚胎的影响，并确定 异常的父核小体对胚胎发育的影响。我们建议父亲 表观基因组，特别是核小体，在调节早期胚胎染色质中起作用， 转录动力学，从而导致适当的胚胎发育。 我们将利用我们的Gcn 5cKO小鼠模型引入精子核小体的异常补充 （1）确定是否存在过量的父本核小体， 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 保留的父本核小体改变植入前胚胎中的染色质动力学和转录。 总之，拟议的研究将提供重要的洞察机制，管理早期胚胎 发育，包括父本表观基因组对染色质动态和合子基因组的影响 activation.它还将提供证据，为什么异常的父亲染色质导致不育， 改变了人类的胚胎发育这些研究的结果有可能最终影响临床 管理被诊断为不孕症的患者。

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.