Epigenetic regulation of histone eviction in spermatogenesis

摘要 在美国，超过15%的夫妇患有不孕症。这些病例中有一半是由于男性 因素如精子计数低，精子形态和活力异常，或原因不明的不良 胚胎发生哺乳动物精子具有高度致密的核和染色质结构，只有5- 正常细胞组蛋白补体的15%。当核小体相关组蛋白 在减数分裂后的雄性生殖细胞内，通过组蛋白乙酰转移酶乙酰化以打开结构， 反过来，允许随后的驱逐和鱼精蛋白的替代-导致深刻的压实， 成熟精子精子中异常的组蛋白滞留可导致不育和胚胎发育不良， 暗示雄性配子的表观遗传调节是生殖的关键因素。我的初步数据 描绘了组蛋白翻译后修饰的动态变化，如乙酰化， 精子发生我的发现进一步证明了减数分裂前组蛋白的条件性缺失 乙酰转移酶Gcn 5导致异常精子，组蛋白保留增加。建议的目标 本研究旨在探讨组蛋白乙酰化在核小体驱逐中的功能和机制， 减数分裂的雄性生殖细胞，并确定是否过量保留的父系组蛋白的贡献， 胚胎转录、发育编程和胚胎发生。为了进行这些实验， 使用条件性小鼠模型通过消融组蛋白功能性干扰组蛋白乙酰化 减数分裂前雄性生殖细胞中的乙酰转移酶Gcn 5（Gcn 5cKO）。在具体目标1中，我将使用Gcn 5cKO小鼠 确定组蛋白乙酰化缺失导致染色质重塑改变的机制 和过量组蛋白保留在成熟精子，以及如何减少组蛋白乙酰化的后果 影响精子发生过程中的基因转录。在具体目标2中，我将研究，和早期的生育能力， 胚胎发育和转录，由此产生的Gcn 5cKO雄性小鼠，以考察 过量的父体组蛋白和相关的改变的父体染色质可及性。据我们所知，这将 是第一个有条件地消除减数分裂前生殖细胞中组蛋白乙酰转移酶的研究， 测试和调查功能后果。作为这些实验的结果，我们希望进一步我们的 了解组蛋白残基的乙酰化和组蛋白驱逐之间的联系，并确定是否 过量的父系组蛋白的传递影响后代的发育。此外，作为小鼠和人类， 精子发生和精子功能是高度保守的，这些实验的发现将是一个 了解，诊断和治疗男性不育症的宝贵信息来源。

ABSTRACT Over 15% of couples in the United States suffer from infertility. Half of these cases are due to male factors such as low sperm count, abnormal sperm morphology and motility, or unexplained poor embryogenesis. Mammalian sperm exhibit a highly compacted nuclear and chromatin structure with only 5- 15% of the normal cellular histone complement. This transition occurs when nucleosome-associated histones within post-meiotic male germ cells are acetylated by histone acetyltransferases to open the structure to, in turn, allow subsequent eviction and replacement with protamines - leading to the profound compaction in mature sperm. Abnormal histone retention in sperm can lead to infertility and poor embryogenesis, thus implicating epigenetic regulation of the male gamete as a critical factor in reproduction. My preliminary data depict dynamic changes in histone post-translational modifications, such as acetylation, during spermatogenesis. My findings further demonstrate that pre-meiotic conditional deletion of the histone acetyltransferase Gcn5 results in abnormal sperm with increased histone retention. The goal of the proposed study is to investigate the functional and mechanistic role of histone acetylation in nucleosome eviction in post- meiotic male germ cells, and to determine whether contribution of excess retained paternal histones affects embryonic transcription, developmental programming and embryogenesis. To conduct these experiments I will use the conditional mouse model to functionally perturb histone acetylation through ablation of the histone acetyltransferase Gcn5 in pre-meiotic male germ cells (Gcn5cKO). In specific aim 1, I will use the Gcn5cKO mice to determine the mechanisms through which loss of histone acetylation leads to altered chromatin remodeling and excess histone retention in mature sperm, and how the consequences of reduced histone acetylation affects gene transcription during spermatogenesis. In specific aim 2, I will examine fertility of, and early embryonic development and transcription, resulting from the Gcn5cKO male mice to investigate the effect of excess paternal histones and associated altered paternal chromatin accessibility. To our knowledge, this will be the first investigation to conditionally ablate a histone acetyltransferase in pre-meiotic germ cells in the testes and investigate the functional consequences. As a result of these experiments we expect to further our understanding of the link between acetylation of histone residues and histone eviction and determine if transmission of excess paternal histones affects offspring development. Furthermore, as mouse and human spermatogenesis and sperm function are highly conserved, the findings of these experiments will be an invaluable source of information for understanding, diagnosing, and treating male infertility.