Germline Silencing of Unpaired Chromatin

描述(由申请人提供)：该提案的总体目标是了解确保产生功能配子和健康后代的分子机制。该提案特别关注线虫线虫中未配对染色质的减数分裂沉默。减数分裂沉默是一种表观遗传调节机制，在哺乳动物和线虫中被描述为在染色质水平上抑制转录。虽然减数分裂沉默的自然目标是雄性染色体，但这一过程将被激活，以抑制任何未配对的染色体或染色体区域，这些染色体或区域是由于雌性或雄性减数分裂过程中的染色体突变或染色体重排而出现的。减数分裂沉默被认为在几种方式中起作用，以促进育性和配子质量。线虫减数分裂沉默的一个特征是与转录沉默相关的组蛋白修饰的积累，组蛋白H3在赖氨酸9(H3K9me2)上的二甲基化。我们发现线虫减数分裂过程中H3K9me2的积累取决于小RNA机制的几个组成部分的活性，包括：Ego-1，一种RNA导向的RNA聚合酶(RdRP)；CSR-1，RNA结合蛋白家族的成员；Ekl-1，一个都铎结构域蛋白；以及DRH-3，一个死盒解旋酶。除了在减数分裂沉默中发挥作用外，我们的遗传证据表明，这四种蛋白参与了生殖系发育和生育所需的功能途径。我们对线虫减数分裂沉默的分析为理解减数分裂沉默在生殖系发育中的作用、异染色质的组织特异性形成以及通过小RNA介导的染色质调节机制提供了一个模型。我们的数据使我们能够为H3K9me2在未配对的染色质上积累的机制生成替代模型。在这里，我们将测试这些模型的具体预测。在目标1中，我们将测试已知的小RNA介导途径的减数分裂沉默成分(EGO-1、CSR-1、EKL-1、DRH-3)如何与配对或未配对的染色体相关的替代假说。在目标2中，我们将测试如何将组蛋白甲基转移酶(HMTase)活性招募到未配对的染色体上的替代预测。这些研究是对目标1的补充。在目标3中，我们将确定H3K9me2在未配对染色体上积累的基因组位置，并利用这些信息来测试EGO-1、CSR-1、EKL-1和/或DRH-3与目标基因座的直接关联。这些研究将使我们能够改进和扩大在目标1中获得的数据。 公共卫生相关性：该提案涉及与生育和疾病抵抗力有关的问题。该项目将研究形成有功能的精子和卵子所需的过程，这也可能有助于降低后代对特定疾病的易感性。

DESCRIPTION (provided by applicant): The overall goal of the proposal is to understand the molecular mechanisms that ensure the production of functional gametes and healthy offspring. The proposal specifically focuses on meiotic silencing of unpaired chromatin in the nematode, Caenorhabditis elegans. Meiotic silencing is an epigenetic regulatory mechanism that has been described in mammals and nematodes as acting at the chromatin level to repress transcription. While the natural targets of meiotic silencing are the male sex chromosomes, the process will be activated to repress any unpaired chromosomes or chromosomal regions that arise due to mutation or chromosomal rearrangement during either female or male meiosis. Meiotic silencing is hypothesized to function in several ways to promote fertility and gamete quality. One hallmark of meiotic silencing in C. elegans, as in mammals, is the accumulation of a histone modification associated with transcriptional silencing, dimethylation of histone H3 on lysine 9 (H3K9me2). We have found meiotic H3K9me2 accumulation in C. elegans depends on the activity of several components of the small RNA machinery, including: EGO-1, an RNA-directed RNA polymerase (RdRP); CSR-1, a member of the Argonaute family of RNA-binding proteins; EKL-1, a Tudor domain protein; and DRH-3, a DEAD-box helicase. In addition to a role in meiotic silencing, our genetic evidence indicate that these four proteins participate in a functional pathway required for germline development and fertility. Our analysis of meiotic silencing in C. elegans provides a model for understanding the function of meiotic silencing in germline develoment, for tissue-specific formation of heterochromatin, and for chromatin regulation via small RNA-mediated mechanisms. Our data have allowed us to generate alternative models for the mechanism of H3K9me2 accumulation on unpaired chromatin. Here, we will test specific predictions of these models. In Aim 1, we will test alternative hypotheses for how known meiotic silencing components of the small RNA-mediated pathway (EGO-1, CSR-1, EKL-1, DRH-3) associate with paired or unpaired chromosomes. In Aim 2, we will test alternative predictions for how histone methyltransferase (HMTase) activity is recruited to unpaired chromosomes. These studies provide a complement to Aim 1. In Aim 3, we will determine the genomic sites of H3K9me2 accumulation on unpaired chromosomes and use this information to test for direct association of EGO-1, CSR-1, EKL-1, and/or DRH-3 with target loci. These studies will allow us to refine and extent the data obtained in Aim 1. PUBLIC HEALTH RELEVANCE: This proposal addresses questions related to fertility and resistance to disease. The project will investigate processes that are required for formation of functional sperm and eggs, and which may also help to reduce the susceptibility of offspring to particular diseases.