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。该建议特别关注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控机制，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男性性染色体，但该过程将被激活以抑制由于女性或男性减数分裂期间突变或染色体重排而产生的任何未配对染色体或染色体区域。据推测，减数分裂沉默在促进生育和配子质量方面有多种作用。与哺乳动物一样，秀丽隐杆线虫减数分裂沉默的一个标志是与转录沉默相关的组蛋白修饰的积累，即赖氨酸9上组蛋白H3的二甲基化（H3K9me2）。我们发现秀丽隐杆线虫的减数分裂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定向RNA聚合酶（RdRP）；rna结合蛋白Argonaute家族成员CSR-1；都铎结构域蛋白EKL-1；以及DRH-3，一种DEAD-box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，这四种蛋白参与生殖细胞发育和生育所需的功能途径。我们对秀丽隐杆线虫减数分裂沉默的分析为理解减数分裂沉默在种系发育中的功能、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生成H3K9me2在未配对染色质上积累的机制的替代模型。在这里，我们将测试这些模型的具体预测。在目的1中，我们将测试小rna介导途径的已知减数分裂沉默成分（EGO-1, CSR-1, EKL-1, DRH-3）如何与配对或未配对染色体相关联的其他假设。在目标2中，我们将测试组蛋白甲基转移酶（HMTase）活性如何被募集到未配对染色体的替代预测。这些研究为目的1提供了补充。在Aim 3中，我们将确定H3K9me2在未配对染色体上积累的基因组位点，并利用该信息检测EGO-1、CSR-1、EKL-1和/或DRH-3与靶位点的直接关联。这些研究将使我们能够完善和扩展Aim 1中获得的数据。

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.