Molecular mechanisms for germline genome activation in C. elegans

项目摘要/摘要 我们正在研究一种广泛的、高度保守但知之甚少的转录调控形式。 在称为基因组激活的过程中，全球水平上的基因表达突然上调。我们的 系统是线虫的发育中的种系，因此我们使用遗传学、基因组学和 细胞学来研究基因组激活是如何被控制的。之前发布的和初步数据来自我们的 研究小组概述了生殖系基因组激活的遗传途径，拓扑异构酶II(TOP-2) 酶以一种信号调节的方式被激活，以产生程序性DNA断裂。这些活动的目的是 Break是将Tip60/RUVB调节因子招募到染色质上，从而使基因组发生解压缩，从而 促进基因组激活。 在这个提案中，我们研究了我们已经发现的基因组激活途径的三个不同的组成部分。 首先，我们研究了原始生殖细胞(PGC)在激活前如何建立基因组结构。我们的 初步数据表明，全基因组异染色化是这种结构被 以及在静息的PGCs中mRNA转录被全局抑制的方法。我们是 不知道在真核生物学中有任何其他例子，在这种情况下异染色质形成 因此，这些实验很可能为基因如何表达提供一个新的范例 在静息细胞中被整体抑制。 我们还将研究TOP-2是如何通过信号继续并在生殖系中诱导DNA断裂而被激活的 基因组。一段时间以来，人们已经意识到，在基因组中发生程序性DNA断裂 减数分裂，然而，我们发现的程序性中断发生在生殖系发育的更早时间是 这是史无前例的，值得详细调查。我们计划研究TOP-2是如何监管的，我们还将 确定断裂发生在基因组的哪个位置。这些实验的成功完成将提供 一种新的程序性断裂形成机制，可能与不同类型的细胞和 有机体。 最后一组实验将研究基因组解压是如何发生的。我们的数据显示，鞋底 TOP-2诱导的中断的目的是将Tip60/RUVB染色质调节因子重新招募到DNA上，从而 可能会发生解压缩。PGCS为什么采取如此极端的措施--故意诱导一种危险形式 它们基因组中DNA损伤的可能性--就染色质分解而言，这是一个有趣的问题。至 为此，我们将研究破碎的DNA如何招募Tip60/RUVB，以及复合体如何分解 染色质。我们将确定工程DNA断裂和靶基因定位的距离阈值 激活。最后，我们将研究核内基因移动性与 解压，这种流动性对转录激活很重要。

Project Summary/Abstract We are investigating a widespread and highly conserved, yet poorly understood, form of transcriptional control whereby gene expression on a global level is abruptly upregulated in a process termed genome activation. Our system is the developing germline of the nematode C. elegans, and thus we use genetics, genomics, and cytology to study how genome activation is controlled. Previously published and preliminary data from our group have outlined a genetic pathway for germline genome activation, whereby the topoisomerase II (TOP-2) enzyme is activated, in a signal-mediated manner, to produce programmed DNA breaks. The purpose of these breaks is to recruit the TIP60/RUVB regulator to chromatin, so that genome decompaction occurs, thereby facilitating genome activation. In this proposal we study three distinct components of the genome activation pathway that we have discovered. First, we study how genome architecture is established in primordial germ cells (PGCs) prior to activation. Our preliminary data suggest that whole-genome heterochromatization is the means by which this architecture is established and the means by which mRNA transcription is globally repressed in resting PGCs. We are unaware of any other examples in eukaryotic biology where heterochromatin formation is employed on such a grand scale, and thus these experiments are likely to supply a novel paradigm for how gene expression can be globally repressed in resting cells. We will also examine how TOP-2 is activated by signaling to go on and induce DNA breaks in the germline genome. It has been appreciated for some time that programmed DNA breaks occur in the genome during meiosis, however our discovery of programmed breaks occurring much earlier in germline development is unprecedented and worthy of detailed investigation. We plan to study how TOP-2 is regulated and we will also determine where in the genome the breaks are made. Successful completion of these experiments will provide a novel mechanism for programmed break formation that is likely to be relevant across cell types and organisms. A final set of experiments will examine how genome decompaction occurs. Our data show that the sole purpose of TOP-2 induced breaks is to recruit the TIP60/RUVB chromatin regulator to DNA so that decompaction can occur. Why PGCs take such extreme measures -- intentionally inducing a dangerous form of DNA damage into their genomes -- for the purpose of chromatin decompaction is a fascinating question. To get at this we will study how broken DNA recruits TIP60/RUVB and how the complex then decompacts chromatin. We will determine distance thresholds for positioning of engineered DNA breaks and target gene activation. Lastly, we will examine the exciting possibility that gene mobility within the nucleus is coupled to decompaction, and that mobility is important for transcriptional activation.