Programmed DNA Elimination in Parasitic Nematodes

项目摘要/摘要 蛔虫在世界许多地区仍然是一个严重的健康问题，感染了近10亿人， 其中许多是儿童，导致严重的发病率。与之相关的寄生线虫弓形虫，引起 在美国，人类弓形虫病也越来越被认为是一个重大的公共卫生问题 各州。这些寄生线虫经历一种新的基因组重组形式，称为程序化 DNA消除。关于这种DNA消除的功能和分子机制，人们知之甚少 进程。我们最近的工作提供了对这一过程的基本见解，证明了DNA消除 在一组寄生线虫中是一个保守的过程，用于沉默种系表达的基因， 特别是睾丸特异基因，通过它们在体细胞中的消除。此外，我们还定义了 全着丝粒染色体被动态重组，以确定将被保留或 被淘汰了。在消除蛔虫DNA的过程中，需要DNA双链断裂来产生染色体 将被消除的碎片。我们确定了72个端粒添加位点，这些位点可能是染色体 就会出现中断。这些位置在不同的体细胞谱系中表现出高度的保真度，蠕虫被限制在3- 6kb的基因组区域，称为染色体断裂区。我们的分析没有发现任何具体的 可能指定或招募分子机制的序列、结构特征或常见的表观遗传因素 到这些地区。然而，这些破碎区的一个显著特征是它们变得更多染色质 就在脱氧核糖核酸消除之前。开放染色质通常是分子过程的结果 基因组，包括DNA复制和RNA转录。在这个提议中，我们假设RNA 转录、R-环和/或DNA复制压力，以及三维基因组组织 可能对启动DNA消除的关键DNA断裂起作用。我们将分三个阶段检验这些假说。 具体目标：(1)。确定DNA断裂和端粒添加在细胞周期中何时发生，从而导致 DNA消除有丝分裂；(2)。确定DNA复制时间和压力、RNA转录和R-环 DNA消除；和(3)。绘制休止区的3D基因组组织图。除了 了解DNA断裂消除蛔虫DNA的分子机制，建议 研究将为寄生线虫染色体和染色质提供新的信息和见解 组织，RNA转录，DNA复制，DNA断裂和末端切除，以及端粒修复。 对这些生物过程的洞察可能会发现可能成为潜在药物的新的分子过程 寄生线虫的靶标。

Project Summary/Abstract Ascaris remains a significant health problem in many parts of the world, infecting close to a billion people, many of them children, leading to significant morbidity. The related parasitic nematode Toxocara, causing human toxocariasis, also is increasingly being recognized as a significant public health problem in the United States. These parasitic nematodes undergo a novel form of genome re-organization, known as programmed DNA elimination. Very little is known regarding the function and molecular mechanisms of this DNA elimination process. Our recent work provides fundamental insights into this process demonstrating that DNA elimination is a conserved process in a group of parasitic nematodes serving to silence germline-expressed genes, particularly testis-specific genes, through their elimination in the somatic cells. Furthermore, we defined how holocentric chromosomes are dynamically re-organized to determine chromosomes regions that will be kept or eliminated. During Ascaris DNA elimination, DNA double-strand breaks are required to generate chromosomal fragments that will be eliminated. We identified 72 telomere addition sites where the presumed chromosomal breaks occur. These sites exhibit high fidelity in different somatic cell lineages and worms constrained within 3- 6 kb genomic regions and are called chromosomal breakage regions. Our analysis did not identify any specific sequence, structural features or common epigenetic factors that might specify or recruit molecular machinery to these regions. However, a prominent feature for these break regions is they become more chromatin accessible just before DNA elimination. Open chromatin is often a result of molecular processes that access the genome, including DNA replication and RNA transcription. In this proposal, we hypothesize that RNA transcription, R-loops, and/or DNA replication stress, as well as the three-dimensional genome organization may contribute to the key DNA breaks that initiate DNA elimination. We will examine these hypotheses in three specific aims: (1). Define when DNA break and telomere addition occurs during the cell cycle that leads to a DNA elimination mitosis; (2). Define DNA replication timing and stress, RNA transcription, and R-loops during DNA elimination; and (3). Map the 3D genome organization of the break regions. In addition to an understanding of the molecular mechanisms of the DNA breaks for Ascaris DNA elimination, the proposed studies will provide new information and insights into parasitic nematode chromosomes and chromatin organization, RNA transcription, DNA replication, DNA breaks and end resection, and telomere healing. Insights into these biological processes may identify novel molecular processes that could be potential drug targets in parasitic nematodes.