Programmed DNA Elimination in Parasitic Nematodes

项目概要/摘要 蛔虫仍然是世界许多地区的一个重大健康问题，感染了近10亿人， 其中许多是儿童，导致发病率很高。相关的寄生线虫弓首线虫， 在美国，人类弓形虫病也越来越被认为是一个重要的公共卫生问题。 States.这些寄生线虫经历了一种新形式的基因组重组，称为程序化重组。 DNA消除。关于这种DNA消除的功能和分子机制知之甚少 过程我们最近的工作为这一过程提供了基本的见解，表明DNA消除 是一组寄生线虫中的保守过程，用于沉默种系表达的基因， 特别是睾丸特异性基因，通过它们在体细胞中的消除。此外，我们还定义了如何 全着丝粒染色体被动态重组以确定将被保留或 淘汰在蛔虫DNA消除过程中，DNA双链断裂需要产生染色体 这些碎片将被清除。我们发现了72个端粒附加位点， 发生断裂。这些位点在不同的体细胞谱系中表现出高保真度，并且蠕虫被限制在3- 6 kb基因组区域，称为染色体断裂区域。我们的分析没有发现任何特定的 序列、结构特征或可能指定或招募分子机制的常见表观遗传因子 到这些地区。然而，这些断裂区域的一个突出特征是它们变得更染色质化 就在DNA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复制应激，以及三维基因组组织 可能有助于启动DNA消除的关键DNA断裂。我们将在三个方面来检验这些假设。 具体目标：（1）.定义在细胞周期中何时发生DNA断裂和端粒增加，从而导致 DNA消除有丝分裂;定义DNA复制时间和压力，RNA转录和R环， DNA消除;和（3）.绘制断裂区域的3D基因组组织图。除了一个 了解DNA断裂的分子机制，蛔虫DNA消除，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愈合。 对这些生物过程的深入了解可能会发现可能成为潜在药物的新分子过程。 寄生线虫中的靶标。

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.