Chromatin diminution in nematodes

 描述(申请人提供)：蛔虫是一种重要的人类病原体，感染约10亿人。了解蛔虫的基因组维持、基因组改变以及这些改变在基因表达中的作用，对于了解其生物学和发病机制具有重要意义。基因组很少发生变化。然而，少数生物经历了被称为染色质减少的大规模基因组变化，这是一种从基因组中消除特定DNA序列的程序化过程。这一现象的许多方面仍然是一个谜。我们以前利用新技术来表征寄生线虫蛔虫染色质的减少，我们证明了在第三到第五次分裂(4到16细胞阶段)期间，寄生线虫猪蛔虫13%的基因组在体细胞谱系中被消除，而种系基因组保持不变。独特的序列(包括~700个基因)在染色质减少以形成体细胞基因组的过程中丢失。我们鉴定的被剔除的基因主要在蛔虫胚系和早期胚胎中表达。这导致我们认为，蛔虫染色质的减少是在体细胞组织中沉默胚系和早期胚胎表达基因的基本的、不可逆转的机制，这是区分胚系和胞体所必需的。在首次发现蛔虫100多年后，如何针对特定的蛔虫染色体区域并选择消除或保留的机制仍不清楚。两个关键的机制问题是：1)染色体断裂和边界区域的位置是如何识别、标记和切割的？2)是什么决定了哪些染色体区域被保留或丢失？我们已经根据我们的初步数据就这些关键问题制定了假设，并建议在特定的目标下检验这些假设。在初步实验中，我们已经证明，在蛔虫染色质减少的过程中，只有那些将被保留的染色体具有广泛的CENP-A沉积(着丝粒形成和着丝粒组装所需的关键因素)，而要被消除的染色体具有很少的CENP-A。这为染色体的特定部分如何标记为保留或消除提供了一种潜在的机制解释。我们建议研究CENP-A的沉积和作用，作为染色质减少中染色体差异分离的机制。我们进一步假设小RNA和Argavies在蛔虫染色质减少中起作用。我们已经证明，专门化的宇航员与将保留在蛔虫减数有丝分裂中的染色体相关联，但不与注定要被消除的染色体相关联。我们建议研究这些Argavies及其相关的小RNA在线虫染色质减少和染色体分离中的作用。对蛔虫染色质减少的分析有望为深入了解基因组稳定性、着丝粒指定、染色体分离以及Argavites和小RNA对染色体功能、分离和染色质减少的贡献提供深刻的见解。我们的研究不仅将提供对DNA消除及其在线虫中的重要性的洞察，而且可能增加我们对生殖系、染色体和一般基因组生物学的理解。最后，更好地了解蛔虫染色体的组织和分离及其不同于人类宿主的小RNA途径，可能会为所需的药物靶点提供新的见解。

 DESCRIPTION (provided by applicant): Ascaris is an important human pathogen infecting ~1 billion people. Understanding genome maintenance, genome alterations, and the role of these alterations in gene expression in Ascaris is important for understanding its biology and pathogenesis. Genomes rarely change. However, a few organisms undergo the wholesale genome change called chromatin diminution, a programmed process that eliminates specific DNA sequences from the genome. Many aspects of the phenomenon remain a mystery. We previously leveraged new technologies to characterize chromatin diminution in the parasitic nematode, Ascaris, We demonstrated that thirteen percent of the genome of the parasitic nematode Ascaris suum is eliminated in somatic cell lineages during the third through fifth cleavage (4 to 16 cell stage), while the germline genome remains intact. Unique sequences (including ~700 genes) are lost during chromatin diminution to form the somatic genome. The eliminated genes we identified are primarily expressed in the Ascaris germline and early embryo. This leads us to suggest that chromatin diminution in Ascaris is the essential, irreversible mechanism for silencing a subset of germline and early embryo expressed genes in somatic tissues required for distinguishing between the germline and soma. More than 100 years after it was first discovered, the mechanisms for how specific Ascaris chromosomal regions are targeted and selected for elimination or retention remain unknown. Two key mechanistic questions are: 1) How are the locations of the chromosomal breaks and boundary regions identified, marked, and cut? 2) What determines which chromosomal regions are retained or lost? We have formulated hypotheses regarding these key questions based on our preliminary data and propose to test these hypotheses in the specific aims. In preliminary experiments, we have shown that during Ascaris chromatin diminution, only those chromosomes that will be retained have extensive deposition of CENP-A (the key factor required for centromere formation and kinetochore assembly), whereas chromosomes destined for elimination have little CENP-A. This suggests a potential mechanistic explanation for how specific portions of chromosomes can be marked for retention or elimination. We propose to investigate the deposition and role of CENP-A as a mechanism for differential chromosome segregation in chromatin diminution. We further hypothesize that small RNAs and Argonautes play a role in Ascaris chromatin diminution. We have shown that specialized Argonautes associate with chromosomes that will be retained in Ascaris diminution mitosis, but not with chromosomes that are destined for elimination. We propose to investigate the role of these Argonautes and their associated small RNAs in nematode chromatin diminution and chromosome segregation. Analysis of Ascaris chromatin diminution promises to provide profound insights into genome stability, centromere specification, chromosome segregation and the contribution of Argonautes and small RNAs to chromosome function, segregation, and chromatin diminution. Our studies will not only provide insight into DNA elimination and its importance in nematodes, but is likely to increase our understanding of the germline, chromosomes, and genome biology in general. Finally, a better understanding of Ascaris chromosome organization and segregation and its small RNA pathways that differ from its human host may provide new insight into needed drug targets.