Chromatin diminution in nematodes

 描述（由申请人提供）：蛔虫是一种重要的人类病原体，感染约10亿人。了解基因组的维护，基因组的改变，以及这些改变在蛔虫基因表达中的作用，对于了解其生物学和发病机制是很重要的。基因组很少改变。然而，一些生物体经历了被称为染色质减少的大规模基因组变化，这是一个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中的染色质减少，我们证明了寄生线虫猪蛔虫基因组的13%在第三至第五次分裂（4至16细胞阶段）期间在体细胞谱系中被消除，而生殖细胞基因组保持完整。独特的序列（包括约700个基因）在染色质减少形成体细胞基因组的过程中丢失。我们鉴定的被消除的基因主要在蛔虫生殖系和早期胚胎中表达。这使我们认为，在蛔虫染色质减少是必要的，不可逆的机制沉默的一个子集的生殖细胞和早期胚胎表达的基因在体细胞组织之间的生殖细胞和索马区分所需的。在首次发现100多年后，特定蛔虫染色体区域如何被靶向和选择消除或保留的机制仍然未知。两个关键的机制问题是：1）如何确定，标记和切割染色体断裂和边界区域的位置？2)是什么决定了哪些染色体区域被保留或丢失？我们已经制定了关于这些关键问题的假设，我们的初步数据的基础上，并建议在特定的目标来测试这些假设。在初步实验中，我们已经表明，在蛔虫染色质减少，只有那些将被保留的染色体有广泛的沉积CENP-A（着丝粒形成和动粒组装所需的关键因素），而注定要消除的染色体几乎没有CENP-A。这为染色体的特定部分如何被标记为保留或消除提出了一个潜在的机械解释。我们建议研究CENP-A作为染色质减少中染色体差异分离的机制的沉积和作用。我们进一步假设小RNA和Argonaute在蛔虫染色质减少中起作用。我们已经表明，专门的Argonautes与染色体，将保留在蛔虫减少有丝分裂，但不与染色体注定要消除。我们建议研究这些Argonautes和它们相关的小RNA在线虫染色质减少和染色体分离中的作用。蛔虫染色质减少的分析有望为基因组稳定性，着丝粒规范，染色体分离和Argonaute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深入了解DNA消除及其在线虫中的重要性，而且可能会增加我们对生殖系，染色体和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与人类宿主不同的小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.