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.
Parasitic nematodes remain a significant public health problem in many parts of the world. Ascaris alone infects upwards of 1 billion people and hinders socioeconomic development in endemic areas. We will carry out studies on an unusual form of programmed DNA elimination in Ascaris, chromatin diminution, that represents a novel drug target in these organisms.
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