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.
Parasitic nematodes remain a significant public health problem in many parts of the world, with Ascaris alone infects upwards of 1 billion people and hinders socioeconomic development in endemic areas. We will carry out molecular and genomic studies on an unusual form of genome re-organization and mechanism of gene regulation in Ascaris, programmed DNA elimination. These studies have the potential to identify novel molecular mechanisms that may represent novel drug targets in these organisms.
