Ascaris is an important human parasite infecting a billion people leading to significant morbidity. Understanding the genome integrity, chromosome dynamics, and the role of programmed DNA elimination in the regulation of gene expression in Ascaris are important for understanding its biology and pathogenesis. In most organisms, the genome remains constant during their life. However, during the development of early Ascaris embryos, major genome changes can occur in pre-somatic cells leading to a reduced somatic genome. Little is known regarding the reorganization of the chromosomes and the molecular mechanisms of this process. We recently demonstrated that during Ascaris DNA elimination, ~13% of the germline genome sequence is thrown out, including ~700 genes. The ~700 eliminated genes are primarily expressed in the Ascaris germline and early embryo, suggesting that DNA elimination in Ascaris is an essential, irreversible mechanism for silencing a subset of germline- and early-embryo-expressed genes in somatic tissues. Chromosome level reference genomes are needed to better understand Ascaris chromosome behavior during DNA elimination. I propose to generate chromosome level genome assemblies for both the Ascaris germline and somatic genomes. Chromosomal assemblies will reveal the distribution and organization of DNA breakpoints along the chromosomes to enable further insight into the DNA elimination process. DNA replication stress is a common and major source of endogenous DNA double-stranded breaks. In Ascaris, endogenous DNA breaks are required for the DNA elimination process. I hypothesize that replication stress may lead to breakpoints in DNA elimination. I propose to establish a DNA replication timing profile during programmed DNA elimination and use the chromosomal assemblies to better define early and late replicating regions. I will test whether replication stress is associated with DNA breaks for elimination by analyzing the replication timing in (1) high transcription regions; (2) difficult to replicate regions; (3) low replication fork density regions; and (4) genomic regions with long genes. DNA replication timing will also provide additional basic information that has not been determined in any nematode and might identify new targets for rational drug design. A related horse parasitic nematode, Parascaris, also undergoes DNA elimination. The chromosome numbers of Ascaris and Parascaris are drastically different in their germline but strikingly similar in their soma, making them an attractive comparison for analysis of the breaks and sequences eliminated at the chromosomal level. Genome comparisons will illuminate the conservation and divergence of the retained chromosomes, eliminated DNA, and breakpoints. Examining conserved features between Ascaris and Parascaris will enable me to identify characteristics of DNA elimination and to gain insights into the mechanisms for DNA breakage and chromosome segregation. This chromosomal level comparison will also allow me to study whether DNA elimination in these parasites is a mechanism to generate a specific set of somatic chromosomes from different numbers of germline chromosomes in the somatic tissues. My proposed studies will (1) define the chromosomal distribution of the DNA breakpoints, chromosomal changes, and the final somatic chromosomes organization after DNA elimination in a parasitic nematode and (2) establish a DNA replication timing profile for Ascaris to determine whether DNA replication stress contributes to the generation of DNA breaks associated with DNA elimination. My genome work will also comprehensively define the genes, regulatory elements, and the organization of genetic elements along Ascaris chromosomes providing a key resource for the nematode community. Development of chromosome level genome assemblies and an understanding of the organization and dynamics of the chromosomes will shed light on an unusual mechanism of gene and genome regulation in this important parasite and will provide an improved genomic resource for other molecular, immunological and cell biology studies on Ascaris.

Public Health Relevance

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. I will carry out comprehensive genome studies on an unusual form of programmed DNA elimination in Ascaris that represents a novel drug target in these organisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI125869-02
Application #
9394772
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Joy, Deirdre A
Project Start
2016-12-07
Project End
2018-11-30
Budget Start
2017-12-01
Budget End
2018-11-30
Support Year
2
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Colorado Denver
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041096314
City
Aurora
State
CO
Country
United States
Zip Code
80045