The long-term goal of research in the O’Neill lab is to gain a better understanding of the mechanisms within the genome that provide and maintain chromosome structural integrity, and the role played by these mechanisms in gene function, cell division, cancer progression, hybrid dysgenesis and species formation. To attain this goal, I have initiated collaborations locally, nationally and internationally to provide a multi-system comparative approach. The initial focus of my research endeavor, and a running theme in my research into genome stability, centers on chromosome biology – both in the context of species evolution and in the context of karyotypic evolution in stem cell growth and cancer. Species-specificity in karyotypes frequently involves centromere-associated rearrangements, including centric shifts, translocations, fusions, and inversions. However, little is known about how the centromere, either as a functioning unit of chromatin or as a specific block of repetitive DNA sequences, acts in the creation of these types of chromosome rearrangements in an evolutionary context. My work endeavors to understand the fundamental processes that influence chromosome stability in an evolutionary context, as well as the manifestation, consequences and potential remediation of disruptions to that process as it relates to human disease.
Noncoding RNAs and Centromere Function
Although only recently discovered, small RNAs have proven to be essential regulatory molecules encoded within eukaryotic genomes. These molecules, represented by four major class sizes ranging from 20nt to 42nt, are participants in a diverse array of cellular processes including gene regulation, chromatin dynamics and genome defense. The genome sequencing initiative for the tammar wallaby has afforded an opportunity to explore the evolution of each of the major classes of small RNAs, siRNAs, miRNAs, piRNAs, and the newest class of small RNAs, crasiRNAs (centromere repeat associated short interacting RNAs). Following our discovery of the larger, 35-42nt class of small RNAs (i.e. crasiRNAs) in the tammar, we have uncovered sequences within this class that are ultra-conserved across vertebrates as well as sequences that are species specific. Through functional studies, we show this class of small RNA is critical for maintaining genome integrity and proper cell division in both the tammar and in human. Most significantly, we have discovered that alteration of the pathway that produces these small RNAs results in a defect in loading of the mo
dified histone CENP-A, a step required for proper centromere integrity and function. Future work will employ chromosome engineering, fluorescent -tagged constructs, small RNA work and deep sequencing to define the action and partners for these small RNAs in the process of CENP-A recruitment and cell stability.
Cancer Susceptibility and Chromosome Instability
The Peromyscus Genetic Stock center established three inbred lines of P. leucopus, the white footed mouse. Although genetically identical, only two of the three inbred lines are susceptible to exceptionally malignant Harderian tumors, which rapidly metastasize to the lymphatic system and lungs. The unique power of the Peromyscus leucopus model system lies in the ability to identify genetic changes and/or epigenetic modifications that mark normal tissues prior to the induction of tumorigenesis, with the long term goal of identifying factors that ultimately predispose susceptible individuals to cancer. In collaboration with the PGSC we are investigating both cytogenetic and molecular defects that predispose P. leucopus to oncogenesis, specifically focusing on the causes of chromosome based abnormalities.
Cancer Susceptibility linked to Viral noncoding RNAs
Despite an incredibly high incidence of infection within the human population (>90%), Epstein Barr Virus (EBV) is maintained as an asymptomatic infection of B lymphocytes in the majority of cases. However, in a small number of cases, EBV is linked to the development of malignancies, including Burkitt’s lymphoma, Hodgkin’s lymphoma, nasopharyngeal carcinoma, gastric carcinoma, Posttransplant lymphoproliferative disorders (PTLD), T cell lymphoma, T/NK nasal type lymphoma and B lymphoproliferative diseases. Interestingly, each of these EBV+ cancers are regionally delimited; for example, Burkitt’s lymphoma is mainly found in equatorial Africa while nasopharyngeal carcinoma is predominantly found in South East Asia, Southern China, North Africa and in the Eskimo population of Alaska. Searches for a link between EBV sequence variants and the observed geographical distribution of cancer types have failed to provide conclusive evidence that EBV diversity contributes to regional EBV+ cancer susceptibility, we are studying the interaction between EBV small RNAs and the host genome in an effort to understand EBV+ cancer susceptibility.
Centromere Drive in Marsupials
Because of their well-studied and relatively simple karyotypes, Macropodine marsupials (the kangaroos and wallabies) offer a unique system in which to study karyotypic diversification and speciation. Characterization of the composition and distribution of centromeric sequences within this group of mammals indicates these sequences have been involved in amplifications, segmental duplications, fissions and fusions. It has been proposed that, while molecular drive is responsible for the convergent and concerted evolution of satellites within one species, genetic conflict and meiotic drive may be responsible for the different centromere satellite sequence suites found between species. Accordingly, as satellite arrays expand, they attract more microtubules in female meiosis, subverting Mendelian chromosomal segregation with distorted transmission of one parental chromosome over the other. In the model proposed by Malik and Henikoff, this Centromere Drive results in rapid evolution of centromere binding proteins selected to restore parity in meiosis (especially if the “driven” centromere is sex-linked). Current models of centromere evolution and molecular drive predict that other species in a phylogeny would experience shifts and expansions of satellite sequences that would result in species-specific satellites. Using a detailed phylogeny with accompanying chromosomal data from the genus Macropus (kangaroos and wallabies), we have shown this not to be the case. Such a system provides an ideal opportunity to test the Centromere Drive hypothesis directly, as opposed to the inferential studies that currently support this theory.