Barbara G. Mellone - US grants

Scientific Research:
A hallmark of successful cell division is the accurate segregation of chromosomes to daughter cells during mitosis and meiosis. Understanding how cells mediate this critical event is central to understanding genome stability and, conversely, how its failure contributes to genetic abnormalities in cells and organisms. The centromere is a specialized structure present on all chromosomes that is absolutely essential for chromosome segregation because it provides the physical location for chromosomes to attach to spindle microtubules and be distributed equally to daughter cells. Even though a large number of centromeric components have been identified to date, particularly in humans, only a subset can be identified in the genome of Drosophila melanogaster. Furthermore, increasing new evidence suggests that the centromeres of Drosophila are regulated differently from those of the other eukaryotes analyzed so far. This raises the important question of whether pathways responsible for centromere function are conserved among different organisms. Characterizing this process in Drosophila, a model system that offers powerful cell biology and genetics, will ultimately contribute to a comparative understanding of centromere biology that will provide further insights into this fundamental phenomenon across species. Specifically, this project will identify novel components involved in centromere structure and function and define their role in pathways that mediate maintenance of the centromere through multiple cell divisions. In addition, an exciting new role for the cell cycle in regulating these processes will be further investigated. These goals will be achieved using an alliance of cell biological, molecular and biochemical methodologies to fill major gaps in our knowledge of the intriguing world of chromosome biology.

Broader Impacts:
This project includes ample opportunities for collaborative research exposing undergraduate and graduate students to the interdisciplinary nature of modern scientific investigation. An additional educational goal is to establish a comprehensive hands-on module that exposes students to cutting-edge cell division research including the latest methods in timelapse microscopy. Key outreach efforts are the promulgation of visual tools for the dissemination of knowledge about chromosome segregation and cell division to middle and high school students, teachers and the general public; the participation in initiatives to attract and develop the next generation of female scientists; and the involvement in programs exposing high school students to laboratory research.

Project Summary Centromeres are essential cis elements present on all chromosomes that direct kinetochore assembly and chromosome segregation in mitosis and meiosis. In humans, the presence of exactly one centromere is an essential prerequisite to prevent chromosomal loss or rearrangement. Neocentromeres are rare, functional centromeres that assemble at atypical (ectopic) locations and that are implicated in developmental abnormalities, miscarriages and cancer progression. The deleterious potential and relative infrequency of neocentromeres suggest that their formation may be prevented in normal cells, which begs the question of whether such mechanisms could be altered in disease. Very little mechanistic information exists about how neocentromeres form and what mechanisms protect against such an occurrence; knowledge gaps that this proposal aims to fill. A major obstacle that has prevented the elucidation of these mechanisms has been the lack of assays that allow the induction of neocentromeres in a complex organism. The research proposed will analyze newly formed neocentromeres, and their propagation, from an epigenetic, structural and functional perspective and will identify the mechanisms that suppress neocentromeres on intact chromosomes, using a novel, inducible neocentromere system in Drosophila. The proposed work will determine: 1) the changes in chromatin structure that occur upon neocentromere formation; 2) the contribution of histone chaperones to this process; 3) the effects of neocentromere formation on genome stability and organism viability and 4) the mechanism and molecules mediating neocentromere inactivation. We hope that these studies will contribute to advances in diagnostic tools or therapies for diseases characterized by the presence of neocentromeres and to the development of synthetic chromosomes for human gene target delivery.

This research will take an evolutionary approach to understand how centromeres assemble. Centromeres are essential elements of chromosomes that are crucial for proper transmission of genetic information from cell to cell during cell division. When centromeres fail to assemble correctly, the result can be an imbalance in chromosome number whereby cells have too many or too few chromosomes. In animals, such an imbalance can lead to visible developmental defects, which in turn can lead to birth defects or death. This study aims to impact our understanding of the conserved aspects of centromere assembly, and hence aspects that are crucial for preventing an imbalance in chromosome number and catastrophic developmental defects. In addition, this research will have a strong educational impact by providing opportunities for undergraduate and graduate students to engage directly in research and by developing a new hands-on summer laboratory workshop for high school teachers to enable them to learn about chromosome biology and take the lessons learned back to their high school classrooms.

In eukaryotes, centromeres assemble by binding special centromere-specific histone H3 proteins, which are escorted to the correct location on the chromosome by chaperone proteins. Recent results have revealed structural and sequence variation in these chaperones, which suggests that during evolution, multiple mechanisms may have arisen for assembling centromeres. This work will focus on analysis of the chaperone proteins using evolutionary, structural and functional studies. Comparisons will be made among a newly discovered chaperone from Drosophila and two well-conserved chaperones from yeast and mouse. The outcomes of the work will provide unique mechanistic and evolutionary insights into the fundamental processes that govern centromere structure and function. The project will also have broad impact from the educational perspective. The research will be carried out by undergraduate and graduate students, many of whom are women, thereby providing opportunities for scientific training, career development and direct participation in the promulgation of scientific findings through publications and conference presentations. In addition, a novel hands-on summer laboratory workshop will be developed for high school teachers (ten per year), focusing on the basic biology of chromosome segregation and cell division and extending to emerging topics such as stem cells and altered genetic states in cancer. The teachers will leave the workshop armed with teaching materials, ideas for lesson plans, and positive research experiences through their direct involvement in experimentation. By enabling the teachers to take what they learn back to their classrooms, the workshops will have a multiplier effect on the scientific education of scores of pre-college level students.

Project Summary Centromeres are essential chromosomal elements that mediate kinetochore assembly and accurate chromosome segregation. Centromere defects lead to chromosome missegregation, with detrimental effects on cell and organism health and fertility. In most multicellular species, centromeres are composed of large regions of highly repetitive DNA marked by chromatin containing the centromere-specific histone variant CENP-A. Previous work demonstrated that both centromeric DNA and CENP-A chromatin have the potential to initiate centromere activity de novo; however, their respective contributions to centromere specification in mitosis and meiosis have remained elusive. The overall goal of this proposal is to determine how centromeric DNA and chromatin contribute to centromere identity. The centromeres of metazoans have been refractory to full sequencing and assembly due their large size and highly repetitive nature, hampering our ability to systematically interrogate the role of centromeric DNA elements. Additionally, there are currently no systems in which to test if de novo centromeres, which are devoid of centromeric DNA, and can sustain centromere function and specification through mitotic and meiotic divisions. Using our unique advancements in Drosophila, which include the identification and assembly of its centromeric sequences and the establishment of an inducible de novo centromere system, this proposal will: 1) test if chromatin-mediated centromeres can sustain chromosome segregation through development and meiosis, effectively replacing endogenous centromeres; 2) test specific hypothesis on how centromeric DNA elements may contribute to CENP-A chromatin establishment or maintenance. Collectively, this work will shed light into centromere specification mechanisms in Drosophila, an exceptional model system that allows centromere studies in the context of animal development and fertility- with broad relevance to other species, including humans.