Sharon Bickel - US grants

genetic regulatory element; chromosome movement; meiosis; Drosophilidae; phosphorylation; genetic crossing over; sperm; chromatin; yeast two hybrid system; fluorescent in situ hybridization;

Intellectual Merit:
Meiosis is a specialized type of cell division that is required for the production of eggs and sperm. A physical connection between each pair of homologous chromosomes is necessary for their accurate segregation during the first meiotic division. If this association is not established or maintained, homologues can missegregate resulting in aneuploid gametes and in most cases, inviable offspring. Therefore, analysis of the mechanisms that ensure proper homologue association will yield critical information about sexual reproduction and species propagation. Normally, meiotic recombination in conjunction with sister-chromatid cohesion establishes a physical connection that holds each pair of homologous chromosomes together until anaphase I. However, some organisms utilize an alternative mechanism that ensures the proper segregation of homologue pairs that fail to recombine. In Drosophila melanogaster (fruit fly) oocytes, accurate segregation of non-recombinant homologues depends on heterochromatin near their centromeres. In addition, recent work in the principal investigator's laboratory has uncovered an unexpected link between sister-chromatid cohesion and heterochromatin-mediated pairing of homologues. These data suggest that sister-chromatid cohesion proteins not only maintain the association of recombinant homologues but also play an important role in promoting the physical association of non-recombinant homologues. This project will define the relationship between sister-chromatid cohesion and heterochromatin in promoting accurate segregation of non-recombinant homologues by completing the following three objectives: 1) Test the hypothesis that the cohesin complex promotes heterochromatin-mediated pairing of non-recombinant homologues; 2) Explore the role of the cohesin regulator WAPL in promoting the association of non-recombinant homologues; 3) Test the hypothesis that the heterochromatin protein HP1 promotes the accurate segregation of non-recombinant homologues.

Broader Impacts:
Accurate chromosome segregation during meiosis is essential for sexual reproduction. This research will expand the understanding of this fundamental biological process by elucidating the mechanisms that ensure proper segregation of non-recombinant chromosomes during female meiosis in Drosophila melanogaster. Given that centromere pairing plays a role in the faithful segregation of non-recombinant chromosomes in both fission and budding yeast and that sister-chromatid cohesion is evolutionarily conserved, these results may also yield valuable information about homologue association and the segregation of non-recombinant chromosomes in other organisms. In addition, this project will integrate research and education at both the undergraduate and graduate levels. Students will be involved in all aspects of the research involved including experimental design, execution and data analysis. The methodology used for each of the objectives lends itself easily to the full participation of undergraduates at both the technical and intellectual level. Both undergraduates and graduate students will attend weekly lab meetings/journal clubs. Learning to effectively communicate their research and ideas will be an essential part of their training. In the past 11 years, the principal investigator has mentored 22 undergraduates and 5 Ph.D. students. The small size of the research group facilitates interaction of the principal investigator with each member of the lab as well as extensive interaction among members. In addition, mentoring undergraduates represents an important component of graduate student training.

This Major Research Instrumentation award to Dartmouth College funds the acquisition of a confocal microscope with a hybrid scanner, spectral unmixing and components that allow for Fluorescence Lifetime Imaging (FLIM) and Fluorescence Correlation Spectroscopy (FCS) measurements. This system will significantly advance basic science research and teaching infrastructure at Dartmouth and throughout New Hampshire and Vermont. The research enabled by the new system addresses fundamental questions in cell biology and developmental biology, and utilizes a diverse array of model systems (bacteria, fungi, algae, plants, worms, flies and mice). In all cases, the new confocal system will substantially improve and expand the imaging capabilities and allow Dartmouth investigators to continue to conduct leading-edge research and to train the next generation of life scientists. Each of the faculty using the new microscope is committed to teaching science at all levels and to preparing successful future scientists and science teachers. The new system makes state-of-the-art imaging technology more accessible to undergraduate students, graduate students and post-doctoral scholars, as well as students and teachers in our local secondary schools. Furthermore, Dartmouth faculty members have an extensive history of including undergraduate interns in their research programs and incorporating the latest scientific approaches and data into their courses. The results of these research and teaching efforts will be broadly disseminated through abstracts and peer reviewed publications, as well as by active participation of students and faculty at professional meetings.

DESCRIPTION (provided by applicant): The goal of this project is to understand the mechanisms that regulate maintenance of meiotic cohesion during prophase I. In humans, female meiosis is especially error-prone and the incidence of chromosome segregation errors increases dramatically as women age. Sister-chromatid cohesion holds sisters together from the time of their synthesis until they segregate to opposite poles and is therefore essential for accurate chromosome segregation during mitosis and meiosis. In addition, meiotic cohesion along the arms of sister chromatids provides an evolutionarily conserved mechanism to keep recombinant chromosomes associated until anaphase I and thereby ensure their accurate segregation during meiosis I. Because human oocytes undergo meiotic recombination during fetal development and remain suspended in a prolonged dictyate (diplotene) arrest until ovulation, the continuous association of homologous chromosomes demands that meiotic sister-chromatid cohesion be maintained for decades. Therefore, one of the factors that may contribute to age-dependent nondisjunction in human oocytes is deterioration of meiotic cohesion with age. Using Drosophila as a model system, we have tested this hypothesis and our recent work supports the model that as oocytes age, normal meiotic cohesion weakens during prophase I and this leads to increased nondisjunction of recombinant chromosomes. Despite its essential role, little is known about the mechanisms that ensure the maintenance of cohesion during meiotic prophase I or those that contribute to its demise. The experiments outlined in this proposal focus on these two fundamental issues. Our specific aims are to: 1) Test the hypothesis that re-establishment of cohesion during prophase I is required for chiasma maintenance;2) Investigate the role of chromatin modifiers/remodelers in the regulation of meiotic sister- chromatid cohesion during prophase I;and 3) Test the hypothesis that oxidative damage contributes to loss of meiotic cohesion. In many respects, Drosophila is an ideal organism to investigate the mechanisms that control cohesion maintenance during meiotic prophase in metazoans and the genetic and cytological tools we have developed will be critical to address these questions. The information gained from the proposed experiments will significantly advance our understanding of the mechanisms that are required to maintain cohesion during meiotic prophase as well as yield valuable insight into the factors that cause meiotic cohesion to deteriorate with age. Given the conserved role of meiotic cohesion in holding recombinant chromosomes together until anaphase I, this work also promises to contribute to our understanding of why the fidelity of chromosome segregation decreases as human oocytes age. PUBLIC HEALTH RELEVANCE: Meiosis is a specialized type of cell division that gives rise to eggs and sperm. In humans, errors during meiosis are the leading cause of birth defects and miscarriages. The experiments in this proposal are designed to elucidate the mechanisms that operate during normal meiosis, and to understand the defects that give rise to errors during human meiosis.

ABSTRACT The long-term goals of my laboratory are to elucidate the mechanisms that regulate sister chromatid cohesion and chromosome segregation during meiosis and to understand why meiotic chromosome segregation errors in human oocytes increase as women age. During a woman's thirties, the incidence of meiotic segregation errors increases exponentially such that the risk of an aneuploid pregnancy for a woman in her early forties exceeds 30%. Such errors are the leading cause of birth defects and miscarriages in humans. Although the link between maternal age and meiotic nondisjunction (NDJ) is well- established, the molecular mechanisms underlying this phenomenon remain poorly understood. One prerequisite for accurate chromosome segregation is sister chromatid cohesion, the protein-mediated linkages that hold sister chromatids together. Meiotic cohesion must remain intact for decades, from the time it is established in the fetal ovary until ovulation triggers resumption of meiosis and anaphase I. Several lines of investigation support the model that loss of cohesion as oocytes age contributes to the maternal age effect. Using Drosophila as a model system, we have recently discovered that accurate chromosome segregation requires a ?rejuvenation? program that establishes new cohesive linkages during prophase I. Aim 1 will use a number of approaches to define the molecular events that underlie cohesion rejuvenation in Drosophila oocytes. Consistent with the proposal that accumulation of oxidative damage in aging oocytes may contribute to the maternal age effect, our recent work provides the first in vivo demonstration that oxidative stress induces meiotic segregation errors. Aim 2 will further delineate the mechanism(s) by which oxidative stress causes NDJ by identifying oocyte proteins that incur oxidative damage. In addition, using our standardized age-dependent NDJ assay, we will determine whether loss of cohesion in Drosophila oocytes that undergo aging can be suppressed by increased levels of Superoxide Dismutase in the oocyte or by nutritional strategies known to reduce oxidative damage and/or extend lifespan. Sirtuins regulate several aspects of cellular homeostasis and reduced Sirtuin activity is associated with aging. Aim 3 will test the hypothesis that reduced Sirtuin activity during meiotic prophase causes premature loss of cohesion that leads to NDJ and determine whether Sirtuin knockdown elicits an increase in oxidative damage and/or modulation of Superoxide Dismutase activity in oocytes. We also will determine whether dietary supplements known to increase Sirtuin activity can suppress age-dependent NDJ. Our experiments will provide a better understanding of the normal pathway(s) that keep meiotic cohesion intact, the mechanisms that lead to loss of cohesion with age and insight regarding the ability of nutritional strategies to suppress age-dependent NDJ. As such, the proposed work addresses an unresolved reproductive health issue that has a major impact on the pregnancy outcomes of older women.