Michael A. Lampson - US grants

DESCRIPTION (provided by applicant): Regulation of cell division is necessary to maintain genome integrity. Defective regulation leads to unequal chromosome segregation and aneuploidy, which is strongly associated with human cancer in mitotic divisions and pregnancy loss and developmental defects in meiosis. Much of the complex dynamics of cell division is controlled by a few key regulatory kinases. One of these kinases, Aurora B, localizes to the centromere until anaphase and regulates the attachments of chromosomes to the mitotic spindle. At anaphase onset Aurora B redistributes to the spindle midzone and controls anaphase microtubule dynamics and cytokinesis. Changes in kinase localization and interactions with other regulatory proteins suggest varying patterns of phosphorylation that would allow a single kinase to regulate multiple processes at different times and places. This proposal focuses on examining phosphorylation dynamics with high temporal and spatial resolution in living cells to test models for how these dynamics are controlled. A key role of Aurora B is to ensure that all chromosomes attach to spindle microtubule in the correct configuration, so that chromosomes segregate accurately in anaphase. Signals must be generated to distinguish correct and incorrect attachment, so that errors can be corrected. The first specific aim will test a model for differential Aurora B signaling at individual centromeres. Forces exerted by spindle microtubules create tension across the centromere, which may regulate phosphorylation of a kinetochore substrate by physically separating the substrate from the kinase, which is concentrated at the inner centromere. The second specific aim will examine the interplay between Aurora B and other mitotic kinases at the centromere. There is evidence for interactions between Aurora B, BubR1, Plk1, and Chk1. By combining quantitative measurements of phosphorylation dynamics in living cells with kinase inhibition, using small molecule inhibitors and RNAi, a model in which multiple kinases act in a signaling network at the centromere will be tested. Aurora B function and localization change dramatically in anaphase, suggesting that complex spatial and temporal phosphorylation patterns may allow a single a kinase to regulate a multiple cellular process. The third specific aim will determine how both Aurora B and opposing phosphatases contribute to a switch in site- specific phosphorylation dynamics in anaphase. These studies will contribute to an understanding of (1) basic mechanisms regulating cell division and (2) the effects of Aurora B inhibitors, which are in clinical trials for cancer therapy. Public Health Relevance: Proper regulation of cell division ensures that daughter cells inherit the correct genetic material. Errors during division lead to cells with genetic abnormalities that are strongly associated with human cancer, pregnancy loss, and developmental defects. The goal of this proposal is to understand the function of a key regulatory protein, which is a promising target for cancer therapy, in cell division.

An increase in aneuploidy is a major cause for the marked decline in human female fertility commencing 35 years-of-age; the incidence of aneuploidy in eggs from women increases to 35% around 40 years-of-age, and is likely to be even higher because aneuploidy leading to a spontaneous abortion is frequently not recognized. Aneuploidy is a leading cause of pregnancy loss, and when development goes to term, an aggravating source of developmental disabilities and mental retardation. Most aneuplodies associated with increased maternal age are due to non-disjunction and meiotic errors that occur during meiosis. Remarkably, the underlying molecular mechanisms that lead to the age-associated increase in aneuploidy are poorly understood. Results of our previous studies suggest that defects in the spindle assembly checkpoint (SAC) and kinetochore function are likely causes for the age-associated increase in aneuploidy. The SAC is one pathway that prevents segregation errors by blocking the onset of anaphase until all chromosomes make proper attachments to the spindle. Using mouse as a model system and imaging of live individual oocytes, Specific Aim 1 will test the hypothesis that the robustness of the SAC in oocytes decreases with age . Another process that prevents errors is regulation of connections between kinetochores and spindle microtubules that results in a spindle with chromosomes correctly attached. Our expression profiling also reveals changes in expression of kinetochore proteins involved in chromosome congression. Specific Aim 2 will examine chromosome congression and molecular mechanisms that underlie correct spindle microtubule-kinetochore attachment, and test the hypothesis that these mechanisms are compromised in oocytes obtained from old females. Specific Aim 3 will test whether specific centromere and kinetochore proteins identified from our expression profiling studies are required for accurate chromosome segregation during MI. Results of experiments proposed in this application will provide a plethora of information regarding molecular bases that underlie the age-associated increase in the incidence of aneuploidy, as well as basic mechanisms required for accurate chromosome segregation. Such findings may suggest experimental interventions that could alleviate the propensity of oocytes obtained from older women to become aneuploid.

DESCRIPTION (provided by applicant): Violations of Mendel's First law occur when segregation of homologous chromosomes in meiosis is nonrandom, termed meiotic drive, which applies to female meiosis because of its inherent asymmetry: only chromosomes that segregate to the egg go into a gamete. Any bias away from random segregation is therefore under strong positive selection and has significant consequences for centromere and karyotype evolution and speciation. The mechanistic basis for the phenomenon is unknown. Nonrandom segregation of Robertsonian (Rb) fusions, which occur between two acrocentric chromosomes (centromere at one end) to form a metacentric (centromere in the middle), can determine whether a species has an acrocentric or metacentric karyotype. Moreover, the direction of the preferential segregation can reverse and drive changes in karyotype and speciation. The overall goal of this proposal is to determine how functional differences between centromeres and meiotic spindle asymmetry lead to nonrandom chromosome segregation, and how the direction of drive is determined. Rb fusions pair with the two homologous acrocentric chromosomes to create a trivalent in meiosis I. Meiotic drive requires preferential orientation of the trivalent on an asymmetric spindle and an asymmetric cell division such that one spindle pole preferentially enters the polar body. Mouse oocytes provide an ideal system to address the underlying mechanisms, which are not understood, because it is well established that the fusion preferentially segregates to the polar body in most strains. Based on our preliminary results, we propose that the fusion centromere preferentially captures microtubules from the pole that has more astral microtubules, which determines the orientation of the trivalent. Aim 1 will distinguish between two models for differences in centromere strength. Aim 2 will test the hypothesis that differential microtubule behavior at asymmetric spindle poles drives trivalent orientation and spindle orientation. Aim 3 will address how the direction of meiotic drive is determined. Multiple Rb fusions have become fixed in the Zalende mouse strain, indicating that the direction of drive is almost certainly reversed relative to common lab strains, which provides an ideal experimental system. We will test two possibilities: either spindle orientation relative to the cortex or trivalent orientation on the spindle could reverse (but not both). The results of the proposed experiments will provide the first insight into mechanisms underlying meiotic drive in animals and establish a link between the basic cell biology of chromosome segregation in individual cells and karyotype evolution and speciation in populations. Moreover, the proposal is relevant to human health because Rb fusions are the most common chromosomal abnormality in humans, occurring in ~ 0.1% of meiotic divisions, and are associated with infertility. Rb fusions preferentially segregate to the egg in humans, which means that the abnormalities persist in families that carry them.

The centromere drive hypothesis invokes genetic conflict to explain the paradox that both centromere DNA sequences and centromere-binding proteins have evolved rapidly, despite highly conserved centromere function across eukaryotes. Genetic conflict at centromeres is grounded in the asymmetry inherent in female meiosis I (MI). In this reductionist cell division, one chromosome from each homologous pair remains in the egg and can be transmitted to the next generation, while the other is degraded in the polar body. Natural selection strongly favors any allele that can increase its chance of remaining in the egg, in violation of Mendel's First Law (Law of Segregation). Such biased chromosome segregation in meiosis does occur and is a form of meiotic drive. The first part of the centromere drive hypothesis is that rapid evolution of centromere DNA is driven by competition to orient towards the spindle pole that will remain in the egg. The model is that expansion of repetitive sequences at a centromere leads to formation of a larger kinetochore and preferential retention in the egg. The second part of the hypothesis explains the evolution of centromere proteins through conflict between individual centromeres, which expand to gain a reproductive advantage, and the reproductive fitness of the organism. If differences between centromeres of homologous chromosomes cause defects in male meiosis, this fertility cost provides selective pressure favoring alleles of centromere-binding proteins that equalize centromeres and suppress drive by binding independent of sequence. The centromere drive hypothesis has had a major impact on the centromere field because it provides a conceptual framework for understanding the evolution of centromere DNA and centromere proteins, but the underlying cell biological mechanisms are unknown. This proposal addresses three major gaps in our understanding of centromere drive. First, how does centromere DNA sequence influence centromere function? Centromeres are defined epigenetically in most organisms, and the contribution of sequence has long been unclear. Second, how is biased segregation in MI achieved? The mechanism by which one centromere preferentially remains in the egg is unknown. Third, is there a fertility cost in male meiosis? Direct evidence for this crucial component of the drive hypothesis is scant. If there is a cost, what is the mechanistic basis? To address these questions, we have established an experimental system in which we observe drive, using a hybrid mouse model created by crossing two strains with different centromeres. Genetic conflict has shaped many aspects of our genomes, and centromeres are a particularly fascinating case because of the implications for non-Mendelian inheritance. The outcomes of our experiments will provide the first mechanistic insight into the cell biology underlying centromere drive. With broad consequences for reproductive biology and chromosome evolution, this project represents a unique contribution to the field of evolutionary cell biology.

Chromosomal abnormalities, particularly aneuploidies, are prevalent during the earliest cell cycles in pre- implantation human embryos. The high incidence of mitotic errors is puzzling ? stable chromosome transmission represents a fundamental process ostensibly honed by natural selection. However, many of the underlying proteins, including centromere proteins that direct chromosome segregation and telomere proteins that preserve chromosome ends, evolve rapidly under positive selection. This paradox of conserved cellular processes supported by unconserved machinery suggests recurrent innovation. A proposed but largely untested resolution to this paradox is that rapid evolution of repetitive DNA drives the evolution of proteins that package this DNA. Under this co-evolution model, constantly changing repetitive DNA compromises viability and/or fertility, spurring adaptation at chromosomal proteins that preserve genome stability. Data from non- mammalian model organisms implicates the very earliest embryonic cycles. Here we consider the distinct challenges posed by sperm-deposited DNA, which enters the egg highly compact and inert and is transformed into competent chromosomes by maternal proteins. We hypothesize that maternally-deposited proteins evolve rapidly to remodel and establish centromeres and telomeres on ever-evolving paternal repetitive DNA. Using mouse as a mammalian model system, we exploit both natural variation in Mus centromeric and telomeric repetitive DNA content and divergent maternal proteins from M. musculus relatives to study the cell biological consequences of ?mismatched? paternal repetitive DNA and maternally provisioned proteins. Our hypothesis predicts that maternally-provisioned proteins adapted to repetitive DNA in one species will not function optimally when confronted with divergent paternal centromeres and telomeres of another species. Our specific aims are to (1) establish an in vitro fertilization (IVF) scheme to systematically vary the paternal DNA and (2) replace rapidly-evolving maternal proteins with diverged versions from related species. In each case, we will determine the consequences for centromere and telomere packaging and embryonic genome stability. This innovative, evolution-guided functional approach reveals otherwise invisible genetic and epigenetic determinants of early embryonic viability. Our overall goal is to establish an integrated experimental system that allows us to challenge diverged, maternally provisioned proteins with paternal genomes of varying repeat number and sequence, providing crucial support for a future R01 that investigates how the zygote restores epigenetic symmetry between essential chromosomal loci that diverge genetically between the maternal and paternal genomes. Defining the centromere and telomere factors at the interface of dynamic evolution with cognate repetitive DNA will expose an underappreciated co-evolutionary process in the pre-implantation embryo. Under this model, the often ignored repetitive DNA composition of paternal and maternal genomes imperils genome stability and transmission, a hallmark of failed human IVF and early pregnancy loss.

Synthetic mammalian artificial chromosomes (MACs) represent a new frontier in genome technology, with the potential to transform chromosome and synthetic biology and stimulate the development of numerous radical advances in medicine. Human Genome Project-Write aims to generate an entire set of synthetic human chromosomes. Short of this ambitious goal, MACs have enormous potential for breakthroughs in biotechnology and medicine, such as creating humanized animal models for drug development or for harvesting patient- personalized organs for transplantation. Furthermore, building MACs from minimal components will advance our fundamental understanding of what comprises a mammalian chromosome. As vehicles for genetic inheritance, fully functional chromosomes are faithfully transmitted through mitosis and the specialized meiotic divisions underlying eukaryotic sexual reproduction and Mendelian inheritance. Our goal is to construct the first MACs that achieve faithful inheritance through the germline, using mouse as a model system. One obstacle is the centromere, the locus on each chromosome that directs transmission through both mitosis and meiosis. Because mammalian centromeres are not encoded in the DNA sequence, it is unclear how to build synthetic chromosomes containing this crucial element. There are additional challenges to create MACs that pair and recombine as homologous chromosomes in meiosis. To solve these problems, we will hijack the existing cellular machinery for assembling centromere chromatin and incorporate additional genetic elements to ensure meiotic pairing and recombination. This effort requires innovation at multiple levels: designing MAC vectors encoding key functional elements, assembling large synthetic DNA constructs, and ultimately creating animals to test MACs in vivo. The proposed work builds on recent advances from the co-investigators? teams in all of these areas, and we have key tools and expertise in place to build the necessary DNA templates, introduce them into embryos, analyze the outcomes, and develop alternative strategies as necessary. The most meaningful preliminary data would be to show a synthetic artificial chromosome that is successfully transmitted through mitosis and meiosis in vivo, but achieving this step is a major goal of our proposal and will require substantial investment of time and effort. Thus, we are requesting support for this project without the preliminary data that would demonstrate high likelihood of success, justifying consideration of our proposal as part of the T-R01 mechanism. We use mouse as a relatively rapid and tractable mammalian model system with outstanding opportunities for testing and debugging MACs, and our advances should readily transfer to other species for applications in biotechnology and medicine. Success in this project will represent a quantum leap in the development of synthetic artificial chromosome that are fully functional in vivo, providing unprecedented genome engineering capabilities in animal models and enabling diverse synthetic biology applications.