Daniel Joseph Burke - US grants

DESCRIPTION (provided by applicant): The long-term goal of this research is to understand the molecular mechanism of non-random chromatid segregation in mouse embryonic stem cells. The immortal strand hypothesis - posits that stem cells segregate the "oldest" DNA strands of all chromosomes asymmetrically to the self-renewing stem cells as a mechanism to prevent the stem cell from inheriting any errors in DNA replication that could lead to a cancerous state. Stem cells use this strategy on a chromosome by chromosome basis to direct the segregation of specific chromatids. Specifically, mouse embryonic stem cells selectively segregate chromatids of chromosome 7 and we propose to determine the mechanism. This proposal has two Aims. The first Aim is to test the hypothesis that non-random chromatid segregation depends on the replicative age of the DNA using chromosome oriented fluorescence in situ hybridization (CO-FISH). The second aim will test the hypothesis that LRD, a homolog of outer-arm 2-axonemal dynein heavy chain, functions as a molecular motor to direct non-random chromatid segregation. PUBLIC HEALTH REVELANCE: Stem cells are unique in having the ability to "self renew" and at the same time to be capable of differentiating into every cell in the body. It is critical that stem cells have mechanisms that protect their genetic integrity so that they may maintain their stem cell identity while faithfully executing the instructions needed to adopt many different cellular fates. One mechanism that stem cells use is to selectively segregate chromosomes after they are replicated so that specific copies are kept together. This is a mysterious process since we generally believe that replicated chromosomes are segregated randomly. Clearly, stem cells are different in this regard. The goal of this research is to understand the mechanism of selective chromosome segregation in stem cells.

DESCRIPTION (provided by applicant): Errors in chromosome segregation are the major source of aneuploidy and a driving force in the development of tumors. The spindle assembly checkpoint (SAC) is one of the mechanisms that prevents aneuploidy and is an important regulator of genomic stability. The SAC is orchestrated by the kinetochore and a major unresolved problem, addressed in this proposal, is to determine how the kinetochore measures the lack of tension and lack of occupancy and how the kinetochore transfers that information to the cell cycle machinery. We will complete high resolution mapping of the tension branch of the SAC within the kinetochore using loss-of- function mutants. We have developed a phospho-specific antibody to Mad3 that is the first biochemical marker for the tension branch of the SAC and we propose to use it to dissect this important component of SAC regulation. We propose a new model for the role of the kinetochore in the tension checkpoint. We will determine if subsets of Mad3-containing proteins are phosphorylated, where Mad3 is phosphorylated in the cell and which kinetochore proteins are needed to transmit the tension signal. We have isolated novel mutants that are specific to the occupancy branch of the SAC. We will dissect the role for the SAC in the DNA damage pathway which requires Mec1 and Tel1 phosphorylation of SAC proteins and represents a novel pathway of mitotic regulation. Together, the experiments in this proposal will provide important insights into how SAC signaling is initiated and integrated into the cell cycle. PUBLIC HEALTH RELEVANCE: Errors in chromosome segregation result in imbalances in chromosome number (aneuploidy), a hallmark of some kinds of birth defects and a major driving force in the development of cancer. The spindle assembly checkpoint is one of the mechanisms that prevents aneuploidy and guards against the diseases resulting from genomic instability. The experiments in this proposal will provide important insights into how the spindle checkpoint is organized, how it is regulated and how it is integrated into the cell cycle. The long term goal is to improve human health by preventing aneuploidy that results in birth defects and cancer.

DESCRIPTION (provided by applicant): TOR (target of rapamycin) is evolutionarily conserved from yeast to humans and has a pivotal role of controlling cell growth in all cell types. There is fundamental gap in our understanding of the physiological pathways that interact with TOR and how modulating those pathways regulate lifespan and healthspan. There is an urgent need to identify the full spectrum of interactions that describes TOR activity. The long-term goal is to have a complete understanding of how TOR regulates cellular physiology. The immediate goal of this application is to use a systems approach and the budding yeast Saccharomyces cerevisiae to fill important gaps in our knowledge of TOR activity. It is the evolutionary conservation of the TOR complexes coupled with the outstanding genetic tools that are available for studying budding yeast that are central to this application. The central hypothesis i that there are physiological pathways that interact with TOR or are regulated by TOR that are unidentified and will be evolutionarily conserved from yeast to humans. The rationale is that novel pathways will be discovered using a new and under-utilized genetic approach called complex haplo-insufficiency (CHI) that generates novel information about genetic interactions. Guided by strong preliminary data, the proposed research will be guided by two specific aims: 1). Identify and characterize novel targets of TORC1 and TORC2. We will perform CHI using a novel genome-wide approach that incorporates essential and non-essential genes. We will prioritize novel interactions in yeast prioritized by human homologs and unidentified pathways or genes. We will characterize the genes in yeast and human cells 2). Identify novel rapalogs. We will use a computational approach to compare data from CHI with TORC1 to publicly available data and predict potential rapalogs by similarity in genetic interactions, concentrating on human homologs. We will characterize the potential rapalogs in yeast and human cells. The proposed research is innovative because it uses a novel genetic approach in the most tractable model organism and applies it for the first time to TOR biology. The proposed research is significant because it is expected to identify new interactions with TOR and ultimately identify compounds that can be used for medical intervention to modulate TOR activity and impact both healthspan and lifespan. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because there is an urgent need to identify the physiological pathways that interact with TOR and identify new chemical compounds that target the TOR pathway for medical intervention. Thus the research is relevant to the NIH mission and is ultimately expected to positively impact lifespan and healthspan and reduce the burdens of human disabilities.