Iain M. Cheeseman - US grants

centromere; biomedical resource; Caenorhabditis elegans; molecular biology; molecular genetics;

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are currently investigating human kinetochore composition and organization using an affinity tagging strategy to isolate known and novel human kinetocore proteins and kinetochore protein complexes. We have developed a combined approach to examine the cellular localization of a protein and conduct a high affinity purification of that protein from human tissue culture cells (Localization and Affinity Purification - LAP tag). Using this approach, we have isolated more than 20 human kinetochore proteins, including at least 10 novel kinetochore proteins. This ongoing analysis will attempt to characterize the full range of human kinetochore proteins and examine their interactions and organization.

DESCRIPTION (provided by applicant): Each cell in the human body contains 46 different chromosomes, large units of DNA that encode instructions for that cell to grow, divide, and carry out its specialized functions. During mitosis, when a cell divides, each of these chromosomes must be accurately distributed to the two new daughter cells. If this process occurs incorrectly for even a single chromosome, the resulting daughter cells will lose or gain thousands of genes and the instructions that they contain. This type of error in chromosome segregation can result in the death of the cell and is thought to contribute to tumorigenesis. Indeed, as many as 70% of tumors are observed to have abnormal numbers of chromosomes. To facilitate the segregation of DNA during mitosis, chromosomes must generate physical attachments to rod-like polymers termed microtubules that provide the structure and forces to move the chromosomes. Anti-mitotic drugs that disrupt the ability of these microtubules to connect with the chromosomes are routinely used for cancer chemotherapy. However, many of these drugs have deleterious secondary affects due to additional roles for microtubules in the nervous system. A key player in chromosome segregation is a large proteinaceous structure termed the kinetochore that forms the interface between the chromosomes and the microtubules. Inhibition of kinetochore activities is predicted target cancer cells while avoiding the dose-limiting neuronal toxicity associated with microtubule-binding chemotherapy drugs. Indeed, inhibitors against several kinetochore proteins are currently in clinical trials. Determining the specific activities of each human kinetochore protein is crucial to provide a context for their functions in chromosome segregation, to evaluate the best targets for the diagnosis and treatment of disease, and to generate assays suitable for the isolation of small molecule inhibitors. The proposed work will analyze the function and regulation of the human kinetochore proteins that are required to generate interactions with microtubules. This work will focus on two key, recently identified groups of kinetochore-associated proteins that bind to microtubule polymers directly. These studies will define the properties of these proteins and determine the mechanisms by which these proteins interact with microtubules, dissect their regulation by upstream kinases that control kinetochore-microtubule attachments, and examine their functions in human cells. In total, these studies will define the basis for kinetochore-microtubule interactions that will ultimately provide the foundation for experiments on the diagnosis and treatment of cancer. PUBLIC HEALTH RELEVANCE: Project Narrative Defects in mitosis that result in errors in chromosome numbers can cause the death of a cell and are thought to contribute to tumor progression. Understanding the means by which these units of DNA, and the genetic information that they contain, are evenly distributed to new cells is critical for the diagnosis and treatment of cancer. This proposed work will determine the mechanisms that direct and control chromosome segregation in human cells.

? DESCRIPTION (provided by applicant): The goal of this proposal is to define the nature of the kinetochore-DNA interface and determine how centromere-specific chromatin directs the assembly of a functional kinetochore structure in human cells. During each cell division, the entire complement of genetic material must be accurately partitioned to the daughter cells. Even a single chromosome mis-segregation event can be catastrophic, resulting in the loss or gain of hundreds of genes, with severe consequences for development and disease, including tumorigenesis. The central player in directing chromosome segregation is the kinetochore, a large macromolecular structure that mediates attachments between spindle microtubules and a region of each chromosome termed the centromere. Determining the molecular basis for kinetochore function is crucial to understand the defective processes that can give rise to tumor cells, and to evaluate the best targets for the diagnosis and treatment of disease. In vertebrates, centromeres are specified by sequence-independent epigenetic mechanisms that involve the targeted deposition of nucleosomes containing the histone H3-variant, CENP-A. A fundamental unanswered question is how the remainder of the kinetochore is assembled downstream of CENP-A. Our previous work demonstrated that CENP-A is not sufficient to direct assembly of a complete, functional kinetochore structure in human cells. We have identified a heterotetrameric complex comprised of the histone fold proteins CENP-T, -W, -S, and -X as a critical additional component of the kinetochore-DNA interface. The CENP-T-W-S-X complex displays structural similarity to a nucleosome and possesses sequence-independent DNA binding activity. In addition, CENP-T interacts directly with outer kinetochore microtubule-binding proteins to direct kinetochore assembly. Thus, the CENP-T-W-S-X complex plays a pivotal role in connecting the DNA and microtubule interfaces at kinetochores. However, it remains unknown how the CENP-T-W-S-X complex is targeted exclusively to centromeres to generate a single, functional microtubule attachment site on each chromosome. This proposed work will define the mechanisms that direct the centromere localization of the CENP-T-W-S-X complex by assessing: 1) The intrinsic sequence features of the CENP-T-W-S-X complex that are required for its localization, 2) The extrinsic factors that associate with the CENP-T-W-S-X complex to deposit or maintain it at centromeres, and 3) The regulatory modifications and chromatin features that control CENP-T-W-S-X complex localization.

Project Summary/Abstract The goal of my laboratory is to define the molecular mechanisms by which accurate cell division occurs. Our efforts focus on the kinetochore, the central player in directing chromosome segregation. The kinetochore is a macromolecular structure that connects chromosomes to the microtubule polymers that power their movement. Our goal is to generate a coherent model for how the kinetochore functions as an integrated molecular machine. To direct faithful chromosome segregation, kinetochores must form two key interaction interfaces. First, kinetochores must associate with a single site on each chromosome to direct the assembly of a stable kinetochore structure. In vertebrates, this site is defined epigenetically by the presence of a specialized histone variant termed CENP-A, and through contributions of a 16-subunit Constitutive Centromere-Associated Network (CCAN). Together, these proteins form the interface with centromeric chromatin. Despite the identification of these molecules, it remains unclear how the CCAN is established and reorganized during the cell cycle, and also how these processes are modulated during different cell division programs, such as in the context of meiosis and early development. In addition, centromeres must have a specific open chromatin environment to facilitate proper kinetochore function, but the relationship between the CCAN and centromere chromatin is poorly defined. Second, kinetochores must form robust interactions with dynamic microtubule polymers and harness the force generated by depolymerizing microtubules to direct chromosome segregation. To understand this elegant interface, it is critical to define the individual contributions of key outer kinetochore microtubule-binding complexes and also assess their integrated activities. The kinetochore must also sense and correct microtubule attachments to ensure high fidelity chromosome segregation, requiring the functions from the spindle assembly checkpoint components. To understand these critical kinetochore activities and the functional requirements for chromosome segregation, it is also important to define the complete complement of human genes that are required for chromosome segregation. The advent of CRISPR/Cas9-based genome editing has transformed the capability to conduct functional genetics experiments in human cells. This includes the ability to systematically screen gene targets for their loss of function phenotypes using cell biological assays and genome-wide functional genetics screening to analyze context-dependent essentiality to define synthetic lethality relationships. For the work in this proposal, our lab will investigate the fundamental mechanisms of chromosome segregation and kinetochore function, focusing on three related areas: 1) Specification and formation of the centromere- DNA interface, 2) Generation and regulation of dynamic kinetochore-microtubule interactions, 3) Functional genetic approaches to analyze chromosome segregation. We will analyze key open questions in these important areas using combined cell biological, biochemical, proteomic, and functional genetics approaches.

Project Summary/Abstract Centromeres remain perhaps the most enigmatic regions of the genome, even though they provide the foundation for chromosome segregation during every eukaryotic cell division. Despite the ?complete sequencing? of the human genome more than a decade ago, it was only recently that the first assembly of a human centromere was completed. In addition, due to the highly repetitive nature of the centromere sequences in humans and most other organisms, data from centromeres is excluded from almost all genome-wide studies that typically require sequencing reads to map to a single locus. If we are to truly understand the genome, it is critical to address this important gap in our understanding to reveal the nature of centromeres. Breaking through these challenges requires a unique conceptual framework, and set of tools and technologies that combine genome technologies (such as longer sequencing read lengths), computational and bioinformatics approaches, and together with complementary functional biological studies. The goal of the Centromere Biology conference is to substantially advance our understanding of the centromere regions of each chromosome by convening researchers of diverse disciplines focusing on this genomic locus. This meeting will bring together a diverse group of outstanding scientists to build a community interested in understanding diverse aspects of centromere properties including the sequence, nature, organization, evolution (including sequence diversity across the human population), and function of this incredibly important and unique genomic locus. Speakers will represent the diversity of disciplines that this community attracts including genomics, bioinformatics, genetics, epigenetics, biochemistry, biophysics, structural biology, cell and molecular biology, as well as theory and modeling. We value this meeting as part of a larger effort to build an inclusive and diverse the centromere community, including strong representation from women and early career researchers.

Project Summary/Abstract The Whitehead Institute requests $600,000 for the purchase of a critically-needed Zeiss LSM 980 with Airyscan 2 laser scanning confocal microscope (?Zeiss 980AS2?), to be located in the W.M. Keck Facility for Biological Imaging (?Keck Facility?) at the Whitehead Institute for Biomedical Research (?Whitehead?). The instrument requested is of great importance, as it will replace a nine year old Zeiss LSM 710 NLO (non-linear optics; ?Zeiss710?) that is currently the most heavily used microscope in the Keck Facility and provide transformative new imaging technologies that are currently unavailable at Whitehead or in the Keck Facility. For each of the past 3 years, the Zeiss710 averaged 1400 hours of use by 40 researchers from 25 different labs. Zeiss has informed us that guaranteed service for the Zeiss710 will end in 2022. Replacement of this critical instrumentation prior to that time is important to minimize interruptions caused by excessive maintenance issues. Other microscopy facilities in our area are unable to accommodate the substantial amount of use the Zeiss710 currently supports, and also lack the critical new capabilities required for the proposed research projects. The Zeiss 980AS2 will provide robust confocal capabilities for the diverse research projects described in this proposal, such as enabling optical sectioning to generate high-contrast images in thick specimens, generating 3D datasets, and conducting photomanipulation to analyze molecular dynamics, such as FRAP (fluorescence recovery after photobleaching). Critically, the requested Zeiss 980AS2 will provide new cutting-edge technologies essential for addressing many outstanding questions from the 10 NIH-funded researchers presented in this proposal. The Airyscan 2 detector and software will bring the first super-resolution capabilities to the Keck Facility. The 32-channel spectral detector will allow simultaneous imaging of up to ten fluorophores, enabling studies that investigate cell type differentiation in organoids and tissues. The high-efficiency GaAsP detectors will enable imaging of dim samples and reduce photobleaching and phototoxicity during live-cell imaging. The Airyscan 2 8Y Multiplex option and Z-piezo stage allow for extremely fast image collection, enabling larger sample sizes for large volume scanning. The Zeiss ZEN Blue System software that controls the Zeiss 980AS2 will substantially simplify currently arduous tasks, such as multi-position tile scanning and finding rare events. By expanding the experimental approaches and capabilities available to researchers, the Zeiss 980AS2 will greatly enhance and facilitate the success of many NIH-funded projects, including the analysis of neural degeneration, cancer, germ cell differentiation, metabolic regulation, cell division, and stem cell identification. Acquisition of the Zeiss LSM 980 with Airyscan 2 will allow researchers to address these important biomedical issues, and the outcomes from this research will include new diagnostics and therapeutics, in accordance with the mission of the NIH.