Gary Karpen - US grants

The genetic material present in chromosomes is organized in a non-random fashion within the nucleus. In most eukaryotes, the ends of chromosomes (telomeres) are associated with the nuclear membrane, and often associate with other telomeres. Whole chromosomes, and specific parts of chromosomes, can occupy unique domains within a nucleus, and in some organisms homologous chromosomes are paired in somatic cells. There are some indications that nuclear organization is required for normal cellular functions. For example, defects in telomere structure and function appear during aging, and in cancer cells., and alteration of domain structures can negatively impact gene expression and, thus, cell and organismal viability. However, in multicellular eukaryotes there are few proteins known to be involved in the organization of chromosomes within the nucleus, and very few examples of systems where cytologically-visible changes in nuclear and chromosome organization can be directly linked to altered biological functions. The powerful genetic, molecular and cell biological approaches available in the Drosophila (fruit fly) model system combines many of the molecular-genetic approaches available in yeasts with the cytological resolution, multicellularity and chromosome structure of mammals. In addition, there is a well-defined, manipulable Drosophila minichromosome (Dp1187) that has been successfully utilized in the study of higher eukaryotic centromeres and other aspects of chromosome structure and function. The experiments described in this proposal use Drosophila cytology, genetics and molecular biology to identify proteins and mechanisms involved in nuclear organization in metazoans, and to understand their regulation, properties and in vivo functions. We will 1) identify candidate Drosophila genes involved in nuclear organization and function with a genetic screen, clone the protein products, and determine their biological functions, and 2) investigate the function of candidate centromere protein genes and determine their roles in the cell using cell biological, genetic and molecular methodologies. The results of these studies will provide new information about the trans-acting components responsible for chromosome and nuclear organization in Drosophila, and will serve as a model system for further investigations in other eukaryotes. Elucidating basic information about nuclear organization components and mechanisms in multicellular eukaryotes has intrinsic interest, but is also likely to have applications to the diagnosis and treatment of aging and aneuploidy in human populations.

[unreadable] DESCRIPTION (provided by applicant): The division of chromosomes into euchromatic and heterochromatic regions is an enigmatic aspect of genome organization in multicellular eukaryotes. Heterochromatin is a major component of genomes as diverse as humans and fruit flies, and plays critical roles in chromosome inheritance and metabolism. In the absence of a detailed analysis of heterochromatin organization and sequence, our ability to investigate chromosome function, heterochromatic genes, and genome evolution remains limited. In the previous granting period, the Drosophila Heterochromatin Genome Project (DHGP) used genomic resources available in Drosophila melanogaster to generate basic information about the structure and sequence composition of the heterochromatin, specifically focusing on gene- and transposable element- rich regions that do not contain long arrays of highly-repeated satellite DNAs. We have assembled 12.5Mb of finished or nearly finished sequence and a BAC-based physical map that spans 16.5Mb of the heterochromatin, and linked sequences and clones to specific locations in the cytological maps. Annotation of 49Mb of sequence identified 700 gene models and other genetic elements, as well as orthologs in other Drosophilids, and demonstrated that >85% of the target sequences contains repetitive DNA. Here, we propose to extend the D. melanogaster heterochromatin sequence and physical maps to encompass most or all of the non-satellite regions, and to initiate studies of satellite regions. We will also to improve annotations and public data displays, examine evolution of heterochromatin through comparative analysis with other Drosophila species, and develop tools to facilitate heterochromatin assemblies and analysis. Achieving these aims will provide information and tools that will further our understanding of higher eukaryotic genome structure, and will lay the groundwork for more complete analysis of heterochromatin structure and function in Drosophila and other eukaryotes, including humans. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Chromosome replication and transmission are essential for the inheritance of genetic traits, but the mechanisms responsible for these processes remain poorly understood in multicellular eukaryotes. The centromere, which appears as a constriction in metaphase chromosomes, is required for kinetochore formation, which serves as the key attachment site to the spindle during mitosis and meiosis. Defects in centromere or kinetochore function result in aneuploidy, which is a hallmark of human cancers and is responsible for many birth defects. A pressing question in the centromere field today is how centromere identity is propagated from one generation to the next in multicellular eukaryotes. Elucidating the determinants of centromere identity, propagation and function requires identification of the gene products that promote centromere formation and function, and determining the mechanisms responsible for assembly of centromeric chromatin. The multifaceted approaches required to address these complex questions in higher eukaryotes are likely to succeed in Drosophila. There is over a century of experimental analyses and biological information that facilitates sophisticated in vivo analyses. Here, we propose genetic, molecular, cell biological and biochemical experiments designed to identify and characterize gene products that promote the assembly and propagation of centromeric chromatin, and to determine their properties and functions. Our entry point into centromeric chromatin is a conserved histone H3-like protein (CID, for Centromere IDentifier) that localizes exclusively to functional centromeres. We will capitalize on results obtained in the previous funding period, which identified key regulators of CID localization and assembly, as well as a link between cell cycle regulation and centromere formation. We propose to investigate the interactions and functions of centromere regulators to elucidate molecular mechanisms of centromere assembly and its regulation through the cell cycle. These studies will address specific hypotheses and provide the groundwork for future analysis of inheritance and centromere function in Drosophila and other higher eukaryotes, such as humans. PUBLIC HEALTH RELEVANCE: Human cancers uniformly contain massive numbers of chromosome rearrangements and other consequences of genome instability. Centromere dysfunction is one cause of genome instability, and misexpression of the centromeric histone we study has been observed in human colon and breast tumors. The results of this project will provide key information about the normal regulation of centromere assembly, which will be important to the development of cancer diagnostic and treatment tools.

bioimaging /biomedical imaging; technology /technique development

DESCRIPTION (provided by applicant): The generation of complete or nearly complete eukaryotic genome sequences has produced an explosion of insights into the coding potential and evolution of genomes. However, we have less information about how key processes involving DMA are regulated, including transcription, replication, repair, and chromosome segregation. DMA sequence alone cannot be expected to reveal the mechanisms involved in genome regulation and inheritance, because these processes do not act on 'naked'DMA. In the context of the cell, DMA is packaged as chromatin, whose composition and organization regulate the accessibility and function of DMA sequences. Chromatin displays increasingly complex levels of organization and composition, starting with the basic nucleosome unit and progressing through higher order structures. Chromatin organization is critical for utilizing information stored in the genome;failure to accurately target or maintain chromosomal proteins and chromatin components results in aberrant patterns of gene expression and chromosome behavior, and is associated with many human diseases, most notably cancer. We propose to participate in the MODENCODE project by determining the locations of 125 chromosomal proteins and histone modifications across the Drosophila melanogaster genome. The proteins and modifications under study are involved in basic chromosomal functions such as DNA replication, gene expression, gene silencing, and inheritance. We will perform Chromatin ImmunoPrecipitation (ChIP) with antibodies obtained commercially and generated and validated by this project, isolate and label the precipitated DNA, and apply the probes to genomic tiling arrays. Data generated by scanning the hybridized arrays will be analyzed by statistical methods, and the array data will be validated by independent analyses in cells and animals. We will initially assay localizations using chromatin from three cell lines and two embryonic stages, and will then extend the analysis of a subset of proteins to four additional animal tissues/stages. We will then perform a variety of comparisons between protein 'landscape'data sets, including analyses of combinatorial patterns of modifications and chromosomal proteins, tissue-specific differences, and interactions among proteins involved in the same epigenetic pathways. Finally, all validated data and analyses will be made available to members of the ENCODE project and scientific community. Successful completion of this project will provide basic information about the distributions of chromatin components across the Drosophila genome sequence, which will serve as a foundation for future functional studies. In addition, the data and analysis are highly likely to provide information critical to understanding the roles of chromatin in human cells and diseases.

DESCRIPTION (provided by applicant): The goal of this project is to understand how DNA repair mechanisms contribute to genome stability within the heterochromatic regions of the genome. Replication, repair and recombination of repeated DNAs can result in chromosome rearrangements and other types of genome instability, which are associated with cancer progression and other human diseases. Using the Drosophila model system, we have shown that 1) chromatin proteins required for heterochromatin establishment and maintenance are required to preserve the stability of repeated sequences, 2) heterochromatin undergoes a dramatic expansion immediately after irradiation, 3) repair of damage in heterochromatin requires the homologous recombination (HR) pathway, and 4) early steps in HR repair occur within the heterochromatin domain, but late events only occur after foci associated with repeated sequences translocate outside the heterochromatin domain. Our working hypothesis is that the potentially damaging consequences of HR repair of repeated sequences are avoided by the unusual spatial and temporal dynamics of heterochromatin repair, which are mediated by heterochromatin components. We propose to use Drosophila animals and tissue culture cells to identify the molecules and mechanisms that regulate the response to DNA damage in heterochromatic, repeated sequences. Specifically, we will use a combination of live and fixed cell imaging, mutant analysis, protein biochemistry, and genomic approaches to determine how heterochromatin expansion, repair foci dynamics, and homologous recombination are regulated at repeated DNAs, and how these events contribute to genome stability. The results of these studies will greatly improve our understanding of how the heterochromatin environment ensures the stability of repeated sequences, which has important applications to the diagnosis and treatment of human diseases. PUBLIC HEALTH RELEVANCE: PROJECT NARRATIVE Human cancers contain massive numbers of chromosome rearrangements and other types of genome instability. Repeated sequences compose a large percentage of the fly and human genomes, and pose significant problems to the maintenance of genome stability. The results of this project will provide key information about how chromatin regulates the stability of repeated sequences, which will be important to the development of cancer diagnostic and treatment tools.

PROJECT SUMMARY/ABSTRACT The long term goal of this project is to elucidate the composition, architecture, and biophysical properties of heterochromatin, and to understand how they contribute to nuclear functions. Heterochromatin is enriched in repeated DNAs, is concentrated in pericentromeric and telomeric regions, and forms a distinct and dynamic 3D domain inside nuclei. Heterochromatin is required for normal sister chromosome pairing and segregation, nuclear architecture, recombination suppression, transposon silencing, and gene silencing. Heterochromatin recruitment is regulated by epigenetic components and mechanisms, specifically di- and tri- methylation of histone H3 lysine 9 (H3K9me2/3) by specific methyltransferases. Heterochromatin Protein 1 (HP1) binds this `mark' and recruits many proteins and complexes to the heterochromatin. We currently lack a clear understanding of the fine structure and organization of the heterochromatin domain, and the biophysical properties responsible for its functions and behaviors. Our preliminary studies in Drosophila have revealed unexpected structural complexity and biophysical properties of heterochromatin that raise questions about our current understanding of the structure and function of this domain, and suggest that heterochromatin may form and function through biophysical mechanisms that have not been associated with chromatin structure and function. In particular, our findings led to the novel hypothesis that the heterochromatin domain forms through a phase separation mechanism, which has recently been shown to compartmentalize functional molecular networks into structures that lack constraining membranes, but has not until now been applied to chromatin domains. We will capitalize on these novel findings and apply advanced imaging, epigenomics, biochemical and biophysical approaches to elucidate: 1) the structural, biochemical and biophysical properties of the heterochromatin domain, 2) the components and mechanisms responsible for heterochromatin formation, and 3) the ways that heterochromatin substructure and biophysical properties contribute to nuclear and organismal functions. Testing the phase separation hypothesis will elucidate important information about the organization and function of heterochromatin in cells and animals, offering the potential of providing a paradigm-shifting foundation for understanding how other chromatin domains form and function. In addition, defective heterochromatin produces genome instability and altered gene expression, contributing to cancer, birth defects, and aging. Understanding how human diseases and conditions alter the biophysical properties that underlie heterochromatin formation and function will ultimately impact the approaches to their diagnosis and treatment.

? DESCRIPTION (provided by applicant): Chromosome replication and transmission are essential for the inheritance of genetic traits, but the mechanisms responsible for these processes remain poorly understood in multicellular eukaryotes. The centromere is required for kinetochore formation, which serves as the key attachment site to the spindle during mitosis and meiosis. Defects in centromere or kinetochore function result in aneuploidy, which is a hallmark of human cancers and is responsible for many birth defects. A pressing question in the centromere field today is how centromere identity is propagated from one generation to the next in multicellular eukaryotes. Our published and unpublished results demonstrate that the levels of centromeric chromatin proteins and their regulators are tightly regulated by gene expression and proteolytic mechanisms to ensure faithful centromere and chromosome propagation. This proposal integrates genetic, molecular, cell biological and biochemical approaches to identify the molecules and mechanisms that regulate the levels of centromeric chromatin proteins, and promote the assembly and propagation of centromeric chromatin, using Drosophila cell culture and animal tissues. The entry point into centromeric chromatin is a conserved histone H3-like protein (CENP-A, CID in flies) that localizes exclusively to functional centromeres. We previously identified key regulators of CENP-localization and assembly, as well as a link between cell cycle regulation and centromere formation, which provide an intellectual and technical foundation for the proposed aims. The specific focus of this proposal is to elucidate the cell cycle regulation of centromeric protein stability and the consequences of misregulation by investigating: 1) how mutual protection and other mechanisms regulate the stability of CID and its chaperone CAL1, 2) the impact of centromere protein misregulation on cells, tissues and the organism, and 3) how centromere protein misregulation contributes to cancer initiation and progression in Drosophila model of glioblastoma. The deeper understanding of the normal regulation of centromere assembly generated by these studies will provide basic information about this essential biological process, insights into mechanisms responsible for the etiology of aneuploidy associated with cancer and birth defects, and ultimately will lead to the development of tools for diagnosis and treatment of human diseases associated with centromere defects, such as cancer.

PROJECT SUMMARY/ABSTRACT Goals: Peri-Centromeric Heterochromatin (PCH) is required for genome stability/DNA repair, chromosome pairing, nuclear architecture, and transposon and gene silencing. Previous studies suggested that histone H3 lysine 9 methylation (H3K9me2/3), Heterochromatin Protein 1 (HP1) binding, HP1-interacting protein recruitment and chromatin compaction are sufficient to explain PCH formation and function. In 2017, my lab and the Narlikar lab published complementary studies suggesting that 3D PCH domains form via liquid-liquid phase separation (LLPS), generating membrane-less condensates with an immobile HP1a core surrounded by a liquid. We proposed that novel properties associated with highly networked, phase separated systems (e.g. liquidity) are critical to understand how PCH, and other chromatin domains, form and regulate essential nuclear functions. However, we lack a mechanistic understanding of the organization, dynamics and biophysical/material properties of PCH components and condensates in a cellular and organismal context. In addition, we need to determine if and how biophysical properties regulate genome functions such as repair, replication and transcription, a current major challenge for the whole field of condensate biology. Approach: This MIRA will interrogate how LLPS and biophysical properties impact the in vivo organization and function of heterochromatin and other associated nuclear bodies. We will capitalize on our preliminary results and knowledge of PCH biology, combined with advanced imaging, biochemical, and experimental and theoretical biophysical approaches, to elucidate 1) the molecular interactions responsible for PCH domain formation; 2) the architectural, biophysical and chemical properties of the domain; and 3) whether or not phase separation and liquidity regulate PCH functions and interplay with other nuclear bodies. Innovation: Although LLPS and biological condensates have become a popular topic for study and discussion in recent years, we know little about in vivo mechanisms and relevance to function in the complex but important cellular and organismal contexts. This is an emerging field, with unique challenges, and an interdisciplinary approach is required to address these key questions. Thus in this MIRA proposal we will combine our decades of experience in PCH biology with the expertise of collaborators in experimental and theoretical biophysics, and advanced bioimaging. Testing our hypothesis will elucidate important information about the organization and function of heterochromatin in cells and animals, potentially providing a paradigm- shifting foundation for understanding how chromatin domains in general form and function. Health Relatedness: Defective PCH causes genome instability and altered gene expression, contributing to cancer, birth defects, and aging. Understanding how biophysical properties that underlie PCH formation and function are altered in human diseases will likely result in novel approaches to diagnosis and treatment.