1997 — 2005 |
Kaufman, Paul D. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Dna Replication-Linked Chromatin Assembly in Yeast @ Univ of Massachusetts Med Sch Worcester
The DNA in eukaryotic chromosomes is bound by histone proteins. Octamers of histone proteins are organized into nucleosomes, the fundamental building blocks of chromatin. Nucleosomes compact the DNA and regulate the accessibility of the genome to all aspects of DNA metabolism. During DNA replication, histones are rapidly assembled onto daughter DNA strands; DNA replication in the absence of histones results in lethality. This ordered deposition is mediated by proteins that bind newly synthesized histones and deposit them onto DNA. Nucleosome assembly proteins include Chromatin Assembly Factor-I (CAF-I) and Asflp, which are ubiquitous among eukaryotic organisms. CAF-I and Asflp act synergistically in vitro to form nucleosomes; in vivo, they build chromatin at specialized regions, such as heterochromatic loci that silence gene transcription. CAF-I is also important for chromatin structure at centromeres. The long-term goals of this work are to understand how CAF-I, Asflp, and other histone deposition proteins function synergically, and how these proteins are differentially regulated at different loci. We study these questions in the budding yeast Saccharomyces cerevisiae, a biochemically and genetically tractable organism. Our specific aims include: 1. Development of reagents to study histone deposition in a defined biochemical reaction. We will test mutant in this system to understand at a mechanistic level silencing phenotypes we have observed. This system will also enable discovery of new proteins that stimulate or regulate histone deposition. 2. Investigation of the special role of histone deposition proteins at centromeres. We will determine which aspects of centromeres. We will determine which aspects of centomeric chromatin are built by histone deposition proteins, and investigate how these are recruited to centromeres. 3. Determination of the cellular distribution of the different histone deposition proteins. We will also explore how proteins involved in DNA damage repair and gene silencing regulate histone deposition differently at different loci. Mechanistic understanding of these highly conserved histone deposition proteins will be applicable to all eukaryotic organisms, including humans. Likewise, discovery of how proteins that sense DNA damage regulate nucleosome formation will improve our understanding of mutagenesis and cancer in metazoans. Proper assembly and function of chromosomes is also important for cell cycle progression and gene expression. Therefore, human homologs of proteins investigated in this proposal are good candidate targets for developing new anti- proliferative drugs.
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0.961 |
2005 — 2007 |
Kaufman, Paul D. |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Structure of Centromere-Specific Protein &Role in Kinetochore Func in Yeast @ University of California San Francisco |
0.948 |
2006 — 2007 |
Kaufman, Paul D. |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Identification of Components of the Hir Complex Required For Chromatin Assembly @ University of Washington |
0.957 |
2007 — 2010 |
Kaufman, Paul D. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Dna Replication Linked Chromatin Assembly in Yeast @ Univ of Massachusetts Med Sch Worcester
DESCRIPTION (provided by applicant): The DNA in eukaryotic chromosomes is bound by histone proteins. Octamers of histone proteins are organized into nucleosomes, the fundamental building blocks of chromatin. Histones are evicted and replaced during all types of polymerase movement, making histone deposition a critical process for all aspects of chromosome biology throughout the cell cycle. In vivo, nucleosome formation requires histone-binding proteins to prevent uncontrolled aggregation of histones and DMA. This proposal is focused on a conserved eukaryotic protein complex important for histone deposition, Chromatin Assembly Factor-1 (CAF-1). CAF-1 interacts with and is stimulated to deposit histones by another histone-binding protein termed Asf1. CAF-1 deposits histones preferentially onto replicating DNA, and thus represent a paradigm for understanding nucleosome formation during S phase of the cell cycle. We will address the following interrelated questions: What auxiliary proteins are required for histone deposition by CAF-1? How is CAF-1 stimulated by Asf1? How is the deposition of different core histone subcomplexes (H3/H4 versus H2A/H2B) coordinated? Are histones exchanged among these complexes, and do multiple assembly complexes contribute histones to the same nucleosome? We will address these questions using biochemical, biophysical, and molecular genetic approaches: We have developed a new in vitro nucleosome assembly assay to facilitate isolation of accessory factors and characterization of reaction intermediates during histone deposition. We will explore how CAF-1 interacts with histones by mapping of interaction sites and analytical ultracentrifugation analyses of CAF-1/histone complexes. We will generate differentially labeled histones to determine whether different complexes contribute to the same nucleosome. Relevance: Inhibition of human CAF-1 results in S phase arrest, apparently due to replication fork collapse. A human Asf1 protein is required for human cellular senescence, a differentiation pathway important for avoidance of tumorigenesis. Therefore, these highly conserved histone deposition proteins directly impact genome stability and growth control, important aspects of human health. The proposed biochemical studies of these proteins are consequently of high priority for cancer research, and these and new proteins discovered in the course of this work may be good candidates in the future for therapeutic intervention.
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0.961 |
2007 — 2010 |
Kaufman, Paul |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Chromatin Proteins That Ensure Dna Replication Fork Stability @ University of Massachusetts Medical School
Abstract:
DNA synthesis is essential during every cell cycle, but it is an intrinsically hazardous event because elongating replication forks can stall or collapse at sites of DNA damage or at protein obstacles. Collapsed forks impede complete genome duplication and activate DNA damage surveillance mechanisms that halt cell cycle progression; an alternative to these scenarios is the activation of recombination events. Thus, agents that disrupt DNA replication forks generally result in dangerous chromosomal translocations or other abnormalities. Although multiple proteins have been implicated in the maintenance of replication fork stability, many aspects of how stalled replication forks are maintained and restarted remain poorly understood.
A highly conserved eukaryotic histone deposition protein termed Asf1 contributes to heterochromatin formation and DNA damage resistance. Funding from the past grant period supported our solution of the three-dimensional structure of the globular domain of budding yeast Asf1. These data were used to guide extensive structure-function analyses, which defined two separate interaction surfaces on Asf1; one for recognition of the HIR chromatin assembly complex, and a different surface for histone recognition. Additionally, a new biological role for Asf1 was discovered: Asf1 is required for the maintenance of DNA replication proteins at stalled forks. Asf1''s role in genome stability depends on its ability to bind histones and promote acetylation of histone H3 on lysine 56 during S phase. In sum, previous work of this lab demonstrated that Asf1 is at the center of a highly regulated pathway that promotes replication fork stability by ensuring deposition of specifically modified histones.
In this project, experiments will address how Asf1 and H3-K56 acetylation affect the DNA structure, protein composition, and kinetic properties of DNA replication forks. These experiments will utilize a variety of biochemical and genetic techniques, focused on detecting aberrant nucleic acid structures and patterns of protein accumulation. For example, replication intermediates will be examined via two-dimensional and alkaline gel electrophoresis to detect broken forks, aberrant recombination intermediates, or nascent strand alterations. Molecular combing of halogen-labeled DNA will be used to assess the efficiency of replication fork movement and restart in the presence of damage. Chromatin immunoprecipitation used to determine whether abnormal recruitment of helicases, nucleases or recombination proteins occurs, and modified in vivo footprinting techniques will be used to test for evidence of loss of leading and lagging strand coordination at the mutant forks.
Broader Impact These experiments will have a wide scientific impact that is not restricted to the study of the yeast Asf1 protein. Asf1 is highly conserved throughout eukaryotic organisms, and serves as the intersection between the histone deposition machinery and the signaling pathway that monitors DNA damage. Therefore, the analyses of Asf1 proposed here will be instructive for understanding DNA replication, DNA repair and nucleosome formation in all eukaryotes. Additionally, we are developing several methodologies for analysis of nascent DNA strands and in vivo protein footprinting at replication forks in order to extend the range of tools available for study of these important structures.
This project also includes a significant educational component, with most of the funding used for research training of laboratory personnel, including funding for lab members to present their data at scientific meetings and via published literature. Furthermore, in previous years, five different undergraduates in this laboratory have co-authored publications resulting from their projects, and we are continuing with our tradition of prioritizing undergraduate research experiences as an integral part of our research via the NSF Research Experience for Undergraduates (REU) program.
In addition to training of undergraduate researchers, in the next year my laboratory will participate in educational opportunities for local high school students. For the last 7 years, a group of four UMMS faculty have been involved in teaching laboratory exercises for an Advanced Placement Biology Course at North High School (Worcester, MA). This has been a rewarding experience for the high school students and provides an opportunity for faculty to interact with and influence nascent scientists and increase participation in community outreach at UMMS.
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0.915 |
2007 |
Kaufman, Paul D. |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Mass Spectrometric Analysis of Histone Modifications @ University of Washington |
0.957 |
2008 — 2010 |
Kaufman, Paul D. |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Identification of Proteins That Regulate the Sin3a Histone Deacetylase Complex @ University of Washington
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. Histone post-translational modifications play a pivotal role in gene regulation, chromatin packaging and cellular differentiation. The histone deacetylase (HDACs) mSin3A complex is involved in transcriptional repression and contains multiple subunits that have been implicated in growth control, including HDAC 1 and 2, the Breast Suppressor of Metastasis 1 proteins (BRMS1 and BRMS1L1), and the p33/ING1 tumor suppressor proteins. However, our preliminary data suggest that mSin3A complex may be compositionally diverse in different cell types and under different growth conditions. Isolation and characterization of different Sin3A subunits is essential for elucidating the role of the various mSin3A complexes. Using mass spectroscopy, we aim to identify associated proteins, and determine how they interact with other subunits. These data will guide functional studies to determine effects on cell proliferation, gene expression and genomic stability.
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0.957 |
2009 — 2010 |
Kaufman, Paul D. |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Hts Development For Targeted Anti-Fungal Small Molecules @ Univ of Massachusetts Med Sch Worcester
DESCRIPTION (provided by applicant): Candida albicans is a widespread human fungal pathogen that causes high rates of mortality during systemic infections (candidiasis). Because fungi are eukaryotic cells, development of antifungal therapeutic compounds that are non-toxic to humans is challenging. A recently discovered fungal histone acetyltransferase (HAT) enzyme, termed Rtt109, acetylates histone H3 lysine 56, and is important for genome stability and resistance to genotoxic agents. Rtt109 is very distantly related the mammalian p300/CBP HAT enzyme, but compounds that inhibit p300/CBP do not inhibit Rtt109. In fungi, H3-K56 acetylation occurs on all newly synthesized molecules, but H3-K56ac is either not abundant or even detectable in mammalian cells. We therefore hypothesized that small molecules can be found that inhibit Rtt109 but do not substantially affect the activity of mammalian HAT enzymes, and thereby display minimal toxicity to mammalian hosts. We have discovered that homozygous rtt109-/- mutant C. albicans lack H3K56 acetylation, and are highly sensitive to genotoxic agents including DNA alkylating agents and reactive oxygen species (ROS) such as hydrogen peroxide. Notably, rtt109-/- mutant cells are much less pathogenic in a mouse model of systemic candidaisis induced by tail vein injection. Together, these data support our hypothesis that Rtt109 is a promising novel target for antifungal therapy. We are particularly encouraged to pursue these studies having generated a preliminary protocol to measure the enzymatic activity of Rtt109 in a microtiter format. Aim 1: Assay Development. We will re-optimize the assay parameters (number of washes, volumes/amounts of reagents used, secondary detection reagent) to determine the optimal Z-factor score in a 384-well plate format. Aim 2: Configuration of Assays for HTS. Based on our optimized assay configuration, we will perform a pilot screen of 30,000 compounds present here at the University of Massachusetts Medical School's Small Molecule Screening Facility. The results of this trial screen will establish the hit rate, the rate of false positives, and the best detection reagent for larger scale screening efforts. We have positive and negative screening criteria planned to prioritize initial candidates, and we will develop the reagents and protocols for these. First, we will require that compounds that inhibit histone acetylation by the Rtt109-Vps75 protein complex will also inhibit acetylation by Rtt109 when it is stimulated by Asf1 rather than Vps75. As a negative selection, we will test preliminary Rtt109 inhibitors for effects on the unrelated picNuA4 HAT enzyme complex, to rule out compounds that broadly inhibit acetyltransferase reactions without specificity for the Rtt109 active site. Finally, we will test the efficacy of identified compounds on C. albicans cells, measuring sensitivity to genotoxic agents and effects on H3K56-ac levels. This will identify compounds best able to permeate cells. We will also identify Rtt109 inhibitors that are toxic to mammalian cells, so that non-toxic candidates can be prioritized. Together, these studies will provide positive control Rtt109 inhibitors for further screening of the larger libraries at the NIH Molecular Libraries. PUBLIC HEALTH RELEVANCE: Candida albicans is a pathogenic fungus that is particularly dangerous to immunocompromised individuals, including AIDS patients. C. albicans infections are also commonly acquired in hospitals, making them a major public health problem. Recently, a new enzyme was discovered that is important for normal growth of fungi, and for pathogenesis by C. albicans. We are developing high-throughput screens for compounds that can inhibit this enzyme, because these will be candidates in our search for new anti-fungal drugs.
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0.961 |
2011 — 2014 |
Kaufman, Paul D. |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Repetitive Chromatin Structures During the Human Cell Cycle @ Univ of Massachusetts Med Sch Worcester
DESCRIPTION (provided by applicant): Chromatin Assembly Factor-1 (CAF-1) is a three-subunit protein complex conserved throughout eukaryotes. CAF-1 is a nucleosome assembly factor important for DNA replication, DNA repair, and heterochromatin formation. CAF-1 protein levels correlate with cell proliferation and cancer prognosis, making these studies a high priority for the medically important processes of genome stability and the maintenance of epigenetic states. Via mass spectrometry, we discovered multiple nucleolar proteins associated with the human CAF-1-p150 subunit. Microscopy detects a subset of cellular p150 associated with nucleoli, the sites of ribosomal RNA (rRNA) synthesis and ribosome assembly. Notably, RNAi-mediated depletion of p150 causes a dramatic loss of nascent rRNA transcripts and spatial redistribution of some nucleolar proteins. Therefore, we have discovered that p150 has a previously unrecognized role in the structure and function of the nucleolus. rRNA synthesis is regulated by energy supply, differentiation, cell cycle progression, tumor suppressors and oncoproteins. Nucleolar alterations are also important for cancer diagnoses, and the rRNA synthesis machinery is increasingly viewed as a therapeutic cancer target. Therefore, these studies are crucial for understanding clinically relevant interactions between DNA replication, gene expression, and growth control. We plan to explore three Aims to explore how p150 functions to regulate rRNA synthesis, and to extend these findings via genome-scale studies: Aim 1. The mechanism of regulation of rDNA transcription by CAF-1 p150. We will test several hypotheses raised by our observations: (a) p150 could be acting as part of a transcriptional activation complex at the rDNA promoter, perhaps distinct from its role as a CAF-1 subunit, (b) p150 could regulate the percentage of transcriptionally accessible rDNA repeats, (c) p150 could promote transcriptional elongation, or (d) p150 might be critical for maintaining the epigenetic modification state of the rDNA repeats. Alternatively, (e) p150 might prevent cryptic transcription events. We note that these possibilities are not mutually exclusive. Aim 2. Molecular analyses of p150 recruitment to repetitive DNAs. We will determine whether specific p150 protein domains are required for association with rDNA, and also assess the role of the new nucleolar interaction proteins in p150 recruitment. We will also determine the extent of cell cycle regulation of these associations. Aim 3. Genome-wide analysis of p150 localization, transcriptional targets, and effects on rDNA conformation. We will test our hypothesis that p150 is a master regulator of three-dimensional interactions of rDNA repeats. We will compare these data to genome-scale analyses of p150's transcriptional targets and genomic localization. Together, these studies will provide candidate direct targets of p150 regulation, and lead us to test dependency relationships for these observations.
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0.961 |
2014 — 2017 |
Kaufman, Paul D. Rando, Oliver J |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Breaking Nucleosomal Symmetry @ Univ of Massachusetts Med Sch Worcester
DESCRIPTION (provided by applicant): Project Summary / Abstract We have developed methods to manipulate for the first time the natural symmetry of nucleosomes, in order to test the extent to which this symmetry is functionally important. These questions cannot be pursued in cells with natural histones. Therefore, we have designed altered histone H3s that have obligate heterodimeric interactions, and which preclude interaction with wild-type H3 molecules. We will now use these altered H3s to measure how nucleosomal asymmetry affects gene expression and histone modification patterns, as follows: Aim 1. Identify the mechanistic basis for epistatic interactions between histone tails. In our preliminary studies, we observed distinct classes of phenotypes upon mutation of modifiable residues: in one case, a single asymmetric H3 point mutation paired with a wild-type partner exhibited all the transcriptional defects of a double point mutant. In another case, genes were only misregulated in symmetric double mutants. We will extend these studies to a large set of histone mutations to understand the mechanistic basis for the epistasis observed between pairs of histone mutants. Aim 2. Determine whether histone crosstalk functions in cis or in trans. A great number of histone modifying enzymes preferentially act on nucleosomes carrying some second modification, a phenomenon often referred to as cross-talk. We will use genetic and biochemical approaches to assess whether crosstalk occurs in cis, on the same tail, or in trans, on opposite tails: we will identify the quantitative difference in gene expression between cells with cis and trans double K->R mutations in the H3 tail, and perform mass spectrometric analysis of purified asymmetric nucleosomes to determine whether second site modifications are lost in cis, in trans, or are unaffected by monomeric histone mutations. Together, these studies will reveal previously unexplored biochemical dependency pathways that alter histone modification patterns, and distinguish gene expression regulatory events that are dependent on one versus two histone H3 N-termini. Notably, because of the extreme conservation of core histones among eukaryotes, this work will open the way to exploring related questions in metazoans. Because histone modifications are central to all aspects of gene expression from yeast to man, and play major roles in human diseases including cancer, these studies will reveal unappreciated regulatory mechanisms that govern human health and growth control.
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0.961 |
2015 — 2019 |
Kaufman, Paul D. Pederson, Thoru |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Nucleolar Genomics During Early Mammalian Development @ Univ of Massachusetts Med Sch Worcester
? DESCRIPTION (provided by applicant): In all eukaryotes, the large ribosomal RNAs are transcribed from repeated ribosomal DNA (rDNA) genes. These rDNA repeats form nucleoli, which are specialized, non-membrane-bound sub-nuclear organelles that are the sites of ribosome assembly. Additionally, nucleoli are dynamic hubs through which numerous proteins shuttle. Less well investigated is the role of nucleoli in organizing the three dimensional structure of mammalian genomes. Long-range chromosome interactions are of great interest because they can regulate the developmental timing or the variegation of gene expression in mammalian cells. Deep sequencing analyses of DNA associated with isolated nucleoli from human somatic cell lines have shown that specific loci, termed nucleolar-associated domains (NADs), form frequent three-dimensional associations with nucleoli. NADs are dynamic, being redistributed to the nuclear periphery or to pericentric heterochromatin foci upon nucleolar alteration via inhibition of rDNA transcription. The human cell lines in which NADs have been studied to date are not suited for answering broad questions about the role of NADs in mammalian development. Early development is a critical period to study NAD biological function, not just because of the fundamental biological events that occur, but also because interactions between pericentromeric chromatin and perinucleolar regions are particularly dynamic during mammalian preimplantation embryonic development. We therefore propose that the biological importance of the 3D genome associations maintained by nucleoli should be explored in a system that allows analysis of mammalian developmental processes; that is, a system in which the functionality of these interactions can be explored in four dimensions. Therefore, we propose for the first time to map of the nucleolar-associated domains (NADs) in the mouse genome, determine how these associations are altered during embryonic stem cell (ESC) differentiation, and develop tools for study of these higher-order chromosome interaction in fixed and live single cells. In keeping with the goals of the NIH Initiative, we intend to produe databases and tools for understanding the 4D regulation of mammalian genome structure and function via NAD interactions, as a comprehensive foundation for the mammalian developmental biology community. Furthermore, we will determine how these associations are altered upon differentiation into each of the three germ layers, and how they are correlated with the global genome reorganization that occurs in post-implantation epiblasts. In addition to these population measurements, we will generate tools for the visualization of the repeat-rich DNAs associated with nucleoli in live, single cells via CRISPR-based targeting. In this manner, our project will be the first to analyze the dynamics of NAD-mediated genome organization during mammalian cell differentiation. In sum, the comprehensive database created by this project will constitute a major new tool for the mammalian developmental biology and genomics communities.
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0.961 |
2018 — 2021 |
Kaufman, Paul D. |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Eukaryotic Nuclear Functions: From Nucleosomes to Chromosomes @ Univ of Massachusetts Med Sch Worcester
Project Summary/Abstract Eukaryotic genomes must simultaneously be packaged to fit into the cell nucleus, but also provide access at specific loci to allow for fundamental biological processes including gene transcription and genome replication. To accomplish these opposing requirements for packaging and access, eukaryotic genomes are regulated at many levels and length scales, from the nucleosome to the higher-order, three-dimensional interactions among chromosomes. My laboratory is investigating two different levels of regulation along this broad but interconnected spectrum: First, we are testing for the first time the extent of regulation of genome function at the level of nucleosome symmetry. Nucleosomes contain two copies of each core histone, held together by a naturally symmetric, homodimeric histone H3-H3 interface. This symmetry has complicated efforts to determine the regulatory potential of this architecture. In other words, is it important whether one or both tails receives a post- translational modification? Answering this question requires the ability to specifically impair modification on a single tail per nucleosome. Through molecular design and in vivo selection, we have generated obligately heterodimeric H3s, providing a unique tool for discovery of the degree to which histone modification symmetry plays a regulatory role in gene expression and other chromosomal functions in living cells. Having validated an asymmetric H3 pair, we are extending these studies to two additional H3 isoforms. First, we recently generated an asymmetric centromeric H3 (Cse4/CENP-A) pair in budding yeast. Using these, we will address long-standing controversies regarding centromeric nucleosome stoichiometry. Second, we are using an asymmetric replication-independent histone H3.3 pair to probe two histone modifications with key roles in chromatin structure and gene regulation. Histone H3.3 is required for repression of endogenous retrovirus transcription and early differentiation in mouse embryonic stem cells, so we plan to investigate the stoichiometry of regulatory relationships for repressive chromatin mechanisms that are absent in yeast, most notably involving H3K9me3 (characteristic of constitutive heterochromatin) and H3K27me3 (characteristic of facultative heterochromatin that is developmentally regulated). Because dominant H3.3 mutations are implicated in several types of cancer, these studies also provide a novel tool for exploration of how these alterations affect epigenomes in living cells. Second, we are exploring interconnections between the three-dimensional organization of the human genome, cell cycle progression, and protection from genotoxic stress. Our experiments have led us to focus on the clinically important proliferation marker protein Ki-67. Ki-67 is required for normal three- dimensional organization of heterochromatic loci around the nucleoli, protects cells from genotoxic stress, and is essential for forming a proteinaceous layer on mitotic chromosomes. It is not understood how Ki-67 contributes to these processes, or how these functions may be interrelated. We recently discovered that in human cells with intact G1/S cell cycle checkpoints, acute depletion of Ki-67 induces cell cycle inhibitor p21, reduces G1/S-regulated RNA levels, and delays S phase entry. These cell cycle phenotypes are accompanied by reduced maintenance of heterochromatin marks (e.g. H3K27me3) on the inactive X (Xi) chromosome in female checkpoint-proficient cells. Notably, all of these phenotypes are absent in cells lacking G1/S checkpoints. In other words, Ki-67 links cell cycle progression and chromosome maintenance in primary cells, and checkpoint-defective tumor cells evade these mechanisms. To begin molecular exploration of these novel functions, we will therefore test for molecular hallmarks of DNA damage upon Ki-67 depletion in checkpoint-proficient cells. We will also map which Ki-67 protein domains are required for its novel activities, and determine if they are separable from previously described roles in mitotic chromosome structure and interphase heterochromatin localization. In this manner, we will be poised to pursue relevant partner proteins on our path to new insights into the coordination of human chromosome structure and function.
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0.961 |
2021 |
Foltz, Daniel Richard Huang, Sui Kaufman, Paul D. |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
The Role of the Nucleolus in Human Genome Organization in Normal and Disease States @ Univ of Massachusetts Med Sch Worcester
7. Project Summary / Abstract In all eukaryotes, the largest nuclear body is the nucleolus, a phase-separated, non-membrane bound organelle specialized for the synthesis of ribosomal RNAs and their assembly into ribosomes. Additionally, the exterior of the nucleolus is a hub for interactions with multiple specific DNA loci, thereby contributing to the three-dimensional architecture of the eukaryotic nucleus. Nucleolus-genome interactions are intimately connected to processes central to human health. For example, nucleolar-associated DNA is highly enriched in centromeric repetitive sequences. Centromeres, the sites of chromosome attachment to mitotic spindles, are fundamentally important for proper chromosome segregation. Several nucleolar proteins have been implicated in centromere-nucleolar interactions, and several centromeric proteins prominently reside in nucleoli in interphase cells. We have found that the nucleolar- centromeric interactions are regulated during cellular differentiation and are greatly increased in cancer cells. However, the mechanisms that regulated these interactions remain unknown. Not only do cancer cells display increased centromere-nucleolar interactions, they also frequently contain a perinucleolar compartments (PNC), a complex cytological feature that is absent in non-tumor cells. PNCs are located on the surface of nucleoli and contain multiple RNA species and RNA-binding proteins. We demonstrate here that these bodies also contain specific DNA loci, some of which encode non-coding RNAs retained within PNCs. A candidate cancer therapeutic termed metarrestin was isolated based on its ability to dissociate PNCs; metarrestin is currently in clinical trials based on its ability to reduce metastasis in human tumor xenograft experiments. Importantly for this proposal, we have observed that metarrestin also perturbs centromere-nucleolar interactions. We also present data that centromere-nucleolus interactions are perturbed in macrophages upon exposure the bacterial lipopolysaccharide (LPS), a canonical stimulus for the innate immune system. We also show that this response is blocked upon inhibition of specific signaling pathways. These changes are accompanied by altered nuclear distribution of the H3K27me3, a histone modification characteristic of facultative heterochromatin. Altogether, the central theme of this proposal is that the factors that govern centromere-nucleolus interactions are important for understanding chromosome missegregation, metastasis, and innate immunity. We plan a series of synergistic experiments to learn more about the underlying mechanisms. For example, we will test whether the centromeric activity of neocentromeres generates nucleolar associations, or if instead that is a property of centromeric satellite repeats regardless of activity. We will take candidate and unbiased approaches to finding centromeric proteins required for nucleolar interactions. We will characterize how metarrestin affects association of DNA loci with PNCs and nucleoli, and we will define cis-acting loci involved in PNC association. We will characterize the signaling pathways required for signaling-mediated disruption of nucleolar-centromeric interactions in macrophages. Results from these studies will allow for subsequent testing of universality. For example, do signaling components in macrophages also operate in tumor cells when treated with the therapeutic metarrestin? In this manner, this collaborative proposal will unite questions from diverse experimental systems to answer questions about the fundamental links between nuclear organization and human health.
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0.961 |