1986 |
Belmont, Andrew S. |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
A 3-D Approach to Chromosome Structure @ University of California San Francisco |
0.905 |
1989 — 1991 |
Belmont, Andrew |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Acquisition of An Optical Sectioning Workstation @ University of Illinois At Urbana-Champaign
A major challenge in cell biological research today is to understand how numbers of component biological molecules interact within complex systems to carry out fundamental cellular functions. Frequently these systems correspond to large, nonsymmetrical structural assemblies, extending from thousands to tens of thousands of angstroms within the cell, whose function is intimately related to their underlying structural architecture, Newly developed techniques of electron microscopy tomography and light microscopy optical sectioning are capable of providing detailed 3-dimensional reconstructions of such assemblies at overlapping resolution. This grant is for the acquisition of a light microscopy optical sectioning workstation to complete an image analysis facility capable of carrying out 3-dimensional reconstructions at both the light and electron microscopy resolution level. The light microscopy station described will allow 3-dimensional structure determination within living cells and tissues. These capabilities represent a vital resource to the research programs of the facility's users. This research is focused on large-scale chromosome structure and nuclear organization, the role of cytoskeleton and function in modulating key events in Drosophila embryonic development (major users), the specific structural coupling between the extracellular matrix and the cytoskeleton and its regulatory role in cell growth, differentiation, and migration, and mapping of neural networks (minor users).
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1 |
1990 — 1994 |
Belmont, Andrew S. |
R29Activity Code Description: Undocumented code - click on the grant title for more information. |
Nuclear and Chromosome Decondensation During G1-S @ University of Illinois Urbana-Champaign
The highest levels of chromatin structure, consisting of the packing of 100 and 300 angstrom chromatin fibers within mitotic and interphase chromosomes, are unknown. Understanding this level of chromatin organization, describing the structure of entire transcription or replication units, is essential to answering fundamental questions related to gene regulation, DNA recombination, cell differentiation, and cell cycle control. This project's long term objectives are to develop a model system allowing dissection of basic structure-function motifs involved in the large-scale chromatin organization of mammalian interphase chromosomes. Specifically, it will focus on the nuclear and chromosomal decondensation occurring during cell cycle progression through Gl into S phase. The strategy will be to develop an in vitro system reproducing decondensation events occurring in vivo, and to exploit this system to analyze their underlying biochemical and structural basis and functional relevance to DNA replication initiation. The grant will lay the foundation for this long term project. A detailed 3-dimensional structural analysis will compare nuclear and chromosome decondensation in vivo during Gl progression with decondensation induced in vitro using cell free extracts. Several 3-dimensional reconstruction methods, including light microscopy optical sectioning, electron microscopy serial sectioning, and electron microscopy axial tomography, will provide overlapping resolution over a complete range of size scales. The specific aims of this grant will include the following: Interphase chromosome decondensation in vivo and in vitro will be described in terms of the intranuclear arrangement of interphase chromosomes, association of chromosomes with nuclear lamins, uncoiling of interphase chromosomes into their component large-scale chromatin structures, and packaging of 300 angstrom chromatin fibers within these individual large- scale domains. The unfolding pathway of early versus late replicating regions will be contrasted, and the architecture of actively replicating chromatin will be determined. Finally, quantitative upper limits on the experimental errors associated with these structural descriptions will be estimated.
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0.958 |
1994 — 1997 |
Belmont, Andrew Stupp, Samuel Greenough, William [⬀] Juraska, Janice (co-PI) [⬀] Abbott, Louise |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Acquisition of 200 Kev Transmission Electron Microscope @ University of Illinois At Urbana-Champaign
The purpose of this proposal is to request funds for the purchase of a Philips CM- 200 Transmission Electron Microscope to be housed in the Visualization Facility (BVF) of the Beckman Institute on the University of Illinois campus. The Principal Investigators will be using this electron microscope for a variety of studies including (i) neuroplasticity and its relationship to underlying cellular and system level processes (William Greenough), (ii) the study of monodisperse rod-coil block copolymers (Samuel Stupp), (iii) analysis of nuclear and chromosome architecture (Andrew Belmont), (iv) sex differences in the organization and behavioral function of the brain (Janice Juraska), and (v) the characterization of morphological and functional abnormalities in cerebellar neural circuitry in a genetic animal model of movement dysfunction (Louise Abbott). In addition to these core projects there are 6 other research projects from a variety of disciplines for which this instrument provides immediate or long term benefits. The purpose of obtaining a new instrument is twofold. Firstly it will provide access to an intermediate voltage instrument for biological research on the UIUC campus. The only intermediate voltage instruments in this region are exclusively devoted to material science research and biologists with projects for which higher voltages are necessary have to travel to remote sites. Secondly it will replace an existing 18 year old instrument currently located in the BVF that is suffering increasingly from age related problems. In addition to providing unique capabilities for imaging thicker specimens this microscope will have a number of other important advantages over the existing instrument. The ability to control the instrument through a computer interface and to collect micrographs using a CCD camera will greatly aid, and in certain cases be essential for several applications. Specifically, for techniques such as serial sectioning, quantitative stereology and single axis tomography which involve the collection or examination of large numbers of images, semi-automatic acquisition procedures can be devised that not only simplify the process but also ensure that the electron dose is minimized and the region of interest is maximized. Furthermore, certain applications of these 3-dimensional reconstruction techniques require literally hundreds of images per data set and these applications for practical considerations will only be feasible given the availability of direct digital data acquisition. These control and imaging capabilities will also greatly improve the routine acquisition of high quality images. Further advantages of the new microscope arise from the advances in technology over the past 20 years. These have led to simplification of alignment procedures, improvements in beam coherence, lenses and goniometer stages and the implementation of techniques for acquiring images at low doses in order to limit beam damage. The microscope will be located in the Beckman Visualization Facility which houses a number of other related instruments (including a confocal microscope, a stereology workstation and various light microscopes) as well as supporting equipment (wet lab, microtomes, darkrooms) and excellent facilities for the digital processing and analysis of images. The location of the microscope in the multi- disciplinary environment of the Beckman Institute, which also includes part of the National Center for Supercomputing Applications, provides an excellent environment to take advantage of some of the latest technology available for the on- line processing and analysis of electron micrographs. We are strongly committed to providing access to this instrument and its associated, special facilities to the campus wide user group whose research wholly or partly depends on the application of electron microscopy. There is enthusiastic commitment to this project from both the Beckman In stitute and the University as a whole. This has been demonstrated by the contribution of 50% matching funds for the project as well as the supporting infrastructure for the equipment provided by the Beckman Institute Visualization Facility.
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1 |
1995 — 1998 |
Belmont, Andrew S. |
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. |
Large Scale Chromatin Structure and Function @ University of Illinois Urbana-Champaign |
0.958 |
1999 — 2002 |
Belmont, Andrew S. |
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. |
Chromatin Domain Structure/Function @ University of Illinois Urbana-Champaign
Current models of gene regulation suggest that interphase chromosomes are divided into large, functionally distinct, chromatin domains, facilitating independent regulation of individual gene loci. Typically these domains are tens to hundreds of kbp in size. The actual structural and molecular mechanisms underlying the establishment of these domains, however, have remained poorly understood. To date, only indirect structural assays have been available to analyze the chromatin structure of these domains. A key limitation has been the technical difficulty associated with directly visualizing the large-scale chromatin folding surrounding specific genes. Our long term objectives are to dissect the large-scale chromatin folding of specific gene loci, to analyze the cis and trans determinants of these folding motifs, and to determine the functional significance of this level of chromatin organization with regard to transcriptional regulation. Our specific aims for this project period will be to address the following questions: 1. What changes in large-scale chromatin organization accompany transcriptional activation? 2. Do SAR/MAR sequences have a direct, structural role in organizing chromatin domains? 3. What is the relationship between domain boundary elements, defined by molecular assays, and sequences associated with cytologically defined domain boundaries? 4. Do LCR sequences alter large-scale chromatin organization? Using novel visualization methods, in vivo dynamics of specific gene loci will be observed directly and light and electron microscopy will be applied to visualize the large-scale chromatin organization and nuclear positioning of these chromosomal regions. These visualization capabilities will allow direct testing of several structural based models of chromatin domain organization and gene regulation. Moreover, these studies are likely to lay the foundation for future mechanistic studies aimed at dissecting the role of cis and trans determinants of chromatin domain structure and function. Insight acquired from these studies should be of help in guiding the design of future constructs and artificial chromosomes used in gene therapy.
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0.958 |
1999 — 2002 |
Belmont, Andrew S. |
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. |
Large Scale Chromatin Structure/Function @ University of Illinois Urbana-Champaign
Within the higher eukaryotic chromosome, DNA is folded into multiple levels of organization. Together these yield greater than a 10,000:1 linear condensation of DNA. The highest levels of chromatin folding, consisting of the folding of 30 nm chromatin fibers, account for up to a 250:1 packing ratio and involve the organization of entire transcription and replication functional domains, their compaction within mitotic and interphase chromatids, and their architecture within interphase nuclei. Essentially uncharacterized at this time, it is this large-scale chromatin structure which is the focus of our research. Our long term objectives are to determine the basic folding motifs underlying large-scale chromatin structure and chromosome architecture, the functional significance of specific conformational changes associated with transcription and replication, and the underlying mechanisms which regulate these conformational transitions. Our specific aims are to answer the following questions: 1) What structural motifs underlie large-scale chromatin condensation/decondensation during the mitotic cell cycle? 2) Which steps in large-scale chromatin condensation during formation of mitotic chromosomes are dependent on SMC protein function? 3) What changes in large scale chromatin structure and intranuclear positioning are associated with DNA replication initiation? 4) What are the DNA sequence requirements for condensed versus extended interphase large-scale chromosome structures? In addition we will explore several experimental approaches to test the role of specific proteins in maintaining specialized large-scale chromatin structures within interphase chromosomes and to search for new proteins involved in large- scale chromatin organization and nuclear architecture. This proposed research will provide a basic description of the folding motifs underlying large-scale chromatin and chromosome organization. Future directions of our work will focus on integrating structural models of large-scale chromatin organization with the underlying biochemistry and molecular biology. In particular we are interested in exploring the mechanistic links between transcription, replication, and large- scale chromatin and nuclear structure. Knowledge gained from this research should provide valuable insight into regulation of transcription and improved design of gene therapy vectors and stable transgene expression.
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0.958 |
2003 — 2021 |
Belmont, Andrew Steven |
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. |
Chromatin Domain Structure and Function @ University of Illinois At Urbana-Champaign
DESCRIPTION (provided by applicant): Despite extensive correlations, a direct functional relationship has not yet been established between the highest levels of chromatin folding, or large-scale chromatin structure, and regulation of transcription. Similarly, despite extensive correlations of changes in intranuclear gene positioning and association with specific nuclear compartments, we still do not know how genes change intranuclear positions and associations, let alone understand the functional consequences of these changes. Our long-term objectives are to determine the large-scale chromatin folding and intranuclear positioning of specific gene loci, to identify the cis and trans determinants of this folding and intranuclear positioning, and to understand the functional significance of this level of chromatin organization with regard to transcriptional regulation. The specific aims of this proposal are to: : 1. Determine the relationship between large-scale chromatin compaction versus transcriptional activation. 2. Determine the relationship between nuclear speckle compartmentalization versus transcriptional activity and/or RNA processing. 3. Determine the relationship between localization to the nuclear periphery and specific epigenetic modifications versus transcription activity and induction. This project will exploit a novel system using BAC transgenes to reconstitute key features of endogenous gene loci as well as new methodologies for visualizing specific chromosome loci in live cells and at the ultrastructural level. Our project will critically test both old and new models for how the highest levels of chromatin folding modulate gene expression. Additional impact on a still wider range of basic science and biotechnology will come from our continued development of new technology for visualizing chromosome structure and dynamics and application of these methods for improved transgene and multi-transgene expression.
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0.958 |
2004 |
Belmont, Andrew S. |
P41Activity Code Description: Undocumented code - click on the grant title for more information. |
Interphase Chromatin Motion Using Fluorescence Microscopy @ University of Illinois Urbana-Champaign
fluorescence microscopy; chromatin; cell cycle; biomedical resource;
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0.958 |
2004 — 2007 |
Belmont, Andrew S. |
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. |
Large-Scale Chromatin Structure and Dynamics @ University of Illinois Urbana-Champaign
DESCRIPTION (provided by applicant): Within the higher eukaryotic chromosome, DNA is folded into multiple levels of organization, yielding alinear compaction of DNA of approximately 20,000 fold. The highest levels of chromatin folding, consisting of the folding of 30 nm chromatin fibers, account for up to a 500:1 packing ratio and involve the organization of entire transcription and replication functional domains, their compaction within mitotic and interphase chromatids, and their architecture within interphase nuclei. Poorly characterized, it is this large-scale chromatin structure which is the focus of our research. Our long term objectives are to determine the basic folding motifs underlying large-scale chromatin structure, the functional significance of specific conformational changes associated with transcription and replication, and the underlying mechanisms which regulate these conformational transitions. Our specific aims are to address the following questions: 1) What structural motifs underlie large-scale chromatin structure of interphase chromosomes? 2) What structural rearrangments in large-scale chromatin structure occur during DNA replication and daughter chromatid segregation? 3) What structural motifs underlie mitotic chromosome condensation and decondensation? 4) Which aspects of large-scale chromatin structure require condensin or cohesin function? This proposed research will provide a basic description of the folding motifs underlying large-scale chromatin and chromosome organization. Future directions of our work will focus on integrating structural models of large-scale chromatin organization with the underlying biochemistry and molecular biology. In particular we are interested in exploring the mechanistic links between large-scale chromatin structure and nuclear architecture to transcription, replication and recombination. Knowledge gained from this research should provide valuable insight into regulation of basic molecular processes and may serve to guide design of future gene therapy vectors and stable transgene expression.
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0.958 |
2007 — 2010 |
Belmont, Andrew S. |
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. |
Chromatin Domain Structure and Funcion @ University of Illinois Urbana-Champaign
[unreadable] DESCRIPTION (provided by applicant): Project Summary: Models of gene regulation suggest large-scale chromatin organization regulates transcription by restricting accessibility of large protein complexes to target sequences, controlling interactions between distant regulatory sequences, and/or modulating intranuclear gene positioning. However, the actual large-scale chromatin organization of transcriptionally active gene loci is unknown, with textbook models based largely on indirect molecular assays. Our long-term objectives are to determine the large-scale chromatin folding and intranuclear positioning of specific gene loci, to identify their cis and trans determinants, and to understand the functional significance of this level of chromatin organization with regard to transcriptional regulation. Several recent experimental developments indicate a high probability for productive investigations. We have developed methods allowing direct visualization of large-scale chromatin decondensation and intranuclear movements accompanying gene activation of BAG transgenes- first in live cells, and then at the ultrastructural level using a novel immunogold labeling procedure. These methods should be applicable to endogenous gene loci. The specific aims for this project period will be to: (1) Directly determine changes in 3-D large-scale chromatin ultrastructure accompanying gene activation; (2) Directly visualize changes in intranuclear positioning of gene loci associated with gene activation / repression and test the dependence of these movements on actin / myosin; (3) Identify the cis and trans determinants of changes in large-scale chromatin structure and intranuclear positioning of gene loci associated with gene activation / repression; (4) Dissect the molecular sequence determinants which determine Drosophila polytene chromosome band and interband organization. Public Health Relevance: Currently a major impediment to development of gene therapy methods is our incomplete understanding of the requirements for ensuring high and sustained levels of expression from transgenes. Insight from our studies should be useful in guiding the design of future gene constructs and artificial chromosomes used in gene therapy. [unreadable] [unreadable] [unreadable]
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0.958 |
2011 — 2014 |
Belmont, Andrew Steven |
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. |
Engineering Stable, Independent Multi-Transgene Expression in Mammalian Cells @ University of Illinois Urbana-Champaign
DESCRIPTION (provided by applicant): Achieving high level, reproducible, stable transgene expression in mammalian cells remains a major bottleneck to critical areas of biomedical research, including production of protein biopharmaceuticals, gene therapy, cellular reprogramming, tissue engineering, as well as basic research into fundamental molecular and cellular biology processes and mechanisms of disease. The rationale for the proposed research is to develop the optimal components for a single and multiple transgene BAC expression system that will overcome long- standing problems in mammalian transgene expression and find applications over a wide range of biomedical research areas. Our long-term goal is to overcome existing problems in mammalian transgene expression in order to achieve the ability to engineer entire synthetic gene networks into human cells for improved ex vivo gene therapy and tissue engineering applications. The specific aims of this proposal are to: 1. Identify appropriate DNA genomic regions, cloned within BACs, and promoters that can be used to drive copy number dependent, position independent expression of single and multiple transgenes. 2. Optimize BAC / promoter combinations through the minimization of BAC size and addition / deletion of appropriate cis regulatory regions. 3. Apply this technology to specific test "driver" applications requiring multi-gene expression, including improved methods for generating induced pluripotent stem cells and facilitated high-throughput screening for chemicals which modulate stem cell pluripotency and differentiation. We propose to develop a general methodology enabling the engineering in a single step any mammalian cell line to express stably any single protein, or set of multiple proteins, at levels comparable to 100s fold higher than endogenous genes. Completion of our proposal should result in an improved methodology for generation of iPS cells and transdifferentiation, and more broadly a new methodology for tissue engineering applications. Our approach is innovative because it builds on special insights derived from our basic science investigations into how 10 and 30 nm chromatin fibers fold into interphase chromosomes, and the relationship between this "large-scale chromatin structure" and transcriptional activation. PUBLIC HEALTH RELEVANCE: This project is relevant to NIH's mission because it directly addresses the critical problem of expressing foreign genes in mammalian cells. This problem currently is a major technical bottleneck limiting progress in multiple applied areas including production of protein biopharmaceuticals, gene therapy, and stem cell engineering.
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0.958 |
2015 — 2019 |
Belmont, Andrew Steven |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Combined Cytological, Genomic, and Functional Mapping of Nuclear Genome Organization @ University of Illinois At Urbana-Champaign
? DESCRIPTION: Decades of microscopy have revealed that the nucleus is not a homogeneous organelle, but rather consists of distinct compartments such as nucleoli, nuclear speckles, the nuclear lamina, among other structures. Increasing evidence indicates that specific genomic regions each associate with these compartments. This genome compartmentalization has been linked to various functions, but these links are still poorly understood. Interestingly, Lamina Associated Domains (LADs) share specific heterochromatin marks, defining chromatin domains with distinct genetic and epigenetic properties. Genomic regions associating with other nuclear compartments may similarly define distinct classes of chromatin domains. One major bottleneck towards a deeper understanding of nuclear organization has been the inability to convert microscopy views of nuclear compartments into genome-wide maps that show which loci are associated with which compartment, and how the chromosomal fiber traverses between compartments. In addition, there is an urgent need for more efficient methods to dissect the mechanisms by which large genomic regions are targeted to specific nuclear compartments. Finally, there is an urgent need for high-throughput approaches that query the functional relevance of genome compartmentalization. For this Center grant, we propose to meet these needs through the following Aims: 1. Develop a strategy that connects microscopy views to genome-wide maps that, together with modeling, reveal the localization and dynamics of genomic regions relative to all major nuclear compartments. 2. Develop methods for efficient manipulation of the genome in order to elucidate mechanisms that target loci to specific compartments. 3. Develop methods to measure, model, and validate the functional relevance of nuclear compartments. The combined results of these approaches will reveal causal relationships now hidden among entangled genomic, epigenetic, and nuclear organization features. Deliverables of this proposal include a wide range of structural and functional maps of nuclear organization, reagents for visualizing endogenous chromosome loci, a powerful pipeline for synthesis of ~100kb DNA fragments, and cell lines facilitating repeated, high-fidelity insertio of these large fragments back into selected sites in the genome. These resources will provide a powerful complement to other 4D Nucleome Consortium efforts. A key strength of this Center proposal is the experience and complementary research capabilities of its five Investigators. Together they will pool their expertise for a concerted investigation into the biological functions of nuclear compartmentalization.
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0.958 |
2015 — 2019 |
Belmont, Andrew Steven |
U54Activity Code Description: To support any part of the full range of research and development from very basic to clinical; may involve ancillary supportive activities such as protracted patient care necessary to the primary research or R&D effort. The spectrum of activities comprises a multidisciplinary attack on a specific disease entity or biomedical problem area. These differ from program project in that they are usually developed in response to an announcement of the programmatic needs of an Institute or Division and subsequently receive continuous attention from its staff. Centers may also serve as regional or national resources for special research purposes, with funding component staff helping to identify appropriate priority needs. |
Biological Validation Development @ University of Illinois At Urbana-Champaign
PROJECT SUMMARY / ABSTRACT ? BIOLOGICAL VALIDATION DEVELOPMENT The ultimate goals of this Module are to demonstrate that we are able to predict chromosome locations and trajectories relative to different nuclear compartments from genome-wide, DamID and TSA-Seq mapping data AND to identify the biological significance of this chromosome positioning. We will accomplish these goals through three major aims. Each will involve iterative cycles of experimentation and computer modeling. The first is to validate predictions for chromosome positioning and dynamics with respect to major nuclear compartments from genome-wide DamID and TSA-Seq data. This involves initially calibrating the output of our genome maps, such that we can estimate contact frequencies and distance distributions relative to each of the major nuclear compartments considered separately. More ambitiously, this calibration data will be used as input for computer modeling in the DAM Module: mapping data for multiple nuclear compartments will be combined with other genomic data to yield more accurate predictions of chromosome location and trajectories. These predictions will then be tested by direct microscopy observations and used to refine our predictive modeling. Second, we aim to identify DNA regions, and ultimately cis elements, responsible for targeting chromosome regions to specific nuclear compartments. Our third and final goal is to predict and test the functional consequences of chromosome loci localization near different nuclear compartments. We will do this through a novel combination of live-cell readout of functional assays as a function of chromosome position AND through a deliberate, rewiring of the trajectories of endogenous chromosomes, followed by readouts using molecular methods. More specifically, we will focus on the following Specific Aims: 1: Validate intranuclear chromosome position as a function of DamID and TSA compartment genome-wide mapping data; 2: Test predicted mechanisms by which intranuclear chromosome compartmentalization is established; 3: Test predicted functional consequences of chromosome localization to different nuclear compartments Completion of these Aims should provide us with the capability and confidence for interpreting the biological significance of changes in genome organization relative to nuclear compartments that we observe in different cell and tissue types. We anticipate that this level of nuclear organization plays a critical but previously unrealized role in establishing and maintaining tissue-specific patterns of gene expression. Therefore the work in this Module will be critical for the future application of our new mapping technologies towards the improved understanding of cell function in normal development and human disease.
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0.958 |
2017 — 2019 |
Belmont, Andrew Kannanganattu, Prasanth Kumar |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Developing Tsa-Rna-Seq For Subcellular Transcriptomics @ University of Illinois At Urbana-Champaign
This project will develop novel ways of finding which RNA molecules are found near a given kind of protein in eukaryotic cells. As the first test case, we will ask what species of RNA are enriched around one kind of formation in the nucleus, called nuclear speckles. The approach that we will develop will be applied later to determine the RNA composition in other cellular domains. This project will serve as a platform for teaching undergraduate, community college, and high school students to appreciate biology, and also for mentoring graduate students.
The localization and targeting of RNAs to specific cellular regions and compartments has been identified as an important mechanism in controlling several cellular processes such as cell migration, neuronal signaling and development in a wide array of model organisms. A major impediment to the understanding of RNA localization studies, however, is the current absence of a good genome-wide method to identify RNAs localized in specific cellular compartments. The objective of the present project is to develop a novel and robust genomic method, TSA-RNA-seq to identify RNAs that are enriched in a specific cellular compartment. TSA-RNA-seq will be developed in the context of a specific sub nuclear compartment called nuclear speckle. Nuclear speckles are dynamic structures enriched with proteins and RNAs involved in mRNA metabolism. Characterization of the mechanism/s that governs specific interactions between speckle-associated genes and RNAs would unravel the principles that define the contribution of nuclear speckles in gene expression and RNA maturation. The primary objectives of the project include: 1) Development of TSA-RNA-seq to identify nuclear speckle-resident RNAs. 2) Development of biochemical purification of speckles and to identify speckle-localized RNA. 3) Validation of speckle-resident RNA. The accomplishment of the objectives in this project would result in development of excellent tools that could be used to identify and to understand the transcriptome associated with any subnuclear domain.
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1 |
2021 |
Belmont, Andrew Steven Han, Kyu Young |
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. |
Identification of the Active Nuclear Niche(S) Using Novel Proteomic, Genomic, Transgenic, and Live-Cell Microscopy Technologies @ University of Illinois At Urbana-Champaign
The study of gene expression and possible role of condensates in regulating gene expression have largely ignored known nuclear structures. This proposal is significant because we propose a novel model for the role of nuclear organization in regulating gene expression: 1) Nuclear speckles and still unknown nuclear compartments/bodies help organize other phase-separated condensates to modulate gene expression; 2) Nuclear speckles together with surrounding nuclear compartments/bodies and associated phase-separated condensates together represent active nuclear niches which may have different functional properties; 3) Small distances matter: gene movements of only a few hundred nm between repressive and these different active nuclear niches may differentially regulate gene expression; 4) Action-at-a distance: component flux into and out of these nuclear compartments will have global effects on gene expression; 5) These same nuclear compartments/bodies may similarly modulate RNA processing and organize nuclear export. Here we propose to: 1) Identify multiple components of known and still unknown nuclear ?active niches?; 2) Map genome-wide the positions and predicted movements of genes relative to these active niches during physiological transitions; 3) Visualize nuclear body/compartment dynamics and fluxes of proteins between nuclear bodies in steady-state and through physiological transitions; 4) Visualize movements of reporter transgenes, endogenous genes, and rewired chromosome loci relative to these nuclear bodies/compartments and temporally correlate changes in gene expression with their dynamic movements and compartment associations; 5) Visualize movements of pre-mRNAs and nuclear mRNAs during RNA processing and export; 6) Measure fluxes of nuclear body components to and from adjacent transcribing chromatin. Additionally, we propose developing relatively low-cost, novel microscope platforms and software specifically designed to facilitate these live-cell imaging goals in our laboratories as well as others. Our Aims will be to: 1. Map proteins, genes, RNAs relative to active nuclear compartment(s) using iterative rounds of TSA-MS-Ratio, validation by light microscopy, and TSA-Seq; 2. Measure dynamics of bodies, components of nuclear bodies using live-cell imaging; 3. Measure temporal correlation between changes in gene expression and gene movement relative to nuclear bodies and visualize the export path of expressed transcripts; 4. Design and deliver two novel microscopes designed to facilitate Aims 1-3 at a modest cost. Successful completion of these Aims should significantly change our current understanding of the role of nuclear organization in regulating gene expression with impact across a wide range of research fields.
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0.958 |