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Developing fluorescent quantum dots as tools to study the dynamic organization of pluripotent chromatin in embryonic stem cells

Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01080
Funds requested: 
$874 792
Funding Recommendations: 
Not recommended
Grant approved: 
No
Public Abstract: 
The nucleus is a membrane-enclosed organelle found in eukaryotic cells and contains most of the cell's genetic material. Genes are organized inside the nucleus in a complex of DNA and proteins that are packaged into units called chromosomes. A central function of the nucleus is to control the activities of the cell by regulating gene expression, which is the basis for cellular division and differentiation and therefore highly relevant to the study of embryonic stem (ES) cells. The genome of ES cells must exhibit high plasticity in order to retain the capacity to enter any one distinct differentiation pathway. The molecular mechanisms for self-renewal, maintenance of pluripotency and lineage specification are poorly understood. Advances in nuclear imaging technologies have revealed a high level of chromatin organization, known as nuclear architecture. Genes from different chromosomes are often in close physical proximity and form discrete foci dubbed transcription factories, which help to orchestrate their transcription and organize the genome in the three-dimensional nuclear space. It has been suggested that a highly dynamic chromatin organization in ES cells is required to maintain plasticity. The in vivo labeling of single endogenous chromosome loci in diploid cells, which would revolutionize the analysis of dynamic genome organization, has not been established. Here we propose to develop a new fluorescence microscopy method to study chromatin dynamics in ES cell self-renewal and differentiation. We will develop quantum dots (QDs), fluorescent semiconductor nanocrystals, as probes to visualize single chromatin sites, without the need to manipulate genomic DNA. QDs offer several advantages over fluorescent proteins such as exceptional brightness and resistance against photobleaching, properties that allow the visualization of a single QD. We will establish a rapid, versatile and efficient strategy to label chromatin with QDs in ES cells. 3D time-lapse microscopy will be used to visualize and track individual sites over time. We plan to investigate the molecular mechanisms of chromatin movement inside the nucleus and to identify ‘dynamic genes’; i.e. genes that change their 3D position inside the nucleus either as a cause or consequence of their activation/repression. Analyzing gene dynamics might establish new principles of epigenetic mechanisms and provide new markers for ES cell identity and differentiation, i.e. identification of characteristic 3D gene patterns marking the transition form a pluripotent to a committed state. The developed QD probes will also be of potential use as diagnostic tools for the identification and functional analysis of stem cells and their derivatives. The proposed strategy might provide new assay platforms to investigate pluripotency and tumorgenicity.
Statement of Benefit to California: 
Embryonic stem (ES) cells possess the unique ability to develop into different cell types of the adult body, and therefore have great potential to be utilized in a wide variety of diagnostic and drug discovery applications and to treat or cure various chronic diseases and injuries. However, significant technical hurdles need to be overcome before stem cell research can be effectively translated to the clinic. One important contribution to our understanding of stem cells includes the development and validation of assays for pluripotency and the capacity to differentiate. Stem cell renewal and differentiation are dynamic processes involving the coordinated expression of specific genes. The nuclei of ES cells have a distinct architecture, especially at chromosome loci involved in maintaining pluripotency. Understanding how the nuclear genome is organized in ES cells provides a framework within which other large-scale chromosome changes that may accompany differentiation can be studied. We propose to study the three-dimensional organization of the nuclear genome and to determine if the dynamic distribution of genes inside the nucleus is relevant for their regulation. In addition, we address the question how the observed intranuclear movements of genes are mediated. Changes in nuclear organization have been linked to aberrant developmental processes, cellular aging and tumorigenesis. In fact, the capacity to regenerate damaged tissues from stem cells declines during aging. The molecular mechanisms that result in age-related defects in nuclear genome organization are largely unknown. Furthermore, many cancers are thought to originate from stem cells and our studies might have a direct impact on cancer research. Our studies might uncover new principles of gene regulation and epigenetic changes in chromatin organization. The development of fluorescent nanocrystals (quantum dots) as specific chromatin stains has the potential to generate markers to further our understanding of pluripotency. This could provide new insights into stem cell-based therapies as well as the selection and manipulation of stem cells. The latter aspect is of particular interest in the context of our work, since nuclear architectural changes are crucial for nuclear reprogramming, a procedure that is central to therapeutic cloning. We envision that using QDs as gene-specific probes might lead to the development of assays to screen for drugs to treat for age-related ES cell defects and other aberrant changes in gene expression caused by dysfunctional nuclear organization. Ultimately, our investigations will lead to a better understanding of regulatory errors in gene expression that influence ES cell self-renewal and differentiation and that contribute to cancer progression. We hope to translate this knowledge into novel strategies to detect nuclei exhibiting aberrant gene activity.
Review Summary: 
This application describes the development of a new fluorescence microscopy technology for investigating changes in the dynamic three dimensional organization of chromatin and the potential role of these changes in epigenetic regulation in pluripotent and committed embryonic stem (ES) cells. The Principal Investigator (PI) proposes to devise tools for labeling specific loci within chromatin that can be visually monitored over time. Initial efforts will focus on the development of efficient methods for delivering bioconjugated Quantum Dots (QD) to stem cell nuclei. Next, the applicants will generate functionalized QD probes to label components of chromatin as well as probes to label specific genes. Finally, the PI will use fluorescence microscopy to map the spatial and temporal patterns of these markers as the stem cells grow and differentiate. The reviewers were enthusiastic about the proposed technology but were uncertain of its potential to advance stem cell biology. While the premise was well conceived and rational, the proposal lacked focus and seemed overly ambitious. The reviewers were ultimately unconvinced of its feasibility due to unanswered questions about the validity of the methods. The reviewers agreed that the proposed technology could be tremendously useful, not only to the stem cell community but also for basic biological research. The ability to globally and gene specifically monitor chromatin dynamics in real time is not currently possible and has the potential to provide new insights into mechanisms of gene regulation, which ultimately dictates the behavior and function of cells. In theory, this effort could lead to the identification of new markers for pluripotency, improved methods for directing the differentiation of cells and possible drug screening approaches. The reviewers were confident in the qualifications of the applicants to conduct the proposed experiments. The overall premise was well rationalized and convincingly supported by preliminary data. Furthermore, appropriate milestones were defined, and the PI provided a good discussion of potential pitfalls and how these might be addressed. The reviewers were particularly appreciative of the PI’s’ description of planned uses for the proposed technology. While acknowledging these positive attributes, the reviewers were uncertain of the overall feasibility of this effort. The reviewers noted that many elements of the research design were unfocused or unclear. The types of targeting molecules to be developed and conjugated to the QD were many. The reviewers suggested that perhaps a focus on a carefully chosen few key targeting molecules to conjugate would be more realistic for a 2 year proposal where only one of the conjugations has been demonstrated. The reviewers also noted that it appeared that more than one targeting agent would be conjugated to the QD and had questions about how this would be controlled as multiple conjugates to a QD were not discussed. Also, the PI proposes to use a particular type of QDs to avoid potential steric issues yet most of the proposed experiments appear to use a different type of QD. While there may have been a rationale for doing so, it was not apparent from the applicants’ discussion. The reviewers were also disappointed that only one line of investigation was proposed to demonstrate normal behavior in labeled cells. As QDs and bioconjugation might be detrimental to function, it would be very important to use a more comprehensive validation strategy so that meaningful data could be obtained. Finally, as the proposed experiments will be performed using a murine cell line, the reviewers were disappointed that the applicants failed to address the potential difficulties in transferring this technology to human cells. The reviewers considered the PI and the team well qualified to conduct the proposed research. The PI has extensive experience with sophisticated microscopic and imaging technology, a strong background in applying these technologies to cell structures and chromatin remodeling and is well poised to lead the imaging experiments to elucidate nuclear architecture. The collaborators provide appropriate complementary expertise in stem cell biology and in an approach to specific gene targeting. One reviewer commented that the PI might be over-extended in terms of balancing commitment to this effort (10%) with additional responsibilities. In summary, while the proposed technology could be useful for a wide variety of applications, the reviewers were uncertain of its feasibility within the award timeframe.
Conflicts: 

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