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Tandem Tudor Domain Probes for Nanoscale Epigenetic Decoding

用于纳米级表观遗传解码的串联 Tudor 结构域探针

基本信息

批准号：
9328107
负责人：
M MITCHELL SMITH
金额：
$ 24.72万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-25 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9328107
关键词：
Acetylation Address Affinity Binding Biochemical Biological Models Biology Bioprobe Bromodomain Cell Nucleus Cell physiology Cells Chromatin Chromatin Fiber Chromatin Structure Clinical Code Color Complex Conflict (Psychology)Cytology DNA Methylation DNA Repair Defect Detection Development Dimensions Electron Microscopy Engineering Environment Enzymes Epigenetic Process Equation Euchromatin Fluorescent Probes Gene Expression Genes Genome Health Sciences Heterochromatin Histones Image Imagery In Situ Label Learning Lysine Maintenance Malignant Neoplasms Measures Methylation Methyltransferase Microscopy Modality Modeling Modification Molecular Molecular Structure Morphology N-terminal Nerve Degeneration Nuclear Nucleoplasm Nucleosomes Pathology Pattern Peripheral Play Polymerase Property Proteins Regulation Reporter Research Resolution Role Shapes Small Interfering RNA Structure Syndrome System Testing Time Work base combinatorial density design drug discovery epigenetic regulation histone methylation histone modification human disease imaging probe improved knock-down light microscopy macromolecule mutant nanometer nanoscale novel personalized approach public health relevance relating to nervous system response stem cell therapy

项目摘要

DESCRIPTION (provided by applicant): The modulation of heterochromatin structure and function plays a critical role in regulating gene expression, and defects in heterochromatin establishment and maintenance are associated with cancer, neural degeneracies, developmental pathologies, and other human diseases. However, many basic aspects of heterochromatin structure are poorly understood and are variously associated with conflicting models. This is due in large measure to experimental limitations. A major challenge in chromatin biology and molecular cytology is how to study the macromolecular structures and dynamics of specific regulated chromatin domains in single cells. We have developed model systems using super-resolution localization microscopy through which we can study specific epigenetic chromatin structures at nanoscale resolution. To date we have successfully applied this system to visualizing active chromatin domains by probing patterns of combinatorial lysine acetylations. Here we propose to extend this approach to develop genetically encoded bioprobes suitable for super-resolution microscopy that will recognize epigenetic features of heterochromatin and DNA damage repair foci. Our preliminary results support a focus on two tudor domain motifs and the research proposed here thus focuses on two principal aims: (1) We will exploit the properties of fluorescent probes based on the tandem tudor domain of Setdb1. Based on current epifluorescence images, we hypothesize that the structures seen reflect the properties of heterochromatin. We will visualize the morphologies of bound chromatin structures, measure their dimensions and densities, and compare the structures observed at the nuclear periphery, the perinucleolar compartment, and internal nucleoplasm. We will test the prediction that our reporters colocalize with known epigenetic marks of heterochromatin and with heterochromatin protein components. We will also test the prediction that reporter-bound chromatin responds in parallel with experimental perturbation of heterochromatin structures. (2) We will develop novel genetically encoded fluorescent probes based on the tandem tudor domain of UHRF1. We will visualize the morphologies of bound chromatin structures, measure their dimensions and densities, and compare the properties of structures within the heterochromatin subcompartments. We will manipulate these probes for multivalent recognition of H3K9me3 and the free N-terminal end of H3 by combining the tandem tudor domain and PHD domains for combinatorial recognition. Together, these aims will result in the development and characterization of novel probes for the super-resolution visualization of critical epigenetic heterochromatin environments labeled in situ. These fundamental advances, in turn, have the potential to radically improve strategies for drug discovery, and yield new treatment modalities for pathologies associated defects in chromatin and epigenetic regulation.