NUCLEAR CAPTURE FOR CELL-TYPE SPECIFIC EPIGENETIC ANALYSIS OF ADIPOCYTES

用于脂肪细胞细胞类型特异性表观遗传分析的核捕获

• 批准号: 8760476
• 负责人: Richard Brian Meagher
• 金额: $ 35.61万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-26 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8760476
• 关键词: Address; Adipocytes; Adipose tissue; Adult; Affect; Affinity; Antibodies; Antibody Affinity; Antigens; Blood; Bone Marrow; Brown Fat; Candidate Disease Gene; Cell Line; Cell Nucleus; Cell Separation; Cells; Chromatin; Chromatin Structure; Cognition Disorders; Companions; Cytometry; DNA Methylation; Data; Development; Diabetes Mellitus; Diagnostic; Diagnostics Research; Disease; Disease Progression; Endocrine Glands; Environment; Epigenetic Process; Epitopes; Fluorescence; Gene Expression; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Goals; Grant; Human; Human Genome; Hybridomas; Individual; Inflammation; Investigation; Malignant Neoplasms; Medical Research; Medicine; Membrane; Membrane Proteins; Metabolic; Metabolic syndrome; Methodology; Methods; Microscopy; Modification; Molecular; Molecular Genetics; Molecular Profiling; Monoclonal Antibodies; Morphology; Mus; Muscle; Mutation; Neurobiology; Neurodegenerative Disorders; Neurons; Nuclear; Nuclear Envelope; Obesity; Play; Production; Property; Proteome; Reagent; Recovery; Research Project Grants; Role; Sampling; Sorting - Cell Movement; Specificity; Staging; T-Lymphocyte; Technology; Testing; Therapeutic; Time; Tissues; Visceral; Work; adipokines; adiponectin; base; cardiovascular disorder risk; cell type; cost; disorder risk; epigenome; genome-wide; insulin sensitivity; mammalian genome; meetings; methylome; public health relevance; technology development; therapeutic development

DESCRIPTION (provided by applicant): This proposal addresses two important issues: the technical issue of isolating specific types of adipocytes from adipose depots and other tissues (e.g., muscle, bone marrow) for genetic and epigenetic analysis, and the issue of determining epigenetic changes to subclasses of adipocytes that are associated with obesity. Epigenetic reprogramming of adipocytes, particularly in visceral adipose depots, plays an important role in the transformation of adipose tissue to a metabolically dysfunctional state contributing to increased systemic inflammation and reduced insulin sensitivity. However, the epigenetic reprogramming of subclasses of adipocytes is essentially unexplored due to the technical difficulty of isolating these cells. We are proposing a powerful technology, Capture by Nuclear Antibody (CANA) that will enable cell type-specific epigenetic analysis of different classes of adipocytes. CANA is based on the fact that mammalian genomes encode more than two thousand nuclear trans-membrane proteins, that cell types differ in the expression of these "nuclear membrane antigens (NMAs)", and NMAs may serve as nuclear immuno-epitope tags for the purification of specific nuclei. We hypothesize that subclasses of adipocytes are epigenetically reprogrammed in obese individuals. The goals of this technology development grant are to (1) generate a battery of adipocyte nuclear membrane antigen-specific monoclonal antibodies (mAbs) to affinity purify distinct classes of human adipocyte nuclei and (2) investigate differences in DNA methylation and RNA expression profiles within two subclasses of human visceral adipocyte nuclei that are associated with obesity. We will meet these goals and set the stage for further testing our hypothesis by pursuing the following Specific Aims: Aim 1. Develop CANA reagent mAbs with clear nuclear subclass specificities. DiSH" (Direct Selection of Hybridomas) technology, which enables the efficient and rapid production of hundreds of mAbs to membrane antigens, will provide more than 200 anti-nuclear (anti-NU) mAbs to nuclei from human visceral adipose tissue and mouse beige and brown adipocyte cell lines. Those with the most useful nuclear subtype specificities will be selected and these mAbs will be used to affinity-purify two subclasses of human visceral adipocyte nuclei. Aim 2. Characterize the genome-wide DNA methylation and RNA expression profiles in two specific subclasses of nuclei affinity purified from the visceral adipose tissue of obese and lean individuals. Epigenetic and gene expression data will be integrated to identify obesity-associated genes. CANA technology addresses one of the greatest challenges to neurobiology today, the difficulty of working with subsets of adult neurons. It will contribute significantly to medical research, diagnostic and therapeutic development in studies of numerous cognitive disorders, including obesity. Once established, the methodology can be expanded to support molecular studies of specific cell types throughout the body.

描述（由申请人提供）：该提案解决了两个重要问题：从脂肪库和其他组织（如肌肉、骨髓）中分离特定类型的脂肪细胞进行遗传和表观遗传分析的技术问题，以及确定与肥胖相关的脂肪细胞亚类的表观遗传变化的问题。脂肪细胞的表观遗传重编程，特别是在内脏脂肪库中，在脂肪组织向代谢功能失调状态的转变中起着重要作用，导致全身炎症增加和胰岛素敏感性降低。然而，由于分离这些细胞的技术困难，脂肪细胞亚类的表观遗传重编程基本上未被探索。我们提出了一种强大的技术，核抗体捕获（CANA），它将使不同类型的脂肪细胞的细胞类型特异性表观遗传分析成为可能。CANA是基于这样一个事实：哺乳动物基因组编码超过2000种核跨膜蛋白，细胞类型在这些“核膜抗原（nma）”的表达上有所不同，nma可以作为核免疫表位标签，用于纯化特定的细胞核。我们假设脂肪细胞亚类在肥胖个体中是表观遗传重编程的。这项技术发展资助的目标是：(1)产生一系列脂肪细胞核膜抗原特异性单克隆抗体（mab），以亲和纯化不同类别的人类脂肪细胞细胞核；(2)研究