NUCLEAR CAPTURE FOR CELL-TYPE SPECIFIC EPIGENETIC ANALYSIS OF ADIPOCYTES

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中分离特定类型脂肪细胞的技术问题（例如，肌肉，骨髓）进行遗传和表观遗传分析，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，在脂肪组织转化为代谢功能障碍状态中起重要作用，从而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。我们正在提出一种强大的技术，即核抗体捕获（CANA），它将使不同类型脂肪细胞的细胞类型特异性表观遗传分析成为可能。CANA基于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）"的表达方面不同，并且NMA可以用作用于纯化特定核的核免疫表位标签的事实。我们推测，脂肪细胞的亚类在肥胖个体中表观遗传学上被重编程。这项技术开发资助的目标是（1）产生一组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核，以及（2）研究 在与肥胖相关的人类内脏脂肪细胞核的两个亚类内的DNA甲基化和RNA表达谱的差异。我们将通过追求以下具体目标来实现这些目标，并为进一步检验我们的假设奠定基础：目标1。开发具有明确核亚类特异性的CANA试剂mAb。DiSH”（杂交瘤直接选择）技术能够高效快速地生产数百种针对膜抗原的mAb，将为人类内脏脂肪组织和小鼠米色和棕色脂肪细胞系的细胞核提供200多种抗核（抗NU）mAb。将选择具有最有用的核亚型特异性的那些，并且这些mAb将用于亲和纯化人内脏脂肪细胞核的两个亚类。目标2.描述从肥胖和瘦个体的内脏脂肪组织中纯化的两个特定核亲和力亚类的全基因组DNA甲基化和RNA表达谱。表观遗传学和基因表达数据将被整合，以确定肥胖相关基因。CANA技术解决了当今神经生物学面临的最大挑战之一，即处理成年神经元子集的困难。它将为包括肥胖在内的许多认知障碍的医学研究、诊断和治疗开发做出重大贡献。一旦建立，该方法可以扩展到支持整个身体的特定细胞类型的分子研究。

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.