NUCLEAR CAPTURE FOR CELL-TYPE SPECIFIC EPIGENETIC ANALYSIS OF ADIPOCYTES

描述(申请人提供)：这项建议涉及两个重要问题：从脂肪仓库和其他组织(例如肌肉、骨髓)中分离特定类型的脂肪细胞以进行遗传和表观遗传学分析的技术问题，以及确定与肥胖相关的亚类脂肪细胞的表观遗传学变化的问题。脂肪细胞的表观遗传重编程，特别是在内脏脂肪储存库中，在脂肪组织向代谢紊乱状态的转变中发挥着重要作用，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，亚类脂肪细胞的表观遗传学重新编程基本上是未知的。我们正在提出一项强大的技术，被核抗体捕获(CANA)，这将使不同类型的脂肪细胞的细胞类型特异性表观遗传学分析成为可能。CANA的基础是哺乳动物基因组编码2000多种核跨膜蛋白，这些核膜抗原(NMA)的表达类型不同，NMA可以作为核免疫表位标签用于特定核的纯化。我们假设肥胖个体的脂肪细胞亚类在表观遗传学上是重新编程的。这项技术发展基金的目标是(1)产生一组脂肪细胞核膜抗原特异的单抗(MAbb)，以亲和纯化不同类别的人类脂肪细胞核和(2)研究 与肥胖相关的两类人内脏脂肪细胞核内DNA甲基化和RNA表达谱的差异。我们将实现这些目标，并通过追求以下具体目标为进一步检验我们的假说奠定基础：目标1.研制具有明确核亚类特异性的CANA试剂单抗。DISH“(杂交瘤直接选择)技术能够高效、快速地生产数百株抗膜抗原的单抗，将从人类内脏脂肪组织以及小鼠米色和棕色脂肪细胞系向细胞核提供200多株抗核(抗Nu)单抗。那些具有最有用的核亚型特异性的单抗将被挑选出来，这些单抗将用于亲和纯化人类内脏脂肪细胞核的两个亚类。目的2.从肥胖者和瘦肉者的内脏脂肪组织中纯化出两种特定的核亲和力亚类，对全基因组DNA甲基化和RNA表达谱进行表征。表观遗传学和基因表达数据将被整合，以识别与肥胖相关的基因。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。它将对包括肥胖在内的众多认知障碍的研究的医学研究、诊断和治疗发展做出重大贡献。一旦建立，该方法可以扩展到支持全身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.