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.
The ability to analyze the epigenome of human adipocytes is limited by the fact that adipocytes are difficult to efficiently separate from other cell types usig standard methods. The goal of this exploratory research grant is to generate monoclonal antibodies to adipocyte-specific nuclear membrane antigens, which can be used to affinity purify adipocyte nuclei for further molecular genetics and epigenetic analysis. Development of this technology will contribute to research, diagnostics and therapeutics on the numerous diseases descending from obesity and demonstrate the utility of using nuclear membrane antigens to purify nuclei for studies in many other fields of medicine, where cell type specific analyses are needed.
