Regulation of adipogenesis in obesityby hydroxymethylation

Yale University, New Haven CT

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Abstract

Project Summary: Regulation of adipogenesis in obesity by hydroxymethylation More than two thirds of adults, and almost one third of adolescents in America are overweight or obese, with combined medical costs amounting to over $147 billion annually. In obesity, white adipose tissue expands due to the growth of existing adipocytes and the differentiation of new adipocytes from adipose stem cell progenitors (APs). It has been suggested that adipogenesis is regulated by hydroxymethylation, a form of epigenetic regulation. Epigenetics refers to heritable gene function changes that occur without nucleotide sequence changes. The Ten-eleven-translocation family of dioxygenases (TET1-3) regulates cell fate decisions by oxidizing 5-methylcytosine (5mC), a marker of gene silencing, to 5-hydroxymethylcytosine (5hmC), a stable marker of gene activation. Understanding the role of hydroxymethylation in regulating adipose differentiation is critical for understanding the mechanisms governing its expansion and may provide insight into novel targets for anti-obesity therapies. I will test the hypothesis that Tet1 catalyzes the hydroxymethylation required to upregulate adipogenic genes necessary for adipocyte progenitor differentiation. My first aim is to characterize the spatiotemporal changes in hydroxymethylation during diet-induced obesity (DIO) and identify the cell types in which these changes occur. Preliminary experiments using murine models of DIO indicate that hydroxymethylation patterns in visceral and subcutaneous adipose change dramatically over the course of obesity. Spatiotemporally mapping these changes will identify when epigenetic changes occur, and which cell types are affected. My second aim is to determine which genes are regulated by hydroxymethylation in adipose progenitor stem cells. In obesity, APs divide and differentiate, causing fat pad growth. This important population of cells can be isolated for study using fluorescence-activated cell sorting. Characterizing how changes in methylation patterns alter gene expression in APs may uncover novel insights into the regulation of adipogenesis. Antibodies that detect epigenetic modifications (5mC or 5hmC) can selectively enrich regions of DNA that have been modified, and combined with next-generation sequencing, this approach will identify genes that are being silenced or activated. These experiments may identify novel regulators of adipogenesis. My third aim is to determine if Tet1 is the enzyme that catalyzes hydroxymethylation to regulate adipogenesis. Preliminary studies in mouse visceral adipose tissue show that Tet1 mRNA increases after high-fat diet feeding and that 5hmC accumulates, but it is currently not known which Tet isoform is responsible. Transgenic mice lacking Tet1 have been generated, but the phenotype of their adipose tissue and their response to high-fat diet have not been explored. These experiments will help determine if Tet1 is required for adipogenesis.