Subsets of Visceral Adipose Tissue Nuclei with Distinct Levels of 5-Hydroxymethylcytosine

Abstract

The reprogramming of cellular memory in specific cell types, and in visceral adipocytes in particular, appears to be a fundamental aspect of obesity and its related negative health outcomes. We explored the hypothesis that adipose tissue contains epigenetically distinct subpopulations of adipocytes that are differentially potentiated to record cellular memories of their environment. Adipocytes are large, fragile, and technically difficult to efficiently isolate and fractionate. We developed fluorescence nuclear cytometry (FNC) and fluorescence activated nuclear sorting (FANS) of cellular nuclei from visceral adipose tissue (VAT) using the levels of the pan-adipocyte protein, peroxisome proliferator-activated receptor gamma-2 (PPARg2), to distinguish classes of PPARg2-Positive (PPARg2-Pos) adipocyte nuclei from PPARg2-Negative (PPARg2-Neg) leukocyte and endothelial cell nuclei. PPARg2-Pos nuclei were 10-fold enriched for most adipocyte marker transcripts relative to PPARg2-Neg nuclei. PPARg2-Pos nuclei showed 2- to 50-fold higher levels of transcripts encoding most of the chromatin-remodeling factors assayed, which regulate the methylation of histones and DNA cytosine (e.g., DNMT1, TET1, TET2, KDM4A, KMT2C, SETDB1, PAXIP1, ARID1A, JMJD6, CARM1, and PRMT5). PPARg2-Pos nuclei were large with decondensed chromatin. TAB-seq demonstrated 5-hydroxymethylcytosine (5hmC) levels were remarkably dynamic in gene bodies of various classes of VAT nuclei, dropping 3.8-fold from the highest quintile of expressed genes to the lowest. In short, VAT-derived adipocytes appear to be more actively remodeling their chromatin than non-adipocytes.

Functional Implications of DNA Methylation in Adipose Biology

“…Similarly, reduced DNA methylation is observed during adipogenesis at the promoters of other adipocyte genes (e.g., Lep [ 30 ], Slc2a4 [GLUT4] [ 30 ], and Peln [ 27 ]). Intriguingly, recent global profiling studies have demonstrated that 5hmC (a cytosine that is an intermediate product of DNA demethylation) colocalizes with PPARγ at enhancers in 3T3-L1 adipocytes ( 31 ), and PPARγ-positive nuclei sorted from visceral adipose tissue from healthy humans are strongly coenriched with 5hmC ( 32 ). Given that the TET proteins, especially TET1 and TET2, are necessary for adipose conversion ( 33 ) and that PPARγ physically interacts with them ( 27 , 34 ) indicate that PPARγ is influencing the methylation pattern.…”

TET2 facilitates PPARγ agonist–mediated gene regulation and insulin sensitization in adipocytes

“…A recent large-scale functional association analysis from GWAS data identified a genetic variant within TET2 (rs9884482) that associates with fasting insulin level, suggesting an impact on insulin resistance [19]. Furthermore, recent global profiling studies have demonstrated that 5hmC colocalizes with PPARγ binding at enhancers in 3T3-L1 adipocytes [20], and PPARγ-positive nuclei sorted from visceral adipose tissue from healthy humans is strongly co-enriched with 5hmC [21]. Together, these studies suggest the presence of functional and physical interactions between TET proteins and PPARγ.…”

Epigenetic regulation of white adipose tissue in the onset of obesity and metabolic diseases

“…Nevertheless, 5hmC levels in VAT have been positively related to age and negatively associated with LDL cholesterol in humans 91 . In VAT, it has been shown that 5hmC levels were higher in the adipocyte (defined by PPARγ positive cells) versus the non‐adipocyte fraction and that the differences in 5hmC levels could have a high impact on transcription 92 . in vitro studies have also demonstrated a possible role of 5hmC in adipocyte differentiation.…”