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Subsets of visceral adipose tissue nuclei with discrete levels of 5-hydroxymethylcytosine

ABSTRACT: 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 Sus scrofa visceral adipose tissue (SsVAT) using the levels of the pan-adipocyte protein, peroxisome proliferator-activated receptor gamma-2 (PPARg2) to distinguish PPARg2-Positive nuclei from PPARg2-Neg (negative) leukocyte, endothelial, and adipocyte progenitor cell nuclei. PPARg2-Postive VAT nuclei showed 2- to 50-fold higher levels of transcripts encoding most of the chromatin-remodeling factors assayed regulating the methylation of histones and DNA cytosine (e.g., DNMT1, DNMT3A, TET2, TET3, KMT2C, SETDB1, PAXP1, ARID1A, KMT2C, JMJD6, CARM1/PRMT4, PRMT5). PPARg2-Positive nuclei have a large decondensed chromatin structure. TAB-seq demonstrated 5´-hydroxymethylcytosine (5hmC) levels were remarkably dynamic in the gene body of PPARg2-Positive nuclei, dropping 3.8-fold from the highest quintile of expressed genes to the lowest.

ORGANISM(S): Sus scrofa

PROVIDER: GSE73684 | GEO | 2016/07/20

SECONDARY ACCESSION(S): PRJNA297667

REPOSITORIES: GEO

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Project description:Adipose tissue is found throughout the human body. The diversity of physiological specialization of fat depots is reflected in the depot-specific alterations seen in lipodystrophies and links between specific patterns of fat distribution and susceptibility to diseases, including Type II Diabetes. We compared gene expression patterns in seven anatomically diverse fat depots and in adipocytes and stromal-vascular cells isolated from each sample. Adipocytes from different depots displayed distinct gene expression profiles. Characteristic patterns of expression of HOX genes distinguished adipocyte samples by site of origin. These depot-specific patterns were recapitulated when adipocyte precursors from each site were differentiated ex vivo, suggesting that these genes may have roles in specifying the depot-specific differentiation of adipocytes. Adipocyte expression of 300 genes with roles in energy metabolism showed both depot-dependent and inter-individual variation. Genes involved in glycogen metabolism and de novo fatty acid synthesis were generally most highly expressed in breast and abdominal subcutaneous adipocytes, suggesting a role for these depots in buffering glucose levels. Indeed, the role of adipocytes in primary metabolism of carbohydrate-rich foods may be considerably more important than has been appreciated. Genes involved in lipid uptake and hormone-stimulated lipolysis were on average most highly expressed in pericolonic, omental, and breast adipocytes; their expression covaried with each other and with that of the adipogenic transcription factor PPARG2. Two isoforms of PPARG, PPARG1 and PPARG2, were expressed in distinct patterns, each paralleling a discrete set of putative targets, suggesting that their differential regulation might influence important depot-specific and inter-individual differences in adipocytes. Dozens of genes encoding secreted proteins and receptors were highly expressed in adipocytes compared to adipose stromal-vascular cells and/or other tissues; many of these genes were not previously associated with adipocyte functions and are candidates for novel roles in sensing and signaling energy status to regulate global energy homeostasis.

Project description:Recents studies in mice and humans demonstrated the relevance of H2S-synthesising enzymes (such as CTH, CBS and MPST) in adipose tissue physiology and preadipocyte differentiation into adipocytes. Here, we aimed to investigate the combined role of CTH, CBS and MPST in the preservation of adipocyte protein persulfidation and adipogenesis. Joint CTH, CBS and MPST gene knockdown was achieved treating fully human adipocytes with siRNAs against these transcripts (siRNA_MIX). Adipocyte protein persulfidation was analyzed by a mass spectrometry label-free quantitative approach coupled with a dimedone-switch method for protein labeling and purification. The proteomic analysis quantified 216 proteins with statistically different persulfidation levels in KD cells compared to control adipocytes. In fully differentiated adipocytes, CBS and MPST mRNA and protein levels were abundant, whereas CTH expression was very low. Of note, siRNA_MIX administration resulted in a significant decrease in CBS and MPST expression, without impacting on CTH. Dual CBS and MPST gene knockdown resulted in decreased expression of relevant genes for adipocyte biology, including adipogenesis, mitochondrial biogenesis and lipogenesis, but increased proinflammatory- and senescence-related genes, in parallel to a significant disruption in adipocyte protein persulfidation pattern. While among less persulfidated proteins, we identified several relevant proteins for adipocyte adipogenesis and function, among the most persulfidated, key mediators of adipocyte inflammation and dysfunction, but also some proteins that might have a positive role of adipogenesis were found. In conclusion, current study indicates that joint knockdown of CBS and MPST (but not CTH) in adipocytes impairs adipogenesis and promotes inflammation, possibly by disrupting the pattern of protein persulfidation in these cells, and suggesting that these enzymes were required for the functional maintenance of adipocytes.

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Subsets of visceral adipose tissue nuclei with discrete levels of 5-hydroxymethylcytosine

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