Subsets of visceral adipose tissue nuclei with discrete levels of 5-hydroxymethylcytosine
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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.
Project description: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. Sus scrofa VAT (SsVAT) nuclei were isolated from SsVAT. SsVAT nuclei were stained with PPARg2 and sorted with fluorescence activated nuclear sorting (FANS) into PPARg2-High, PPARg2-Med (Medium), PPARg2-Low, and PPARg2-Neg (Negative) four populations.TAB-seq data on 5-hydroxymehtylcytosine (Yu, M. et al. 2012. Cell 149, 1368-1380) was collected from genomic DNA isolated from PPARg2-High, PPARg2-Med+Low (pooled PPARg2-Med and PPARg2-Low), and PPARg2-Neg SsVAT nuclei.
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:Single nucleus RNA sequencing (snRNA-seq), an alternative to single cell RNA sequencing (scRNA-seq), encounters technical challenges in obtaining high-quality nuclei and RNA, persistently hindering its applications. Here, we present a robust technique for isolating nuclei across various tissue types, remarkably enhancing snRNA-seq data quality. Employing this approach, we comprehensively characterize the depot-dependent cellular dynamics of various cell types underlying adipose tissue remodeling during obesity. By integrating nuclear RNA-seq data from adipocyte nuclei of varying sizes, we identify distinct adipocyte subpopulations categorized by size and functionality. Specifically, we characterize dysfunctional hypertrophic adipocytes prevalent in visceral adipose tissues during obesity, exhibiting cellular stress, inflammation and impaired metabolic gene expression. Obesity-induced changes in gene expression profiles of adipocyte subpopulations reveal their distinct contributions to adipose tissue pathophysiology. Our study establishes a robust snRNA-seq method, providing novel insights into the mechanisms orchestrating adipose tissue remodeling during obesity, with broader applicability across diverse biological systems.
Project description:Two types of UCP1 positive cells-brown and beige adipocytes exist in mammals. Beige adipocytes are very plastic, and can be dynamically regulated by environment.Beige adipocytes formed postnatally in subcutaneous inguinal white adipose tissue (iWAT) lost thermogenic gene expression and multilocular morphology at adult stage, but cold could restore their “beigeing” characteristics, a phenomenon termed as beige adipocyte renaissance. Our results showed that beige cell maintenance and renaissance in adult mice were regulated by cAMP and HDAC4 signaling in white adipocytes non-cell autonomously. Genetic modulations of various components of this cAMP-HDAC4 cascade (e.g. LKB1) led to persistent browning and reduced adiposity independent of thermogenesis. To further study the mechanisms of beige adipocytes maintenance, we performed RNA-seq with samples from inguinal white adipose tissues of WT, AdipoqCre LKB1 F/F, and AdipoqCre LKB1 F/F; HDAC4 F/F mice.Our studies will move the beige adipocyte field forward and attract clinical applications to target beige adipocyte renaissance.
Project description:Human adipose depots are functionally distinct. Yet, recent single-nucleus RNA-sequencing (snRNA-seq) analyses largely uncovered overlapping/similar cell-type landscapes. We hypothesized that adipocytes subtypes, differentiation trajectories, and/or intercellular communication patterns could illuminate this depot similarity-difference gap. For this, we performed snRNA-seq of human subcutaneous/visceral adipose tissues (5/10 samples, respectively). Of 27,665 adipocyte nuclei in both depots, the majority were “classical”, namely- enriched in lipid metabolism pathways. However, we also observed “non-classical” adipocyte subtypes, enriched in immune-related, extracellular matrix deposition (fibrosis), vascularization/angiogenesis, or ribosomal processes. Pseudo-temporal analysis showed a developmental trajectory from adipose progenitor cells to classical adipocytes via non-classical adipocytes, suggesting that the classical state stems from loss, rather than gain, of specialized functions. Lastly, intercellular communication routes were consistent with the different inflammatory tone of the two depots. Jointly, these findings provide a high-resolution view into the contribution of cellular composition, differentiation, and intercellular communication patterns to human fat depot differences.
Project description:To investigate the effect of ZNF395 on adipogenesis, we tested whether ZNF395 enhance cell conversion from human dermal Fibroblast (FIB) to Adipocyte (ADP). PPARG2 was reported as a master regulator and can induce adipogenesis in non-adipogenic fibroblasts. We transduced PPARG2 with or without ZNF395 in FIB with lentivirus. Interestingly, co-transduction of PPARG2 and ZNF395 showed higher occurrence of adipocyte-like cells as compared with PPARG2 alone. Moreover, genes related with lipid metabolic process and lipid transport was significantly up-regulated in combination of PPARG2 and ZNF395. These results suggest that ZNF395 co-ordinate the transcriptional regulatory pathway with PPARG2, necessary for the induction of adipogenesis.
Project description:Obesity is considered an important factor for many chronic diseases, including diabetes, cardiovascular disease and cancer. The expansion of adipose tissue in obesity is due to an increase in both adipocyte progenitor differentiation and mature adipocyte cell size. Adipocytes, however, are thought to be unable to divide or enter cell cycle. We demonstrate that mature human adipocytes unexpectedly display a gene and protein signature of cell cycle re-entry. Adipocyte cell cycle progression associates with obesity and hyperinsulinemia, with a concomitant increase in cell size, nuclear size and nuclear DNA content. However, chronic hyperinsulinemia in vitro or in patients, is associated with subsequent cell cycle exit, leading to a premature senescent transcriptomic and secretory profile in adipocytes. Premature senescence is rapidly becoming recognized as an important mediator of stress-induced tissue dysfunction. By demonstrating that adipocytes can re-enter cell cycle we define a mechanism for how mature, human adipocytes senesce and demonstrate that by targeting the adipocyte cell cycle program it is possible to impact adipocyte senescence and obesity-associated adipose tissue inflammation.
Project description:The transcriptional mechanisms by which temporary exposure to developmental signals instigates adipocyte differentiation are unknown. During early adipogenesis, we find transient enrichment of the glucocorticoid receptor (GR), CCAAT/enhancer binding protein b (CEBPb), p300, mediator subunit 1, and histone H3 acetylation near genes involved in cell proliferation, development and differentiation, including the gene encoding the master regulator of adipocyte differentiation, peroxisome proliferator activated receptor g2 (PPARg2). Occupancy and enhancer function are triggered by adipogenic signals, and diminish upon their removal. GR, which is required for adipogenesis but need not be active in the mature adipocyte, transiently functions with other enhancer proteins to propagate a new program of gene expression that includes induction of PPARg2, thereby providing a memory of the earlier adipogenic signal. Thus, the conversion of preadipocyte to adipocytes involves the formation of an epigenomic transition state that is not observed in cells at the beginning or end of the differentiation process. Genomic occupancy profiled by high throughput sequencing (ChIP-seq) from 3T3-L1 cells during differentiation for H3K9Ac, CEBPb and GR.
Project description:Beyond its thermogenic potential, brown adipose tissue (BAT) performs important endocrine functions that regulate metabolism. However, the BAT microenvironment and the factors involved in BAT homeostasis and adaptation to cold remain poorly characterized. We therefore aimed to study secreted factors from active brown adipocytes that may be involved in adipocyte function and/or may orchestrate inter-cellular communications. For this, mRNA levels in mature adipocytes of brown, beige and white adipose depots from mice exposed to 21 days of cold were evaluated using RNA sequencing, and bioinformatic analysis was used to predict for potentially secreted factors. Cxcl12 was found to be the most cold-induced C-X-C chemokine in BAT, and Cxcl12 mRNA expression analysis by qPCR and fluorescence in-situ hybridization revealed its enrichment in brown adipocytes upon cold. Cold increased CXCL12 secretion from BAT, yet its level in plasma remained unchanged indicating a potential local action. Cxcl12 knockdown in mature brown adipocytes impaired thermogenesis, estimated by norepinephrine (NE)-induced Ucp1 gene expression, glycerol release and mitochondrial respiration despite unaltered β-adrenergic signaling, suggesting Cxcl12 regulates adipocyte function independently from the β-adrenergic pathway. Importantly, unaltered adipocyte characteristics upon Cxcl12 loss may indicate CXCL12 primarily regulates the NE-induced adipocyte activation. Furthermore, CXCL12 might exert inter-cellular crosstalk via its capacity to promote macrophage chemotaxis and neurite outgrowth. Here we present CXCL12 as a novel brown adipocyte, cold-induced secreted factor involved in adipocyte function and inter-cellular crosstalk within BAT.
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.