Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms
Enhancer-driven transcriptional regulation is a potential key determinant for human visceral and subcutaneous adipocytes
Introduction
A large proportion of the population in industrial countries is burdened with obesity as well as its concomitant diseases, such as type-2 diabetes, hypertension and cancer [1, 2]. Cumulating number of studies suggest that the fat distribution in the body and the specific adipocyte types are critical determinants for the risks to develop obesity-related diseases [3]. In particular, visceral fat dominated obesity is linked to alterations of cardiac-associated structures, which lead to myocardial diseases such as hypertensive, ischemic heart diseases, and diabetic cardiomyopathy. This fat type is also related to secretion of lipids, adipokines, as well as pro-inflammatory and oxidative factors, which may contribute to the development of cancer and other diseases [2, 4]. In contrast, subcutaneous fat is characterized by a high mitochondrial content, leading to anti-inflammatory and anti-apoptotic properties, and is therefore considered to be less harmful [3].
In the past two decades, various therapeutic approaches were implemented to address obesity. Besides changes in lifestyle, increased activity, and implementation of bariatric surgery several newer drugs were introduced, providing promising alternative therapeutic options [5]. For example, administration of PPARγ agonists leads to a reduction of obesity-related phenotypes and cardiovascular failures [6]; possibly due to the trans-differentiation of visceral adipocytes into more subcutaneous-like adipocytes [7, 8]. A better understanding of the underlying molecular mechanisms and biological differences between visceral and subcutaneous fat could reinforce the exploration of novel molecular approaches on a cellular level. However, investigation of the transcriptional and chromatin landscape of adipocytes has predominantly been performed in mouse cells [[9], [10], [11], [12]], while only limited data are available for humans [13, 14]. In addition, the data for human adipocytes were mainly derived from cell lines, limiting their usability. Regarding visceral and subcutaneous fat tissues so far only gene expression [15, 16] and DNA methylation [17] patterns have been investigated, while a characterization of the chromatin landscape has not been performed yet. We reasoned that a comparative investigation of the histone modification patterns of human visceral and subcutaneous adipocytes could provide further insights about differences between visceral and subcutaneous fat, which could allow examining strategies to modulate human adipogenesis.
Herein, we explored whether it is possible to investigate the chromatin landscape of human visceral and subcutaneous adipocytes in vitro and whether the obtained results would be consistent with available data from patients. For this purpose, we initially obtained one sample of primary human visceral and subcutaneous pre-adipocytes, respectively, and differentiated them into mature adipocytes. Subsequently, we investigated their expression profiles and epigenetic landscapes using RNA-Seq and ChIP-seq, respectively, and compared those data with publicly available gene expression data of visceral and subcutaneous fat tissues. We demonstrate that these adipocytes have a differential gene expression pattern that is similar to what can be observed between visceral and subcutaneous human fat depots, supporting that the in vitro differentiated adipocytes are valid study subjects. Further, we found that those cells possess a distinctive pattern of histone modifications, with strongest differences seen at enhancers. Analysis of these data revealed a robust correlation between the chromatin landscapes with the biological properties of visceral and subcutaneous fat tissues. Subcutaneous adipocytes show enrichment for processes involved in general fat metabolism, while visceral adipocytes are associated with processes involved in development, cytoskeleton and innate immune response. Motif analysis of cell type-specific enhancers suggests that the given enhancer landscapes are established by a substantially different set of transcription factors. We further found that the chromatin landscape in both cells are linked to different obesity-related diseases, supporting the previous observation that subcutaneous and visceral adipocytes contribute distinctly to the development of obesity-associated diseases. Therefore, our work suggests that in vitro differentiated adipocytes are suitable study subjects to investigate differences between visceral and subcutaneous human fat tissues. Our data support that the biological differences of these fat types are based on discrete cell types, determined by different gene regulatory networks. An expansion of this initial study could provide valuable information to explore novel strategies to re-program visceral into more subcutaneous-like adipocytes [5], which may help to treat obesity-related diseases.
Section snippets
Cell culture
Human primary pre-adipocytes were obtained from Innoprot (Derio – Bizkaia, Spain) with following specifications: visceral pre-adipocytes (Cat. Number: P10761, Batch #5125, Healthy Caucasian, 15 years, female, sample obtained from tissue close to kidney), subcutaneous pre-adipocytes (Cat. Number: P10762, Batch #9163, Healthy Caucasian, 51 years, male, sample obtained from tissue close to lymph nodes). Cells were grown in poly-l-lysine (Sigma-Aldrich, Deisenhofen, Germany) coated flasks.
The in vitro differentiation of visceral and subcutaneous adipocytes is similar but not identical
We aimed first to identify a suitable approach to study the chromatin landscape of human visceral and subcutaneous fat in an in vitro setting. We hypothesized that in vitro differentiated adipocytes derived from subcutaneous or visceral pre-adipocytes may be sufficiently similar to the respective human fat tissues to study their chromatin landscapes. To explore whether this approach could be applicable, we obtained one sample of primary human visceral and subcutaneous pre-adipocytes,
Discussion
Risk reduction of obesity-associated diseases is a constant challenge in the treatment of patients suffering from obesity. The trans-differentiation of visceral adipocytes into more subcutaneous-like adipocytes in vitro [7] demonstrates that specific cell types can be manipulated into more favorable cell types, which may lead to reduced health risks. However, the underlying molecular mechanisms are mostly unknown. Recent technical advances to investigate the chromatin landscape in cells can
Conclusions
This exploratory study suggests that in vitro differentiated human visceral and subcutaneous adipocytes are potentially suitable study subjects to investigate differences between visceral and subcutaneous fat tissues. Our preliminary analysis suggests that visceral and subcutaneous adipocytes are discrete cell types, which each possess a characteristic chromatin and transcriptional landscape associated with distinct biological functions. The enhancer landscapes appear to be particularly
Limitations
This study aimed to elucidate whether the chromatin landscape of human visceral and subcutaneous fat can be investigated in vitro and to gain initial insights into the gene regulatory mechanisms of those fat types at a genome-wide level. In vitro cultured cells cannot fully reflect the properties of the in vivo situation, suggesting that our results only partially mirror the chromatin landscape of the adipocytes in vivo, and should therefore be considered as preliminary. For this study only one
Abbreviations
- C/EBP
CCAAT/Enhancer Binding Protein
- ChIP-Seq
Chromatin immunoprecipitation followed by DNA sequencing
- ETS
E26 transformation-specific
- GREAT
Genomic Regions Enrichment of Annotations Tool
- GR
glucocorticoid receptor
- GSEA
gene set enrichment analysis
- H3K27ac
Histone 3 Lysine 27 acetylation
- H3K4me3
Histone 3 Lysine 4 trimethylation
- H3K9me3
Histone 3 Lysine 9 trimethylation
- JAK
Janus Kinase
- MACS
Model-based Analysis of ChIP-Seq
- NES
Normalized Enrichment Score
- NF-κB
nuclear factor kappa-light-chain-enhancer of activated B
Funding
This study was supported by the German Research Foundation (DFG, LI 2057/2-1 to RL, and DA1214/2-1 to SD).
Authors' contributions
SD conceived the study. KB performed differentiation, RT-qPCR and ChIP-Seq experiments. MG performed editorial work. RL analyzed and interpreted the data and drafted the manuscript with the help of MG, KB and SD. All authors read and approved the final manuscript.
Transparency document
Acknowledgment
We thank the Transcriptome and Genome Analysis Laboratory (TAL), Core Unit at the University Medical Center Göttingen for performing next generation sequencing.
References (77)
- et al.
Global obesity: trends, risk factors and policy implications
Nat. Rev. Endocrinol.
(2013) Obesity and cancer–mechanisms underlying tumour progression and recurrence
Nat. Rev. Endocrinol.
(2013)Subcutaneous and visceral adipose tissue: structural and functional differences
Obes. Rev.
(2010)- et al.
Adipokines: a link between obesity and cardiovascular disease
J. Cardiol.
(2014) - et al.
Convertible visceral fat as a therapeutic target to curb obesity
Nat. Rev. Drug Discov.
(2016) - et al.
PPARs and the complex journey to obesity
Nat. Med.
(2004) Chronic peroxisome proliferator-activated receptor gamma (PPARgamma) activation of epididymally derived white adipocyte cultures reveals a population of thermogenically competent, UCP1-containing adipocytes molecularly distinct from classic brown adipocytes
J. Biol. Chem.
(2010)- et al.
Turning WAT into BAT: a review on regulators controlling the browning of white adipocytes
Biosci. Rep.
(2013) Dynamic rewiring of promoter-anchored chromatin loops during adipocyte differentiation
Mol. Cell
(2017)Global mapping of cell type-specific open chromatin by FAIRE-seq reveals the regulatory role of the NFI family in adipocyte differentiation
PLoS Genet.
(2011)
Comparative transcriptomic and epigenomic analyses reveal new regulators of murine brown adipogenesis
PLoS Genet.
MLL3/MLL4 are required for CBP/p300 binding on enhancers and super-enhancer formation in brown adipogenesis
Nucleic Acids Res.
Comparative epigenomic analysis of murine and human adipogenesis
Cell
Browning of human adipocytes requires KLF11 and reprogramming of PPARgamma superenhancers
Genes Dev.
Worsening of obesity and metabolic status yields similar molecular adaptations in human subcutaneous and visceral adipose tissue: decreased metabolism and increased immune response
J. Clin. Endocrinol. Metab.
Body mass index-independent inflammation in omental adipose tissue associated with insulin resistance in morbid obesity
Surg. Obes. Relat. Dis.
An analysis of DNA methylation in human adipose tissue reveals differential modification of obesity genes before and after gastric bypass and weight loss
Genome Biol.
Ultrafast and memory-efficient alignment of short DNA sequences to the human genome
Genome Biol.
deepTools: a flexible platform for exploring deep-sequencing data
Nucleic Acids Res.
Galaxy: a platform for interactive large-scale genome analysis
Genome Res.
Cistrome: an integrative platform for transcriptional regulation studies
Genome Biol.
Bioconductor: open software development for computational biology and bioinformatics
Genome Biol.
Model-based analysis of ChIP-Seq (MACS)
Genome Biol.
The human genome browser at UCSC
Genome Res.
Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles
Proc. Natl. Acad. Sci. U. S. A.
Enrichment map: a network-based method for gene-set enrichment visualization and interpretation
PLoS One
Cytoscape: a software environment for integrated models of biomolecular interaction networks
Genome Res.
i-cisTarget: an integrative genomics method for the prediction of regulatory features and cis-regulatory modules
Nucleic Acids Res.
GREAT improves functional interpretation of cis-regulatory regions
Nat. Biotechnol.
The NIH roadmap epigenomics mapping consortium
Nat. Biotechnol.
An integrated encyclopedia of DNA elements in the human genome
Nature
STAR: ultrafast universal RNA-seq aligner
Bioinformatics
Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2
Genome Biol.
An early inflammatory gene profile in visceral adipose tissue in children
Int. J. Pediatr. Obes.
Gene expression profiling in subcutaneous, visceral and epigastric adipose tissues of patients with extreme obesity
Int. J. Obes.
Exploration, normalization, and summaries of high density oligonucleotide array probe level data
Biostatistics
Linear models and empirical bayes methods for assessing differential expression in microarray experiments
Stat. Appl. Genet. Mol. Biol.
Cited by (3)
Weighted gene co-expression network analysis reveals similarities and differences of molecular features between dilated and ischemic cardiomyopathies
2023, Journal of Electronic Science and TechnologyEffect of peroxisome proliferator ⁃ activated receptor γ on skin physiological and pathological processes
2023, Chinese Journal of Dermatology
- 1
Equal contribution.