Enhancer-driven transcriptional regulation is a potential key determinant for human visceral and subcutaneous adipocytes

Highlights

• gene expression of in vitro differentiated human visceral and subcutaneous adipocytes is similar to human fat tissues

• visceral and subcutaneous adipocytes possess distinct chromatin landscapes, linked to different biological processes

• the respective enhancer landscapes are enriched for an opposing set of transcription factor motifs

• predominantly the chromatin landscape of visceral adipocytes is linked to obesity-associated diseases

Abstract

Obesity is characterized by the excess of body fat leading to impaired health. Abdominal fat is particularly harmful and is associated with cardiovascular and metabolic diseases and cancer. In contrast, subcutaneous fat is generally considered less detrimental. The mechanisms that establish the cellular characteristics of these distinct fat types in humans are not fully understood. Here, we explored whether differences of their gene regulatory mechanisms can be investigated in vitro. For this purpose, we in vitro differentiated human visceral and subcutaneous pre-adipocytes into mature adipocytes and obtained their gene expression profiles and genome-wide H3K4me3, H3K9me3 and H3K27ac patterns. Subsequently, we compared those data with public gene expression data from visceral and subcutaneous fat tissues. We found that the in vitro differentiated adipocytes show significant differences in their transcriptional landscapes, which correlate with biological pathways that are characteristic for visceral and subcutaneous fat tissues, respectively. Unexpectedly, visceral adipocyte enhancers are rich on motifs for transcription factors involved in the Hippo-YAP pathway, cell growth and inflammation, which are not typically associated with adipocyte function. In contrast, enhancers of subcutaneous adipocytes show enrichment of motifs for common adipogenic transcription factors, such as C/EBP, NFI and PPARγ, implicating substantially disparate gene regulatory networks in visceral and subcutaneous adipocytes. Consistent with the role in obesity, predominantly the histone modification pattern of visceral adipocytes is linked to obesity-associated diseases. Thus, this work suggests that the properties of visceral and subcutaneous fat tissues can be studied in vitro and provides preliminary insights into their gene regulatory processes.

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.