As one of the world’s greatest health challenges, obesity is usually accompanied by an increased risk for many chronic diseases, including type 2 diabetes, cardiovascular, hypertension and dyslipidemia. Obesity is a multifactorial disorder that is associated with both genetic and environmental factors, including eating behavior and exercise (Inagaki et al., 2016). As the link between genetic variants and environmental factors, the role of epigenetic modifications in obesity, including histone modifications, DNA methylation and non-coding RNA, had gained much attention in the late decade. Metabolic diseases, such as obesity and diabetes, were found to be tightly associated with epigenetic variation (Ling and Ronn, 2019). Among epigenetic locus, same histone could involve various modifications of several chemical groups, such as methylation and acetylation (Okuno et al., 2013), to activate or repress gene expression. For example, the various degrees of methylation of lysine 9 of histone H3 (H3K9me) are associated with transcriptional repression, whereas H3K4me and H3K27ac are associated with transcriptional activation.

Adipose tissues play vital roles in maintaining energy balance and are classified into white adipose tissue (WAT) and brown adipose tissue (BAT). WAT is responsible for energy storage and obesity syndrome, while BAT consumes fat to generate heat, which is essential for body weight and temperature control (Lee et al., 2014). The differentiation and stimulation of adipose tissue were regulated by various transcription factors and epigenetic modifications (Klose et al., 2006; Tateishi et al., 2009; Ohno et al., 2013; Zhou et al., 2014; Inagaki et al., 2016; Sanchez-Gurmaches et al., 2016; Wang and Seale, 2016; Cheng et al., 2018). For example, previous studies demonstrated the important roles of the methylation modifications of histone H3K9 sites in regulating metabolism of adipose tissue and maintaining homeostasis (Klose et al., 2006; Tateishi et al., 2009; Ohno et al., 2013; Cheng et al., 2018). High levels of H3K9me2 inhibited the expression of PPARγ and thereby affected the process of adipogenesis from preadipocytes (Rosen and Spiegelman, 2001; Takada et al., 2009). The deletion of G9a with the methylation of H3K9me1 and H3K9me2 in mouse adipose tissues could increase the expression of PPARγ and thereby promoted adipogenesis by increasing adipogenic gene expression (Tachibana et al., 2005; Wang et al., 2013). In addition, the histone demethylase LSD1 (also known as KDM1) was essential for the initiation of adipogenesis by decreasing H3K9me2 levels and maintaining H3K4me2 levels at promoter regions (Musri et al., 2010; Duteil et al., 2014). Moreover, high-fat diet (HFD) could stimulate the proliferation of preadipocytes in subcutaneous adipose tissue (Joe et al., 2009). A long term HFD could induce changes in epigenetic modifications in WAT, as demonstrated by decreased levels of H3K4me2 and increased levels of H3K36me2 in mice with HFD induced obesity (Nie et al., 2017). These results suggested that changes in epigenetic states could disrupt the functions of adipose tissue and ultimately led to obesity.

Energy metabolic equilibrium and inflammation of adipose tissues are essential during the establishment of obesity syndrome. Usually, less than 10% of macrophages are located in normal adipose tissues, whereas the development of obesity is associated with increases in the number of macrophages and the polarization of M1 macrophages, which are closely related to energy homeostasis and inflammation (Odegaard and Chawla, 2008; Chawla et al., 2011). Macrophages are the most abundant immune cells and proinflammatory cytokines in obese adipose tissues, that could promote insulin resistance of adipose tissue and result in type 2 diabetes and other metabolic diseases (Odegaard and Chawla, 2008; Chawla et al., 2011). However, little is known on the correlation between genes controlled by histone modification and energy metabolism or inflammation of WAT and BAT in HFD induced obesity syndromes.

In this study, to understand the effect of histone modification on gene expression across the whole genome under high fat diet conditions, we analyzed the methylation modifications of histone H3K9 sites (H3K9me2 and H3K9me3) near the transcription start site and H3K4me1 and H3K27ac histone modifications throughout the genome. Because of the fact that the traditional bulk RNA sequencing cannot identify cell type specific heterogeneity due to the mixture of cell types in the specimen, we additionally integrated two bulk RNA-seq and a single-nuclei adipocyte RNA-seq datasets (SNAP-seq, GSE133486) (Rajbhandari et al., 2019) to identify the correlation between genes activated via histone modification and inflammation associated energy metabolism. In this study, we attempted to address the following questions: (1) Which genes are activated or repressed by histone modification in mouse adipose tissues under a long term HFD? (2) What biological processes are associated with genes that are modulated by HFD induced histone modifications? (3) Are there any correlations between genes modulated by HFD induced histone modifications and energy metabolism or inflammation under both short- and long-term HFD conditions? Elucidation of these questions will expand our understanding of the influence of eating behavior on obesity and could assist the identification of putative therapeutic targets for the prevention and treatment of metabolic disorders.

The post-transcriptional modifications of histone tails are essential for the regulation of the chromatin state, which could result in activation or repression of the transcription of the associated genes. For example, the di-methylation and tri-methylation of histone H3 at lysine 9 (H3K9me2 and H3K9me3) are mostly associated with gene repression, whereas histone H3K27 acetylation and H3K4me1 are features of gene activation (Zhang T. et al., 2020). During the growth and differentiation of adipose tissue, histone modifications not only determine the fate of precursor cells but also participate in the regulation of physiological activities and the body’s adaptation to the environment. In this study, H3K9me2 and H3K9me3 modification sites were analyzed in the adipose tissue of SCD and HFD fed mice by chromatin immunoprecipitation and high throughput sequencing. Generally, we found a stronger signal of peaks around the transcription start site in SCD fed mice than HFD-fed group (Figures 1C,D), suggesting the common demethylation and activation of transcription through the whole genome induced by HFD.

The KEGG analysis showed that the genes activated by HFD feeding were mainly associated with lipogenesis, energy metabolism and insulin resistance. For example, the Wnt (Bowers and Lane, 2008), mTOR (Caron et al., 2015) and Hippo signaling pathway (Shu et al., 2020) were major regulators of adipogenesis. The mTOR (Caron et al., 2015), cAMP, AMPK, and PI3K-Akt signaling pathway were closely related to energy metabolism.

Notably, we found that the MAPK signaling pathway was enriched with many H3K9me2 and H3K9me3 de-methylation activated genes (Figure 2A) as well as H3K4me1, H3K27ac and RNA expression activated genes (Figure 2B) upon HFD treatment. MAPK pathways regulated multiple cellular functions, including cell proliferation, migration, differentiation, and apoptosis. A previous study showed that MAPK signaling related genes interacted with dietary factors involved in inflammation and oxidative stress (Slattery et al., 2013). Inflammation of fat adipose tissue could induce insulin resistance and metabolic disease (Acin-Perez et al., 2020), which suggested that activated MAPK might help to improve the understanding of the epigenetic effects of HFD on obesity and metabolic disease.

To illustrate the correlation between obesity, energy metabolism and inflammation, we integrated bulk RNA-seq and single-nuclei RNA-seq dataset of adipocytes from other published study. Single-nuclei RNA-seq could show cell type specific differential gene expression and the results showed that some genes (Map3k5, Met, and Vegfa) in the MAPK signaling pathway activated by a HFD via histone modifications showed high expression levels in the metabolic active adipocyte subtype in WAT though single-nuclei RNA sequencing. These genes might also participate in inflammation (Figures 4, 5) in mice, rat and humans under short-term or long-term HFD (Figure 5). In addition, western blot further confirmed the higher expression of Met and Vegfa genes and the enhanced ratio of p44/42 MAPK phosphorylation under HFD conditions (Figure 6).

Previous studies also showed the potential role of Map3k5, Met, and Vegfa in obesity and metabolic dysregulation (Rudich et al., 2007; Bian et al., 2010; Lu et al., 2012; Elias et al., 2013; Sundaram et al., 2014a, b; Hattori et al., 2016; Haim et al., 2017). For example, Sundaram et al. (2014a, b) found that the expression of Met increased in obese mice with breast cancer compared with their lean controls, suggesting that Met was potentially associated with obesity and immunity (Sundaram et al., 2014a, b). The MAP3K5 gene was upregulated in adipose tissues of obese animal (Haim et al., 2017), and a MAP3K5 mutation that decreased RNA expression was linked to obesity with whole body insulin resistance (Rudich et al., 2007; Bian et al., 2010). In mice, Map3k5 knockout mice became obese by interfering with brown fat function (Hattori et al., 2016). Vascular endothelial growth factor A (Vegfa), a member of the VEGF family, was also highly expressed in adipose tissues (Lu et al., 2012). Lu et al. (2012) found that mice with repressed expression of the Vegfa gene presented a lean phenotype and resistance to HFD induced body weight gain (Lu et al., 2012). Elias et al. (2013) showed that Vegfa overexpression in adipose tissue could enhance PGC-1α and UCP-1 expression, which resulted in increases in thermogenesis and energy expenditure and decreases in obesity, suggesting the important role of Vegfa under HFD conditions (Elias et al., 2013). In addition, increased Vegfa expression might be associated with higher inflammation in adipose tissues through its anti-inflammatory role in adipose tissue, which resulted in increases in activated macrophages (Elias et al., 2013; Schlich et al., 2013). Wang et al. (2006) found that the VEGF increased with the stimulation of pro-inflammatory factor TNFα in isolated progenitor cells of the human adipose, suggesting the potential roles of VEGF in inflammation. Here our study showed the significantly increased level of the phosphorylation of p42/44 MAPK in both BAT and WAT rat under a HFD condition, suggesting a relatively activated state of MAPK pathway in obesity adipose tissues. Overall, the demethylation activated genes Map3k5, Met and Vegfa in the activated MAPK signaling pathway could participate in inflammation and energy metabolism to lead to obesity, and thus, these genes might be promising markers for the prevention and treatment of metabolic diseases.

In summary, ChIP-seq technology was utilized to assess the changes in the modifications of histone H3 sites in BAT and WAT of mice under SCD or HFD conditions. We found that the HFD decreased the level of H3K9me2 or H3K9me3 level in gene promoters crossing the whole genome. Through integrated analyses of the bulk RNA transcriptome and single-nuclei adipocyte RNA sequencing in mouse, rat and human adipose tissues, we found that a series of genes that were activated by histone modification were also upregulated under both long-term and short-term HFD conditions. These genes, including MAP3K5, MET, and VEGFA, were enriched in KEGG pathways involving lipogenesis, energy metabolism, and inflammation pathways. All these findings would help us to improve the understanding of the influence of diet on obesity and human metabolic diseases through epigenetic modifications and could assist the identification of putative therapeutic targets for the prevention and treatment of metabolic disorders in the future.

The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/genbank/, GSE164530.

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Jiao Tong University and the Tongji University.

ZW, MZ, CZh, and ZL conceived and designed the study. ZW, MZ, MW, CZo, SL, YG, and SQ performed the experiments. ZW, MZ, and YG performed the data analysis. All authors participated in data interpretation, manuscript drafting, and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.