Set7/9, a methyltransferase, regulates the thermogenic program during brown adipocyte differentiation through the modulation of p53 acetylation

https://doi.org/10.1016/j.mce.2016.04.022Get rights and content

Highlights

  • The expression level of Set7/9 was decreased during brown adipocyte differentiation.

  • Ectopic expression of Set7/9 led to enhanced expression of key thermogenic genes.

  • Knockdown of Set7/9 led to significantly reduced expression of key thermogenic genes.

  • Set7/9 acts as a fine regulator of the thermogenic program during brown adipogenesis.

Abstract

Brown adipose tissue, which is mainly composed of brown adipocytes, plays a key role in the regulation of energy balance via dissipation of extra energy as heat, and consequently counteracts obesity and its associated-disorders. Therefore, brown adipocyte differentiation should be tightly controlled at the multiple regulation steps. Among these, the regulation at the level of post-translational modifications (PTMs) is largely unknown. Here, we investigated the changes in the expression level of the enzymes involved in protein lysine methylation during brown adipocyte differentiation by using quantitative real-time PCR (qPCR) array analysis. Several enzymes showing differential expression patterns were identified. In particular, the expression level of methyltransferase Set7/9 was dramatically repressed during brown adipocyte differentiation. Although there was no significant change in lipid accumulation, ectopic expression of Set7/9 led to enhanced expression of several key thermogenic genes, such as uncoupling protein-1 (UCP-1), Cidea, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), and PR domain containing 16 (PRDM16). In contrast, knockdown of endogenous Set7/9 led to significantly reduced expression of these thermogenic genes. Furthermore, suppressed mitochondrial DNA content and decreased oxygen consumption rate were also detected upon Set7/9 knockdown. We found that p53 acetylation was regulated by Set7/9-dependent interaction with Sirt1. Based on these results, we suggest that Set7/9 acts as a fine regulator of the thermogenic program during brown adipocyte differentiation by regulation of p53 acetylation. Thus, Set7/9 could be used as a valuable target for regulating thermogenic capacity and consequently to overcome obesity and its related metabolic diseases.

Introduction

Adipocytes play important roles in energy homeostasis in human and other mammals, and are thus closely associated with metabolic disorders such as obesity and type 2 diabetes. There are two distinct types of adipocytes, white and brown adipocytes, which have opposite functions with regard to the energy balance (Bae et al., 2012, Kim et al., 2015a). White adipocytes store excess energy as triglycerides in lipid droplets, whereas brown adipocytes release energy in the form of heat through thermogenesis. Unlike white adipocytes, the thermogenic capacity of brown adipocytes results from the expression of the brown fat-defining marker, uncoupling protein-1 (UCP-1), located in the mitochondrial inner membrane. UCP-1 causes a proton leak across the inner membrane of mitochondria, thereby converting chemical energy into heat. Therefore, brown adipocytes have been considered as the potential target for creating strategies to treat obesity and its related diseases (Park et al., 2014).

Both brown and white adipocytes are derived from mesenchymal stem cells (MSCs) (Park et al., 2014). White adipocyte differentiation is regulated by positive and negative stimuli, including a variety of hormones, growth factors and transcription factors (Kim et al., 2013a, Kim et al., 2013b, Lee et al., 2016; Rosen and Spiegelman, 2000). Although the molecular mechanism underlying brown adipocyte differentiation has not been as extensively studied as that of white adipocyte differentiation (Choi et al., 2013a, Hilton et al., 2015, Rosen and Spiegelman, 2000), the differentiation process of brown and white adipocytes has a similar transcriptional pattern. Peroxisome proliferator-activated receptor-γ (PPAR-γ) and C/EBP-α, master transcriptional regulators in white adipocyte differentiation, are also essential factors in brown adipocyte differentiation, and their expression level has been shown to increase during differentiation of brown adipocytes. Nevertheless, other transcriptional regulators, such as PPAR-γ coactivator-1α (PGC-1α) and PR domain containing 16 (PRDM16), play important roles in brown adipocyte-specific expression of UCP-1 (Seale et al., 2007). These transcription factors and any other factors that regulate thermogenesis are key regulators of the differentiation and functions of brown adipocytes.

Post-translational modifications (PTMs) refer to the covalent and generally enzymatic modifications of protein, and comprise one of the most important approaches for regulation of biological processes via alteration of the physiological behavior of that protein, such as regulation of protein-protein interactions, stability, localization, and/or enzymatic activities (Kim et al., 2012). A number of PTMs occur on non-histone proteins as well as histones (Biggar and Li, 2015, Zhou et al., 2014). The attachment and removal of most PTMs are catalyzed by enzymes. Recently, PTM-regulatory enzymes have emerged as major drug targets. Although phosphorylation, acetylation, and ubiquitination have been extensively investigated, methylation of non-histone proteins has also been known as an important PTM for influencing protein behaviors (Biggar and Li, 2015, Zhang et al., 2012).

We extensively assessed the changes in the expression levels of methyltransferases and demethylases during brown adipocyte differentiation. Several enzymes displaying differential expression patterns were identified. Among these, we focused on methyltransferase Set7/9. Set7/9 catalyzes the methylation at histone H3K4, resulting in gene activation (Nishioka et al., 2002a, Nishioka et al., 2002b). Set7/9 also induces non-histone protein lysine methylation, such as that for p53, TAF10, and DNA methyltransferase 1 (DNMT1) (Couture et al., 2006, Pradhan et al., 2009). In particular, Set7/9 modulates p53 activity via direct interaction (Chuikov et al., 2004) or interaction with Sirt1 (Liu et al., 2011). p53 is known to be required for differentiation into mature brown adipocytes (Molchadsky et al., 2013). Here, we investigated the functional roles of Set7/9 during brown adipocyte differentiation, mainly focusing on the modulation of p53 activity.

Section snippets

Cell culture and brown adipocyte differentiation

Primary brown progenitor cells were obtained from the interscapular brown adipose tissue of 1-to-3-day-old mice and isolated cells were cultured as previously described (Kim et al., 2015b). The immortalized brown preadipocyte cell line was kindly provided by Dr. Shingo Kajimura (UCSF). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 1% antibiotic/antimycotic solution and 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere with 5% CO2. For brown adipogenic

The expression level of Set7/9 decreased during brown adipocyte differentiation

Brown preadipocytes were cultured, and differentiation into mature brown adipocytes was induced by culture in differentiation cocktail media (Choi et al., 2013a, Kim et al., 2015b, Son et al., 2015). To identify the methyltransferases and demethylases involved in brown adipocyte differentiation, we used a qPCR array for samples derived from both preadipocytes and mature brown adipocytes. From a total of 31 methyltransferases and 18 demethylases, several enzymes showed differential expression

Discussion

A number of PTMs occur on non-histone proteins as well as histones and control protein-protein interactions, stability, intracellular localization, and enzymatic activities of proteins involved in various cellular processes. Relatively few non-histone proteins have been reported that can be modified by lysine methylation. The enzymes involved in lysine methylation were first found to target histone and thus were initially termed as histone methyltransferases and histone demethylases. According

Conflict of interest

The authors have no conflicts of interest and declare no competing financial interests.

Acknowledgments

We would like to thank Professors Sayeon Cho, Seung Jun Kim, and Sang J. Chung for the continuous encouragement and helpful advice. This work was supported by grants from the KRIBB and from the Research Program (grants 2006-2004112, 2012M3A9C7050101, 2015M3A9B5030308, and 2015M3A9D7029882) through the National Research Foundation of Korea.

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