Abstract

Objective

   We previously established the scaffold protein 14-3-3ζ as a critical
   regulator of adipogenesis and adiposity, but whether 14-3-3ζ exerted
   its regulatory functions in mature adipocytes or in adipose progenitor
   cells (APCs) remained unclear.

Methods

   To decipher which cell type accounted for 14-3-3ζ-regulated adiposity,
   adipocyte- (Adipoq14-3-3ζKO) and APC-specific (Pdgfra14-3-3ζKO) 14-3-3ζ
   knockout mice were generated. To further understand how 14-3-3ζ
   regulates adipogenesis, Tandem Affinity Purification (TAP)-tagged
   14-3-3ζ-expressing 3T3-L1 preadipocytes (TAP-3T3-L1) were generated
   with CRISPR-Cas9, and affinity proteomics was used to examine how the
   nuclear 14-3-3ζ interactome changes during the initial stages of
   adipogenesis. ATAC-seq was used to determine how 14-3-3ζ depletion
   modulates chromatin accessibility during differentiation.

Results

   We show a pivotal role for 14-3-3ζ in APC differentiation, whereby male
   and female Pdgfra14-3-3ζKO mice displayed impaired or potentiated
   weight gain, respectively, as well as fat mass. Proteomics revealed
   that regulators of chromatin remodeling, like DNA methyltransferase 1
   (DNMT1) and histone deacetylase 1 (HDAC1), were significantly enriched
   in the nuclear 14-3-3ζ interactome and their activities were impacted
   upon 14-3-3ζ depletion. Enhancing DNMT activity with S-Adenosyl
   methionine rescued the differentiation of 14-3-3ζ-depleted 3T3-L1
   cells. ATAC-seq revealed that 14-3-3ζ depletion impacted the
   accessibility of up to 1,244 chromatin regions corresponding in part to
   adipogenic genes, promoters, and enhancers during the initial stages of
   adipogenesis. Finally, 14-3-3ζ-regulated chromatin accessibility
   correlated with the expression of key adipogenic genes.

Conclusion

   Our study establishes 14-3-3ζ as a crucial epigenetic regulator of
   adipogenesis and highlights the usefulness of deciphering the nuclear
   14-3-3ζ interactome to identify novel pro-adipogenic factors and
   pathways.

   Keywords: 14-3-3ζ, Adipogenesis, Energy homeostasis, Chromatin
   accessibility, Adipogenic genes, Epigenetic regulation

Highlights

     * •
       14-3-3ζ influences adiposity and body weight via adipose progenitor
       cells and not mature adipocytes.
     * •
       The 14-3-3ζ interactome is enriched with regulators of chromatin
       remodeling during the early stage of adipogenesis.
     * •
       14-3-3ζ regulates chromatin accessibility to influence the
       expression of pro-adipogenic genes.

1. Introduction

   The expansion of adipocyte number occurs through adipocyte
   differentiation, or adipogenesis, and this process plays a pivotal role
   in the development of obesity [[46][1], [47][2], [48][3]]. The
   differentiation of adipose progenitor cells (APCs) is driven by the
   availability of energy-dense nutrients and a variety of adipogenic
   triggers, and adipogenesis is facilitated by complex signaling pathways
   and tightly regulated transcriptional networks [[49]4,[50]5].

   The canonical model of adipogenesis posits that hormonal and nutrient
   stimuli promote the sequential expression and activation of early
   adipogenic transcription factors (ATFs), which include
   CCAAT-enhancer-binding proteins-β/δ (C/EBP-β/δ), STAT5A signal
   transducer and activator of transcription 5A/B (STAT5A/B), Kruppel-like
   factor 5 (KLF5), and glucocorticoid receptor (GR) and late ATFs, such
   as C/EBPα and Peroxisome proliferator-activated receptor γ2 (PPARγ2)
   [[51]4,[52]5]. Although these events take place during the early stages
   of APC differentiation, little is known about the molecular factors
   that tightly coordinate them in space (i.e., cytosolic to nuclei
   translocation) and time (sequential activation of ATFs). This lack of
   knowledge has contributed to the difficulty in targeting adipogenesis
   with pharmacological approaches to treat obesity.

   Molecular scaffold proteins likely play important roles in coordinating
   signaling pathways that control metabolism [[53]6,[54]7], and of the
   various families of scaffolds, the importance of 14-3-3 proteins in
   signal transduction has become apparent [[55]6,[56]7]. Through
   recognition of specific phosphorylated serine or threonine motifs
   (RSXpS/TXP and RXXXpS/TXP), all seven mammalian 14-3-3 protein isoforms
   interact with a broad variety of enzymes, transcription factors, and
   transporters. Thus, 14-3-3 proteins can regulate diverse cellular
   processes, such as cell cycle progression, apoptosis, secretion, and
   metabolism [[57]7,[58]8].

   Despite a high degree of sequence-homology, 14-3-3 family members can
   perform isoform-specific biological functions, and our group has
   identified critical roles of the 14-3-3ζ isoform in the regulation of
   whole-body adiposity, adipogenesis, adipocyte function, and glucose and
   lipid homeostasis [[59]7,[60]8]. In the context of adipocyte
   differentiation in vitro, silencing of Ywhaz (the gene coding for
   14-3-3ζ) blocked the differentiation of 3T3-L1 preadipocytes [[61]9].
   We also found that systemic deletion of 14-3-3ζ significantly reduced
   visceral adiposity and promoted glucose intolerance and insulin
   resistance in male mice [[62]9]. Postnatal depletion of 14-3-3ζ in
   mature adipocytes was found to reduce adipose tissue Pparg2 mRNA
   expression and impair the lipolytic response of adipose tissue
   [[63]10]. In contrast, whole-body transgenic overexpression of 14-3-3ζ
   potentiated age-dependent and high-fat diet-induced expansion of
   adipose tissue [[64]9]. Taken together, these findings demonstrate
   important roles of 14-3-3ζ in adipocyte development and function.

   With the ability of 14-3-3 proteins to interact with a diverse array of
   phosphorylated proteins, we have discovered that the interactome of
   14-3-3ζ changes in response to physiological and pathophysiological
   stimuli like adipocyte differentiation or obesity, respectively. We
   first used mouse embryonic fibroblasts derived from transgenic mice
   expressing a tandem affinity purification (TAP) epitope-tagged 14-3-3ζ
   molecule combined with affinity proteomics to discover that the
   interactome of TAP-14-3-3ζ is enriched with RNA splicing factors
   following the induction of adipocyte differentiation [[65]11].
   Recently, we have determined that the interactome of 14-3-3ζ in adipose
   tissue is also sensitive to high-fat diet-induced obesity [[66]12].
   Altogether, these findings demonstrate the usefulness of determining
   the 14-3-3ζ interactome in identifying novel regulators of adipocyte
   differentiation or expansion of adipose tissue mass.

   A major limitation of whole-body deletion or overexpression 14-3-3ζ
   mouse models is the inability to distinguish the individual
   contributions of specific cell types, and with our findings that
   14-3-3ζ may regulate adiposity [[67]9,[68]10], whether 14-3-3ζ
   primarily influences mature adipocytes or APCs, let alone other cell
   types, is unclear. Moreover, the recent discovery of adipocyte and APC
   heterogeneity further adds to the complexity in understanding the roles
   of specific proteins in adipose tissue niches. Indeed, adipocytes
   derived from APCs that express different markers such as
   Platelet-derived growth factor receptor (PDGFR)-α and -β, represent
   unique sub-populations that can be influenced by age, anatomical
   localization and nutritional contexts [[69][13], [70][14], [71][15],
   [72][16], [73][17], [74][18]]. For example, subsets of PDGFRα+ cells
   with high or low expression of CD9 were found to be committed to
   pro-fibrotic and adipogenic cells, respectively [[75]14]. Also, CD24+
   progenitors, and not CD24^-, were characterized as having high
   adipogenic potential [[76]15]. The concept of heterogeneity is not
   restricted to rodents, as spatial technologies have revealed
   heterogeneity within human adipose tissues [[77]13]. Although systemic
   14-3-3ζ deletion and over-expression had opposing effects on adiposity,
   it is not clear whether 14-3-3ζ is differentially expressed in APCs or
   mature adipocytes to account for the differences in fat mass.

   Herein, we sought to examine the impact of deleting 14-3-3ζ in Adipoq+
   mature adipocytes (Adipoq14-3-3ζKO) and in Pdgfra+ APCs
   (Pdgfra14-3-3ζKO) on murine adiposity under normal chow and high-fat
   diet conditions. No differences in body weights were found in
   Adipoq14-3-3ζKO mice. However, Pdgfra14-3-3ζKO male mice showed
   moderate reduction in body weight while Pdgfra14-3-3ζKO female mice
   exhibited marked increases in body weight, adiposity and fat mass.
   These observations suggest critical roles of 14-3-3ζ in APCs rather
   than in mature adipocytes. To further define processes regulated by
   14-3-3ζ in the differentiation of APCs, CRISPR-Cas9 genome editing was
   used to generate 3T3-L1 preadipocytes that express a TAP-tagged 14-3-3ζ
   molecule to permit the identification of the nuclear interactome of
   14-3-3ζ during the early stages of adipogenesis. Chromatin remodeling
   was among the highest enriched biological functions, which led to the
   use of Assay for Transposase-Accessible Chromatin with sequencing
   (ATAC-seq) to assess how 14-3-3ζ influences chromatin accessibility. We
   found that the expression of critical adipogenic genes, such as Fabp4,
   Adig, Retn, and Fam83a, are correlated with 14-3-3ζ-regulated chromatin
   accessibility. Taken together, our study highlights the importance of
   14-3-3ζ during the early stages of adipogenesis and a novel role by
   which it regulates adipocyte differentiation.

2. Results

2.1. Adipose expression of Ywhaz correlates with body fat mass and insulin
resistance in mice

   With the finding that extreme differences in Ywhaz expression are
   associated with opposite effects on adiposity [[78]9], we first wanted
   to determine if natural variations in Ywhaz mRNA expression are
   similarly correlated with adiposity and other metabolic parameters.
   Thus, the Hybrid Mouse Diversity Panel (HMDP) was used to explore the
   association of Ywhaz mRNA expression in perigonadal adipose tissue
   samples with metabolic traits. The HMDP is composed of one hundred
   inbred strains of male and female mice that were fed a high-fat and
   high-sugar (HF/HS) diet for 8 weeks and assessed for metabolic
   parameters such as body weight, lean and fat masses, fasting glycemia,
   and insulinemia [[79]19]. A significantly positive correlation between
   Ywhaz expression and insulin resistance, as measured by HOMA-IR, was
   detected in male and female mice after HF/HS feeding (bicor >0.4, P <
   10^−9, [80]Figure 1A,D). Percent fat mass before and after HF/HS diet
   feeding in males ([81]Figure 1B,C) and females ([82]Figure 1E,F) were
   also found to be significantly correlated with Ywhaz mRNA expression.

Figure 1.

   [83]Figure 1
   [84]Open in a new tab

   Adipose tissue expression of Ywhaz is positively correlated with body
   fat mass and insulin resistance in male and female mice. Correlation of
   (A–C) male and (D–F) female perigonadal adipose tissue (VAT) gene
   expression of Ywhaz with HOMA-IR (A and D) and whole body fat mass
   before (B and E) and after (C and F) high-fat, high-sucrose feeding,
   using the Hybrid Mouse Diversity Panel (HMDP) resource [[85]19]. (G)
   Timeline of the experiment performed on 3T3-L1 cells transfected with
   siCTL or siYwhaz (10 nM each) prior to standard MDI or MDIR
   differentiation protocols [[86]9,[87]10,[88]87]. After treatments,
   cells were subjected to Oil Red-O staining (H), measurement of Oil
   Red-O incorporation level by absorbance at 490 nm (I), and qRT-PCR to
   measure Pparg2(J), Adipoq(K) and Ywhaz(L) mRNA levels. Significant
   differences between experimental conditions are indicated by ∗P < 0.05
   or #P < 0.05 (calculated by Student’s t-test). (For interpretation of
   the references to color in this figure legend, the reader is referred