GNE-049

Inhibition of the acetyl lysine-binding pocket of bromodomain and extraterminal domain proteins interferes with adipogenesis

Olivier Goupille, Tipparat Penglong, Zahra Kadri, Marine Granger-Locatelli, SuthatFucharoen, Leila Maouche-Chrétien, Stéphane Prost, Philippe Leboulch, Stany Chrétien

Abstract

The bromodomain and extraterminal (BET) domain family proteins are epigenetic modulators involved in the reading of acetylated lysine residues. The first BET protein inhibitor to be identified, (+)-JQ1, a thienotriazolo-1, 4-diazapine, binds selectively to the acetyl lysine-binding pocket of BET proteins. We evaluated the impact on adipogenesis of this druggable targeting of chromatin epigenetic readers, by investigating the physiological consequences of epigenetic modifications through targeting proteins binding to chromatin. JQ1 significantly inhibited the differentiation of 3T3-L1 preadipocytes into white and brown adipocytes by down-regulating the expression of genes involved in adipogenesis, particularly those encoding the peroxisome proliferator-activated receptor (PPAR-γ), the CCAAT/enhancer-binding protein (C/EBPα) and, STAT5A and B. The expression of a constitutively activated STAT5B mutant did not prevent inhibition by JQ1. Thus, the association of BET/STAT5 is required for adipogenesis but STAT5 transcription activity is not the only target of JQ1. Treatment with JQ1 did not lead to the conversion of white adipose tissue into brown adipose tissue (BAT). BET protein inhibition thus interferes with generation of adipose tissue from progenitors, confirming the importance of the connections between epigenetic mechanisms and specific adipogenic transcription factors.

Keywords: BET protein inhibitor; Epigenetic modulators; STAT5; White and brown adipose tissues.

1. Introduction

The specificity, timing, and order of gene expression during differentiation are critical to organogenesis and homeostasis. Recent progress, including the discovery of epigenetic regulation of chromatin structure and the description of super-enhancers involved in concentrating cell type-specific transcription factors close to genes that are activated or repressed during the differentiation program, open new fields of investigation [1,2]. Extensive research linking cell identity with epigenetic regulation has been published: it shows that epigenetic readers, including members of the bromodomain and extraterminal (BET) domain family, are at the interface between chromatin remodeling and transcription factors [2,3].
The pharmacologic compound (+)-JQ1 is the first inhibitor described to bind selectively to BET proteins (BRD2, BRD3, BRD4 and BRDT) [3]. (+)-JQ1 was first shown to regulate the expression of c-Myc in various tumor cell types [4,5]. Actually, several new targets and applications of JQ1 have been described [3,6]. Our laboratory has shown that JQ1 can reinitialize the erythroid program of the erythroleukemia UT7 cell line [7] or can induce STAT5 inhibition and purges the quiescent leukemic stem cells in chronic myeloid leukemia [8].
Here, we studied the effect of this molecule on adipogenesis. Adipogenesis is the commitment of pluripotent precursors to preadipocytes then to adipocytes. This last step requires two stages: (1) adipocyte determination or early differentiation, in which several rounds of cell division are observed (mitotic clonal expansion), and (2) the terminal differentiation, in which peroxisome proliferator activated receptor (PPAR) -γ and CCAAT/enhancer-binding protein (C/EBP) -α activate the transcription of adipocyte genes, conferring the ability to accumulate intracellular lipid droplets [9-11]. Over expression of PPAR-γ directs fibroblast differentiation into adipocyte [12]. Both PPAR-γ and C/EBPα colocalized on the chromatin and are required for expression of the genes coding for adipocyte-specific late markers, including the adipocyte fatty-acid-binding protein Fabp4 and Adipsin genes [13]. STAT5 is involved in the clonal expansion and also at the end of adipocyte differentiation whereas PPAR-γ is required at the last step of adipogenesis [14-16]. There are two types of adipose tissue: white adipose tissue (WAT) for fat storage and brown adipose tissue (BAT) that burns fat to generate heat via lipid oxidation. BAT adipocytes are characterized by a multilocular lipid droplet structure, numerous mitochondria and overexpression of the mitochondrial brown fat uncoupling protein 1 (UCP1) in the inner mitochondrial membrane [17]. The natural molecular programs that govern white to brown adipose conversion remain not totally understood and morphological identification of BAT in cell culture is not easy. Histone modifications are involved in adipogenic determination [18]. Epigenetic marks and chromatin-modifying proteins control adipogenesis by regulating gene expression. The key regulators, PPAR-γ C/EBPα and STAT5A and B, bind to BRD2 proteins on chromatin [2,19] and may couple a transient epigenomic state and differentiation progression [20,21]. To investigate the impact of chromatin modification, we used JQ1 to target the chromatin epigenetic reader and studied the consequences for adipogenesis.

2. Materials & methods

2.1. Reagents

The vector pRcRSV-puro, both empty and carrying a fragment encoding mouse STAT5B1*6 (STAT5Bca) were kindly provided by Dr F. Gouilleux. LY294002 was from Cayman Chemicals (70920), (-)-JQ1 and (+)-JQ1 were synthesized by Sigma-Aldrich (SML0974), RVX-208 was from Selleckchem (S7295), Phenyl-Acetate (PA) from SantaCruz Biotechnology (sc-296401). Rosiglitazone was from Sigma-Aldrich (R-2408) and T3 (3,3’,5-Triiodo-L-Thyronine) was from Sigma-Aldrich (T2752).

2.2. Cell Culture

Mouse pre-adipocyte 3T3-L1 cells (white adipocyte) were maintained in DMEM-High glucose with 10% Donor FBS (cat. 10270, Gibco, Invitrogen). Murine stromal OP9 cells were cultured in MEM-α (Invitrogen) supplemented with 20% Donor FBS. For adipose differentiation, cells were induced in 24-well plates with 10% FBS (cat. 16030, Gibco, Invitrogen) and a IDM cocktail of 0.17 mM insulin (Sigma-Aldrich), 1 µM dexamethasone (Sigma) and 0.5 µM 1-methyl-3-isobutylxanthine (IBMX, I7018; Sigma-Aldrich), ± 1 µM Rosiglitazone (R), 2 days after confluence was reached (day 0). At day two, the media was changed to 10% FBS with 0.17 mM insulin ± 1 µM Rosiglitazone. This medium was changed every 48h until differentiation (day 6-8). Controls were cultivated with the inactive enantiomer (-)-JQ1 at 500 nM. (+)-JQ1 was added at indicated concentrations and was in contact with the 3T3-L1 either from day 2 for 4 days or from day 0, day 2 or day 4 each for 48h. Other inhibitors were added on day 2 at the following concentrations: RVX-208 20µM, LY294002 20 µM and PA 10mM. For BAT differentiation, T3 (1 M) was added to differentiation medium containing rosiglitazone (1 M). Undifferentiated cells were grown for the whole period in a basal medium consisting of DMEM supplemented with 10 % FBS.

2.3. Establishment of stable populations of 3T3-L1 applied.

scraping in RIPA lysis buffer containing protease inhibitor mixture (Roche), sodium orthovanadate (1 mM), and sodium fluoride (10 mM). The protein concentration was determined with a Pierce BCA protein assay kit (Thermo Scientific). Aliquots of 50 µg of total proteins were separated on a 10 % SDS-polyacrylamide gel and transferred to nitrocellulose membrane. Primary antibodies against phospho-STAT5 (9358s), total STAT5 (9363s), PPAR- (2435s; all from Cell Signaling) and -actin (MAB1501R; Millipore) as a loading control were applied overnight at 4°C at a dilution of 1:1000 or 1:10000 for -actin. Immunoblot was performed with the respective primary antibodies and Horseradish

2.7. Statistical analysis.

Results are expressed as means ± sd. The comparisons were performed using unpaired Student’s t-test was used for single comparisons, and Student’s t-test with Bonferronni posttest α=0.05 for multiple comparisons. One way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test was used to check significance, as indicated in figure legends. *, # and + indicate p<0.05, **, ## and ++ indicate p<0.01 and ***, ### and +++ indicate p<0.001. Statistical analysis was performed with Graph Pad Prism V6.0 PC. 3. Results 3.1. Effect of (+)-JQ1 on lipid accumulation in 3T3-L1 and OP9 cells 3T3-L1 cells are derived from the 3T3 Swiss albino mouse MEF line and are used as an in vitro model of adipogenesis. We evaluated 3T3-L1 preadipocyte differentiation into white adipocytes upon exposure to inducers (insulin and rosiglitazone) for 7 days in the presence of (-)-JQ1 as a negative control or of various concentrations of (+)-JQ1. The JQ1 treatment began at the end of the clonal expansion (Day 2) (see Supplementary Fig. 1A). After eight days of treatment, differentiation was assessed by staining lipid droplets with Oil red O and microscopy (Supplementary Fig. 1B). Intracellular lipids were quantified during adipogenesis (Supplementary Fig. 1C): at 500 nM (+)-JQ1, the intracellular lipid droplet content decreased by 50% (IDM condition) and by 70% (IDMR condition) from that in the ()-JQ1 vehicle treated cells. Similar results were obtained for (+)-JQ1 at 250 nM and 1000 nM; (+)-JQ1 treatment at 500 nM was used for subsequent experiments. The expression of PPAR-γ, C/EBPα Fabp4 and Adipsin adipogenic genes was inhibited by (+)-JQ1 (Supplementary Fig. 2). To identify the critical time when JQ1 inhibition occurs during differentiation, we performed the same experiments but cells were treated with (+)-JQ1 at different times (Fig. 1A). The maintenance of (+)-JQ1 for day 0 to 6 prevented the formation of lipid droplets and treatment during clonal expansion (day 0-2) totally inhibited terminal differentiation. The presence of (+)-JQ1 at the end of terminal differentiation (day 46) decreased cell maturation but less than treatment between days 2 and 6 or days 2 and 4 (Fig. 1B-C). The findings of adipogenic gene expression levels were coherent with these results, although Fabp4 expression in the rosiglitazone (+R) condition was less strongly inhibited than expected (Supplementary Fig. 3). The formation of lipid droplets in OP9 murine stromal cells was similarly inhibited by (+)-JQ1 (Supplementary Fig. 4). Thus, (+)-JQ1 impacted the clonal cell expansion, suppressed the accumulation of intracellular lipids and reduced the expression of adipogenic genes. 3.2. Bromodomain 1 or 2 of BET family proteins is involved in 3T3-L1 adipogenesis All BET family of proteins recognizes acetylation marks through two conserved bromodomains (BD1 and BD2). (+)-JQ1 binds to the acetylated lysine pocket of both BD1 and 2. Compounds that can distinguish between BD1 and BD2 of BET proteins have been developed (Supplementary Fig. 5B): RVX-208 binds preferentially to BD2 [23] and LY294002 has a dual kinase and BET-bromodomain inhibitory activity, inhibiting acetyllysine binding by BD1 [24]. We treated 3T3-L1 cells with these two drugs. RVX-208 and LY294002, like (+)-JQ1, decreased the accumulation of intracellular lipid droplets whereas phenylacetate (PA), an activator of PPAR-γ, increased the lipid content (Supplementary Fig. 5). Thus, inhibition of either BD1 or BD2 of BET proteins impedes adipogenesis. 3.3. (+)-JQ1 decreases STAT5 expression, but adipogenesis is not restored after expression of a constitutively activated mutant form of STAT5 To investigate mechanism by which (+)-JQ1 impedes adipocyte differentiation (Fig. 2A), we studied the phosphorylation of STAT5 and expression of STAT5 and PPAR-γ (Fig. 2B). At 20 mn, (+)-JQ1 did not inhibit phosphorylation of STAT5, suggesting that the suppressive function of (+)-JQ1 on adipocyte differentiation is independent of STAT5 activation. However, by day 6 of differentiation, we observed strong inhibition of both STAT5 and PPAR-γ expressions by (+)-JQ1 (Fig. 2B). The expression of genes regulated by STAT5 or PPAR-γ was also strongly affected (Fig. 2A and C), consistent with previous results [25,26]. We tested the effect of the expression of a dominant active form of STAT5 (STAT5Bca) that has constitutive STAT5B transcriptional activity independent of BRD2 association [19]. 3T3-L1 cells were transfected with a plasmid encoding STAT5Bca and puromycin-resistant cells were selected, treated with JQ1 and subjected to the differentiation protocol. STAT5Bca expression strongly enhanced intracellular lipid droplet formation in controls ((-)-JQ1 condition) but did overcome, at the same level, the inhibitory effect of (+)JQ1 (Fig. 3). Therefore, the STAT5/BRD2 association is not the direct target of (+)-JQ1 in the inhibition of adipogenesis. Thus, as described previously (+)-JQ1 appears to be a strong inhibitor of a pan-BRD associated proteins but we still cannot identify the details of the mechanism of its inhibition of adipogenesis. 3.4. (+)-JQ1 decreases brown adipogenesis After treatment of 3T3-L1 cells by (+)-JQ1, we suspected some multilocular lipid droplet structures characteristic of brown adipocytes in +R cultures treated by (+)-JQ1 (Fig. 4A). We investigated the relevance of (+)-JQ1 to convert WAT to BAT: 3T3-L1 cells were grown in conventional conditions (IDMR) or in brown fat conditions (T3) in the presence or absence of (+)-JQ1. Mitochondrial abundance was evaluated by mitotracker red staining. Treatment of 3T3-L1 in white fat condition with (+)-JQ1 increased red staining to the same level as that in 3T3-L1 cultivated in brown fat conditions (T3) (Fig. 4B). However, UCP1 was exclusively expressed in the T3 condition and was strongly inhibited by (+)-JQ1 treatment. In ex vivo cultures of stromal vascular cells isolated from interscapular BAT (see supplementary materials and methods), (+)-JQ1 decreased BAT differentiation (Fig. 4D-F). Therefore, (+)JQ1 does not promote the conversion of white to brown fat but interferes strongly with the adipogenesis, whether white or brown. 4. Discussion The epigenetic control of adipogenesis and the possibility to converse white fat to brown fat have recently emerged as fields of interest in the search for new strategies, new targets and molecules for the fight against obesity [17,18]. The expression of master transcription factors of adipogenesis, PPAR-γ and C/EBPα, is regulated by chromatin remodeling and histone modification [11,21]. Association of PPAR-γ with BRD2 is involved in a transient epigenomic state necessary for the progression of differentiation [27]. BRD2 is a scaffolding protein that recruits transcription regulatory factors such as E2F-1 [28], STAT5 [29], and more recently the histone arginine demethylase JMJD6 [30], to regulate gene transcription and adipogenesis [15,30,31]. BRD2 also binds acetylated-lysine of histones to the chromatin through two bromodomains and only this function was inhibited by JQ1. Here we show that JQ1 inhibits adipogenesis (white and brown) of 3T3-L1 preadipocytes and primary stromal cells, and does not convert white fat to brown fat. Disruption of Brd2 in mice causes severe obesity with a relative increase of BAT and knock-down of BRD2 in 3T3-L1 increases PPAR-γ transcriptional activity and adipogenesis [27]. Thus, the binding of BET proteins to acetylated-lysine promotes adipogenic differentiation whereas BRD2 association with specific transcription factors (PPAR-γ or STAT5, see below) inhibits adipogenesis. The dual functions of BET proteins explain this apparent contradiction: the bromodomains interpret the histone code and the extraterminal domain controls the activity of the transcription complexes. The new concept of the super-enhancer, where BET proteins were required for the initial step of differentiation concentrating transcription factors at a strategic site on the chromatin, may explain the inhibition of adipogenesis by JQ1 [32]. The strong inhibition of 3T3-L1 cell differentiation in presence of JQ1 during the phase of clonal expansion (Fig. 1) is consistent with this hypothesis. We went on to consider the transcription factor STAT5. STAT5 regulates the early phase of adipogenesis: activation of STAT5 induces adipocyte differentiation and excessive activation of STAT5 inhibits the differentiation of 3T3-L1 cells [16]. The function of STAT5 expression in the late phase of adipogenesis was poorly understood [25,26]. In acute T-cell lymphoblastic [19] and chronic myeloid leukemias [8], JQ1 treatment decreases STAT5 activity in synergy with tyrosine kinase inhibitors (TKI) inducing apoptosis or G0 progression of leukemic hematopoietic stem cells respectively. BRD2 binds to STAT5 independently of STAT5 acetylation and JQ1 inhibits STAT5-mediated transcription in correlation with histone acetylation and occupancy [29]. Experiments with a constitutively active form of STAT5B demonstrated that STAT5 acetylation is not necessary for its transcriptional activity and that the recruitment of STAT5/BRD2 to acetylated chromatin is required for the initial activation of STAT5 regulated genes. This activation is abolished by deacetylase inhibitors [29] or by JQ1 treatment (our results). Our results indicate that JQ1 inhibits adipogenesis from the beginning of preadipocyte clonal expansion (hyperplasia phase) and during differentiation. However, as (+)-JQ1 is a pan-BRD-domain inhibitor, it remains unclear which of the associations of BRD2 with STAT5, PPAR-γ or others factors (E2F, JMJD6…) is the most important. 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