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Tannic acid, a novel histone acetyltransferase inhibitor, prevents non-alcoholic fatty liver disease both in vivo and in vitro model

Objective We examined the potential of tannic acid (TA) as a novel histone acetyltransferase inhibitor (HATi) and demonstrated that TA prevents non-alcoholic fatty liver disease (NAFLD) by inhibiting HAT activity. Methods The anti-HAT activity of TA was examined using HAT activity assays. An in vitro NAFLD model was generated by treating HepG2 cells with oleic and palmitic acids. Male C57BL/6J mice were fed a control diet (CD) or Western diet (WD) with or without supplementation with either 1% or 3% TA (w/w) for 12 weeks. Finally, the possibility of interacting p300 and TA was simulated. Results TA suppressed HAT activity both in vitro and in vivo. Interestingly, TA abrogated occupancy of p300 on the sterol regulatory element in the fatty acid synthase and ATP-citrate lyase promoters, eventually inducing hypoacetylation of H3K9 and H3K36. Furthermore, TA decreased acetylation at lysine residues 9 and 36 of histone H3 protein and that of total proteins. Consequently, TA decreased the mRNA expression of lipogenesis-related genes and attenuated lipid accumulation in vivo. We observed that NAFLD features, including body weight, liver mass, fat mass, and lipid profile in serum, were improved by TA supplementation in vivo. Finally, we demonstrated the possibility that TA directly binds to p300 through docking simulation between ligand and protein. Conclusions Our findings demonstrate that TA, a novel HATi, has potential application for the prevention of NAFLD.


INTRODUCTION
Non-alcoholic fatty liver disease (NAFLD) covers a wide spectrum of various liver diseases [1]. The clinical burden of NAFLD is not only limited to liver-related morbidity and mortality but also involves several extrahepatic dysfunctions, indicating that NAFLD is a multisystem disease [2,3]. Currently, no effective treatment is available for NAFLD. To develop efficacious preventive and therapeutic options, researchers have investigated the cellular and molecular mechanisms, particularly genetic factors, underlying the development of NAFLD. Although genetic factors contribute to the development of NAFLD, environmental factors, including diet and exercise, which have an influence on epigenetic factors, are also key determinants [4,5]. The environment regulates the expression of genes involved in carbohydrate and lipid metabolism by altering histone acetylation patterns in the chromatin [6]. Histone acetylation is a representative epigenetic mechanism, which is regulated by histone acetyltransferases (HATs) and deacetylases (HDACs). An imbalance between HAT and HDAC activities is associated with various human diseases, including cancer, inflammation, and metabolic disorders [7]. Although many small molecules that modulate HDAC activity have been extensively studied [8], little information is available regarding the modulation of HAT activity, even though HAT enzymes are involved in the onset of various diseases. In NAFLD models, the "first hit" is liver fat accumulation, which causes insulin resistance, whereas the interplay of inflammatory cytokines, which causes inflammation, acts as the representative "second hit" [9]. However, recently, the "multiple hit" hypothesis has taken into consideration that multiple insults, including insulin resistance, nutritional factors, gut microbiota, and genetic and epigenetic factors, act collectively to induce NAFLD and provides a more accurate explanation of NAFLD pathogenesis [10]. The p300 HAT enzyme, which functions as a transcriptional coactivator, is co-recruited with nuclear factor-kB to regulate various inflammatory signaling pathways [11]. Carbohydrate-responsive element-binding protein (ChREBP) is a transcriptional activator of lipogenic genes, which plays a major role in the development of NAFLD [12]. p300 increases ChREBP transcriptional activity through acetylation of Lys-672, which, in turn, increases p300 occupancy on target gene promoters [13]. Furthermore, p300 overexpression results in NAFLD, insulin resistance, and inflammation [13]. However, little is known regarding the role of HATs in the development of NAFLD. During an ongoing screening study using an in vitro HAT assay system to identify phytochemical compounds with HAT inhibitory activity, we identified tannic acid (TA) as a novel HAT inhibitor (HATi) with specificity for major HAT enzymes. TA, a plant-derived hydrolysable tannin polyphenol, is a gallic acid polymer glucoside found in many dietary plant products such as coffee, tea, cocoa, and sorghum grain [14]. Various studies have revealed the beneficial effects of TA [15e17]. Recent studies have shown the anti-obesity effects of TA [18,19]; however, no explanation has been provided regarding the exact molecular mechanism of TA activity on signaling pathways. Furthermore, no study has shown beneficial effects of TA on the development of NAFLD, even though this disease is closely associated with components of the metabolic syndrome, such as obesity, insulin resistance, and dyslipidemia [20]. In the present study, we investigated the potential of TA as a novel HATi and demonstrated that TA prevented the development of NAFLD via its HATi activity, both in vitro and in vivo.

Cell culture
HepG2 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in a humidified atmosphere of 5% CO 2 at 37 C in high-glucose DMEM supplemented with 10% fetal bovine serum and antibiotics (Welgene, Daegu, Korea). To establish a NAFLD model using HepG2 cells, we used a non-fat BSA-conjugated combination of oleic acid (SigmaeAldrich, St. Louis, MO, USA) and palmitic acid (Sigma) at a ratio of 4:1 (OPA). To evaluate the effect of TA on hepatic lipid accumulation, HepG2 cells were concurrently treated with TA and OPA at the indicated concentrations for 18 h. Epigallocatechin-3-gallate (EGCG) was used under the same conditions.

Cytotoxicity
HepG2 cells were seeded in 24-well plates at 5 Â 10 4 cells/well and, after reaching approximately 70% confluence, were treated with TA at 10, 25, 50, or 100 mM, in the presence or absence of OPA. After a 24-h incubation, the cells were treated with 10 mL of a water-soluble tetrazolium salt (WST-1) solution (Enzo Life Sciences, Inc., Farmingdale, NY, USA) for 3 h. Thereafter, 100 mL of the supernatant was transferred to a 96-well plate, and the absorbance was measured at 450 nm (Molecular Devices, Sunnyvale, CA, USA).

Lipid content
HepG2 cells were seeded in 24-well plates at 5 Â 10 4 cells/well. After reaching approximately 70% confluence, they were treated in the presence or absence of OPA, with TA at 10 or 25 mM. After a 24-h incubation, the cells were washed with 200 mL of PBS, and fixed with 200 mL of 4% paraformaldehyde for 15 min at room temperature.
The cells were then washed three times with PBS and incubated with 200 mL of 60% isopropanol for 5 min, followed by staining with 200 mL of 0.1% Oil red O staining solution (SigmaeAldrich) for 1 h. After additional washing with water, images were captured under a light microscope (Olympus IX51; Olympus Corporation, Central Valley, PA, USA). For lipid quantification, isopropanol was added to each well to dissolve the lipid-stained red dye. After 10 min, the absorbance was measured at 510 nm (Molecular Devices).

Histone extraction
Cells were seeded at a concentration of 5 Â 10 6 in 10-mm dishes. When cells had reached approximately 70% confluence (w2 Â 10 8 cells), they were treated with OPA with or without TA at the indicated concentrations for 18 h. HepG2 cells were lysed according to the manufacturer's protocol (Abcam, Cambridge, MA, USA). The cells were washed with PBS and lysed with pre-lysis buffer to isolate the nuclear fraction. The nuclear pellet was resuspended in lysis buffer, incubated for 30 min on ice, and centrifuged at 10,000 rpm for 5 min at 4 C. The supernatant was transferred to a clean tube, to which DTT buffer was added. Samples were stored at À70 C until analysis.
2.5. In vitro HAT and HDAC assays HeLa cell nuclear extract was purchased from BioVision Biotechnology (Milpitas, CA, USA). HAT and HDAC activities in the nuclear extracts were assessed using a commercially available kit according to the manufacturer's protocol (BioVision Biotechnology). To examine the inhibitory effect of TA on specific HATs, we used 100 ng of CREBbinding protein (CBP), p300, or p300/CBP-associated factor (pCAF) glycerol, 2 mM DTT, and protease inhibitor cocktail] was added and the pellets were resuspended five times using a syringe, incubated on ice for 20 min, and then centrifuged at 13,000 rpm and 4 C for 20 min. Liver tissues obtained at the end of the animal experiments were washed with ice-cold PBS and lysed according to the manufacturer's protocol (Abcam, ab113474). Briefly, tissues were weighed and cut into small pieces. To obtain the cytosolic fractions, tissue pieces were homogenized in pre-extraction buffer containing DTT using a glass tissue homogenizer (Thomas Scientific, NJ, USA). Extraction buffer was added to the pellets, the extracts were incubated on ice for 15 min, and then further homogenized using a Teflon pestle (Thomas Scientific). The suspensions were then centrifuged for 10 min at 14,000 rpm and 4 C and the supernatants were used as nuclear fractions. Each nuclear fraction was used for the HAT assays.  Table 3. Body weights were measured at the beginning of the experiment and subsequently at 1-week intervals for 12 weeks. Similarly, the food consumption of each group was recorded at weekly intervals for 12 weeks. At the end of the experiment, mice were sacrificed by cervical dislocation under anesthesia (100 mg/kg ketamine þ 5 mg/kg xylazine) and blood samples were collected via abdominal heart puncture for serum isolation. After laparotomy, liver, retroperitoneal fat, and epididymal fat were weighed, and a portion of the liver from each mouse was fixed in 4% formalin solution for hematoxylin and eosin staining (H&E staining). The remaining livers were used for experiments as indicated.
2.11. Hematoxylin and eosin staining Liver specimens were fixed in 4% buffered formalin, embedded in paraffin, and cut into 4e5 mm-thick sections, which were stained with hematoxylin and eosin (H&E). Lipid accumulation in livers was assessed by microscopic observation using an Eclipse 80i microscope (Nikon Instruments, Inc., Melville, NY, USA).

Measurement of triglycerides, total cholesterol, and LDL cholesterol
The levels of triglycerides (TGs), total cholesterol, and LDL cholesterol (LDL-C) in the serum were measured enzymatically using a commercial kit (Asan Pharm, Seoul, Korea).
2.13. Proteineligand docking simulations for the E1A binding protein p300 (EP300) To identify the active site in EP300, the three dimensional X ray crystallographic structure of EP300 was downloaded from the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do) (PDB ID: 4BHW) [26]. Therefore, the reported 3D structure of EP300 was applied to docking simulation. The 4BHW structure used for docking simulation contains a catalytic core in p300, and the bromo (aa 1048e1167), RING (really interacting new gene) (aa 1168e1242), PHD (plant homeodomain) (aa 1243e1285), and HAT (aa 1285e1664) domains. The bromodomain regulates gene transcription via recruitment of different molecular partners and has attracted considerable interest as a promising new epigenetic target in diverse human diseases [27]. The RING and PDH domains negatively regulate p300 HAT activity and the removal of this inhibitory function results in increased HAT activity [28,29]. The HAT domain remodels chromatin to "heterochromatin" its superstructure and enables transcription of proximal genes [30]. These domains function collectively to manifest HAT activity [28]. Prior to docking TA to EP300, we investigated the docking site in other EP300-like proteins. The EP300-like proteins were searched for using the sequence alignment method of the HHblits program [31] and the position of the bound ligands of these similar proteins was mapped to pocket sites of the EP300 core active sites measured using the VMD [32] module (protein pocket calculation program, Pck; http://schwarz. benjamin.free.fr/Work/Pck/home.htm). We then selected the most conserved pocket position based on overlapped positions between mapped ligand binding residues and the EP300 pocket sites for the TA docking site (Supplemental Figure 1). The TA used as a ligand for docking was downloaded as a 2D structure from the PubChem site (CID: 16129878) (https://pubchem.ncbi.nlm.nih.gov) [33] and converted to a 3D structure using the MarvinSketch 15.7.20.0 program (https://chemaxon.com/products/marvin) (Supplemental Figure 2). The docking simulations were run using the Autodock Vina 1.1.2 program (http://vina.scripps.edu) [34], which can dock flexible ligands into a target protein, while keeping the protein fixed. We docked TA around the backbone residues, and the Ca coordinates in each backbone binding residue of the protein receptor were used for the center of the docking space. We used default options for docking simulations except box size (30 Å 3 ). This is because TA has a large volume and thus necessitates a large binding space. The docked ligand was selected in order of binding energy up to 20 ligands per box. Thus, in the case of EP300 with 15 binding residues, up to 20 Â 15 docked ligands are generated. The docked ligand is clustered in cluster size 1 Å to classify similar forms. From the docking results, the best docked model, which has the lowest docking energy, was selected to represent its most favorable binding mode. When selecting the lowest energy ligand in a cluster, free energy (lowest energy þ (-kTlnN), k: Boltzman constant, T: Kelvin temperature, N: the number of structure in each cluster) was used to account for the number of ligands in the population.
2.14. Statistical analysis Data were analyzed using one-way analysis of variance with Tukey's multiple comparison test, and values are expressed as the mean AE SD. Statistical analyses were conducted using SPSS ver. 20 (SPSS, Inc., Chicago, IL, USA). Differences were considered statistically significant at p < 0.05.

TA has potential as a novel HATi
To examine whether TA modulates HAT activity, we initially measured HAT activity in a cell-free system. TA at various concentrations was incubated with a HeLa nuclear extract (NE), and epigallocatechin-3gallate (EGCG), a well-known specific HATi [35], was used for comparison (Supplemental Information Figure 1, left panel). As shown in Figure 1A (left panel), TA significantly inhibited the HAT activity. Interestingly, the IC 50 values for HAT inhibition by TA ( Figure 1A considerably stronger inhibitory effect than that shown by EGCG. To confirm the anti-HAT activity of TA, we examined the inhibitory effect of TA on HAT using a radiolabeled H4 tail peptide. Consistent with the results of the cell-free HAT assay, TA showed a HAT inhibitory effect in an autoradiography assay ( Figure 1B). To ensure that the activity of TA was specifically directed against HATs, we initially sought to assess the effect of TA on HDAC activities. When HeLa NEs were used as a source of HDAC enzymes, deacetylase activity was not affected by the presence of TA, whereas Trichostatin A (TSA) efficiently blocked nuclear HDAC activities (Supplemental Figure 4). This result indicates that TA has no specificity for HDACs. Taken together, these data show that TA has potential as a novel HATi. Next, we examined the enzyme specificity of TA. The HAT activities of p300, CREB-binding protein (CBP), and p300/CBP-associated factor (PCAF) HATs were measured either in the absence of TA or in the presence of increasing concentrations of TA. As shown in Figure 1C, TA was found to be a highly efficient inhibitor of p300 acetyltransferase activity, with an IC 50 value of 3.886 mM (Supplemental Figure 5, (Figure 2A). To exclude the possibility that the effect was due to TA cytotoxicity, we performed a cell viability assay (Supplemental Figure 6). TA did not cause obvious cytotoxicity even at the maximum concentration used. Next, qRT-PCR was performed to determine whether TA is involved in regulation of the lipogenesis-related genes sterol regulatory elementbinding protein 1c (SREBP-1c), ATP-citrate lyase (ACLY), fatty acid synthase (FASN), and peroxisome proliferator-activated receptor gamma (PPARg). As expected, OPA significantly increased the mRNA expression of these genes, whereas TA prevented the increase and also abrogated their protein expression ( Figure 2B,C). To elucidate the relationship between mRNA expression of the lipogenic genes and HAT activity, we incubated HepG2 cells, in the presence or absence of OPA, with C-646, a specific p300/CBP inhibitor, at the indicated concentrations ( Figure 2D). mRNA expression of the lipogenic genes was abrogated by blocking the p300/CBP activity using C-646. Collectively, our data indicate that TA ameliorates lipid accumulation through inhibition of lipogenic genes, via its anti-HAT activity.
3.3. TA attenuates OPA-induced hyperacetylation of both histones and total proteins through its anti-HAT activity To evaluate whether the anti-lipogenic properties of TA are correlated with its anti-HAT activity, we initially examined HAT activity in HepG2 cells. Cells were cultured with OPA in the presence or absence of TA at the indicated concentrations, and total HAT activity was measured in isolated nuclear fractions. HAT activity in the OPA-treated cells was significantly higher than that in the control group, and was inhibited by TA ( Figure 3A). To examine whether the TA-mediated reduction in HAT activity affected protein lysine acetylation, we evaluated acetylation at Lys residues in a whole-cell lysate and a histone extract from HepG2 cells. Acetylation at Lys residues in total proteins was increased by OPA, whereas TA effectively attenuated this ( Figure 3B). Moreover, compared with that in the control, TA inhibited the acetylation of histones H3K9 and H3K36 ( Figure 3C), indicating that the anti-HAT activity of TA effectively blocks the acetylation of both total proteins and histone H3 tails, which are involved in NAFLD [36,37].

TA blocks hyperacetylation of H3K9 and H3K36 in the promoters of both FASN and ACLY
Lysine acetylation of histones in promoter regions neutralizes their positive charge and induces conformational changes in DNA, thereby increasing the transcriptional activation of genes [38]. Hyperacetylation of histones in the promoter regions of the FASN and ACLY genes is responsible for upregulation of the transcriptional activity of these genes, resulting in lipid accumulation. To elucidate whether TA modulates the acetylation of histones in the promoters of the FASN and ACLY genes, we performed chromatin immunoprecipitation (ChIP) assays and also examined the occupancy of p300, a transcriptional   (Figure 4, lower panel). It was also observed that the occupancy of p300, which is responsible for histone acetylation, differed between the two genes. p300 was bound to the FASN promoter with a pattern similar to the H3K9 and H3K36 acetylation status of the FASN promoter region, whereas binding of p300 to the ACLY promoter was also diminished by 25 mM TA (Figure 4, upper and lower panels, respectively). Collectively, our results indicate that TA abrogates occupancy of p300 in the SRE of both FASN and ACLY genes, subsequent to inhibition of the OPA-induced hyperacetylations on histone H3K9 and H3K36 in this region.

TA ameliorates the pathogenic features of NAFLD in vivo
To confirm that TA attenuates lipid accumulation and ameliorates the various pathogenic features of NAFLD, we fed mice either a CD or WD with or without 1% or 3% TA supplementation (Supplemental Table 3). Mice in the WD group showed a higher average body weight compared with those in the CD group, whereas the mice group fed the WD containing 3% TA had a low average body weight that was comparable to that of mice in the CD group ( Figure 5A). Although food intake was increased in the WD groups in the presence of TA (Figure 5E), the average body weight gain in the 3% TA group was lower than that in the WD-fed group ( Figure 5B). To determine whether this observed weight gain was associated with an increase in the size and weight of the liver, epididymal fat, and retroperitoneal fat, we compared these measurements among the groups. As shown in Figure 5C, not only was the size of these tissues enlarged but also their weights were significantly increased in the WD group at 12 weeks (p < 0.01). Furthermore, in H&Estained liver tissue, lipid accumulation was observed to be higher in the WD-fed mice than in the CD group, and the liver tissues in mice fed the 3% TA-supplemented diet exhibited reduced lipid droplet accumulation compared with that in the WD-fed mice. Lipid accumulation in the 3% TA-supplemented mice was also comparable to that in the CD-fed mice ( Figure 5D). The triglyceride (TG), total  cholesterol, and LDL cholesterol (LDL-C) levels were significantly lower in both 1% and 3% TA-supplemented groups than in the WD control group ( Figure 5F). The increased levels of aspartate transaminase (AST) and alanine transaminase (ALT) in the WD-fed mice group were also improved by supplementation with 3% TA ( Figure 5F). These results establish that dietary supplementation with TA ameliorates NAFLD pathogenic features in vivo.
3.6. TA decreased the mRNA expression of lipogenesis-related genes by hypoacetylation of H3K9 and H3K36 via its anti-HAT activity in vivo Finally, to confirm that the TA-mediated improvement of NAFLD in vivo was due to its anti-HAT activity, we examined the anti-HAT activity of TA in vivo. Initially, the HAT activity was measured in NEs prepared from mouse livers after dietary supplementation of the CD or WD with or without TA for 12 weeks. Consistent with the previous in vitro results ( Figure 3A), HAT activity was increased in the WD-fed mice, and TA attenuated this increase ( Figure 6A). In addition, the Lys acetylation levels in the whole-cell extracts from mouse livers were significantly higher in the WD-fed mice than in the CD group mice and were attenuated by TA supplementation ( Figure 6B). The acetylation of histones H3K9 and H3K36 was also significantly lower in the 3% TA-supplemented group than in the WD-fed mice ( Figure 6C). Moreover, mRNA expression of mouse lipogenic genes such as DGAT2, FASN, SREBP-1c, and PPARg was also decreased in the TA-supplemented mice ( Figure 6D). Taken together, our findings indicate that dietary supplementation with TA induces hypoacetylation of histone and non-histone proteins through its anti-HAT activity, resulting in the mRNA expression of lipogenesis-related genes in vivo.
3.7. Docking simulation between EP300 and TA Finally, we searched the possible ligand binding site of EP300-like proteins to determine the TA docking position of EP300 because protein structures with similar sequences are often consistent in function and shape. As a result of the sequence alignment, we found a total of 1,189 template structures (EP300-like proteins) and examined the binding sites of 483 ligands in the template structures (Supplemental Figure 6). The most probable binding site from the EP300-like protein was located in a pocket for the bromo domain, and the most conserved ligand binding positions from EP300-like protein is the pocket number 66 (Supplemental Figure 6, orange color of right panel), and the pocket residues are PRO1074, PHE1075, GLN1077, VAL1079, LEU1084, ILE1086, TYR1089, MET1097, ASP1098, MET1124, ASN1127, ALA1128, TYR1131, ASN1132, and VAL1138, and then we docked the TA to these residues (see the methods and materials for detail information). We identified that the top five docked ligands bound to around the bromo domain of EP300 in buried form ( Figure 7A), which have the free energy values between À8.78 kcal/mol and À9.17 kcal/mol (Supplemental Figure 7). Particularly, the most possible binding residues of EP300 for TA are containing ASN1126, TRP1129, LEU1130, TYR1139, LYS1140, SER1143, LYS1144, SER1146, GLU1147, GLU1150, LYS1167, GLU1169, PHE1170, SER1171, ARG1187, ASP1188, LYS1235, ASP1237, LEU1239 and PRO1453 (Figure 7 B), forming 13 hydrogen bonds with the EP300 protein to form a stable structure ( Figure 7C and Supplemental Figure 8). Altogether, TA binds to a pocket between bromo and RING domain of p300, possibly reducing its HAT activity through inducing conformational change of p300.

DISCUSSION
Epigenetic dysregulation is known to initiate the development of various human diseases and to contribute to their progression. Although epigenetic modulation including HDACi and DNA methyltransferase inhibition has been extensively studied, and several are currently being assessed in clinical trials [39], there is little information available on HAT inhibitors. HAT is a plausible target for preventive or therapeutic agents [40,41], and there are now several known HATi derived from natural products [42e44] that have favorable safety profiles [45]. EGCG, the major polyphenol found in green tea, has potent anti-HAT activity, with global specificity for the majority of HAT enzymes [35]. In addition, previous studies have shown that garcinol inhibits p300 and PCAF in vitro and in vivo, whereas anacardic acid inhibits TIP60 as well as p300 and PCAF, and curcumin inhibits p300 and PCAF, suggesting that dietary compounds are potent epigenetic regulators, particularly via HAT inhibition [42]. However, to our knowledge, epigenetic regulation by TA has not previously been investigated, and thus the present study is the first to indicate that TA is a potent and specific HATi. Initially, we demonstrated the anti-HAT activity of TA. TA showed global specificity for the majority of HAT enzymes examined, but not other epigenetic enzymes, i.e., HDACs, illustrating that TA is a specific HATi. A previous acetylation study identified an astonishing 978 acetylated proteins in human liver tissue [46], indicating that protein acetylation is involved in a broad range of cellular activities in the liver. To date, substantial evidence has indicated the involvement of HAT activity in NAFLD [13,47]. Using qRT-PCR analysis, we observed that C-646 treatment leads to a decrease in the mRNA expression of lipogenic genes related to NAFLD, supporting the notion that p300-induced HAT activity causes NAFLD. Although TA has been shown to have an inhibitory effect on lipid accumulation in the liver [48,49], there has been no evidence that TA suppresses the HAT-mediated acetylation of histone and non-histone proteins and the subsequent expressions of lipogenesis-related genes. In the present study, in vitro and in vivo HAT assays clearly showed the TA inhibits HAT-induced histone acetylation. The acetylation of histone H3K9 is well known as an epigenetic marker for NAFLD [47,50], and, as previously remarked, acetylation of proteins is associated with dynamic cellular activities in the liver [46]. In addition, we showed that TA attenuated the lipid accumulationinduced increase in acetylation levels of total proteins and histones H3K9 and H3K36 in vitro and in vivo. Thus, our study provides firm evidence to support the notion that TA suppresses HAT-mediated acetylation of both histone and non-histone proteins. The hyperacetylation of histone proteins by HATs is known to be associated with gene activation [51]. In particular, p300, which is a transcription factor having HAT activity, links activators to the transcription machinery at promoters [52]. The p300/CBP complex dynamically regulates hundreds of different transcription factors, including SREBP-1c and PPARg [53,54]. Glucose-induced p300/CBP activation contributes to the development of NAFLD through enhanced activation of lipogenic genes [55]. Indeed, numerous studies have shown the relationship between lipogenic gene expression and NAFLD, as well as the preventive effects of natural compounds [56,57]. Our data indicated that the mRNA expression of lipogenic genes was increased during lipid accumulation in vitro and in vivo. In addition, consistent with some previous studies [48,58,59], we observed that TA downregulated the mRNA expression of lipogenic genes. Importantly, in contrast to previous studies, we provide direct evidence to explain the observed responses based on an epigenetic mechanism. A recent Original Article 44 study showed that the HAT enzyme p300, bound to the FASN promoter, increases histone acetylation in this region, and eventually increases mRNA expression of the FASN gene [60]. Consistent with these observations, our data also showed that histone acetylation was increased in the FASN promoter during lipogenesis and, using ChIP assays, we further demonstrated that TA modulated this process. These findings provide important evidence in support of our hypothesis. Unexpectedly, however, not only was the recruitment of p300 to the SRE region of the ACLY promoter not inhibited but also acetylation of H3K9 and H3K36 was not significantly abrogated by treatment with 10 mM of TA, suggesting that the ACLY gene is less vulnerable to epigenetic regulation by TA. Another plausible explanation is that increased ACLY expression promotes histone acetylation by generating acetyl-CoA [61], which plays an essential role in determining the overall status of histone acetylation in mammalian cells [46,62]. Finally, we adopted a WD-induced NAFLD model to confirm whether TAinduced inhibition of HAT activity prevents development of the pathogenic features of NAFLD. Two previous studies have reported that histone acetylation is influenced by a WD [63,64], suggesting the possibility of preventing WD-induced diseases through the regulation of HAT activity. As previously mentioned, our results showed that dietary TA supplementation not only ameliorated WD-induced hyperacetylation of histone and non-histone proteins in the liver tissues but also attenuated NAFLDinduced phenotypes and clinicopathological characters, even though food intake was increased in the TA-fed mice, thereby supporting our hypothesis that WD-induced disease could be regulated by the control of To identify the active site in p300, the three dimensional X ray crystallographic structure of p300 was downloaded from the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do) (PDB ID: 4BHW). The 3D structure of TA (PubChem CID: 16129878), the ligand to be used for docking, was generated using Marvin sketch (https://chemaxon.com/products/marvin). The docking simulation was run using Autodock Vina 1.1.2 (http://vina.scripps.edu). All structures were drawn using Chimera software.
HAT activity. Animals fed high-TA-containing diets have a higher total feed intake than those fed a low-TA-containing diet, and the groups on high-TA diets showed a significant decrease in weight gain [65,66]. In this regard, a study on the biochemical mechanism underlying the activity of TA demonstrated that the major effect of TA was not related to the inhibition of food consumption. Instead, it has been demonstrated that the effects of TA are mediated via the efficiency with which digested and absorbed nutrients are converted into new body substances [67], indicating that this phenomenon could be due to a direct inhibitory effect on a key metabolic pathway or indirect effect related to the diversion of metabolism by TA. On the basis of the findings of these previous studies and our present data, it can be postulated that the preventive effects of TA on the development of NAFLD in vivo are related to TA-induced epigenetic regulation of lipid metabolic pathways. We have also demonstrated a feasible mechanism by which the relative occupancy of p300 in the promoter regions of SREBP1c-regulated genes determines overall FASN-or ACLY-mediated mRNA expression, indicating that TA, as a novel HATi, has the important effect of regulating lipid metabolism. By extension, to explain the mode of action of TA as a HATi, we predicted a possible p300-TA docking model ( Figure 7, and Supplemental Figures 3e6). The results showed that TA interacts with a catalytic pocket, bromodomain, and RING domain in p300, and regulates gene transcription by binding directly to histone H3 and negatively regulates itself via an autoinhibitory function [28], indicating that TA may interrupt the function of p300 by inducing a conformational change. Taken together, the results of this study suggest the potential application of a new class of TA nutraceuticals having HATi activity.
In summary, we have provided evidence that TA, a novel HATi, inhibits the acetylation of histone and non-histone proteins and activation of the mRNA expression of lipogenesis-related genes both in vitro and in vivo (Figure 8), indicating that inhibition of HAT activity by TA is necessary for the prevention of NAFLD. Nevertheless, despite our growing awareness of the value of TA as a phytochemical, issues regarding its practical application remain to be addressed. In this regard, the present study can be considered to have certain limitations. First, notably, we have not provided an explanation for the nuclearspecific effects of TA. TA rarely penetrates the plasma membranes of intact cells, but here we should emphasize that TA enters previously injured cells only through the damaged portions of the cell membrane [68]. Accordingly, to explain the nuclear-specific effects of TA, it would be possible to envisage a scenario whereby TA penetrates membranes damaged by WD-induced excessive oxidative stress [69,70] and thereafter forms a complex with p300 in cytoplasm, and this complex is subsequently translocated to the nucleus. In this regard, a recent study has shown the translocation of p300 from the cytoplasm to nucleus via activation of the RhoA-Akt axis pathway, which is closely associated with lipid metabolism [71]. Second, although cytotoxicity was not observed at the concentrations used in our in vivo and in vitro studies, it is conceivable that the concentrations of TA used in the present study lack physiological relevance. Lastly, we did not elucidate the causes that induce discordance between food intake and weight gain in the mice fed TA-containing diet, although the possibility was addressed based on the previous reports showing the same result with our study. However, it is important to determine the energy where is being absorbed and expended metabolically. Given the importance of the aforementioned limitations of this study, further relevant in-depth studies should be conducted.