Novel insights for PI3KC3 in mediating lipid accumulation in yellow catfish Pelteobagrus fulvidraco

In this study, the transcriptional regulation of PI3KC3 by three transcription factors (PPARγ, PPARα, and STAT3) and the potential role of PI3KC3 in mediating lipid accumulation were determined in yellow catfish Pelteobagrus fulvidraco. The 5’-deletion assay, overexpression assay, site-mutation assay, and electrophoretic mobility shift assay suggested that PPARα, PPARγ, and STAT3 negatively regulated the promoter activity of pi3kc3. Moreover, the transcriptional inactivation of pi3kc3 was directly mediated by PPARα and PPARγ under fatty acid (FA) treatment. Using primary hepatocytes from yellow catfish, FA incubation significantly increased triacylglyceride (TG) content, non-esterified fatty acid (NEFA) content, and lipid drops (LDs) content, the mRNA level of pparα, pparγ, stat3, and dnmt3b, the protein level of PPARα, PPARγ, and STAT3, and the methylation level of pi3kc3, but significantly reduced the mRNA and protein level of PI3KC3. Our findings offer new insights into the mechanisms for transcriptional regulation of PI3KC3 and for PI3KC3-mediated lipid accumulation in fish.


Introduction
Phosphatidylinositol-3 kinases (PI3Ks) are the key signaling molecules, which control many cellular processes including cell growth, proliferation, differentiation, survival, intracellular trafficking, and nutrient metabolism (Foster et al. 2003;Liu et al. 2006  , but the relationship between PI3KC3 and lipid metabolism has not been investigated. PI3KC3 belongs to the type III PI3K families, and its cDNA sequence and core promoter have been cloned from yellow catfish in our previous study (Zhuo et al. 2017;. We only found that FOXO1 positively regulated the transcription of pi3kc3 due to the short length of pi3kc3 promoter sequence we have obtained . However, the underlying transcriptional mechanism and the function of PI3KC3 were still limited to known. The expression of gene was regulated by the interaction of transcription factors with promoter elements. Signal transducers and activators of transcription proteins (STATs) belong to a family of latent cytoplasmic transcription factors, which participate in gene regulation. STAT3 is a member of STAT family, which modulates the expression of many target genes involved in lipid metabolism (Wu et al. 2016;. Peroxisome proliferator-activated receptor alpha and gamma (PPARα and PPARγ) are the two important transcription factors that modulate the expression of many target genes involved in lipid metabolism (Zheng et al. 2015a, b). Several previous studies have suggested that PI3K pathway activated PPARα and PPARγ, and played an important role in the regulation of cellular lipid metabolism Yang et al. 2018). However, limited studies reported whether the transcription of PI3K was regulated by the transcription factors related to lipid metabolism, such as STAT3, PPARα, and PPARγ.
The methylation of DNA belongs to one of the most important epigenetic mechanisms, which represses gene expression by recruiting proteins or by preventing the binding of the transcription factors to DNA sequences (Nagase and Ghosh, 2008;Moore et al. 2013). DNA methylation is primarily modulated by DNA methyltransferases (DNMTs), including DNMT1, DNMT3A, and DNMT3B (Nagase and Ghosh, 2008). Many studies suggested that aberrant DNA methylation was correlated with disorders and dysregulation of lipid accumulation. Nutritional factors including dietary high-fat or fatty acid supplement could modify specific gene transcription through the alteration of DNA methylation status (Ge et al. 2013;Marco et al. 2014;Kim et al. 2015;Zhang et al. 2017;Li et al. 2018;Parsanathan et al. 2019;Hunter et al. 2019). DNA methylation often happened on the CpG islands within the promoter region of the gene. Interestingly, two CpG islands were predicted on the promoter of pi3kc3, which attracting our great interest to study whether DNA methylation was involved in the PI3KC3 of yellow catfish.
Yellow catfish, an omnivorous freshwater fish, is widely distributed in the inland freshwater waters in China. Gong et al. (2018) published its genomic sequences, which provided good basis for exploring the regulatory mechanism of lipid metabolism and for amplifying the long length sequence of pi3kc3 promoter. Moreover, our previous study suggested that PI3K pathways were involved in regulating lipid metabolism. HEK293T is a good cell line, with very high transfection efficiency during the transfection, which has been widely used to study the function of fish promoters Chen et al. 2020a, b;LV et al. 2021). To further investigate the function of PI3KC3 promoter and the regulatory mechanism of PI3KC3 in mediating lipid accumulation in yellow catfish. In this study, the transcriptional regulation of pi3kc3 by three transcript factors (PPARγ, PPARα, and STAT3) was studied by using HEK293T. Our results suggested that the promoter activity of pi3kc3 was negatively regulated by PPARα, PPARγ, and STAT3, and the transcriptional inactivation of pi3kc3 was directly mediated by PPARα and PPARγ under FA treatment. Meantime, by using primary hepatocytes from yellow catfish, we found that FA incubation disturbed the methylation and gene expression of pi3kc3. Our study elucidated innovative insights into the regulatory mechanism of PI3KC3 in fish.

Experimental animals and reagents
To eliminate gender-differentiated response, the mixed sex yellow catfish (body weight: 22.5 ± 4.4 g, male: female = 1:1) were obtained from a local commercial farm. HEK293T cell lines were purchased from the Cell Resource Center in the Fishery College of Huazhong Agricultural University. Dulbecco's Modified Eagle's Medium (DMEM), 0.25% trypsin-EDTA, and fetal bovine serum (FBS) were obtained from Gibco/Invitrogen, USA. Dimethyl sulfoxide (DMSO), penicillin, palmitic acid, oleic acid, streptomycin, trypan blue, and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). We ensured that the experiments were performed in accordance with the experimental protocols of Wuhan Polytechnic University (WHPU) and were approved by the ethics committee of WHPU.

Experimental treatment
Two experiments were carried out. Exp. 1 was conducted to study the transcriptional regulation of pi3kc3 promoter. Exp. 2 was conducted to determine the potential role of PI3KC3 in influencing lipid accumulation in the hepatocytes from yellow catfish under FA incubation.
Exp. 1: transcriptional regulation assay of pi3kc3 promoter Promoter cloning and plasmids construction The genomic DNAs were extracted from the liver of six yellow catfish (male: female = 1:1) by using a commercial DNA extracted kit (Omega, Norcross, GA, USA). The promoter sequence of pi3kc3 was obtained by RT-PCR (reverse transcription-polymerase chain reaction) according to the genome of yellow catfish (Gong et al. 2018). The primers for pi3kc3 promoter cloning are presented in Table S1. For generating the luciferase reporter construct, we subcloned different plasmids with pi3kc3 promoter into pGl3-Basic vectors (Promega, USA) by using SacI and HindIII restriction sites. On the basic of the distance from its TSS, we named the plasmid as pGl3-1781/ + 59 of pi3kc3 promoter. Then, we used pGl3-1781/ + 59 vector as the template to produce the plasmids pGl3-1361/ + 59, pGl3-848/ + 59, and pGl3-381/ + 59 of pi3kc3 vectors. We used ClonExpress II One Step Cloning Kit (Vazyme, Piscataway, NJ, USA) to ligate all of the products. We performed the PCR via the TaKaRa PrimeSTAR® HS DNA Polymerase kit (TaKaRa, Tokyo, Japan). Finally, we sequenced all these plasmids in the Tsingke Company (Wuhan, China). The primers for the plasmids construction are presented in Table S2. In addition, the overexpression plasmids of PPARα, PPARγ, and STAT3 were obtained from our previous studies (Lv et al. 2021).
Plasmid transfections and assays of luciferase activities HEK293T cells were cultured in DMEM medium with the 10% FBS (Gibco, Carlsbad, CA, USA) in an incubator (5% CO 2 and 37 °C). Prior to the transfection, HEK293T cells were seeded at a density of 1.2 × 10 5 in 24-well plate. They were cultured until the 70-80% confluence. Lipo-fectamine™2000 (Invitrogen) was utilized to transfect all these plasmids into HEK293T cells, based on the manufacture's protocol. The 500 ng overexpression plasmids, 400 ng reporter plasmids, and 20 ng pRL-TK (the internal control with a Renilla luciferase reporter vector) were co-transfected into HEK293T cells. After 4 h, we replaced the transfection medium by 10% FBS-DMEM or 10% FBS-DMEM + 0.6 mM FA. FA was added as a mixture of palmitic acid and oleic acid at a ratio of 1:1. The form and the concentration of FA were selected according to our pilot trial and the publications of the in vitro studies (Wu et al. 2019Chen et al 2020a;Song et al 2020). Then, after 24-h incubation, cells were collected to determine the promoter activity, based on the manufacturer's instruction of the Dual-Luciferase Reporter Assay System (Promega). The relative luciferase activities were obtained by calculating the ratio of Firefly luciferase activity to Renilla luciferase activity. We conducted all these experiments in triplicates and three independent experiments were carried out.

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Vol:. (1234567890) Site-mutation analysis of binding sites on the pi3kc3 promoter To identify the corresponding binding sites on the regions of pi3kc3 promoter, we used QuickChange II Site-Directed Mutagenesis Kit (Vazyme, Piscataway, NJ, USA) to perform site-directed mutagenesis analysis. Several mutations were performed at the sites of -1621/-1611 bp, -1603/-1594 bp, -922/-907 bp, -1083/-1076 bp, and -245/-230 bp of pi3kc3 promoter. The primers used for mutagenesis are shown in Table S3. The DNA sequencing was utilized to confirm these mutations. Then, the Lipofectamine 2000 reagent (Invitrogen) was utilized to co-transfect the plasmids into HEK293T cells. After 4-h transfection, the medium was substituted with 10% FBS-DMEM or 10% FBS-DMEM + 0.6 mM FA. After 24-h incubation, we harvested the cells to determine the luciferase activities, based on the procedures mentioned above. We conducted all these experiments in triplicates and three independent experiments were carried out.

Electrophoretic mobility shift assay (EMSA)
The EMSA was conducted to confirm the functional PPARα, PPARγ, and STAT3 binding sites on the pi3kc3 promoter according to our and other recent publications Zhuo et al. 2018;Chen et al. 2020b). Nuclear and cytoplasmic extracts were extracted according to the method of Read et al. (1993). Protein contents were determined by the BCA method (Smith et al. 1985). The oligonucleotide probes were synthesized in the Tsingke Company (Wuhan, China). Nuclear extracts (10 µg) were incubated for 30 min at room temperature by using the binding buffer (20 mM HEPES, pH7.9, 1 mM MgCl 2 , 0.5 mM DTT, 4% Ficoll, 110 mM KCl, 0.2 μg Poly(dI-dC)). Then, the biotin-labeled double-stranded oligo nucleotides (Table S4) were added. The reaction continued for 30 min and then the electrophoresis was performed on 6% native polyacrylamide gels. For the competitive binding analysis, a 100-fold excess of unlabeled double-stranded DNA oligo with mutant binding site (Table S4) was added with the corresponding labeled one.
Exp2: FA incubation with hepatocytes of yellow catfish Hepatocytes were isolated from yellow catfish (each independent experiment six fish were used, male: female = 1:1) according to our previous studies and were cultured in M199 medium containing 1 mmol/L L-glutamine, 5% (v/v) FBS, penicillin (100 IU/mL), and streptomycin (100 g/mL) in a humidified atmosphere with 5% CO 2 at 28 °C ). Hepatocytes were counted using a hemocytometer based on the trypan blue exclusion method and only more than 95% cell viability were used for the present experiment. Hepatocytes were plated onto 25 cm 2 flasks at the density of 10 6 cells/mL, and then they were incubated with PBS (control) and 0.6 mM FA. Each treatment was performed in triplicate and three independent experiments were carried out. After 48 h, the hepatocytes were gathered for the following analysis.
Triacylglyceride (TG), non-esterified fatty acid (NEFA), and lipid drops (LDs) assay TG and NEFA contents were determined with commercial kits (Nanjing Jian Cheng Bioengineering Institute, China), according to the manufacturer's instructions. Bodipy 493/503 staining was used to assess the changes of intracellular LDs. Briefly, hepatocytes were cultured in 12-well plates and treated with the corresponding treatments for the required period, and then they were washed twice with PBS. After that they were incubated with 5 mg/ mL Bodipy 493/503 (D3922; Thermo Fisher Scientific Waltham, MA, USA) for 30 min, followed by 3 PBS washes. Then the hepatocytes were observed with a laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) to visualize the intensity of fluorescence. The green dots were defined as LDs, which were quantified with a CytoFlex flow cytometer (Beckman Coulter, Brea, CA, USA). Data analysis was performed with FlowJo v.10 software (Ashland, OR, USA). mRNA level determination by real-time quantitative PCR (RT-qPCR) Total RNA was isolated using Trizol reagent (TaKaRa, Dalian, China) according to the manu-Fish Physiol Biochem (2022) 48:571-583 574 facturer's instruction. cDNA was then reverse-transcribed from normalized RNA using oligo (dT) primers and M-MLV reverse transcriptase (TaKaRa, Dalian, China). The mRNA levels of pparα, pparγ, stat3, dnmt1, dnmt3a, dnmt3b, and pi3kc3 were examined by RT-qPCR. RT-qPCR assays were performed in a quantitative thermal cycler (MyiQ™ 2 TwoColor Quantitative PCR Detection System, BIO-RAD, USA) with a 20 μL reaction volume containing 10 μL SYBR Premix Ex Taq™ II (TaKaRa, Japan), 1 μL of diluted cDNA (tenfold), 10 mM each of forward and reverse primers (0.4 μL), and 8.2 μL H 2 O. Primers are given in Table S5. The qPCR parameters consisted of initial denaturation at 95 °C for 30 s, followed by 40 cycles at 95 °C for 5 s, 57 °C for 30 s, and 72 °C for 30 s. All reactions were performed in duplicates and each reaction was verified to contain a single product of the correct size by agarose gel electrophoresis. The melting curve was generated for every PCR product to confirm the specificity of the assays. A set of seven common housekeeping genes (β-actin, 18 s-rrna, gapdh, rpl7, hprt, ubce, and tuba) were selected from the literature (Vandesompele et al. 2002) in order to test their transcription stability. Two most stable control genes (gapdh and 18srrna, M = 0.35) were selected by using geNorm software. The relative expression levels were calculated with the "delta-delta Ct" method (Pfaffl 2001), and normalized in terms of the geometric mean of two genes by geNorm.

Analysis of protein expression by western blot
Western blotting was performed according to the previous study . Hepatocytes were lysed in RIPA buffer (Sigma, USA). Equal amounts of protein were separated on 12% SDS-PAGE, transferred onto PVDF membranes, and then blocked with 8% (w/v) dry milk. After that, the membranes were incubated with primary antibodies as follows: rabbit anti-PPARα (15,540-1-AP, Proteintech, USA), rabbit anti-PPARγ (16,643-1-AP, Proteintech, USA), rabbit anti-STAT3 (10,253-2-AP, Proteintech, USA), rabbit anti-PI3KC3 (AbClone, A12295, USA), and anti-GAPDH (10,494-1-AP; Proteintech, USA) overnight at 4 °C. Then, HRP-conjugated anti-rabbit secondary antibody (CST, USA) was used to probe with. Finally, the protein bands were visualized with enhanced chemiluminescent (ECL) and quantified by Image J software . Methylation analysis of CpG island of pi3kc3 promoter Genomic DNA from hepatocyte was extracted using AxyPrep DNA Kit (Axygen Biotechnology, Hangzhou, China) according to the manufacturer's instructions. The genomic DNA extracted above was modified according to the manufacturer's protocol using the DNA Methylation Gold Kit (Zymo research, Orange, CA). Two CpG islands on the pi3kc3 promoter were predicted. The bisulfite-modified DNA was amplified by nest PCR with two BSP (bisulfite sequencing PCR) specific primer pairs (list in Table S6), under the following conditions: 95 °C denaturation for 3 min; 30 cycles of 95 °C for 30 s, 53 °C for 30 s, and 72 °C for 40 s; and 72 °C extension for 5 min. The PCR products were gel purified and were subjected to cloning into a pMD 19-T Vector (TaKaRa, Dalian, China). After the cloning, total of 20 clones from each treatment were randomly selected for DNA sequencing. Sequencing chromatogram analysis of partial bisulfite-modified DNA after amplification is shown in Fig. S1. The methylation level was analyzed by the online website (http:// quma. cdb. riken. jp/).

Statistical analysis
We used SPSS 19.0 software for all these statistical analyses. All of these data were expressed as means ± SEM (standard errors of means). Before statistical analysis, we evaluated all data for normality using the Kolmogorov-Smirnov test. In order to test the homogeneity of variances, we performed Bartlett's test. We analyzed data with Duncan's multiple or Student's t-test where appropriate. Difference was considered significant at p < 0.05.

Site-mutation analysis
To further elucidate whether the regions of pi3kc3 promoter possessed PPARα, PPARγ, and STAT3 response elements, site-directed mutations were performed. Overexpressed PPARα markedly reduced the promoter activity of the widetype pi3kc3 plasmid, and its inhibitory effect was completely abolished when the -245/-230 bp PPARα site was mutated, suggesting that the -245/-230 bp PPARα site negatively controlled pi3kc3 transcription ( Fig. 2A).

EMSA analysis of binding sequence of transcription factors
Next, we examined whether PPARα, PPARγ, and STAT3 functionally bind with their corresponding regions of pi3kc3 promoter. For the PPARα binding assay, the 100-fold unlabeled PPARα binding sequence competed for the binding when we used biotin-labeled PPARα binding sequence (-245/-220 bp of pi3kc3 promoter) as the probe, while the 100-fold unlabeled mutated PPARα binding sequence markedly reduced this competition, indicating that PPARα binding sequence was functionally bound by Values are presented as mean ± SEM (n = 3). Asterisk (*) indicates significant difference between PPARα, PPARγ, and STAT3 overexpression and the control (p < 0.05). Hash symbol (#) indicates significant difference between two plasmids under the same treatment (p < 0.05). Mut is the abbreviation of mutagenesis Fish Physiol Biochem (2022) 48:571-583 577 PPARα (Fig. 3A). Similar results are also found for the PPARγ binding sequence (-922/-900 bp of pi3kc3 promoter, Fig. 3B) and STAT3 binding sequence (-743/-720 bp of pi3kc3 promoter, Fig. 3C), suggesting that both PPARγ and STAT3 were also functionally bound by pi3kc3 promoter.
Effect of FA incubation on the promoter activity of pi3kc3 promoter To investigate the response of promoters induced by FA, we used 0.6 mM FA to incubate HEK293T for 24 h. Compared with the control group, FA incubation significantly reduced the luciferase activities of pGl3-1781/ + 59, pGl3-1361/ + 59, pGl3-848/ + 59, and pGl3-381/ + 59 of pi3kc3 plasmids (Fig. 4A). To further determine whether FA induced the decreasing of pi3kc3 promoter activity could be mediated by PPARα, PPARγ, and STAT3 elements, we conducted the site-directed mutation at their corresponding sites of pi3kc3 promoter and used FA to incubate the cells.
Compared to the wild-type pGl3-1781/ + 59 vector of pi3kc3 promoter, FA-induced transcriptional inactivation of pi3kc3 was subdued after the mutation of PPARα and PPARγ elements, but not after the mutation of STAT3 element (Fig. 4B). Effect of FA incubation on lipid accumulation in the hepatocytes from yellow catfish Compared to the control group, FA incubation significantly increased the TG content, NEFA content, and LDs content in the hepatocytes from yellow catfish (Fig. 5).
Effect of FA incubation on gene expression, protein expression, and methylation level For the mRNA expression assay, compared to the control group, FA incubation notably up-regulated the mRNA level of pparα, pparγ, stat3, and dnmt3b, but down-regulated the mRNA level of pi3kc3, and showed no effect on the mRNA level of dnmt1 and dnmt3a in the hepatocytes from yellow catfish (Fig. 6A). For the protein expression assay, compared to the control group, FA incubation significantly increased the protein level of PPARα, PPARγ, and STAT3, but decreased the protein level of PI3KC3 (Fig. 6B,C).

Discussion
At present, the underlying transcriptional mechanism of PI3KC3 and the role of PI3KC3 in regulating lipid metabolism in yellow catfish remain largely unknown. In order to identify the role of pi3kc3 in regulating lipid metabolism, it is very important to explore the transcriptional regulation of pi3kc3 by transcription factors related to lipid metabolism. PPARα and PPARγ are the two important nuclear transcription factors that regulate lipid metabolism (Zheng et al. 2015a;. Yang et al. (2018) reported that PIK3R3 mediated hepatic lipid homeostasis through PPARα. Several other studies also revealed that PPARα and PPARγ positively regulated the expression of PI3K regulatory subunit in mammals (Rieusset et al. 1999;2001a;. However, to our best known, the regulations of PPARα and PPARγ on the expression of PI3K catalytic subunit have never been reported. In this study, we found that overexpression of PPARα and PPARγ significantly decreased the transcriptional activity of pi3kc3, and subsequent sitemutation and EMSA assay demonstrated that PPARγ and PPARα directly mediated transcriptional activity of pi3kc3, implying that PPARγ and PPARα negatively regulated the transcriptional activity of pi3kc3. STAT3 belongs to a family of transcriptional regulator, which modulates the expression of many target genes related to lipid metabolism ). It has been reported that PI3K and STAT3 were interdependent in many cellular processes Hart et al. 2011;Chu et al. 2014). In this study, we found that overexpression of STAT3 reduced the transcriptional activity of pi3kc3, and subsequent site-mutation and EMSA assay demonstrated that STAT3 directly mediated the transcriptional activity of pi3kc3, implying that STAT3 also negatively regulated the transcriptional activity of pi3kc3. Conversely, Abell et al. (2005) reported that STAT3 positively regulated the expression of PI3K regulatory subunit in mammary gland tissue. Taken together, our study found that PPARα, PPARγ, and STAT3 negatively regulated the transcriptional activity of pi3kc3 from yellow catfish.
Fatty acid is the direct factor that regulated intracellular lipid level. In the present study, we found that FA incubation markedly increased lipid and NEFA level in hepatocytes from yellow catfish, in agreement with other studies (Wu et al. 2019Chen et al. 2020a, b;Song et al. 2020). In addition, FA incubation significantly reduced the mRNA and the protein levels of PI3KC3, but increased the mRNA and the protein levels of PPARα and PPARγ. Similarly, Zhong et al. (2019) also reported that fatty acid stimulated lipid droplets formation and PPARα expression in HepG2 cells. Our studies also demonstrated that FA-induced transcriptional inactivation of pi3kc3 was subdued after the mutation of PPARα and PPARγ elements, implying that FA decreased the expression of pi3kc3 directly through PPARα and PPARγ in yellow catfish. Moreover, our study revealed that FA-induced hepatocellular TG accumulation coincided with the decreasing expression of PI3KC3. However, previous studies have suggested that there was a positive relationship between PI3K activity and TG accumulation Wang and Sul 1998). Thus, we speculated that the decreasing expression of PI3KC3 may result in the increasing of PI3KC3 activity. For example, other studies pointed out that increasing the expression of PI3KCa and PI3KCb resulted in the decreasing of their activities in human (Pankow et al. 2006). Meantime, we also found that FA incubation up-regulated the mRNA level and protein level of STAT3. However, FAinduced transcriptional inactivation of pi3kc3 was not changed after the mutation of STAT3 element, indicating that STAT3-PI3KC3 is probably not the prioritized binding under FA treatment.
On the other hand, DNA methylation has a specific effect on gene expression, and methylation of the CpG islands on the promoter region of gene directly represses gene expression (Bird 2002;Chan 2007). Recently, it has been reported that PI3K pathway regulated DNA methylation in several specific gene loci . Barberio et al. (2019) also reported that the methylation and expression of the gene in the upstream or downstream of PI3K signaling pathway were altered in obesity. However, to our best knowledge, the methylation status of PI3K itself has yet not been investigated. In this study, for the first time, we found that FA induced the hypermethylation of pi3kc3, but reduced its mRNA expression. Similarly, other previous studies also pointed out that methylation of CpG islands impaired transcription factor binding to its targets and accordingly led to silence of gene expression (Siegfried and Simon 2010;Moore et al. 2013). Moreover, we found that FA induced the hypermethylation of pi3kc3 promoter along with the up-regulation of mRNA expression of dnmt3b. Our previous study also found that high-fat induced the expression of dnmt3b, but not dnmt1 and dnmt3a in the ovary of yellow catfish (Zhuo et al. 2019). Some other studies also pointed out that high-fat or fatty acid supplement induced the global and gene-specific DNA methylation along with the increased expression of the dnmts in mice both in vivo and in vitro (Kim et al. 2015;Hunter et al. 2019;Parrillo et al. 2016). Together, these results indicated that DNA methylation participated in FA-induced expression of pi3kc3 expression in the hepatocytes of yellow catfish.
In summary, we identified that three transcription factors (PPARα, PPARγ, and STAT3) negatively regulated the transcription activity of pi3kc3 promoter. The transcriptional inactivation of pi3kc3 was directly mediated by PPARα and PPARγ under FA treatment. Furthermore, for the first time, we found that FA-induced expression of PI3KC3 was regulated by DNA methylation in the hepatocytes of yellow catfish.
Author contribution MQ Zhuo designed the experiments; MQ Zhuo and J Chen performed the experiments, and analyzed the samples with the help of ML Wu and WB Wang; J Chen and ML Wu analyzed the data; and J Chen and MQ Zhuo wrote the manuscript. MQ Zhuo revised the manuscript. All the authors approved the manuscript.