Gene 561 (2015) 157–164

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Fatty acid synthase promoter: Characterization, and transcriptional regulation by sterol regulatory element binding protein-1 in goat mammary epithelial cells Jun Li, Jun Luo ⁎, Huifen Xu, Miao Wang, Jiangjiang Zhu, Hengbo Shi, Abiel Berhane Haile, Hui Wang, Yuting Sun Shaanxi Key Laboratory of Molecular Biology for Agriculture, College of Animal Science and Technology, Northwest A&F University, Yangling, 712100, Shaanxi, PR China

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Article history: Received 16 October 2014 Received in revised form 22 January 2015 Accepted 12 February 2015 Available online 14 February 2015 Keywords: Fatty acid synthase promoter Lipogenesis Sterol regulatory element binding protein-1 Transcriptional regulation

a b s t r a c t Fatty acid synthase (FASN) is the central enzyme of the de novo fatty acid biosynthesis pathway. Although the FASN transcriptional regulatory mechanism has been elucidated clearly in many tumor cells, its mechanism is still not clear in the ruminant mammary gland. In this study, we cloned and sequenced a 1.8-kb fragment of the FASN 5′ flanking region from goat genomic DNA. Multiple alignment analysis demonstrated that the entire 1.8-kb fragment has little homology but that the sub-section nearest the transcriptional start site (−203 to +1) is more conserved across species, in particular the binding motifs for transcriptional regulation. Deletion analysis revealed a putative core promoter region located in −297/−14 bp upstream of the transcription site within the high homology domain. Mutations of sterol response elements (SRE1 and SRE2) and the nuclear factor Y (NF-Y) binding site appeared to significantly down-regulate the FASN promoter activity in goat mammary epithelial cells (P b 0.05). Further analysis showed that both SRE sites responded to sterol regulatory element-binding protein 1 (SREBP-1). SREBP-1 overexpression and knockdown by small interference RNA influenced the abundance of endogenous FASN. These data suggested that SREBP-1 may regulate FASN expression at the transcriptional level in the lactating goat mammary gland. Hence, the current work will contribute valuable information to understanding the molecular regulatory mechanisms of FASN during lactation. © 2015 Published by Elsevier B.V.

1. Introduction The high content of short- and medium-chain fatty acids confers a high nutrition value to goat milk (Hansen et al., 1984; Haenlein, 2004). Their biochemical nature greatly influences the flavor and quality of the milk (Chilliard et al., 2003). Fatty acid synthase (FASN), a key enzyme in the process of the de novo fatty acid synthesis of milk, catalyzes all of the reaction steps for synthesizing saturated fatty acids from acetyl-CoA and malonyl-CoA in an NADPH-dependent manner (Smith et al., 2003). The acetyl-CoA and malonyl-CoA transacylase (AT/MT) domain of the FASN gene in the goat mammary gland show medium-chain thioesterase activity, which reportedly influences the specific fatty acid composition of goat milk (Grunnet and Knudsen, 1978, 1979). Therefore, studying the regulation of FASN in goat Abbreviations: ACC, acetyl-CoA carboxylase; FASN, fatty acid synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GMECs, goat mammary gland epithelial cells; LXR, liver X receptor; NF-Y, nuclear factor Y; PPARα, peroxisome proliferator-activated receptor α; SCD, stearoyl-CoA desaturase; siRNA, small interference RNA; SRE, sterol response elements; SREBP-1, sterol regulatory binding protein 1; Sp1, specificity protein 1; USF, upstream stimulatory factor. ⁎ Corresponding author at: College of Animal Science and Technology, Northwest A&F University, No. 3 Taicheng Road, Yangling, Shaanxi, PR China.

http://dx.doi.org/10.1016/j.gene.2015.02.034 0378-1119/© 2015 Published by Elsevier B.V.

mammary epithelial cells (GMECs) will provide a theoretical and practical basis for genetically regulating beneficial fatty acids in milk. Although FASN is regulated by allosteric effectors or covalent modification, its regulation occurs mainly at the transcriptional level (Griffin et al., 2007). In non-ruminants, FASN gene transcription is under tight control by nutritional and hormonal conditions (such as insulin) in the liver and adipose tissues (Hillgartner et al., 1995). Response elements, such as insulin (Moustaid et al., 1994), cAMP (Rangan et al., 1996), and sterol response element (SRE) (Magana and Osborne, 1996), have been identified in human and rat FASN promoters. Several independent studies have confirmed the presence of sterol regulatory element-binding proteins belonging to the bHLH/LZ family in the promoters of their target genes, which can stimulate transcription by binding to SREs (Ericsson et al., 1996; Lopez et al., 1996; Guan et al., 1997; Shimano, 2001). An SRE/E-box element at −65 bp was found to be crucial for FASN promoter response to nutritional signals and insulin in adipose cells (Latasa et al., 2000). Insulin regulates FASN gene expression through SREBP-1 by causing it to bind the promoter of FASN in adipocytes (Kim et al., 1998). However, unlike rodents, ruminant mammary gland lipogenesis in lactating cows is not responsive to or controlled by insulin (McGuire et al., 1995). Marked species differences exist in the organs principally responsible for lipogenesis. Little information

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has been reported on the transcriptional regulation of goat FASN in lactating mammary glands. Although many investigations have been conducted on the transcriptional regulation of the FASN gene in non-ruminants, the role of the goat FASN promoter in the lactating mammary gland has not yet been characterized. The aim of this study was to characterize the goat FASN promoter and investigate the transcriptional regulatory mechanisms of goat FASN in GMECs. Our findings suggest that goat FASN is regulated by SREBP-1 in GMECs. 2. Materials and methods The Animal Care and Use Committee of the Northwest A&F University approved all procedures and experiments. 2.1. Cloning of the FASN promoter region Genomic DNA, used for amplifying the 5′ flanking sequence of the FASN gene, was extracted from blood samples of three Xinong Saanen dairy goats (lactating 100 days) using a genomic DNA extraction kit (Tiangen, Beijing, China). The 5′ flanking sequence of the goat FASN gene was amplified by PCR (the primers are listed in Table 1) using goat genomic DNA as a template. PCR of the FASN promoter was performed in 25-μL reaction mixtures containing 1 μL of DNA template, 1 μL of each 10 μM primer, 2 μL of 2.5 μM dNTP mix, 12.5 μL of 2× GC Buffer I (Takara, Japan), and 1.25 U Prime STAR (Takara, Japan). The PCR cycling conditions were performed according to the manufacturer's instructions. Amplified products were cloned into the pMD19-T vector (Takara, Japan) and sequenced (Invitrogen, Shanghai, China). 2.2. Bioinformatics analysis The promoter region was predicted using Promoter 2.0 (http:// www.cbs.dtu.dk/services/Promoter/). Similarly, putative transcription factor binding sites were predicted using MatInspector (http://www. genomatix.de/), AliBata 2.1 (http://www.gene-regulation.com/pub/ programs.html), TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCH. html), and the TRES (Transcription Regulatory Element Search) tool (http://bioportal.bic.nus.edu.sg/tres/). The 5′ flanking sequence of the dairy goat FASN gene was compared to that of bovine (Bos taurus, AF285607), human (Homo sapiens, AF250144) and rat (Rattus norvegicus, X54671) using the BioXM 2.6. 2.3. Deletion analysis and plasmid construction The FASN promoter fragment spanning from − 1524 to + 322 bp was excised and inserted upstream of the luciferase gene in a pGL3Basic vector (Promega, Madison, WI, USA). To generate 5′ deletion plasmid derivatives −1524, −1044, −838, −591, −397, −293, −79, and −14, PCR primers were designed to hybridize at the corresponding positions and coupled with the common downstream primer at + 129

(Table 1). The primers were introduced with MluI and BglII restriction enzyme sites at their terminal ends, such that the PCR products were digested at the corresponding restriction sites and ligated to the multiple cloning sites of the pGL3-Basic vector digested with the same enzymes. All of the plasmids were confirmed by DNA sequencing. 2.4. Site-directed mutagenesis Site-directed mutagenesis of the FASN promoter was carried out by overlap extension PCR with the specific mutagenic primers in Table 2. The SRE1, NF-Y and SRE2 sites were mutated by site-directed mutagenesis methods using overlap extension PCR. For each mutant promoter, PCR was first used to generate two DNA fragments that both contained the designated mutations in the overlapping regions. The two DNA fragments were then used together as a PCR template to generate a third DNA fragment. SRE1, NF-Y and SRE2 mutants were investigated using pGL-(− 293/+ 129) constructs as templates. These fragments were then fused with the luciferase sequence after digestion with the same restriction enzymes, MluI and BglII, to create mutant constructs pSRE1-mut, pNF-Y-mut and pSRE2-mut. The sequences of all mutated constructs were confirmed by DNA sequencing. 2.5. Construction of the SREBP-1 expression vector The coding domain of the goat transcription factor SREBP-1 was amplified by reverse transcription PCR. The primers used were as follows: forward 5′-GCGTCGACGCCACCATGGACGAGCCACCCTTCAA-3′ and reverse 5′-TTGCGGCCGCCTAGGCAGACACCAGGTCCT-3′. RNA, used for RT-PCR, was extracted from a lactating mammary gland tissue of Xinong Saanen dairy goats using Trizol (Invitrogen, USA). First strand cDNA was synthesized by the Reverse Transcription Kit (Takara, Japan). Amino acids 1–403 from the SREBP-1 cDNA were amplified and subcloned into the shuttle vector pAdTrack-CMV. Eventually, the sequence was confirmed by restriction enzyme digestion and sequencing. 2.6. SiRNA experiment SREBP-1 knockdown was performed using specific siRNAs targeting SREBP-1 predesigned by Invitrogen (USA). The SREBP-1 siRNA sequence was as follows: 5′-GCUCCUCACUUGAAGGCUUTT-3′. Scrambled siRNA, a functional non-targeting siRNA provided by the same company, was used as a negative control. Briefly, in a 24-well plate, 100 nmol SREBP-1 or control siRNA was transfected with siRNA using Lipofectamine™ RNAiMAX (Invitrogen, USA). After incubation at 37 °C with 5% CO2 for 48 h, the cells were collected and prepared for analysis by real-time quantitative PCR (RT-qPCR). 2.7. Cell culture and transfection The isolation of goat primary GMECs was described previously (Wang et al., 2010; Prpar et al., 2012; Lin et al., 2013a). GMECs were

Table 1 Primers for isolation and deletion of goat fatty acid synthase promoter constructs. Primer name

5′deletion primers

Forward Reverse F1 F2 F3 F4 F5 F6 F7 F8 Reverse

Primer sequence (5′ → 3′)

Binding region

GAAGGAAAAGAAAATCCCACAAAGT GTCTCGTGGGACTCGAACATACA ATTACGCGTGAAGGAAAAGAAAATCCCA ATTACGCGTTGTTTTCAGTCTGGAAAGTC ATTACGCGTAAGAGGTGTCCGTGCATAGG ATTACGCGTGCCCGGCCCATCACCCTATC ATTACGCGTTGACCCTCAGAGTGACCGAAGT ATTACGCGTGCACGGAACGGAAGTTGG ATTACGCGTTCAGCCCATGTGGCGTGTC ATTACGCGTGGAGCCAGAGAGACAGTAGC GGAAGATCTGGGTTCCCGACTCACAACT

−1524 +322 −1524 bp⁎ −1044 bp −838 bp −591 bp −397 bp −293 bp −79 bp −14 bp +129 bp

* Indicates the number of bases upstream (-) and downstream (+) from the start site of transcription.

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2.10. Statistical analysis

Table 2 Primers used in site-directed mutagenesis. Primer name

Primer sequence (5′ → 3′)

S1 SRE SRE-mut SRE-anti-mut NF-Y NF-Y-mut NF-Y-anti-mut SRE/E-box SRE/E-box-mut SRE/E-box-anti-mut A1

ATTACGCGTGCCCGGCCCATCACCCTATC CGGCGCGCCGCATgACggCACTGG CGGCGCGCCGCATAACTTCACTGG CGCCGCCGCCAGTGAAGTTATGCG AGCCCCGACGCTCATtggCCTGGGC AGCCCCGACGCTCATGAACCTGGGC CTGCGCCGCCCAGGTTCATGAGCG CAGCCAAGCTGTCAGcccATGTGGCGTGTC CAGCCAAGCTGTCAGTTTATGTGGCGTGTC CGTGCGGACACGCCACATAAACTGACAG GGAAGATCTGGGTTCCCGACTCACAACT

cultured in a basal medium supplemented with 5 μg/mL insulin, 0.25 μmol/L hydrocortisone, 10 ng/mL epidermal growth factor, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum. To promote lactogenesis, GMECs were cultured in a lactogenic medium as reported by Kadegowda et al. (2009). The lactogenic medium was prepared as the basal medium, supplemented with 2 μg/mL prolactin. MCF-7 cells were used to confirm the results, these were cultured in DMEM/F12 medium (Invitrogen, USA) supplemented with 10% fetal bovine serum (Hyclone, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator (5% CO2, 37 °C). The cells were seeded in 96-well culture plates and cultured overnight or until 80%–90% confluent. Transient transfection was performed with FuGENE HD following the manufacturer's instructions (Roche, USA). Cells in antibiotic-free, serum-free DMEM/F12 were transfected with 0.2 μg of total DNA per well for 6 h before replacing it with a fresh culture medium containing 10% FBS and antibiotics. As an internal control, Renilla luciferase vector (pRL-TK) was included in all transfections to normalize transfection efficiency. Transfection complexes contained a 25:1 ratio of pGL-FASN construct to pRL-TK vector. A promoter-null plasmid, pGL3-Basic, was also included in the transfection assay as a negative control. All transfections were carried out in triplicate and repeated at least thrice in independent experiments.

2.8. Luciferase assays After 48 h of incubation transfection, cells were harvested to assay their luciferase activity. Transfected cells were washed twice with phosphate buffer saline (PBS), lysed with Promega passive lysis buffer, and assayed for Firefly and Renilla luciferase activities in a luminometer by the Dual-Luciferase Reporter Assay System (Promega, Madison, USA) according to the manufacturer's instructions. The luciferase readings of each sample were first normalized against the pRL-TK levels.

2.9. RNA extraction and RT-qPCR Total RNA was extracted from GMECs using RNAprep Pure Cell/ Bacteria Kit (Tiangen, China), and first-strand cDNA was synthesized using a PrimeScript™ RT Reagent Kit (Takara, Japan). RT-qPCR was performed using the SYBR®Premix Ex Taq™ II Kit (Takara, Japan) in a CFX96™ Real-Time PCR Detection system (Bio-Rad, USA). The primers of FASN, SREBP-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and PCR reactions used in the study were reported previously (Lin et al., 2013a). GAPDH was selected as an internal control. Although we recognized that a single internal control gene is not ideal for normalization of gene expression data, GAPDH has been used previously in GMECs (Lin et al., 2013b). The data were analyzed using the relative quantification (2−△△Ct) method (Livak and Schmittgen, 2001). Each sample was performed at least in triplicate.

The results represent the mean ± S.D. of at least three independent experiments. The results were analyzed for significant differences using Student's t-test (unpaired and two-tailed) or one-way ANOVA by SPSS 17.0, as appropriate. P b 0.05 was considered statistically significant. 3. Results 3.1. Cloning, sequencing and characterization of the 5′ regulatory region of the goat FASN gene We amplified 1846 bp of the 5′ flanking sequence (GenBank accession no. KP749922) containing 1524 bp upstream of the transcription start site (+1), the first exon (+1 to +79), and part of the first intron (Fig. 1A). The transcription start site (+ 1) was identified by 5′RACE analysis in our laboratory (DQ915966). Sequence analysis revealed that the FASN 5′ flanking region is a highly GC-rich region characterized by the presence of GC box motifs (Fig. 1B). Moreover, it contains a TATA box-like sequence located at approximately −35 bp. Bioinformatics analysis of the 5′ flanking region using TFSEARCH and AliBata 2.1 revealed various consensus binding sites for transcription factors, including peroxisome proliferatoractivated receptor α (PPAR α, − 569 bp), activating protein 2 (AP-2, −476, −425 bp), liver X receptor (LXR, −392 bp), estrogen receptor (ER, − 381 bp), C/EBP (− 259 bp), specificity protein 1 (Sp1, − 309, −249, − 169, −90 bp), and nuclear factor Y (NF-Y, −102 bp). It also contains two elements including SRE (SRE1 at − 150, SRE2 at −72 bp) and E-box (−68 bp) (Fig. 1B). The goat FASN 5′ upstream sequence shared extensively conserved regions (−203/+1) with bovine, human and rat sequences (Fig. 2). 3.2. Luciferase assays for confirmation of the FASN core promoter region To determine the core sequences of the FASN promoter and identify cis-regions responsible for promoter activity, we first created a series of truncated luciferase constructs by progressive deletions at the 5′ upstream of the 1653-bp fragment (− 1524/+ 129) to produce pGL(− 1524/+ 129), pGL-(− 1044/+ 129), pGL-(− 838/+ 129), pGL(− 591/+129), pGL-(− 397/+129), pGL-(− 293/+ 129), pGL-(− 79/ + 129) and pGL-(− 14/+ 129) vectors (Fig. 3, left). These deletion constructs were transiently transfected into GMECs and MCF-7 cells. The luciferase activity was not diminished by deleting DNA up to position −397 (pGL − 397/+129); on the contrary, an increase in promoter activity was observed for this construct compared to the original pGL-(−1524/+129) reporter construct. However, two statistically significant and gradual reductions in promoter activity were observed using constructs pGL-(− 293/+ 129) and pGL-(− 79/+ 129). Further deletions, including the removal of the TATA box, abolished promoter activity in the constructs pGL-(− 14/+ 129). These data suggest that the sequences between − 397 and + 129 bp in pGL- (− 397/+ 129) constructs bear the cis-functional elements required for maximal FASN transcriptional activation. Due to the observed promoting and inhibiting effects on the relative luciferase activity, regions from −1524 to −397 and from −397 to −293 together with −293 to −79 might each contain additional negative and positive cis-regulatory elements (Fig. 3, right). Collectively, these results indicate that the core region of the FASN promoter spans from −293 to −14 bp to confer basal transcriptional activity. 3.3. Analysis of cis-acting elements regulating FASN promoter activity To identify functional elements affecting FASN expression, we constructed mutant versions for each component, the SRE1, NF-Y, and SRE2 sites, and measured their promoter activities to evaluate their contribution. These mutant and wild-type constructs were then transiently

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Fig. 1. Analysis of FASN promoter region by bioinformatics. A. Schematic representation of FASN 5′ flanking region. +1 represents transcription start sites (TSS). B. Analysis of the putative transcription factors of the 5′ flanking region of the goat FASN. The consensus sequences for putative binding sites of transcription factor are underlined, and the names are indicated above the sequence. The transcription start sites (TSS) are marked by bent arrows.

transfected into GMECs for luciferase reporter assays (Fig. 4). The results indicated that pSRE1-mut, pNF-Y-mut and pSRE2-mut exhibited a decrease of 63% (P b 0.01), 39% (P b 0.01) and 35% (P b 0.05) in promoter activity, respectively, compared with the wild-type construct pGL(−293/+129). The mutation of both SRE sites abolished the promoter activity, despite the fact that other transcription factors might be able to bind to the intact sites (P b 0.0001). This result suggests that the SREs are critical for mediating FASN promoter activation.

3.4. SREBP-1 activates the FASN promoter and endogenous FASN expression To assess the role of SREBP-1 in the regulation of transcriptional activity of the goat FASN promoter, we co-transfected the FASN promoter luciferase reporter construct pGL-(−397/+129) with pCMV-SREBP-1 or an empty vector into GMECs and analyzed the luciferase activity. The results indicated that SREBP-1 increased the activation of the FASN promoter approximately 2.5-fold compared to the empty expression vector (P b 0.001; Fig. 5A). The mRNA level of SREBP-1 in GMECs increased approximately 60-fold (P b 0.0001; Fig. 5C), while the FASN mRNA increased approximately 5.5-fold (P b 0.001; Fig. 5B). This result indicated that FASN could be up-regulated by SREBP-1. To further examine this effect, we designed small interference RNA targeted against SREBP-1 to evaluate the role of endogenous SREBP-1 in the regulation of FASN promoter activity in GMECs. After a 48-h incubation with siRNA-SREBP-1 transfected at a final concentration of 100 nM, the transcriptional expression of the SREBP-1 gene was abolished (P b 0.001; Fig. 6C). FASN promoter activity decreased 60% in the group transfected with siRNA-SREBP-1 relative to cells transfected with the scramble control siRNA (P b 0.01; Fig. 6A). RT-qPCR analysis confirmed that the FASN mRNA level decreased 38% in GMECs transfected with siRNA-SREBP-1 compared to cells transfected with the negative control siRNA (P b 0.01; Fig. 6B). Taken together, these results demonstrated that SREBP-1 modulated the transcriptional activity of FASN in GMECs.

3.5. Response of two SREs in the FASN promoter to SREBP-1 The core region of the goat FASN promoter contains one NF-Y site and two SREs. To further examine the role of these elements in the regulation of FASN by SREBP-1, we tested the effect of SREBP-1 on the 293-bp wild-type FASN promoter or analogous promoter constructs in which the SREs were individually or both mutated. GMECs were cotransfected with the mutant reporter gene constructs and pCMVSREBP-1 or pCMV-GFP (Fig. 7). The results showed that the activation of SREBP-1 markedly enhanced the wild-type FASN promoter activity by 2.5-fold (P b 0.01), and its stimulatory effect was not abolished when each of these binding sites was individually mutated. This result is due to the alternative binding ability of transcription factor SREBP-1 to the intact SRE sites. In contrast, mutated constructs of either the −150-bp SRE1 or −71-bp SRE2 resulted in a reduction of promoter activity by 40% and 30% compared to the wild type construct, respectively. The mutation of both SREs caused a marked reduction of FASN promoter activity and completely abolished the stimulatory effect of SREBP-1. This result clearly indicates that the activation of the FASN promoter requires the presence of the two DNA binding sites for SREBP-1. Additionally, NF-Y is known as a co-regulator of SREBP-mediated induction of target gene expression (Shimano, 2001). However, in this study, the mutation of NF-Y binding sites did not reduce FASN promoter activity, which was induced by SREBP-1 activation.

4. Discussion In this study, we isolated the 5′ flanking region of the goat FASN gene. Alignment analysis with other species showed a high degree of homology between goat and bovine sequences. Although the degree of homology among ruminants, rats and humans was low, the core region of the promoter was highly conserved. This region was previously reported to be important for the response of the human and rat FASN

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Fig. 2. Alignment of the human, rat, bovine and goat FASN promoter sequences using BioXM 2.6. The arrow indicates published transcription start sites (+1) for the different species. The human, rat, and bovine sequences are from the database, whereas the goat sequence was generated during this study. An asterisk (*) indicates agreement across all sequences.

Fig. 3. Deletion analysis of the goat FASN promoter. Goat FASN promoter fragments pGL-1524, pGL-1044, pGL-838, pGL-591, pGL-397, pGL-293, pGL-79 and pGL-14 were generated as described under the Materials and methods section, and subcloned upstream of the Firefly luciferase reporter gene in the pGL3-Basic vector (LUC). GMECs and MCF-7 cell lines were transfected with equimolar amounts of a series of 5′-deletion fragments with the promoter-less plasmid pGL3-Basic (LUC) (n = 8). Results are presented as normalized relative luciferase activity (Firefly/Renilla).

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Fig. 4. The effect of mutagenesis of transcription factor binding sites on the FASN promoter activity (n = 5). Transcription factor binding sites were subjected to site-directed mutagenesis using the primers outlined in Table 1. *P b 0.05, **P b 0.01 difference relative to the wild type group.

promoter to insulin and sterols (Bennett et al., 1995; Wang and Sul, 1997). It contains several potential transcription factor binding sites including Sp1 and NF-Y and two perfectly conserved sequences identified as SRE sites. The truncation of the goat promoter to exclude SRE1, NF-Y, and Sp1 (from −293 to −79) markedly reduced promoter activity, particularly in the GMECs. Continued truncation of the SRE2 and E-box (from −79 to −14) almost abolished FASN promoter activity. This result indicates that the binding of these transcription factors to this region is important to maintaining FASN expression in the mammary gland. In non-ruminants, SREBP-1 directly controls the expression of FASN (Ericsson et al., 1996; Lopez et al., 1996; Magana and Osborne, 1996). Latasa et al. investigated the importance of the SREBP-1 binding site at −150 and concluded that this particular site is mostly responsible for nutritional responsiveness. In their studies, further analysis of the indicated region by site-directed mutagenesis evidently eliminated feedinginduced activation (Latasa et al., 2000). Although our study supports the conclusion that the recognition sites at −150 bp are important under basal conditions and upon ectopic expression of SREBP-1, our studies indicate that the abrogation of the − 150-bp site reduced, but did not eliminate, SREBP-1 responsiveness. We discovered that a second site at −71 bp also contributes to the induction of FASN upon SREBP-1 activation. Latasa et al. also reported that the mutation of either the −150 SRE or the −65 E-box abolished the feeding-induced activation of the FASN promoter and concluded that the FASN promoter is activated

during re-feeding via the induced binding of SREBP to the −150 SRE. Moreover, USF binding to the − 65 E-box is also required for SREBP binding and the activation of the FASN promoter in transgenic mice (Latasa et al., 2003). In this study, the mutation of both SRE sites abolished the basal and SREBP-1-induced promoter activity. Our results are also in agreement with similar studies performed in 3T3-L1 adipocytes using the rat FASN promoter, which differs from the goat and bovine FASN promoter in the − 65-bp region, as it contains one SRE site rather than two (Wang and Sul, 1997). In addition to the SRE sites that appear to be important for mediating SREBP1-induced activation, the FASN promoter also contains several other transcription factor binding sites that may be important for its basal activation or activation through other pathways. Interestingly, in rodents, the SRE site is found to be frequently stationed within a region of DNA termed the “SRE complex” in many promoters of lipogenic genes, which contains several other consensus transcription factor binding sites, including a site for NF-Y, an E-box, and a Sp-1 site (Amemiya-Kudo et al., 2000; Griffin et al., 2007). The SRE complex has been shown to play a role in the regulation of other SREBP-responsive genes, such as FASN (Griffin et al., 2007) and stearoyl-CoA desaturase (Bene et al., 2001) in non-ruminants. The SREBP1-mediated induction of target gene expression occurs in concert with the binding of additional co-regulatory NF-Y to the promoter, resulting in synergistic levels of transcriptional activation (Shimano, 2001). Ishimoto et al. have found that the human lipin 1 promoter was not induced by the overexpression

Fig. 5. The effect of nuclear sterol regulatory element binding protein (SREBP)-1 (nSREBP-1) overexpression on goat FASN promoter activity and endogenous expression of goat FASN mRNA in GMECs. (A) Overexpression of nSREBP-1 enhanced FASN promoter activity (n = 5). GMECs were co-transfected with the FASN reporter plasmid pGL-(−293/+129)-luc and either the pCMV-nSREBP-1 expression plasmid or empty vector for 48 h. Cells were transfected with Renilla luciferase to control for transfection efficiency. (B) and (C) Endogenous nSREBP-1 and FASN expression levels (n = 3). GMECs were co-transfected with the pCMV-nSREBP-1 expression plasmid or empty vector, and total RNA was prepared 48 h later for analysis of nSREBP and FASN mRNA levels by RT-qPCR. *P b 0.05, **P b 0.01 difference relative to the control.

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Fig. 6. The effect of nuclear sterol regulatory element binding protein (SREBP)-1 (nSREBP-1) knockdown on goat FASN promoter activity and endogenous expression of goat FASN mRNA in mammary epithelial cells. (A) FASN promoter activity is reduced by suppression of endogenous nSREBP-1 (n = 5). GMECs were co-transfected with the FASN reporter plasmid pGL(−293/+129)-luc and either scrambled or nSREBP-1 specific small RNA oligos (siNC and siSREBP, respectively) for 48 h. Cell extracts were assayed for luciferase activities. (B) and (C) siSREBP reduced endogenous nSREBP-1 and FASN mRNA expression levels (n = 3). GMECs were treated in the same manner as that for A, and total RNA was prepared 48 h later for analysis of nSREBP and FASN mRNA levels by RT-qPCR. *P b 0.05, **P b 0.01 difference relative to the control.

of SREBP-1 when the nearby NF-Y binding site was mutated and concluded that SREBP-1 and NF-Y coordinately control the transcriptional activity in the human lipin 1 promoter by sterol depletion (Ishimoto et al., 2009). In our study, although the NF-Y and Sp-1 sites are present in close proximity to SREs in the FASN promoter, the mutation of the NF-Y site had no effect on SREBP-1 response, which may be due to species differences between ruminants and rodents. The function of these sites and whether they coordinate each other to regulate FASN expression in goat mammary epithelium are under investigation. Therefore, further research is necessary to explore the exact regulatory mechanism of FASN by the SREBP-1 complex and to determine if other key regulators exist, such as the transcription factor USF, that have roles in the control of lipogenesis in goat lactating mammary gland. SREBP-1 is thought to be a critical transcription factor regulating genes involved in milk fat synthesis in the lactating dairy cow and rat (Peterson et al., 2004; Harvatine and Bauman, 2006; Bionaz and Loor,

2008; Rodriguez-Cruz et al., 2011). It contributes to the coordinated regulation of pathway components by directly binding to the promoters of target genes, including ACC and FASN (Shimano, 2001). In the current study, we examined the effects of SREBP-1 on FASN promoter activity and mRNA levels through overexpression and RNAi of SREBP-1, and as our main finding, we elucidated the involvement of SREBP-1 as the primary regulator of the FASN gene in lactating mammary epithelial cells of goat. This finding is supported by several lines of evidence (Rodriguez-Cruz et al., 2011; Wang et al., 2012). Further research addressing the mechanism of SREBP-1 regulation of target genes involved in milk fat synthesis and fatty acid metabolism will bring new insights into the effects of fatty acid composition on the flavor of goat milk. Regulatory mechanisms other than SREBP-1 may also exist. To elucidate such mechanisms, further studies on FASN regulation are required using regions far upstream of this gene. In this study, functional analyses for various 5′ deletions of the FASN promoter indicated that repressor elements may exist far upstream of the transcription start site. However, the identity of the transcription repressors that are recruited by the FASN promoter remains to be elucidated. Therefore, identifying the inhibitory mechanisms down regulating the FASN gene may help improve milk fatty acid synthesis in the mammary gland. In conclusion, we determined the goat FASN core promoter with a high degree of homology among goats, bovines, rats, and humans. Alternate and joint mutations of the two SRE sites reduced and abolished the activity of the FASN promoter, respectively. The overexpression of SREBP-1 can increase FASN promoter activity and endogenous FASN mRNA expression levels. Meanwhile, the knockdown of SREBP-1 inhibited FASN promoter activity and endogenous FASN mRNA levels. The mechanism of SREBP-1 regulation of FASN is through the binding of two SREs to the FASN promoter. Further analysis of the FASN promoter might reveal a new aspect of complex regulatory mechanisms of milk fatty acids in GMECs. To our knowledge, this is the first study examining the FASN promoter in GMECs. Conflict of interest

Fig. 7. nSREBP-1 enhances FASN promoter activity through SRE2 and SRE2. nSREBP-1 enhances wild type and single mutant FASN promoters, but not both SRE and E-box mutant promoter. GMECs were transfected with the wild type pGL-(−293/+129) or with mutant constructs in which the indicated transcription factor binding sites were disrupted, with or without plasmid expressing nSREBP-1 (n = 5). Forty-eight hours post-transfection, cell lysates were assayed for luciferase expression. *P b 0.05, **P b 0.01 difference relative to the control.

The authors declare no conflict of interest. Acknowledgments This research was jointly supported by the “National Natural Science Foundation of China (31072013)”, the “Transgenic New Species

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Breeding Program of China (2014ZX08009-051B)”, and the “Special Fund for Agro-scientific Research in the Public Interest (201103038)”.

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Fatty acid synthase promoter: characterization, and transcriptional regulation by sterol regulatory element binding protein-1 in goat mammary epithelial cells.

Fatty acid synthase (FASN) is the central enzyme of the de novo fatty acid biosynthesis pathway. Although the FASN transcriptional regulatory mechanis...
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