- Research article
- Open Access
Subfunctionalization of peroxisome proliferator response elements accounts for retention of duplicated fabp1 genes in zebrafish
© The Author(s). 2016
- Received: 5 February 2016
- Accepted: 30 June 2016
- Published: 16 July 2016
In the duplication-degeneration-complementation (DDC) model, a duplicated gene has three possible fates: it may lose functionality through the accumulation of mutations (nonfunctionalization), acquire a new function (neofunctionalization), or each duplicate gene may retain a subset of functions of the ancestral gene (subfunctionalization). The role that promoter evolution plays in retention of duplicated genes in eukaryotic genomes is not well understood. Fatty acid-binding proteins (Fabp) belong to a multigene family that are highly conserved in sequence and function, but differ in their gene regulation, suggesting selective pressure is exerted via regulatory elements in the promoter.
In this study, we describe the PPAR regulation of zebrafish fabp1a, fabp1b.1, and fabp1b.2 promoters and compare them to the PPAR regulation of the spotted gar fabp1 promoter, representative of the ancestral fabp1 gene. Evolution of the fabp1 promoter was inferred by sequence analysis, and differential PPAR-agonist activation of fabp1 promoter activity in zebrafish liver and intestine explant cells, and in HEK293A cells transiently transfected with wild-type and mutated fabp1promoter-reporter gene constructs. The promoter activity of spotted gar fabp1, representative of the ancestral fabp1, was induced by both PPARα- and PPARγ-specific agonists, but displayed a biphasic response to PPARα activation. Zebrafish fabp1a was PPARα-selective, fabp1b.1 was PPARγ-selective, and fabp1b.2 was not regulated by PPAR.
The zebrafish fabp1 promoters underwent two successive rounds of subfunctionalization with respect to PPAR regulation leading to retention of three zebrafish fabp1 genes with stimuli-specific regulation. Using a pharmacological approach, we demonstrated here the divergent regulation of the zebrafish fabp1a, fabp1b.1, and fabp1b.2 with regard to subfunctionalization of PPAR regulation following two rounds of gene duplication.
- Peroxisome proliferator activated receptor (PPAR)
- Dual luciferase assay
- Fatty acid-binding protein
- Teleost fishes
- Gene promoter evolution
- Spotted gar
Gene duplication is thought to facilitate increasing organismal complexity, but evolution does not accommodate redundancy. Duplication of genes can occur by unequal crossing-over during meiosis, replication slippage, retrotransposition, aneuploidy, or whole genome duplication . The common fate of duplicated genes is loss of one copy owing to accumulated mutation and functional decay (non-functionalization) [2, 3]. Alternatively, both copies of a duplicated gene may be retained if one of the duplicates acquires a novel function (neo-functionalization), or the functions of the ancestral gene are subdivided between the duplicates (subfunctionalization) [2–4]. Non-, neo-, and sub-functionalization represent three possible fates of duplicated genes as described in the duplication degeneration complementation (DDC) model . Both mutation of protein coding regions and the loss or gain of cis-acting regulatory elements in the promoters of duplicated genes may account for altered function of duplicated genes. Mutations in regulatory elements of promoters may affect tissue-, developmental stage- and stimulus-dependent transcript levels of duplicated genes [2–5].
Fatty acid binding proteins (Fabp), which belong to the multigene family of intracellular lipid-binding proteins, function as carriers of fatty acids, eicosanoids and other hydrophobic ligands to effectors in the cytosol and nucleus . Previously, we observed that the promoters of the tandemly duplicated fabp genes of zebrafish, fabp1b.1 and fabp1b.2, differ in their regulation by peroxisome proliferator activated receptors (PPARs), where fabp1b.1 promoter activity was induced by PPAR, but fabp1b.2 promoter activity was not induced by PPAR . The zebrafish fabp1a and fabp1b genes were generated by duplication of ancestral fabp1 gene owing to a whole genome duplication (WGD) event that occurred in the ray-finned teleost lineage approximately 325 mya [8–11]. Subsequently, the zebrafish fabp1b.1 and fabp1b.2 genes arose by tandem duplication of fabp1b, most likely by misaligned cross-over of homologous chromosomes during meiosis [12–14]. The zebrafish fabp1b.1 and fabp1b.2 genes are the only tandem duplicates of the multigene family of intracellular lipid-binding protein genes identified, thus far, in teleost fishes . As a result, the zebrafish genome contains three extant fabp1 genes, fabp1a, fabp1b.1, and fabp1b.2. Spotted gar (Lepisosteus oculatus, order Lepisosteiformes) belongs to an order of teleost fishes that did not undergo a WGD, therefore, its genome contains a single copy of the fabp1 gene.
Zebrafish fabp1b.1 and fabp1b.2 differ in their responsiveness to dietary fatty acids: fabp1b.1 mRNA levels are increased in the intestine of linolenic acid-fed zebrafish, whereas fabp1b.2 mRNA levels are unaffected by linolenic acid . Zebrafish fabp1a, fabp1b.1, and fabp1b.2 also differ in their responsiveness to the non-selective PPAR agonist, clofibrate . fabp1a mRNA levels are increased in the liver of clofibrate-fed zebrafish, fabp1b.1 mRNA levels are increased in the heart of clofibrate-fed zebrafish, while fabp1b.2 mRNA levels are unaffected by clofibrate . These findings implicate the PPARs in the differential regulation of the fabp1a, fabp1b.1 and fabp1b.2 genes in zebrafish [14, 15].
PPARs are nuclear receptor transcription factors that bind, and are activated by, free fatty acids and eicosanoids [16–18]. Upon activation, PPARs heterodimerize with the retinoid X receptor (RXR) and bind to a PPAR response element (PPRE) located in the promoters of many vertebrate genes, including fabp genes [16–18]. The consensus sequence for the vertebrate PPRE is defined as 5′-CAAAACAGGTCANAGGTCA-3′ [16–18]. Binding of the PPAR to a PPRE may cause increased or decreased gene expression, depending on the gene [16–18]. Three PPAR isoforms have been identified across vertebrate species: PPARα, PPARγ, and PPARß/∂ [16–18]. While PPARα and PPARγ are expressed in many vertebrate tissues, PPARß/∂ expression is limited to the skin, adipose, and brain [16–18]. A PPRE may be PPAR isoform-selective (i.e., a PPRE that preferentially binds PPARα relative to PPARγ) [16, 17]. A PPRE with high sequence identity in the 5′ flanking region (5′FR) (underlined: 5′-CAAAACAGGTCANAGGTCA-3′) to the consensus PPRE exhibits greater activation of transcription at promoters by the isoform PPARα compared to the isoform PPARγ, whereas PPARγ binding is less-dependent on the 5′FR than PPARα [16–18]. Both PPARα and PPARγ bind to the direct repeat element (DR1) (underlined 5′-CAAAACAGGTCANAGGTCA-3′) of the PPRE to activate transcription [16–18]. A PPRE with low sequence identity in the 5′FR and high sequence identity in the DR1, therefore, may be PPARγ-selective [16–18], as is apparent for fabp1b.1 promoter activity, which displays PPARγ-selectivity in liver and intestine explant tissue and fabp promoter-reporter gene constructs in the human embryonic kidney cells, HEK293A .
The objective of this study was to investigate divergent, PPAR-dependent transcriptional regulation at the promoters of the zebrafish (Danio rerio) fabp1a, fabp1b.1 and fabp1b.2 genes, and the spotted gar fabp1 gene (representative of the ancestral fabp1 gene) in order to determine the molecular mechanisms that led to the retention of the three fabp genes in zebrafish following the teleost-specific WGD event and subsequent local (tandem) duplication event. To define teleost fabp1 promoter evolution, the regulation of zebrafish fabp1a, fabp1b.1, and fabp1b.2 gene promoters was investigated by three approaches: (1) assay of gene transcripts in liver and intestine explant cultures treated with PPAR-agonists; (2) identification of putative PPREs in the zebrafish fabp1a, fabp1b.1, and fabp1b. and the spotted gar fabp1 promoters by in silico analysis; and (3) in HEK293A cells using wild-type and mutagenized zebrafish fabp1a, fabp1b.1, and fabp1b.2, and spotted gar fabp1 promoters fused to the luciferase reporter gene, to determine the promoter-specific regulation of fabp1 genes by PPARα and PPARγ. We applied a comparative pharmacological approach to spotted gar fabp1 and zebrafish fabp1a, fabp1b.1, and fabp1b.2 promoter activity across a wide range of PPAR agonist concentrations in the absence or presence of PPAR antagonists. In this way, it was possible to model evolutionary processes for PPAR isoform-selectivity through readily quantifiable measurements of agonist potency, efficacy, and specificity.
Differential induction of zebrafish fabp1a, fabp1b.1 and fabp1b.2 transcription by PPAR agonists in zebrafish liver and intestine explant culture
In silico analyses
Promoter sequences of the zebrafish fabp1a, fabp1b.1, and fabp1b.2, and spotted gar fabp1 genes, 5′ of their transcription start sites (TSS), were analyzed in silico for the presence of putative PPREs. Sequences of 3,308 bp for zebrafish fabp1a, 3,059 bp for zebrafish fabp1b.1, 3,218 bp for zebrafish fabp1b.2, and 3,283 bp spotted gar fabp1 were retrieved from databases using the conserved non-coding sequence (CNS) discovery pipeline (v. 3.0)  (Additional file 1) and putative PPREs identified by the algorithm, MatInspector (v. 8.1). The length (in bp) of the promoter fragments retrieved was chosen by the CNS discovery pipeline as the region within 4,000 bp 5′ upstream of the TSS containing > 60 % of transcription factor binding motifs with > 60 % sequence identity to the vertebrate transcription factor binding site .
Four putative PPREs were identified in the spotted gar fabp1 promoter that had 75.6 % (indicated by the purple rectangle in Fig. 3), 64.7 %, 74.3 % (indicated by the red rectangle in Fig. 3), and 64.8 % sequence identity to the consensus sequence of the vertebrate PPRE (5′–3′, respectively). Similar to the PPRE in the zebrafish fabp1a gene, the putative PPRE present at −1,953 bp relative to the TSS of spotted gar fabp1 displayed high sequence identity to the PPRE consensus sequence in the 5′FR (henceforth referred to as PPRE-1). Like the PPRE in zebrafish fabp1b.1, the putative PPRE at −539 bp relative to the TSS of spotted gar fabp1 displayed high sequence identity to the PPRE consensus in the DR1 region (henceforth referred to as PPRE-2) [16, 17, 20, 21]. Taken together, these data suggest that the spotted gar fabp1 promoter might be regulated by both PPARα- and PPARγ-selective PPREs [16, 17, 20, 21]. Furthermore, these data suggested that the PPREs with preferential binding affinity for PPARα and PPARγ, respectively, in the ancestral (spotted gar) fabp1 gene were subdivided between fabp1a and fabp1b.1 subsequent to the WGD event that occurred in ray-finned fish.
Analyses of zebrafish fabp1a, fabp1b.1 and fabp1b.2 and spotted gar fabp1 promoter activity in HEK293A cells
Pharmacological characterization of PPAR induction of zebrafish fabp1a and fabp1b.1 promoter activity
E max (RLU)
152.80 ± 3.44
152.00 ± 7.80
147.90 ± 5.30
136.7 ± 11.1*
133.4 ± 4.94
152.80 ± 3.43
149.60 ± 3.66
151.70 ± 6.22
135.30 ± 7.11
144.40 ± 2.67
E max (RLU)
152.30 ± 5.57
151.30 ± 4.91
148.7 ± 5.27
146.4 ± 3.52
152.80 ± 7.24
152.30 ± 5.67
148.30 ± 4.87
150.70 ± 7.96
147.80 ± 2.02
178.50 ± 8.68**
Treatment of HEK293A cells transfected by the fabp1b.1 promoter construct with WY14643 induced fabp1b.1 promoter activity (Fig. 4c). WY14643-dependent fabp1b.1 promoter activity was inhibited by GW6471, but not by T0070907 (Table 1, Fig. 4c). Treatment of HEK293A cells transfected by the fabp1b.1 promoter construct with rosiglitazone also increased fabp1b.1 promoter activity (Fig. 4d). Rosiglitazone-induced fabp1b.1 promoter activity was inhibited by T0070907, but not by GW6471 (Table 1, Fig. 4d). Rosiglitazone was a more potent agonist of fabp1b.1 promoter activity than WY14643, indicating that the zebrafish fabp1b.1 promoter contained a functional, PPARγ-selective PPRE (Table 1). Efficacy (E max) did not differ between agonists or promoters. Thus, differences in agonist activity between the fabp1a and fabp1b.1 promoters were attributed to disparate potencies (i.e. EC50) of the PPAR isoforms acting at the fabp1a and fabp1b.1 promoter PPREs (Table 1).
Treatment of HEK293A cells transfected by the fabp1a Δ5′FR promoter construct with WY14643 shifted the CRC to the right compared to non-mutated fabp1a (Table 1, Fig. 5c). Treatment of HEK293A cells transfected with the fabp1a ΔDR1 promoter construct with WY14643 shifted the CRC to the right compared to non-mutated fabp1a and fabp1a Δ5′FR (Table 1, Fig. 5c). Treatment of HEK293A cells transfected by the fabp1a Δ5′FR promoter construct with rosiglitazone shifted the CRC compared to non-mutated fabp1a (Table 1, Fig. 5d). The CRC was shifted to the right in HEK293A cells transfected with the fabp1a ΔDR1 promoter construct and treated with rosiglitazone compared to non-mutated fabp1a and and fabp1a Δ5′FR (Table 1, Fig. 5d). These data demonstrate that the PPRE at −2,710 was functional and regulated, in part, by PPARα as mutagenesis of the 5′FR consistently affected PPAR-dependent induction of promoter activity . Moreover, the DR1 element of the PPRE is required for the binding of all PPARs, a finding further supported by the functionality of the PPRE at −2,710 bp of the fabp1a promoter fragment [16–18].
WY14643 did not change the CRC for promoter activity in HEK293A cells transfected by the fabp1b.1 Δ5′FR promoter construct compared to non-mutated fabp1b.1 promoter construct (Table 1, Fig. 5e). WY14643 treatment shifted the CRC to the right in HEK293A cells transfected by the fabp1b.1 ΔDR1 promoter construct compared to non-mutated fabp1b.1 promoter (Table 1, Fig. 5e). Although the EC50 was shifted slightly to the right, the rosiglitazone produced a potent and fully efficacious response in HEK293A cells transfected by the fabp1b.1 Δ5′FR promoter compared to non-mutated fabp1b.1 promoter (Table 1, Fig. 5f). The rosiglitazone CRC was shifted to the right in HEK293A cells transfected with the fabp1b.1 ΔDR1 promoter construct by 2.5 orders of magnitude compared to non-mutated fabp1b.1 promoter (Table 1, Fig. 5e). These observations provide compelling evidence that the zebrafish fabp1b.1 promoter region contains a functional, PPARγ-selective PPRE at −1,232 bp as rosiglitazone was a more potent agonist of PPAR induction of fabp1b.1 promoter activity than WY14643, and the DR1 element, not the 5′FR, was the major regulator of PPAR potency in these assays (Table 1). Since neither mutagenesis of the 5′FR or DR1 in fabp1a or fabp1b.1 abolished transcriptional induction of these fabp genes by PPAR agonism, and no change in E max was observed, additional, functional PPREs are likely present in both the fabp1a and fabp1b.1 promoters.
Pharmacological characterization of PPAR agonist and antagonist regulation of spotted gar fabp1 promoter activity
E max (RLU)
80.6 ± 7.40
97.5 ± 3.78
74.2 ± 9.63
E min (RLU)
10.9 ± 4.64
8.86 ± 3.50
8.93 ± 4.72
24.7 ± 4.88**
21.6 ± 4.13**
E max (RLU)
56.3 ± 2.40
57.4 ± 2.44
42.5 ± 4.50
E min (RLU)
5.28 ± 4.80
−0.42 ± 2.73
1.21 ± 7.80
Pharmacological characterization of spotted gar fabp1 promoter PPRE mutants
E max (RLU)
101 ± 2.56
70.6 ± 2.17*
102 ± 8.65
E min (RLU)
1.32 ± 2.91
–1.64 ± 3.08
–17.4 ± 18.2
27.6 ± 4.76**
E max (RLU)
64.9 ± 2.34
61.9 ± 6.66
62.1 ± 5.07
E min (RLU)
−7.47 ± 3.79
1.17 ± 0.05
2.94 ± 4.03
In this study, we employed a pharmacological approach to define the PPAR selectivity, potency, and efficacy of PPAR-dependent regulation in the promoters of the zebrafish fabp1a, fabp1b.1 and fabp1b.2 genes. We observed that the zebrafish fabp1a promoter contained a functional, PPARα-selective PPRE, while the zebrafish fabp1b.1 promoter contained a functional, PPARγ-selective PPRE. The spotted gar fabp1 promoter contained two functional PPREs: a PPARα-selective PPRE (PPRE-1) and a PPARγ-selective PPRE (PPRE-2). These results are consistent with previously published conclusions that: (1) the steady-state level of fabp1a and fabp1b.1 mRNA and hnRNA levels are induced in adult zebrafish fed a linolenic acid- or clofibrate-rich diets, and this transcriptional activation is mediated by PPAR [14, 15], and (2) that the fabp1a and fabp1b.1 promoters are functionally-selective for PPARα and PPARγ, respectively as described here and in a previous report .
The spotted gar fabp1 promoter served as a representative of the ancestral fabp1 gene that gave rise to fabp1a and fabp1b following the teleost WGD . The two functional PPREs identified in the spotted gar fabp1 promoter were oriented such that PPRE-1 was PPARα-selective and PPRE-2 was PPARγ-selective. Pharmacological analyses and site-directed mutagenesis demonstrated that both the spotted gar PPRE-1 of fabp1 and the similarly-aligned zebrafish PPRE of fabp1a (Fig. 1) were more responsive to PPARα-agonists and antagonists than to PPARγ-agonists and antagonists, based on promoter activity assays, suggesting that the fabp1a PPRE at −1,953 bp was derived from the PPARα-selective PPRE-1 in the ancestral fabp1 prior to the teleost WGD. Furthermore, pharmacological analyses and site-directed mutagenesis demonstrated that both the PPRE-2 of spotted gar fabp1 and the similarly-aligned zebrafish PPRE of fabp1b.1 (Fig. 1) were both more responsive to PPARγ agonists and antagonists than to PPARα agonists and antagonist as assayed by the induction of promoter activity, suggesting that the fabp1b.1 PPRE at −539 bp was derived from an ancestral PPARγ-selective PPRE-2 present in the spotted gar (ancestral) fabp1 gene prior to its duplication following the teleost WGD.
Previous studies have focused on non-quantitative or semi-quantitative data derived from electrophoretic mobility shift assays to determine the specificity of PPARs interaction with PPREs [16–18, 22]. The unique pharmacological approach used in this study to define the regulation and its evolution of promoter activity provided quantitative data, which supports the contention that the 5′FR is directly involved in PPARα-, but not PPARγ-, dependent promoter activation . Furthermore, this work supports earlier findings that the DR1 regulates general PPAR-dependent promoter activation [16–18, 22].
The present subfunctionalized state of PPAR responsiveness in the zebrafish fabp1a, fabp1b.1, and fabp1b.2 promoters may represent a form of segregation avoidance such that three gene products sharing similar function are expressed in different tissues, under different developmental or environment conditions [27, 28]. These data demonstrate the divergent, PPAR isoform-specific regulation of zebrafish fabp1a, fabp1b.1, and fabp1b.2 in relation to their subfunctionalization across evolutionary history using a unique pharmacological approach.
Zebrafish and spotted gar fabp1 promoter sequences
Promoter sequences for zebrafish fabp1a, fabp1b.1, and fabp1b.2 and spotted gar fabp1 genes were obtained using the CNS Discovery Pipeline (v. 3.0) created and described by Turco et al. . The source code for the CNS Discovery Pipeline 3.0 is available for download at https://github.com/gturco/find_cns with instructions for installation at (https://github.com/gturco/find_cns/blob/master/INSTALL.rst) . CNS Discovery Pipeline was run using default settings except that the filter for promoter regions containing gene-coding regions was removed. The input was the zebrafish Zv9 whole genome assembly (GenBank Assembly ID GCA_000002035.2) and spotted gar Linkage group LG2 LepOcu1 representative genome assembly (GenBank Assembly ID GCA_000242695.1, Gene symbol LOC102694982) . The length of promoter fragments retrieved by the CNS discovery pipeline was determined as the region within 4,000 bp 5′ of the TSS containing > 60 % sequence identity to the consensus of vertebrate transcription factor binding motifs . The resulting “.fasta” output files for the fabp promoters and their corresponding genes were used to design PCR primers to clone fabp promoter fragments (Additional file 1).
Identification of putative PPREs in teleost fabp1 promoters by in silico analysis
Promoter sequences were analyzed for putative PPREs using MatInspector (v. 8.1) with the Genomatix ElDorado genomes database and the vertebrate matrix group. The PPRE was defined as 5′-CAAAACTAGGTCANAGGTCA-3′ [16–18]. The mismatch threshold was set to 35 % (i.e. transcription factor sites were identified if they were 65 % similar to the corresponding IUPAC string).
Primary zebrafish cell culture methods were adapted from Kan et al. . Primary explant cell cultures of zebrafish liver and intestine were obtained from adult male fish. Fish were euthanized with tricaine (10 % v/v) and rinsed with 70 % ethanol in sterile phosphate-buffered saline (PBS). The liver and intestine were dissected, rinsed once with PBS, and incubated in 0.25 % trypsin-EDTA (Gibco, Oakville, ON) for 5 min at room temperature. Tissue was suspended in trypsin-EDTA by pipette and centrifuged at 500 x g for 5 min at room temperature. Cells were resuspended in media containing 50 % Leibovitz’s L-15, 35 % high glucose DMEM, 15 % Ham’s F-12, 5 % FBS, 0.15 g/L sodium bicarbonate, 15 mM HEPES, 0.01 mg/mL bovine insulin, and 50 ng/mL human EGF (Gibco) and maintained 28 °C, 100 % atmospheric air on poly-D-lysine-coated cell culture plates. Primary zebrafish cells were maintained for 48 h prior to drug treatment. Media was changed daily. All protocols were in accordance with the guidelines outlined by the Canadian Council on Animal Care. All animal protocols were approved by the Carleton Animal Care Committee at Dalhousie University prior to start of this study.
Human embryonic kidney 293A (HEK293A) cells were obtained from Cedarlane (Burlington, ON). HEK293A cells were maintained at 37 °C, 5 % CO2 in DMEM containing 10 % FBS and 104 U/mL Pen/Strep. HEK293A cells express PPARα and γ , which was confirmed by sequencing the RT-PCR products (data not shown).
Cloning of zebrafish fabp1a, fabp1b.1, and fabp1b.2, and spotted gar fabp1 promoter fragments into the pGL3-basic plasmid
DNA fragments containing the zebrafish fabp1a, fabp1b.1 and fabp1b.2, and spotted gar fabp1 promoter region were amplified from genomic DNA by PCR. Genomic DNA was isolated from frozen liver using the GenElute Genomic DNA Miniprep kit according to the manufacturer’s instructions (Sigma-Aldrich, Oakville, ON). The PCR contained: 2 mM MgCl2, 0.5 μM forward and reverse primers (Additional file 2), 0.3 mM dNTPs, 1 U Taq DNA polymerase, and 40 ng genomic DNA. PCR conditions were: 95 °C for 10 min; 35 cycles of 95 °C for 30 s, 57 °C for 30 s, 72 °C for 6 min; and 72 °C for 10 min. PCR products were resolved by gel electrophoresis and purified using the GenElute Gel Extraction kit (Sigma-Aldrich). Purified fabp1a, fabp1b.1, and fabp1b.2 PCR products were digested with MluI and HindIII according to the manufacturer’s instructions (Fermentas, Burlington, ON). The purified spotted gar fabp1 PCR product was ligated into pGEM-T easy vector (Fermentas) at 16 °C overnight using T4 DNA ligase according to the manufacturer’s instructions (Invitrogen, Burlington, ON). The spotted gar fabp1 promoter fragment was excised from pGEM-T by digestion with NcoI and SacI according to the manufacturer’s instructions (Fermentas). fabp1a, fabp1b.1, and fabp1b.2 PCR products were ligated into pGL3-Basic (Promega, Madison, WI) at 16 °C overnight using T4 DNA ligase according to the manufacturer’s instructions (Invitrogen). The resulting plasmids (pfabp1a, pfabp1b.1, pfabp1b.2, and pfabp1) were propagated in ampicillin-resistant DH5α competent E. coli (New England Biolabs, Whitby, ON) and purified using the GenElute Plasmid Midiprep kit (Sigma-Aldrich). The pHRL-TK plasmid was obtained from Promega.
pfabp1a, pfabp1b.1, and pfabp1 mutant plasmids were generated by PCR-based site-directed mutagenesis. The pfabp1a PPRE 5′FR at −2,710 bp was mutated from 5′-CAAAAC-3′ to 5′-TGGGGT-3′ and the PPRE DR1 at −2,710 bp was mutated from 5′-ACAAGT-3′ to 5′-CGTGGA-3′. The pfabp1b.1 PPRE 5′FR at −1,232 bp was mutated from 5′-CTAAAC-3′ to 5′-TCGGGT-3′ and the PPRE DR1 at −1,232 bp was mutated from 5′-TAAGGT-3′ to 5′-CGGAAC-3′. The pfabp1 PPRE-1 (−1,953 bp) was mutated from 5′-CCCTA-3′ to 5′-TTTCG-3′ and PPRE-2 (−539 bp) was mutated from 5′-AGGACA-3′ to 5′-TAAAGT-3′. Reactions were composed of 2 mM MgCl2, 0.5 μM forward and reverse mutagenic primers (Additional file 2), 0.3 mM dNTPs, 1 U Taq DNA polymerase, and 40 ng plasmid DNA. PCR conditions were: 95 °C 1 min, 18 cycles of 95 °C 50 s, 60 °C 1 min, and 68 °C 8 min, followed by a final extension at 68 °C for 10 min. Input plasmid was removed by digestion with the methylation-insensitive DpnI (5 U) in 1X FastDigest Green Buffer® in a final volume of 20 μL (Fermentas, Burlington, ON) for 1 h at 37 °C. The constructs of wild-type and mutagenized zebrafish and spotted gar promoters was confirmed by DNA sequencing of the promoter-reporter gene constructs prior to transfection of HEK293A cells (data not shown).
Transfection, PPAR agonist and antagonist treatment, and the dual luciferase assay
Transfections of HEK293A cells was performed using lipofectamine 2000 reagent according to the manufacturer’s instructions (Invitrogen) with 400 ng of pfabp1a, pfabp1b.1, pfabp1b.2, pfabp1, or pGL3-Basic (background control), and 200 ng pHRL-TK. The luciferase activity of the pHRL-TK plasmid containing the Renilla luciferase gene under the regulation of the cytomegalovirus thymidine kinase (TK) promoter was used to normalize firefly luciferase activity under the regulation of zebrafish promoters. Luciferase activity was quantified according to the manufacturer’s instructions (Promega).
HEK293A and primary zebrafish cells were treated with rosiglitazone (PPARγ agonist), WY14643 (PPARα agonist), T0070907 (PPARγ antagonist), GW 6471 (PPARα antagonist), or vehicle (0.5 % DMSO) at the concentrations and times indicated [32, 33]. All PPAR agonists and antagonists were purchased from Sigma-Aldrich.
Quantitative reverse transcriptase PCR
RNA was extracted from HEK239 cells using Trizol® (Invitrogen). Reverse transcription reactions were carried out with SuperScript III® reverse transcriptase (+RT; Invitrogen), or without (−RT) as a negative control for use in subsequent PCR experiments according to the manufacturer’s instructions. Two micrograms of RNA were used per RT reaction. qRT-PCR was conducted using the LightCycler® system and software (version 3.0; Roche, Laval, QC). Reactions were composed of a primer-specific concentration of MgCl2 (Additional file 2), 0.5 μM each of forward and reverse primers (Additional file 2), 2 μL of LightCycler® FastStart Reaction Mix SYBR Green I, and 2 μL cDNA to a final volume of 20 μL with dH2O (Roche). The PCR program was: 95 °C for 10 min, 50 cycles of 95 °C 10 s, a primer-specific annealing temperature (Additional file 2) for 5 s, and 72 °C for 10 s. Experiments always included sample-matched –RT controls, a no-sample dH2O control, and a standard control containing product-specific cDNA of a known concentration. cDNA abundance was calculated using the ΔΔCT method and normalized to GAPDH levels .
CRCs were fit using non-linear regression analyses [variable slope (four parameters) and Bell-shaped] in GraphPad Prism (v. 5.0). Statistical analyses were conducted by one-way ANOVA followed by Tukey’s post-hoc test or two-way ANOVA follow by Bonferroni’s post-hoc test, as indicated. Homogeneity of variance was confirmed using Bartlett’s test. All results are reported as the mean ± standard deviation (SD) or standard error of the mean (SEM), as indicated, from at least three independent experiments.
5′FR, 5′ flanking region; CNS, conserved non-coding sequence; CRC, concentration-response curve; DDC, duplication-degeneration-complementation; DR1, direct repeat region; fabp, fatty acid-binding protein; HEK, human embryonic kidney cell line; PBS, phosphate-buffered saline; PPAR, peroxisome proliferator-activated receptor; PPRE, peroxisome proliferator-activated receptor response element; RT, reverse transcriptase; SEM, standard error of the mean; SD, standard deviation; sg, spotted gar; TGD, tandem gene duplication; TK, thymidine kinase; TSS, transcription start site; WGD, whole genome duplication; zf, zebrafish
This work was supported by an National Sciences and Engineering Research Council Canada (NSERC) Grant to JMW, and a Bridge Funding Grant from Dalhousie University to EMD-W. RBL is supported by studentships from the Canadian Institutes of Health Research (CIHR), the Huntington Society of Canada, Killam Trusts, and Nova Scotia Health Research Foundation. None of these funding bodies influenced the design of the study or the collection, analysis, or interpretation of data presented in the manuscript.
Availability of data and material
RBL, EMD-W, and JMW conceived the study. RBL conducted the experiments and analyzed the data. RBL, EMD-W, and JMW contributed to the writing of the manuscript. All authors read and approved the final manuscript.
RBL is currently a postdoctoral fellow at in the Department of Molecular Therapeutics The Scripps Research Institute, Florida campus. EMD-W is a Professor in the Department of Pharmacology at Dalhousie University. JMW is a Professor in the Department of Biology at Dalhousie University.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
All protocols requiring euthanized zebrafish were in accordance with the guidelines detailed by the Canadian Council on Animal Care. Zebrafish animal protocols were approved by the Carleton Animal Care Committee at Dalhousie University prior to beginning the study.
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