Comparative Biochemistry and Physiology, Part B 213 (2017) 81–90

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Comparative Biochemistry and Physiology, Part B

journal homepage: www.elsevier.com/locate/cbpb

Differential regulation of the duplicated fabp7, fabp10 and fabp11 genes of MARK
zebrafish by peroxisome proliferator activated receptors
Robert B. Laprairiea, Eileen M. Denovan-Wrighta, Jonathan M. Wrightb,⁎
a
Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada
b
Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada

A B S T R A C T

In the duplication-degeneration-complementation model, duplicated gene-pairs undergo nonfunctionalization

(loss from the genome), subfunctionalization (the functions of the ancestral gene are sub-divided between du-
plicate genes), or neofunctionalization (one of the duplicate genes acquires a new function). These processes
occur by loss or gain of regulatory elements in gene promoters. Fatty acid-binding proteins (Fabp) belong to a
multigene family composed of orthologous proteins that are highly conserved in sequence and function, but
differ in their gene regulation. We previously reported that the zebrafish fabp1a, fabp1b.1, and fabp1b.2 pro-
moters underwent subfunctionalization of PPAR responsiveness. Here, we describe the regulation at the du-
plicated zebrafish fabp7a/fabp7b, fabp10a/fabp10b and fabp11a/fabp11b gene promoters. Differential control at
the duplicated fabp promoters was assessed by DNA sequence analysis, responsiveness to PPAR-isoform specific
agonists and NF-κB p50 antagonists in zebrafish liver and intestine explant tissue, and in HEK293A cells
transfected with fabp promoter-reporter constructs. Each zebrafish fabp gene displayed unique transcriptional
regulation compared to its paralogous duplicate. This work provides a framework to account for the evolutionary
trajectories that led to the high retention (57%) of duplicated fabp genes in the zebrafish genome compared to
only ~3% of all duplicated genes in the zebrafish genome.

1. Introduction
Gene duplication occurs by unequal crossing-over during meiosis,
replication slippage, retrotransposition, aneuploidy, and whole genome
duplication (Zhang, 2003). Duplication of genes is thought to con-
tribute to increasing organismal complexity. The common fate of du-
plicated genes is loss of one copy through accumulated mutation and
functional decay (i.e. nonfunctionalization) (Force et al., 1999; Lynch
and Conery, 2000). Alternatively, both copies of a duplicated gene may
be retained in the genome if regulatory elements controlling the ex-
pression of one duplicate acquires a novel function(s) (neofunctionali-
zation), or the regulatory elements of the ancestral gene promoter are
subdivided between duplicates (subfunctionalization) (Force et al.,
1999; Lynch and Conery, 2000; Taylor and Raes, 2004). Mutations in
regulatory elements of promoters may affect developmental stage-,
tissue-, and stimulus-dependent transcript levels of duplicated genes
(Holland et al., 1994; Force et al., 1999; Lynch and Conery, 2000;
Taylor and Raes, 2004).
Fatty acid binding proteins (Fabp) belong to the multigene family of
intracellular lipid binding proteins. Fabps serve as carriers of saturated
and unsaturated long-chain fatty acids, eicosanoids and other hydro-
phobic ligands to effector molecules in the cytosol and nucleus (Esteves
et al., 2015). Previously, we reported that the promoters of the zebra-
fish fabp1a, fabp1b.1, and fabp1b.2 genes had undergone divergence
relative to the ancestral fabp1 promoter, such that fabp1a was induced
preferentially by the peroxisome proliferator activated receptor (PPAR)
PPARα (defined hereafter as PPARα-selectivity), while fabp1b.1 was
induced by PPARγ (defined hereafter as PPARγ-selectivity), whereas
fabp1b.2 was not induced by either PPARα or PPARγ (Laprairie et al.,
2016a, 2016b). Zebrafish fabp1a and the ancestral fabp1b genes are
duplicates of an ancestral fabp1 gene that arose owing to a whole
genome duplication (WGD) event that occurred early in the ray-finned
teleost lineage approximately 230–400 mya (Robinson-Rechavi et al.,
2001; Glasauer and Neuhauss, 2014). A subsequent tandem duplication
of the ancestral fabp1b during misalignment of homologous chromo-
somes during meiosis generated the extant fabpb.1 and fabp1b.2 genes
in the zebrafish genome (Karanth et al., 2009a, 2009b).
In addition to the fabp1 genes, the zebrafish genome contains the
fabp gene duplicates, fabp7a/fabp7b, fabp10a/fabp10b, and fabp11a/
fabp11b (Karanth et al., 2009a, 2009b; Venkatachalam et al., 2012).

⁎
Corresponding author at: Department of Biology, Dalhousie University, 1355 Oxford Street, PO BOX 15000, Halifax, Nova Scotia B3H 4R2, Canada.
E-mail addresses: robert.laprairie@dal.ca (R.B. Laprairie), emdenova@dal.ca (E.M. Denovan-Wright), jmwright@dal.ca (J.M. Wright).
URL: http://biology.dal.ca/ (J.M. Wright).

http://dx.doi.org/10.1016/j.cbpb.2017.08.003
Received 8 May 2017; Received in revised form 20 August 2017; Accepted 20 August 2017
Available online 24 August 2017
1096-4959/ © 2017 Elsevier Inc. All rights reserved.
R.B. Laprairie et al.
Each of these gene duplicates displays unique developmental stage- and
tissue-specific patterns of expression (Karanth et al., 2009a, 2009b;
Venkatachalam et al., 2012). Moreover, transcriptional initiation of
each of these gene duplicates is affected by changes in dietary fatty
acids and the non-specific PPAR agonist, clofibrate, thereby implicating
PPARs in the transcriptional regulation of these genes (Karanth et al.,
2009b; Venkatachalam et al., 2012). PPAR transcription factors re-
spond to changing levels of lipid in the cell and in turn regulate ex-
pression of genes for lipid storage and metabolism (Palmer et al., 1995).
Peroxisome proliferator response elements (PPREs), specific DNA se-
quences that bind PPAR, are preferentially bound by one of three PPAR
isoforms, PPARα, PPARβ/δ or PPARγ (Palmer et al., 1995). The pri-
mary physiological role of PPARα is to increase the expression of genes
that facilitate uptake and utilization of fatty acids in tissues such as the
liver (Juge-Aubry et al., 1997; Hsu et al., 1998; Ricote and Glass, 2007).
PPARγ activation increases fatty acid uptake and adipogenesis in adi-
pocytes and other tissues (Juge-Aubry et al., 1997; Hsu et al., 1998;
Ricote and Glass, 2007). PPARβ/δ is known to regulate lipid accumu-
lation and cell polarization, but its physiological functions are less-
clearly defined (Juge-Aubry et al., 1997). PPREs with high sequence
similarity in the 5′ flanking region (5′FR) (underlined: 5′-CAAAACAG-
GTCANAGGTCA-3′) to the consensus PPRE exhibit greater activation of
transcription at promoters by PPARα compared to PPARγ (Palmer et al.,
1995; Juge-Aubry et al., 1997; Hsu et al., 1998). PPARγ binding is less-
dependent on the 5′FR than PPARα (Palmer et al., 1995; Juge-Aubry
et al., 1997; Hsu et al., 1998). Both PPARα and PPARγ bind to the direct
repeat element (DR1) (underlined 5′-CAAAACAGGTCANAGGTCA-3′) of
the PPRE to activate transcription (Palmer et al., 1995; Juge-Aubry
et al., 1997; Hsu et al., 1998). A PPRE with low sequence similarity in
the 5′FR and high sequence similarity in the DR1, therefore, may be
PPARγ-selective (Laprairie et al., 2016b). PPARs may also facilitate
repression of gene transcription through the recruitment of, and inter-
action with, other transcription factors, including AP-1 and NF-κB p50
(Ricote and Glass, 2007).
NF-κB p50 binds to NF-κB response elements (NRE) that may reg-
ulate fabp gene transcription as fabp ligands are known to mediate in-
flammatory and immune responses in teleost fishes (Calder, 2001;
Karra et al., 2015). Transcription factor NF-κB p50 interacts with PPAR
to inhibit promoter activity and mRNA transcription (Ricote and Glass,
2007). NF-κB p50 inhibits gene transcription by recruiting histone
deacetylases and, thereby, altering chromatin conformation (Ricote and
Glass, 2007; Elsharkawy et al., 2010). NF-κB p50 may, therefore, serve
as an essential signaling pathway between inflammation and lipid
sensing systems in teleost fishes.
The objective of this study was to investigate the divergent, PPAR-
dependent, transcriptional regulation of the zebrafish (Danio rerio)
fabp7a/fabp7b, fabp10a/fabp10b, and fabp11a/fabp11b genes to define
the molecular mechanisms that led to the retention of these genes in the
zebrafish genome following their duplication. To define elements that
control transcription of teleost fabp genes, zebrafish fabp gene pro-
moters were studied by three methods: (1) identification of putative
PPREs in the zebrafish fabp promoters by in silico analysis; (2) assay
firefly luciferase reporter gene expression under the control of fabp gene
promoters to determine how each fabp gene was preferentially regu-
lated by either PPARα and PPARγ in a heterologous expression system,
i.e., zebrafish genes in human embryonic kidney 293A (HEK293A) cells;
and (3) analysis of fabp gene transcript levels in an endogenous system
using zebrafish liver and intestine explant cells treated with PPAR
agonists and a NF-κB p50 antagonist. By this approach, it was possible
to determine PPAR isoform- and tissue-selectivity through readily
quantifiable measurements of agonist potency, efficacy, and specificity.
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Comparative Biochemistry and Physiology, Part B 213 (2017) 81–90
2. Materials and methods
2.1. Retrieval of promoter sequences
Promoter sequences for zebrafish fabp7a, fabp7b, fabp10a, fabp10b,
fabp11a, and fabp11b genes were obtained using the CNS Discovery
Pipeline (v. 3.0) as described by Turco et al. (2013). CNS Discovery
Pipeline is a sequence analysis tool that identifies putative gene pro-
moters as regions containing a relatively high density of transcription
factor binding sites with proximity to known transcription start sites
(Turco et al., 2013). CNS Discovery Pipeline was run as described
previously (Laprairie et al., 2016a, 2016b) using default settings except
that the filter for promoter regions containing gene-coding regions was
removed and introns for the genes of interest were excluded. The input
was the zebrafish GRCz10 whole genome assembly (GenBank Assembly
ID GCA_000002035.3) (Benson et al., 2013). The resulting. FASTA
output files for the fabp promoters and their corresponding genes were
used for subsequent analysis and the design of PCR primers used to
clone the fabp promoter fragments (S1 File).
2.2. Transcription factor analysis
Promoter sequences identified using the CNS discovery pipeline
were analyzed for putative PPREs and NREs using MatInspector (v. 8.1)
with the Genomatix ElDorado genomes database and the vertebrate
matrix group. The PPRE was defined as 5′-
CAAAACTAGGTCANAGGTCA-3′ (Palmer et al., 1995; Juge-Aubry et al.,
1997; Hsu et al., 1998). The NRE was defined as 5′-GGGRATTTCC-3′
(Turco et al., 2013). The mismatch threshold was set to 35% (i.e.
transcription factor sites were identified if they were 65% similar to the
corresponding IUPAC string).
2.3. Cell culture
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 (Zagranichnaya et al., 2005) and this was confirmed by RT-PCR (data
not shown). Primary zebrafish cell culture methods were adapted from
Kan et al. (2009). Primary cell cultures of zebrafish liver and intestine
were obtained from adult male fish. Fish were euthanized with tricaine
[stock solution 0.4 g tricaine salt (Sigma-Aldrich, Oakville, ON), 21 mM
Tris in dH2O adjusted to pH 7.0] diluted 10% v/v in sterile phosphate-
buffer saline (PBS). Fish were rinsed with 70% ethanol in sterile PBS.
The liver and intestine were dissected, rinsed once with phosphate-
buffered saline, 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 × g for 5 min at room tem-
perature. Cells were re-suspended 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 at 28 °C, 100% atmo-
spheric air on poly-D-lysine-coated cell culture plates. Primary zebrafish
cells were maintained for 48 h prior to treatment with WY14643 or
rosiglitazone (Lehmann et al., 1995; Keller et al., 1997). Media was
changed daily. All protocols were in accordance with the guidelines
detailed by the Canadian Council on Animal Care (CCAC). All animal
protocols were approved by the Carleton Animal Care Committee at
Dalhousie University prior to beginning the study.
2.4. Cloning zebrafish promoter fragments into the pGL3-basic plasmid
Zebrafish fabp7a, fabp7b, fabp10a, fabp10b, fabp11a, and fabp11b
promoters were amplified from genomic DNA by PCR. Genomic DNA
was isolated from liver using the GenElute Genomic DNA Miniprep kit
according to the manufacturer's instructions (Sigma-Aldrich). PCR
R.B. Laprairie et al.
reagent concentrations were: 2 mM MgCl2, 0.5 μM forward and reverse
primers (S1 Table), 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, 55 °C for 30 s, 72 °C for 6 min; and 72 °C for 10 min. PCR
products were resolved by agarose gel electrophoresis and purified
using the GenElute Gel Extraction kit (Sigma-Aldrich). Purified fabp7a,
fabp7b, fabp10a, fabp10b, fabp11a, and fabp11b PCR products were li-
gated into pGEM-T easy vector (Fermentas, Burlington, ON) at 16 °C
overnight using T4 DNA ligase according to the manufacturer's in-
structions (Invitrogen, Burlington, ON). pGEM-T easy vectors con-
taining zebrafish promoters were propagated in ampicillin-resistant
DH5α competent E. coli (New England Biolabs, Whitby, ON) and pur-
ified using the GenElute Plasmid Midiprep kit (Sigma-Aldrich).
Zebrafish fabp promoter fragments were excised from pGEM-T by di-
gestion with SacI and NcoI. The pGL3-basic plasmid (Promega,
Madison, WI) was similarly digested with NcoI and SacI (Fermentas).
Zebrafish fabp promoter fragments were ligated into pGL3-Basic at
16 °C overnight using T4 DNA ligase (Invitrogen). The resulting plas-
mids (pGL3-fabp7a, pGL3-fabp7b, pGL3-fabp10a, pGL3-fabp10b, pGL3-
fabp11a, and pGL3-fabp11b) were propagated in ampicillin-resistant
DH5α competent E. coli (New England Biolabs) and purified using the
GenElute Plasmid Midiprep kit (Sigma-Aldrich). All plasmids were se-
quenced from both strands of DNA (GeneWiz, Camden, NJ) (S1 File)
using pGL3 forward and reverse primers (S1 Table). The plasmid,
pHRL-TK (Promega), contains the Renilla luciferase gene under the
regulatory control of the cytomegalovirus thymidine kinase (TK) pro-
moter. Renilla luciferase under the control of the TK promoter (pHRL-
TK), widely used to normalize firefly luciferase activity, was employed
in the dual luciferase assays (Laprairie et al., 2016a, 2016b).
2.5. Transfection, treatment, and the dual luciferase assay
Transfections of HEK293A cells were performed using
Lipofectamine 2000 reagent with 400 ng pGL3-fabp, or pGL3-Basic
(background control) and 200 ng pHRL-TK (Invitrogen). The luciferase
activity of the pHRL-TK plasmid containing the Renilla luciferase gene
under the regulation of the TK promoter served as a control to nor-
malize firefly luciferase activity under the regulation of zebrafish pro-
moters.
HEK293A and primary zebrafish cells were treated with WY14643
(PPARα agonist), rosiglitazone (PPARγ agonist), or vehicle (0.5%
DMSO) at the concentrations and times indicated (Lehmann et al.,
1995; Keller et al., 1997). PPAR agonists were purchased from Sigma-
Aldrich. Luciferase activity was quantified by the dual luciferase assay
according to the manufacturer's instructions (Promega).
2.6. Quantitative reverse transcriptase PCR
RNA was harvested from cells using the Trizol® extraction method
(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.
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 con-
centration of MgCl2 (S1 Table), 0.5 μM each of forward and reverse
primers (S1 Table), 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, 59 °C
for 5 s, and 72 °C for 10 s. Experiments always included sample-mat-
ched − 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 (Livak and
Schmittgen, 2001) and normalized to GAPDH levels (Zagranichnaya
et al., 2005).
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Comparative Biochemistry and Physiology, Part B 213 (2017) 81–90
2.7. Statistical analyses
Promoter activity quantified in the dual luciferase assay was fit to a
non-linear regression (4 parameter) model using Prism (v. 6.0,
GraphPad, La Jolla, CA) in order to determine EC50 (compound con-
centration at half-maximal response) and Emax (maximal response).
Statistical analyses were conducted by one-way ANOVA followed by
Tukey's post-hoc test or two-way ANOVA followed by Bonferroni's post-
hoc test, as indicated. Homogeneity of variance was confirmed using
Bartlett's test. The level of significance was set to P < 0.001
(Colquhoun, 2014), and all results are reported as the mean ± the
standard error of the mean (SEM) from at least three independent ex-
periments. For experiments with HEK293A cells, an independent ex-
periment (n) was defined as being from separate cultures of cells grown
on different days using new dilutions of treatment compound(s). For
experiments with cultured zebrafish liver and intestine, data for each
independent replicate (n) was obtained from 2 zebrafish liver or in-
testine samples, pooled. Experiments were designed so that 1 sample
was used for 6 different treatments. The total number of zebrafish used
was 24.
3. Results
3.1. Characterization of zebrafish fabp promoters
Promoter sequences for the zebrafish fabp7a, fabp7b, fabp10a,
fabp10b, fabp11a, and fabp11b genes, 5′ upstream of their transcription
start sites (TSS), were identified in silico using the CNS discovery pi-
peline (Turco et al., 2013). Using this approach, sequences of 3069 bp
for fabp7a, 1083 bp for fabp7b, 3550 bp for fabp10a, 2519 bp fabp10b,
2028 bp for fabp11a, and 3210 bp for fabp11b were retrieved from the
zebrafish GRCz10 whole genome assembly (GenBank Assembly ID G-
CA_000002035.3) (S1 File).
We have previously observed that zebrafish fabp transcription is
responsive to the general PPAR agonists, such as clofibrate (Karanth
et al., 2009a, 2009b; Venkatachalam et al., 2012; Laprairie et al.,
2016a, 2016b). The identified fabp7a, fabp7b, fabp10a, fabp10b,
fabp11a, and fabp11b promoters were analyzed for the presence of
putative PPAR response elements (PPREs) using MatInspector (v. 8.1).
A single putative PPRE displaying high percentage sequence identity
(> 80%) to the consensus sequence for the vertebrate PPRE was
identified in each of the zebrafish duplicated fabp promoter fragments
analyzed: fabp7a: 85% (− 1892 bp relative to TSS), fabp7b 86.9%
(− 961 bp relative to TSS), fabp10a 86% (− 2927 bp relative to TSS),
fabp10b 76% (−1607 bp relative to TSS), fabp11a 89% (− 1367 bp
relative to TSS), and fabp11b 88% (− 1474 bp relative to TSS). The
orange rectangles shown in Fig. 1 indicate putative PPREs identified by
the in silico analysis (S1 File).
The 5′FR and DR1 regions of a PPRE confer PPAR-isoform se-
lectivity to PPARα and PPARγ, respectively (Palmer et al., 1995; Juge-
Aubry et al., 1997; Hsu et al., 1998). The PPREs identified in the fabp
promoter fragments varied in their percentage sequence identity to
consensus sequences for the 5′FR and DR1 regions of vertebrate PPREs
(Table 1). The predicted PPAR subtype specificity for the fabp7a,
fabp7b, fabp10a, fabp10b, fabp11a, and fabp11b promoters, based on
these in silico analyses alone, is given in Table 1.
3.2. PPAR-dependent regulation of zebrafish fabp promoters in HEK293A
cells
Zebrafish fabp promoter sequences identified by in silico analysis
were amplified from genomic DNA (Fig. 1) and cloned into the multiple
cloning site of pGL3-Basic plasmid, 5′ of the firefly luciferase gene.
Plasmids were transiently transfected into HEK293A cells as these cells
serve as a well-characterized heterologous expression system for the
study of PPAR activity (Zagranichnaya et al., 2005; Laprairie et al.,
R.B. Laprairie et al. Comparative Biochemistry and Physiology, Part B 213 (2017) 81–90

induced by WY14643 and rosiglitazone treatment in HEK293A cells

(Table 2; Fig. 2D, E). Zebrafish fabp11a and fabp11b promoter activity
was also induced in HEK293A cells by WY14643 and rosiglitazone
treatment (Table 2; Fig. 2F, G). No significant difference was observed
in fabp11a promoter activity by WY14643 or rosiglitazone in HEK293A
cells (Table 2). WY14643 and rosiglitazone did not differ with respect
to potency in the induction of fabp11a and fabp11b promoter activity
(Table 2).

3.3. Differential regulation of fabp transcripts by PPAR agonists in zebrafish

liver and intestine explant cells

Steady-state levels of fabp mRNA transcripts were quantified in

Fig. 1. Identification of PPREs and NREs in zebrafish fabp7a, fabp7b, fabp10a, fabp10b,
fabp11a, and fabp11b promoters. fabp promoter fragments are indicated by black (fabp7a,
fabp10a, fabp11a), or red (fabp7b, fabp10b, fabp11b) rectangles. A single PPRE was
identified in each of fabp7a, fabp7b, fabp10a, fabp10b, fabp11a, and fabp11b. PPREs are
indicated with orange boxes. Text below each gene provides the PPRE sequence and
position relative to the TSS. Multiple NREs were identified in each of fabp7a, fabp7b,
fabp10a, fabp10b, fabp11a, and fabp11b. In this figure, the NRE closest to each PPRE is
shown. NREs are indicated with green boxes. The TSS for each gene coding region is
indicated by a black arrow. fabp gene coding regions are indicated by the grey horizontal
rectangle and exons (1–4) are indicated by dark grey boxes. Red arrows indicate the
primer-binding sites of PCR primers used in the amplification of zebrafish fabp promoter
fragments. Data were obtained from zebrafish Zv10 whole genome assembly (GenBank
Assembly ID GCA_000002035.3 and analyzed in MatInspector v. 8.1.)
2016a, 2016b). Under basal conditions, all the zebrafish fabp gene
promoters, fabp7a/fabp7b, fabp10a/fapb10b and fabp11a/fabp11b, were
active in HEK293A cells relative to the TK promoter luciferase gene
construct (Fig. 2A). The promoter activity of the zebrafish fabp7a gene
was induced by the PPARα agonist, WY14643, and by the PPARγ
agonist, rosiglitazone, in HEK293A cells (Fig. 2B, C; Table 2). Rosigli-
tazone was a more potent (lower EC50) and efficacious (higher Emax)
inducer of fabp7a promoter activity than WY14643 (Table 2; Fig. 2B,
C). fabp7b promoter activity was induced by WY14643 and rosiglita-
zone (Table 2; Fig. 2B, C). fabp10a and fabp10b promoter activity was
zebrafish liver and intestine explant tissue treated with WY14643
(PPARα agonist) or rosiglitazone (PPARγ agonist) to determine if fabp
mRNA levels were modulated by PPAR agonists in an endogenous
setting like promoter regulation in the HEK293A heterologous system.
Transcripts for fabp7a, fabp7b, fabp10a, fabp10b, fabp11a, and
fabp11b were detected in both liver and intestinal explant tissue con-
sistent with their detection in intact liver and intestine of adult zebra-
fish (Karanth et al., 2009a, 2009b; Venkatachalam et al., 2012). Pre-
viously, we reported that fabp7a and fabp7b mRNA levels are induced in
the liver and intestine of zebrafish fed the non-specific PPAR agonist
clofibrate (Karanth et al., 2009a, 2009b; Venkatachalam et al., 2012).
WY14643 and rosiglitazone treatment increased fabp7a mRNA levels in
liver explant tissue, WY14643 did not increase fabp7a mRNA levels in
intestine explant tissue, and rosiglitazone did increase fabp7a mRNA
levels in intestine (Fig. 3A). Unlike fabp7a, fabp7b mRNA levels were
not changed in liver following treatment with either WY14643 or ro-
siglitazone (Fig. 3B). WY14643 and rosiglitazone did increase fabp7b
mRNA levels in intestine (Fig. 3B). fabp10a mRNA levels were induced
by WY14643 and rosiglitazone in liver and intestinal explant tissue cells
(Fig. 3C). In both liver and intestine, fabp10b mRNA levels were in-
creased by WY14643 treatment, but not by rosiglitazone treatment
(Fig. 3D). WY14643 and rosiglitazone treatment increased fabp11a
transcript levels in liver explant tissue but not intestine (Fig. 3E).
fabp11b mRNA levels did not change in liver following treatment with
either WY14643 or rosiglitazone (Fig. 3F). fabp11b mRNA levels were,
however, decreased 50% by WY14643, and 95% by rosiglitazone in
intestinal explant tissue (Fig. 3F). This repression of fabp11b mRNA
levels was specific to the intestinal explant tissue and was PPARγ-se-
lective (Fig. 3F). Transcriptional regulation of fabp11a, therefore, ap-
pears not to be PPAR-isoform selective, but was liver-specific, whereas
repression of fabp11b transcript levels by the PPARγ agonist was in-
testine-specific (Fig. 3E, F). PPARγ agonist treatment had induced the
fabp11b promoter activity in HEK293A cells (Fig. 2G). Based on these
observations, we sought to determine how PPAR agonism led to re-
pression of fabp11b transcript levels in intestinal explant tissue.

Table 1
Putative PPREs in fabp7a, fabp7b, fabp10a, fabp10b, fabp11a, and fabp11b promoter fragments.

5'FRa DR1a

Sequence Fraction similar % similar Sequence Fraction similar % similar Predicted PPAR selectivity HEK293A cells Zebrafish explant tissue

Consensus CAAAC AGGTCANAGGTCA

fabp7a CATAA 3/5 0.6 GAGCATAGAACTT 6/13 0.5 α γ γ

fabp7b AAAGA 2.5/5 0.5 AAGGCAATTTTCA 8.5/13 0.7 γ γ γ
fabp10a CAAAA 4/5 0.8 GGACAATATGTCA 9.5/13 0.7 α α None
fabp10b CAACA 3/5 0.6 TGGTCAATGGACC 9/13 0.7 γ α α
fabp11a GAATG 2/5 0.4 ATGTAAAAGGTCA 11/13 0.8 γ None None
fabp11b ATAAA 2/5 0.4 AGTACATAAGTGT 8.5/13 0.7 γ None γ (intestine, repression)

a
All sequences are shown 5′–3′. Similarity to the consensus sequences was calculated such that matching nucleotides were assigned a score of ‘1’ and conservative substitutions (i.e.
purine to purine and pyrimidine to pyrimidine) were assigned a score of ‘0.5’. Bold text in the table denotes the consensus sequences: 5' flanking region (FR): 5'-CAAAC-3', and direct
repeat 1 (DR1): 5'-AGGTCANAGGTCA-3' for PPREs.

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R.B. Laprairie et al.
3.4. Identification of peroxisome proliferator-activated receptor and NF-κB
p50 response elements in zebrafish fabp promoters
We considered the potential role of NF-κB p50 in the regulation of
the transcriptional initiation of the zebrafish fabp11b gene. The steady
state levels of fabp11b transcripts were repressed by PPAR agonists in
zebrafish intestinal explant tissue (Fig. 3F), but fabp11b promoter ac-
tivity was increased – and not decreased – by PPAR agonists in tran-
siently transfected HEK293A cells. Remodeling of normal chromatin
conformation in the region of the fapb11b promoter is unlikely on
transiently-transfected plasmid DNA in HEK239A cells (Reeves et al.,
1985; Mearini et al., 2004; Mladenova et al., 2009; Tanny, 2014). A
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Comparative Biochemistry and Physiology, Part B 213 (2017) 81–90
Fig. 2. PPAR agonist-dependent regulation of
zebrafish fabp7a/fabp7b, fabp10a/fabp10b, and
fabp11a/fabp11b promoter activity in HEK293A
cells. Promoter activity was measured at basal le-
vels (A) and in cells treated with 1 nM–1 mM
WY14643 (PPARα-selective) (B, D, F) or rosiglita-
zone (PPARγ-selective) (C, E, G) for 24 h. Firefly
luciferase activity driven by fabp7a or fabp7b (B,
C), fabp10a or fabp10b (D, E), and fabp11a or
fabp11b (F, G) promoters was normalized to Renilla
luciferase activity driven by the TK promoter in
HEK293A cells. n = 4.
conformational transition of chromatin would be required for NF-κB
p50 to repress zebrafish fabp11b promoter activity in HEK293A cells.
Although transiently-transfected plasmid DNA is incorporated into
chromatin when complexed with histones to form nucleosome-like
particles, these do not appear to be remodeled into condensed/un-
condensed chromatin conformations (Reeves et al., 1985; Mearini et al.,
2004; Mladenova et al., 2009; Tanny, 2014). The duplicated zebrafish
fabp7a/fabp7b, fabp10a/fabp10b and fabp11a/fabp11b gene promoters
(Fig. 1) were analyzed for putative NRE using MatInspector (v. 8.1).
Multiple NREs were identified in each of the zebrafish fabp7a/fabp7b,
fabp10a/fabp10b and fabp11a/fabp11b gene promoters (S1 File). Those
NREs most-proximal to a putative PPRE are illustrated as green boxes in
R.B. Laprairie et al. Comparative Biochemistry and Physiology, Part B 213 (2017) 81–90

Table 2 and 50 ng/mL SN50 demonstrated that induction of promoter activity

Induction of zebrafish fabp promoter activity by PPARα and PPARγ agonists in HEK293A was not changed by SN50 treatment (Fig. 5). We concluded that the
cells.
absence of PPAR- and NF-κB p50-mediated fabp11b repression in
WY14643 Rosiglitazone HEK293A cells may be the result of transiently transfected plasmid not
being regulated through chromatin remodeling (Reeves et al., 1985;
fabp7a Mearini et al., 2004; Mladenova et al., 2009; Tanny, 2014).
EC50 (nM) (95% CI) 1233 (405–3760) 11.0 (4.51–25.7)⁎
Emax ± SEM 31.7 ± 2.68 49.6 ± 2.02⁎
4. Discussion
fabp7b
⁎
EC50 (nM) (95% CI) 1053 (185–5990) 44.9 (3.62–55.6)
Emax ± SEM 27.3 ± 3.12 43.6 ± 4.27⁎
In this study, we report the differential regulation of the duplicated
fabp genes fabp7a, fabp7b, fabp10a, fabp10b, fabp11a, and fapb11b by
fabp10a
PPARα, PPARγ and NF-κB p50 in transfected HEK293A cells and in
EC50 (nM) (95% CI) 28.3 (8.5–94.0) 277 (103–746)⁎
Emax ± SEM 42.7 ± 2.14 53.9 ± 3.17⁎ explant liver and intestinal tissue from zebrafish. Each zebrafish du-
plicated fabp gene described in this study exhibited unique transcrip-
fabp10b
EC50 (nM) (95% CI) 38.1 (6.82–123) 357 (186–1440)⁎ tional regulatory parameters controlled by a combination of PPARα,
Emax ± SEM 60.1 ± 3.89 52.5 ± 4.49 PPARγ and/or NF-κB p50 compared to that of their paralog (Table 1,
fabp11a Fig. 4).
EC50 (nM) (95% CI) 11.6 (2.09–64.1) 92.5 (23.8–360)
Emax ± SEM 67.3 ± 3.51 50.7 ± 3.47 4.1. Comparison of HEK293A cell and zebrafish tissue explant models
fabp11b
EC50 (nM) (95% CI) 290 (104–810)^ 209 (52.5–1610) In initial experimental work, PPAR-dependent regulation of zebra-
Emax ± SEM 67.2 ± 3.80 43.8 ± 4.39⁎ fish fabp gene transcription was assessed by fabp promoter-luciferase
⁎
reporter gene constructs transfected into HEK293A cells followed by
P < 0.001 compared to WY14643 within promoter tested.
^
P < 0.001 compared to duplicate promoter, as determined by non-overlapping C.I.s
PPAR isoform-specific agonist treatment. The advantage of this ap-
(EC50) or two-way ANOVA followed by Bonferroni's post-hoc analysis (Emax). n = 4. proach was that PPAR regulation of fabp promoter activity was quan-
tifiable and data were amenable to pharmacological determination of
Fig. 1 (fabp7a 48 bp 5′; fabp7b 9 bp 3′; fabp10a 97 bp 3′; fabp10b 389 bp potency (EC50) and efficacy (Emax) (Laprairie et al., 2016a, 2016b).
3′; fabp11a 149 bp 3′; fabp11b 78 bp 3′). Based on the proximity of the Based on the results of these functional fabp promoter assays, hy-
NREs and PPREs revealed by in silico analysis and by previous studies potheses were made regarding transcriptional regulation of fabp genes
(Ricote and Glass, 2007), transcriptional initiation at the fabp7a, by specific PPAR-isoforms, which were then tested in zebrafish liver
fabp7b, fabp10a, and fabp11b promoters may be regulated by interac- and intestinal explant tissue. The two different experimental systems
tions between PPAR and NF-κB p50. employed in this study were selected as they provided complementary
approaches to evaluate PPAR-dependent regulation at the duplicated
zebrafish fabp7a/fabp7b, fabp10a/fabp10b and fabp11a/fabp11b gene
3.5. Differential regulation of fabp transcripts by NF-κB p50 antagonism in promoters. Functional fabp promoter analyses in HEK293A cells re-
zebrafish liver and intestine present a heterologous system where regulation of promoter activity
occurs on transiently transfected plasmids that are not affected by
mRNA levels were quantified in explant tissue derived from zebra- chromatin conformation (Sherf et al., 1996; Smith et al., 1997; Hebbar
fish liver and intestine treated with vehicle (0.5% DMSO), 1 μM and Archer, 2008). In contrast, zebrafish explant tissues are an en-
WY14643, or 1 μM rosiglitazone, with or without 50 ng/mL of the NF- dogenous system in which we could confirm that the PPAR-dependent
κB p50 inhibitor SN50. A concentration of 50 ng/mL of SN50 was regulation of fabp genes was tissue specific, and we were able to de-
chosen as it corresponds to the concentration at which 80% of NF-κB monstrate the transcriptional repression of several zebrafish fabp genes
p50 is inhibited (i.e. IC80) in mouse NIH 3T3 cells (Lin et al., 1995). by NF-κB p50 on fabp, which appears to be dependent on appropriate
SN50 treatment did not change fabp7a mRNA levels in liver or in- chromatin conformation in the region of the specific zebrafish fabp
testinal explant tissue cells compared to basal transcript levels, or promoter DNA (Ricote and Glass, 2007). PPAR regulation of fabp pro-
transcript levels induced by the PPARα or PPARγ agonists (Fig. 4A). SN- moters was largely the same with the notable exceptions of fabp10a (no
50 treatment alone did not change fabp7b mRNA levels in liver or in- specificity in zebrafish tissue) and fabp7b and fabp11b which were
testine (Fig. 4B), but WY14643 or rosiglitazone with SN-50 increased regulated by NF-κB p50 in zebrafish explant tissue. These key differ-
fabp7b mRNA levels compared to either PPAR agonist alone (Fig. 4B). ences between model systems are likely accounted for by chromatin-
SN50 ± PPAR agonist treatment did not change fabp10a mRNA levels dependent effects as well as species differences between model systems
in liver or intestinal explant tissue compared to basal or PPAR agonist- (Sherf et al., 1996; Smith et al., 1997; Ricote and Glass, 2007; Hebbar
mediated mRNA levels, respectively (Fig. 4C). SN50 alone did not and Archer, 2008). To confirm that NF-κB p50 repression of fabp11b
change fabp10b mRNA levels in liver or intestine, nor did SN50 treat- transcriptional initiation in zebrafish liver and intestinal explant tissue,
ment affect the induction of fabp10b transcript levels by PPAR agonists but not in HEK293A cells, is due to appropriate chromatin conforma-
(Fig. 4D). SN50 ± PPAR agonist did not change fabp11a mRNA levels tion at the fabp11b promoter will require extensive future studies, e.g.,
in liver or intestine compared to basal levels or PPAR agonist-stimu- production of antibodies to zebrafish transcription factors, chromatin
lated levels (Fig. 4E). Treatment with SN50 alone increased fabp11b immunoprecipitation (ChIP) (Lindeman et al., 2009; Trompouki et al.,
mRNA levels in liver and intestine (Fig. 4F). In the liver, WY14643 or 2011). Using these two complementary approaches provided evidence
rosiglitazone with SN50 did not change fabp11b mRNA levels compared of divergent transcriptional regulation that may account for the reten-
to SN50 alone (Fig. 4F). In the intestine, WY14643 or rosiglitazone tion of these duplicated fabp genes in the zebrafish genome.
+ SN50 did not change fabp11b mRNA levels compared to SN50 alone,
but no PPAR agonist-dependent repression was observed for fabp11b 4.2. PPAR isoform-specific promoter regulation
either (Fig. 4F).
HEK293A cells have been shown to express NF-κB p50 (Geiger et al., Previous studies of PPAR isoform specificity have focused on non-
2012). Assay of HEK293A cells transiently transfected with zebrafish quantitative or semi-quantitative data derived from electrophoretic
fabp gene promoters and treated with 1 mM WY14643 or rosiglitazone mobility shift assays to determine the specificity of PPAR isoform

86
R.B. Laprairie et al.
interaction with PPREs (Palmer et al., 1995; Juge-Aubry et al., 1997;
Hsu et al., 1998; Wolfrum et al., 2001). In a previous study, we quan-
tified PPAR-isoform selectivity of the zebrafish fabp1a and fabp1b.1
promoters (Laprairie et al., 2016a, 2016b). Preferential regulation of
fabp1a and fabp1b.1 promoter activity was attributed to PPARα or
PPARγ using site-directed mutagenesis of the 5′FR and DR1 regions of
each promoter's PPRE (Laprairie et al., 2016a, 2016b). Based on our
previous characterization of fabp promoter PPAR responsiveness
(Laprairie et al., 2016a, 2016b), we conclude that the PPAR agonist
concentration-mediated induction of fabp promoter activity here oc-
curred via PPAR. The PPARα-selectivity of the zebrafish fabp1a pro-
moter was attributed to the 5′FR of a PPRE in the fabp1a promoter that
was highly similar to the 5′FR consensus sequence of vertebrate PPREs
(Palmer et al., 1995; Juge-Aubry et al., 1997; Hsu et al., 1998; Laprairie
et al., 2016a, 2016b). The PPARγ-selectivity of the zebrafish fabp1b.1
promoter was attributed to the DR1 of a PPRE in the fabp1b.1 promoter
that resembled the DR1 consensus sequence of vertebrate PPREs and a
5′FR that was less-similar to the 5′FR consensus sequence (Palmer et al.,
1995; Juge-Aubry et al., 1997; Hsu et al., 1998; Laprairie et al., 2016a,
2016b). Unlike fabp1a and fabp1b.1, the fabp genes assessed in this
87
Comparative Biochemistry and Physiology, Part B 213 (2017) 81–90
Fig. 3. PPAR agonist-dependent regulation of
zebrafish fabp7a/fabp7b, fabp10a/fabp10b, and
fabp11a/fabp11b mRNA levels in zebrafish liver
and intestine explant tissue. Liver and intestine
cells were cultured ex vivo for 24 h before being
treated with 1 μM WY14643 (PPARα-selective
agonist) or 1 μM rosiglitazone (PPARγ-selective
agonist) for 24 h. fabp7a (A), fabp7b (B), fabp10a
(C), fabp10b (D), fabp11a (E), or fabp11b (F)
mRNA levels were quantified by qRT-PCR using
the ΔΔCT method and normalized to GAPDH.
*P < 0.001 compared to vehicle treatment
within tissue, †P < 0.001 compared to WY14643
treatment within tissue, ^P < 0.001 within drug
treatment between tissues, as determined via two-
way ANOVA followed by Bonferroni's post-hoc
test. n = 4.
study did not differ in PPAR isoform selectivity between duplicates.
Based on the in silico analysis of PPREs present within the zebrafish fabp
promoters, we predicted that fabp7a and fabp10a would exhibit PPARα-
selectivity, whereas fabp7b, fabp10b, fabp11a, and fabp11b would dis-
play PPARγ-selectivity (Figs. 2–4; Table 1) (Palmer et al., 1995; Juge-
Aubry et al., 1997; Hsu et al., 1998). Contrary to these predictions, data
derived from fabp promoter activity assays in HEK293A cells and
quantification of steady-state levels of fabp transcripts in zebrafish liver
and intestinal explant tissue indicated that fabp7a promoter activity
was PPARγ-selective, fabp10b promoter activity was PPARα-selective,
and fabp11a promoter activity was not PPAR isoform-selective. PPAR
isoform selectivity cannot, therefore, be consistently predicted based on
PPRE sequence alone. The quantitative data produced here demonstrate
PPAR isoform specificity of fabp transcriptional control in heterologous
cell culture and explant tissue culture systems (Juge-Aubry et al.,
1997).
It is important to note the species differences that exist within this
study. Zebrafish express five PPAR isoforms: Pparaa, Pparab, Pparda,
Ppardb, and Pparg; whereas the HEK293A cells used in this study ex-
press only 1 isoform each of human PPARα and PPARγ (Den Broeder
R.B. Laprairie et al. Comparative Biochemistry and Physiology, Part B 213 (2017) 81–90

Fig. 4. NF-κB p50 antagonist-dependent regulation of zebrafish fabp7a/fabp7b, fabp10a/fabp10b, and fabp11a/fabp11b mRNA levels in zebrafish liver and intestine explant tissue. Liver
and intestine cells were cultured ex vivo for 24 h before being treated with 50 ng/mL SN50 (NF-κB p50 antagonist) with or without 1 μM WY14643 (PPARα-selective agonist) or
rosiglitazone (PPARγ-selective agonist) for 24 h. fabp7a (A), fabp7b (B), fabp10a (C), fabp10b (D), fabp11a (E), or fabp11b (F) mRNA levels were quantified via qRT-PCR using the ΔΔCT
method and normalized to GAPDH. Data for vehicle, WY14643, and rosiglitazone are repeated from Fig. 3 and provided here for comparison. ⁎P < 0.001 compared to vehicle treatment
within tissue, †P < 0.001 compared to equivalent drug treatment without SN50 and within tissue, ^P < 0.001 within drug treatment between tissues, as determined via two-way
ANOVA followed by Bonferroni's post-hoc test. n = 4.

Fig. 5. NF-κB p50 antagonism did not change fabp7a/

fabp7b, fabp10a/fabp10b, and fabp11a/fabp11b promoter
activity in HEK293A cells. Promoter activity was measured
in cells treated with 1 mM WY14643, 1 mM rosiglitazone,
50 ng/mL SN50, 1 mM WY14643 + 50 ng/mL SN50, or
1 mM rosiglitazone + 50 ng/mL SN50 for 24 h. Firefly lu-
ciferase activity driven by fabp7a, fabp7b, fabp10a, fabp10b,
fabp11a, or fabp11b promoters was normalized to Renilla
luciferase activity driven by the TK promoter in HEK293A
cells. Data for vehicle, 1 WY14643, and rosiglitazone are
from independent experiments to those described in Fig. 2.
n = 3.

et al., 2015). Although a high degree of sequence similarity (67–74%
mRNA, 88–94% amino acids in DNA binding domain, 74–83% amino
acids in ligand binding domain) and functionality exists between the
zebrafish PPARα isoforms and human PPARα, and the zebrafish PPARγ
isoforms and human PPARγ (Maloney and Waxman, 1999; Den Broeder
et al., 2015; Zhao et al., 2015), the present study does not account for
species differences in PPARs. Indeed, some of the discrepancies ob-
served between HEK293A cells and zebrafish tissue may be explained
by such differences.
The selectivity of the PPAR agonists used here must be considered

88
R.B. Laprairie et al.
for both human and zebrafish PPAR isoforms. WY14643 and rosiglita-
zone are well-characterized, selective PPARα and PPARγ agonists, re-
spectively (Staels et al., 1998; Young et al., 1998). In zebrafish,
WY14643 is a potent and selective agonist of both Pparaa and Pparab
relative to other Ppar isoforms, with similar potency for the zebrafish
and human PPARα orthologs (Gu et al., 2014). Rosiglitazone is an
agonist of both human PPARγ and zebrafish Pparg (d'Alençon et al.,
2010; Tingaud-Sequeira et al., 2011; Laprairie et al., 2016a, 2016b),
albeit with reduced efficacy at zebrafish Pparg as compared to human
PPARγ (Grimaldi et al., 2015). In this study, a relatively high con-
centration of rosiglitazone (1 μM) was used in zebrafish tissue to acti-
vate Pparg. The selectivity of rosiglitazone for Pparg compared to other
zebrafish Ppar isoforms is not known, but given the observation of ro-
siglitazone-specific effects in zebrafish tissue here, and previously
(Laprairie et al., 2016a, 2016b), rosiglitazone likely displays some de-
gree of Pparg selectivity.
4.3. NF-κB p50-dependent promoter regulation
We were surprised to observe a PPAR agonist-dependent repression
of fabp11b mRNA levels in zebrafish intestine, which had not been
observed in previous studies of the fabp1a and fabp1b.1 promoters
(Laprairie et al., 2016a, 2016b), or other promoters included in the
present study. PPARs may inhibit the transcription of genes that they
regulate by interacting with other transcription factors, including NF-κB
p50 (Ricote and Glass, 2007). PPARα is known to reduce the formation
of NF-κB p50/C/EBP complexes and consequently reduce C-reactive
protein (CRP) levels in a PPAR agonist-dependent manner (Kleemann
et al., 2003, 2004). Similarly, PPARγ is known to reduce IL-12 gene
expression in a PPAR agonist dose-dependent manner through direct
interactions with NF-κB p50 when a PPRE exists within proximity to an
NRE (Delerive et al., 1999; Chung et al., 2000; Zingarelli et al., 2003),
which is consistent with the results of fabp11b transcriptional control by
PPARs (Fig. 4F) and NF-κB p50 observed in the explant tissue culture
cells reported here (Fig. 3E, F; Fig. 4E, F) and, indirectly, by in silico
analyses (Fig. 1). Treatment of zebrafish explant intestinal cells with the
NF-κB p50 inhibitor, SN50, alleviated NF-κB p50-dependent inhibition
of fabp11b mRNA. Addition of PPAR agonists yielded PPAR-dependent
induction of fabp11b mRNA levels in explant tissue, consistent with the
effect of PPAR agonists on the fabp11b promoter in HEK293A cells. We
conclude that NF-κB p50 repressed fabp11b promoter activity in zeb-
rafish intestine, and PPAR was only able to activate fabp11b promoter
activity in the absence of NF-κB p50 as observed in the functional
promoter assays in HEK239A cells owing to aberrant chromatin con-
formation of transiently zebrafish fabp promoter-luciferase plasmid
constructs as reported by previous studies (Reeves et al., 1985; Lin
et al., 1995; Mearini et al., 2004; Mladenova et al., 2009; Elsharkawy
et al., 2010; Tanny, 2014). In contrast, SN50 treatment alone had no
effect on fabp7b promoter activity in explant tissue, but when used with
a PPAR agonist, these two compounds did induce fabp7b promoter ac-
tivity, indicating that NF-κB p50 maintained a basal level of fabp7b
promoter activity in zebrafish, and PPAR was only able to activate
fabp7b promoter activity in the absence of NF-κB p50. In addition to the
PPAR- and tissue-specific differential regulation of the zebrafish
fabp7a/fabp7b, fabp10a/fabp10b and fabp11a/fabp11b genes described
here, NF-κB p50-dependent inhibition of fabp7b and fabp11b represents
an additional regulatory element by which sister duplicate fabp genes
have been subfunctionalized (Lynch and Conery, 2000; Taylor and
Raes, 2004; Conrad and Antonarakis, 2007). Differences in the mag-
nitude of gene response to PPAR and NF-κB stimuli in explant cells
(Fig. 4) may be the consequence of the numerous other transcription
factors regulating fabp gene responsiveness that are beyond the scope of
the present study. The consequence of NF-κB p50 regulation may be
that expression of selected fabp genes is limited by inflammatory sti-
muli. Many fabp substrates, such as linoleic acid, mediate inflammatory
signaling in the adipocytes, lymphocytes, and hepatocytes of
89
Comparative Biochemistry and Physiology, Part B 213 (2017) 81–90
vertebrates (Calder, 2001; Karra et al., 2015). As such, the data re-
ported here provides interesting evidence to suggest a link between the
subfunctionalized promoter regulation and expression of selected fabp
genes, fabp7b and fabp11b, and inflammatory stimuli.
5. Conclusions
In this study, we used a quantifiable approach to define the differ-
ential transcriptional regulation at the promoters of the zebrafish fabp
genes. Three levels of subfunctionalization of the zebrafish fabp7,
fabp10, and fabp11 gene duplicates were defined: (1) tissue-specific
regulation; (2) PPAR-isoform specific; (3) and NF-κB p50-dependent
repression. Each of the fabp genes investigated here displayed a unique
profile of tissue-, PPAR isoform- or NF-κB p50-specific regulatory
parameters relative to their paralogous sister duplicate, and to all other
zebrafish fabp gene promoters examined to date (Laprairie et al.,
2016a, 2016b). The preservation and unique regulation of the fabp gene
duplicates described here likely provides for tissue- and stimuli-specific
control over fabp activity, which in turn provides a physiological con-
trol of the trafficking of hydrophobic ligands to effector molecules
(Esteves et al., 2015). In the duplication degeneration-complementation
model, preservation of gene duplicates is more likely when mutations
arise in the regulatory elements of duplicated genes that sub-divide
functions of the ancestral gene (Force et al., 1999). In addition to PPAR
isoform-specific induction and NF-κB p50 repression of zebrafish fabp
gene transcription, additional experimental work is required to account
for the tissue-specific induction of fabp transcript levels. These studies
might provide insight into the evolution of other regulatory mechan-
isms that may account for the retention of fabp7, fabp10, and fabp11
gene duplicates in the zebrafish genome by subfunctionalization
(Karanth et al., 2009a, 2009b; Venkatachalam et al., 2012). The work
reported here, and earlier studies on the differential regulation of the
zebrafish fabp1 genes (Laprairie et al., 2016a, 2016b), in part, define
the evolutionary trajectories of duplicated genes that led to the reten-
tion of 57% of duplicate fabp genes in the zebrafish genome compared
to only ~3% of all duplicated genes in the zebrafish genome following
the teleost-specific whole genome duplication event (Ohno, 1970;
Lynch and Conery, 2000; Taylor and Raes, 2004; Woods et al., 2005;
Smathers and Peterson, 2011).
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.cbpb.2017.08.003.
Acknowledgements
This work was supported by funds from the Natural Sciences and
Engineering Research Council of Canada (NSERC) (ROP-44248) to
JMW and a Bridge Funding grant from Dalhousie University (44248) to
EMD-W. RBL was supported by studentships from the Canadian
Institutes of Health Research, the Huntington Society of Canada, and
Killam Trusts. The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Nucleotide sequence data accessed for this study are available in the
GenBank databases under the accession numbers GRCz10 Assembly ID
GCA_000002035.3, AY145893.1, AL845421.9, CR293507.10,
FP102515.5, CT027607.10, and BX248082.1.
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