OPEN
SUBJECT AREAS:
EVOLUTIONARY
ECOLOGY
ENVIRONMENTAL SCIENCES
Received
23 October 2014
Accepted
21 January 2015
Published
25 February 2015
Correspondence and
requests for materials
should be addressed to
Y.Z. (zhaoyb@pku.
edu.cn) or J.H. (hujy@
urban.pku.edu.cn)
Families of Nuclear Receptors in
Vertebrate Models: Characteristic and
Comparative Toxicological Perspective
Yanbin Zhao1, Kun Zhang1, John P. Giesy2,3,4 & Jianying Hu1
1
MOE Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences, Peking University, Beijing 100871,
China, 2Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon,
Saskatchewan, Canada, 3Department of Zoology, and Center for Integrative Toxicology, Michigan State University, East Lansing,
MI, USA, 4Department of Biology & Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong, Kowloon,
Hong Kong, SAR, China.
Various synthetic chemicals are ligands for nuclear receptors (NRs) and can cause adverse effects in
vertebrates mediated by NRs. While several model vertebrates, such as mouse, chicken, western clawed frog
and zebrafish, are widely used in toxicity testing, few NRs have been well described for most of these classes.
In this report, NRs in genomes of 12 vertebrates are characterized via bioinformatics approaches. Although
numbers of NRs varied among species, with 40–42 genes in birds to 66–74 genes in teleost fishes, all NRs had
clear homologs in human and could be categorized into seven subfamilies defined as NR0B-NR6A.
Phylogenetic analysis revealed conservative evolutionary relationships for most NRs, which were consistent
with traditional morphology-based systematics, except for some exceptions in Dolphin (Tursiops
truncatus). Evolution of PXR and CAR exhibited unexpected multiple patterns and the existence of CAR
possibly being traced back to ancient lobe-finned fishes and tetrapods (Sarcopterygii). Compared to the
more conservative DBD of NRs, sequences of LBD were less conserved: Sequences of THRs, RARs and RXRs
were $90% similar to those of the human, ERs, AR, GR, ERRs and PPARs were more variable with
similarities of 60%–100% and PXR, CAR, DAX1 and SHP were least conserved among species.
N
uclear receptors (NRs) are one of the largest groups of transcription factors in vertebrates, and serve
important functions in regulation of a range of physiological functions including growth and differentiation of cells, metabolic processes, reproduction, development and overall homeostasis. Transcriptional
activities of NRs are regulated by binding of endogenous small lipophilic compounds1,2. There is growing
evidence that diverse chemicals that occur in the environment, including synthetic molecules such as pharmaceuticals, endocrine disrupting chemicals and some industrial compounds, can mimic endogenous small compounds that can bind to ligand binding domains (LBDs), activate NR-mediated signals that then lead to toxic
responses3,4. Typically, interactions of some pesticides and industrial chemicals with estrogen (ER) and androgen
(AR) receptors have been linked to a number of adverse effects including birth defects, developmental neurotoxicity, both male- and female-factor reproductive health, such as decreased quality of sperm, and increased
incidences of cancers5–7.
A series of in vitro bioassays, based on signaling of endocrine receptors including well-studied steroid hormone
receptors such as ER, AR, glucocorticoid receptors (GRs), and progesterone receptor (PR) and the less wellstudied retinoic acid receptor (RAR), retinoid X receptor (RXR), and thyroid hormone receptor (THR), have been
established or are under assessment by OECD and/or US EPA8–10. Due to their relatively clear physiological
functions and responses to environmentally-relevant organic micropollutants, these NR-based assays have been
used in assessment of toxicological effects of chemicals in the environment. For example, ERs, AR and THRs,
involved in development and maintenance of the endocrine system, have been demonstrated to be targets of
alkylphenols, phthalates (PAEs), dichlorodiphenyltrichloroethane and some metabolites of polychlorinated
biphenyls (PCBs) and polybrominated diphenyl ethers (PBDE)11–13. Besides endocrine receptors, PXR and
CAR, NRs that participate in metabolism of both endobiotics and xenobiotics to detoxify or bioactivate chemicals,
can be activated by a variety of pharmaceuticals such as rifampicin, pesticides such as chlorpyrifos and methoxychlor, and other synthetic chemicals used in industry, such as PBDEs and BPA14–17 In addition to these wellknown NRs, there are more NRs, that, during the past decade, have been identified in genomes of several
SCIENTIFIC REPORTS | 5 : 8554 | DOI: 10.1038/srep08554
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vertebrates. These include 48 NR genes in human (Homo sapiens), 47
genes in rat (Rattus norvegicus), 49 genes in mouse (Mus musculus)
and 68 genes in the teleost puffer fish Fugu rubripes18,19. Specifically,
structures of 48 NRs in the human have been identified and categorized, based on sequence homology, into seven different subfamilies
NR0B-NR6A20. Except for two NRs in the subfamily NR0B which
lack a DNA binding domain (DBD), all 46 NRs contain the following
six functional domains: (A–B) variable N-terminal regulatory
domain; (C) conserved DNA-binding domain; (D) variable hinge
region; (E) conserved ligand binding domain (LBD) and (F) variable
C-terminal domain20. In addition, sets of NRs described in humans
offered a better understanding of characteristics of NRs, and provided insight for uncovering novel molecular and signal targets and
mechanisms of action of synthetic toxicants. For instance, it has been
found that some widely used pharmaceutical drugs that are found in
the environment, including thiazolidine diones, trichloroacetic acid
and toxaphene are ligands for human RORa, PPARa and ERRa,
respectively21–23. Compared with the extensive understanding of
NRs in human, fewer NRs have been identified in other vertebrates
used as models to screen chemicals for toxic potencies, such as reptiles, amphibians and teleost fishes. While in recent years, due to
extensive information about their developmental biology and
molecular genetics and now the availability of completed sequencing
of their genomes, these vertebrate species have been much used as
toxicological models such as western clawed frog (X. tropicalis), zebrafish (Danio rerio), and freshwater Japanese medaka (Oryzias
latipes)24–26, information on NRs in these vertebrates were still limited to ERs, AR, GR, PXR, RARs and PPARs, though studies on some
novel NRs, such as VDR, FXR and NURR are in progress27–29.
Additionally, since sets of NRs in human, mouse and rat that have
been identified in previous studies were based on their genomes
assembled a decade ago18, there is also a need to reevaluate the characteristics of NRs in these genomes due to the constantly updated
sequence data and annotations. In addition to the sequences of genomes, predicted transcriptomes and proteomes, now available for all
of these species in Genebank and Ensembl, provide useful databases
that can be further used to uncover and characterize additional NRs.
Therefore, comprehensive descriptions of NRs and their families for
these vertebrates used as models to screen for toxic potencies of
chemicals, will be helpful for their further development and interpretation of results of studies of synthetic chemicals of environmental significance.
In this study, complete sets of NRs were described for genomes of
12 vertebrates used as models in studies of toxic potency and
mechanisms of action of chemicals. Several bioinformatics
approaches were applied to four mammals (human, Homo sapiens;
mouse, Mus musculus; rat, Rattus norvegicus and dolphin, Tursiops
truncatus), two birds (chicken, Gallus gallus and mallard (wild duck),
Anas platyrhynchos), a reptile (Chinese softshell turtle, Pelodiscus
sinensis), an amphibian (Western clawed frog, Xenopus tropicalis)
and four teleost fishes (zebrafish, Danio rerio; medaka, Oryzias
latipes; tilapia, Oreochromis niloticus and stickleback, Gasterosteus
aculeatus). The locations of NRs on chromosomes, phylogenetic
analysis and DBD and LBD sequence conservations among species
were also analyzed to better understand the characteristics of these
NRs in these vertebrates.
Results and Discussion
Identification of NRs in 12 vertebrates. Substantial and continuous
information gathered from developmental biology and molecular
genetics, together with the complete sequencing of genomes has
placed a series of vertebrate species in attractive positions for use
in toxicological research. Twelve species were chosen for description
and complete sets of NR genes within their genomes were identified
by use of a systemic bioinformatics approach. In total, 42–74 NR
genes were uncovered within these vertebrates and a large number of
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variations were observed among classes (Fig. 1A, Table S2).
Comparisons of sequences showed that all of these NRs displayed
significant similarity to NRs of the human and could be categorized
into the seven subfamilies NR0B-NR6A, with no novel subfamilies.
For mammals, there were 48, 49, 49 and 47 NRs identified in human,
mouse, rat and dolphin genomes, respectively (Fig. 1A). Compared
to the human, one more gene (NR1H5) was observed for mouse and
rat and one (NR2F2) was absent from dolphin (Fig. 2). Sets of NRs in
human and mouse were consistent with previous reports18, while two
more NRs (NR1D2 and NR2E3) were newly identified for the rat.
The absences of these two NRs in rat in previous study18 were due to
the existence of sequence gaps in the rat genome which was
assembled in 2003.
The numbers of NRs in birds were less than those in human,
though there were some unique genes observed. There were seven
NRs (NR1B3, NR1D1, NR1H2, NR1I2, NR2B2, NR3B1 and NR4A1)
present in the human that were absent from the chicken. Similarly,
there were nine NRs (NR1B3, NR1D1, NR1H2, NR1I2, NR1I3,
NR2B2, NR2E3, NR2F1 and NR3B1) present in the human that were
absent from the mallard, though there were three new NRs (NR1F3,
NR1H5 and NR2A3) were identified that were unique to chicken and
mallard (Fig. 2). Similar absences were observed in the genomes of
turkey (Meleagris gallopavo), flycatcher (Ficedula albicollis) and
zebra finch (Taeniopygia guttata), where 9, 5 and 6 NRs, respectively,
that are present in the human genome were absent from these birds
(Fig. 3C). These results demonstrated that a cluster of NRs were
indeed absent from genomes of the class aves, especially in galloanserae, that were deleted during the course of evolution.
Some NRs present in the human were absent from turtle and
western clawed frog while some others were unique in these species.
In the one species of turtle, 48 NRs were identified with four genes
absent (NR1B3, NR1H2, NR1I2 and NR2B2) and four new genes
gained (NR1F3, NR1H5, NR2A3 and NR2F1) compared with those
in human. Similarly, 52 NRs were identified in western clawed frog
with 2 genes absent (NR1H2 and NR4A3) and six additional genes
(NR1F2, NR1H5, NR2A3, NR2F5, NR3B3 and NR4A2) appeared
which were not present in the human (Fig. 2).
For the four teleost fishes studied, there were many additional NRs
uncovered in this study. Specifically, 73 and 74 NRs were identified in
zebrafish and tilapia, respectively (Fig. 1A), which were consistent with
those reported for Fugu rubripes (68 NRs identified)19. The additional
NRs were mainly due to the paralogue genes exist in their sets of NRs
(Fig. 1C). In zebrafish, two or more paralogues were identified to
correspond with one of 20 NRs in human and with one of 18, 22
and 17 NRs in medaka, tilapia and stickleback, respectively. Existences
of paralogue genes in teleost fishes were not random but focused on
some specific NR units. For instance, NR1F3 (RORc) was the most
abundant NR, with a total of seven paralogue gene copies in these four
teleost fishes. The NRs NR1A1, NR1B3, NR1C1, NR1I1, NR2B2,
NR2F6, NR3A2, and NR3B3 were also rich in paralogues, with one
paralogue gene copy in each of the four teleosts (Fig. 3D).
Characteristics of NRs families. Genomic locations of NRs in seven
vertebrate genomes (human, mouse, rat, chicken, zebrafish, medaka
and stickleback) were retrieved via the Ensemble annotations. In
general, distributions of NRs on chromosomes were more
widespread in teleost fishes than those of mammals and birds
(Fig. 1B). This is possibly due to the existence of more paralogue
genes in teleosts. For example, NRs in zebrafish, medaka and
stickleback were distributed throughout their genomes except for
1–2 chromosomes. The most abundant clusters of NRs were
observed on chromosomes 8 and 16 in zebrafish, each with 6 NRs;
on chromosomes 7 and 16 in medaka, each with 7 NRs; and on
chromosome 12 in stickleback, with 8 NRs. The narrowest
distribution of NRs was observed for species of chicken, in which
44 NRs were distributed in 61% (19/31) chromosomes.
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Figure 1 | Identification of NRs in genomes of 12 toxicological vertebrate models. (A) Total number of NRs in each vertebrate genome (B) the
genomic distributions of NRs in seven vertebrate species (C) the number of NRs for each type (NR0B-NR6A) and the paralogous gene numbers (P.G.) in
total.
Phylogenetic analyses, based on their full amino acid sequences
and DBD plus LBD compositions of NRs, were performed for 48
types of NRs among these 12 vertebrates. The Neighbor-Joining (NJ)
and Maximum-Likelihood (ML) phylogenetic analyses showed similar patterns, while the Neighbor-Joining algorithm gave better resolution at the base of the phylogram. Conservative evolutionary
relationships were observed for most NRs, i.e. the evolutionary relationships were generally consistent with the traditional morphologybased systematics (Fig. S1). As exemplified for NR3A1 (ERa), closer
relationships were observed within each class and the traditional
teleost-amphibian-reptile-bird and mammal evolutionary relationships were followed (Fig. 3A). This was verified by the similarity of
sequences of the LBD of ERa among species (Fig. 4). In details, about
82–93% sequence similarities among teleost, 99% between birds and
98–99% among mammals was observed and the sequence similarities
among classes were relatively small (Fig. 5). Some exceptions were
observed in Dolphin such as NR2A1 and NR2A2 (Fig. S1). Though
dolphin, diverged from artiodactyls approximately 50 million years
ago30, was thought to show the closest relationship with human
among the 12 vertebrates, there were 32% NRs that showed closer
relationships between rodents and human compared with those in
dolphin. Similarities between sequences of the DBD and LBD also
confirmed this likely historical divergence. In rodents, 13% of
sequences of amino acids of DBD and 26% of those of the LBD
exhibited relationships more similar to those of the human than
dolphin (Fig. 3B). These variations in NRs in dolphin were possibly
due to the results of positive Darwinian selection, the major driving
force for adaptive evolution and diversification among species, to
adapt their radical habitat transition from land to a marine environment. Though increasing toxicological research has been preformed using dolphins and extrapolations from dolphin to human
were thought to be more significant, results of the present study
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demonstrated more variations, indicating more genetic characteristics should be taken into account when assessing toxicities of chemicals based on results of studies with dolphins. In addition, since PXR
and CAR displayed the largest variations and were absent in several
vertebrates used in this study (Fig. 2 and 4), more comparisons
among species were conducted. Existence of NR1I (VDR, PXR and
CAR) genes were demonstrated in 35 vertebrate species (20 mammals, 5 birds, 2 reptile, 1 amphibian and 7 teleost fishes) with for
which complete sequences of genomes were available and unexpected patterns were showed for their evolutions. VDR genes appeared
in all vertebrate genomes, a result which was consistent with those in
previous reports that VDR could be detected in mammals, birds,
amphibians, reptiles, teleost fishes, and even the sea lamprey31.
PXR appeared in most teleost fishes (expect for stickleback), amphibians and mammals (also known as SXR), but were totally absent
from reptiles and birds. Though CAR also appeared in all mammals,
it exhibited quite different patterns in other classes. CAR was mostly
absent in birds (expect for chicken), but retained in reptiles and
amphibians, and appeared in lobe-finned fishes and tetrapods
(Sarcopterygii) (Fig. 3E). Since Sarcopterygii appeared nearly 400
million years ago during the Devonian, and are widely accepted as
ancestors of all tetrapoda, including amphibians, reptiles, birds and
mammals32, the appearance of CAR in Sarcopterygii possibly indicated that the existence of CAR was much earlier than previously
thought. In general, these results revealed a novel evolutionary relationship for PXR/CAR. These two NRs likely coexisted in ancient
Sarcopterygii, first due to the duplication events, descended into
amphibians and then to mammals, but one of them was absent from
reptiles and both were absent from most birds (Fig. S2).
Alignment of sequences of DBD and LBD. Since cross-species
extrapolations from surrogate vertebrate species to humans are
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Figure 2 | Nuclear receptor families in 12 model vertebrates. Each nuclear receptor is presented as a colored block. The white spaces indicate that no
ortholog was identified. Nuclear receptor family for each vertebrate species was marked with different color. From left to right: human ‘‘
’’;
mouse ‘‘
’’; rat ‘‘
’’; dolphin ‘‘
’’; chicken ‘‘
’’; duck ‘‘
’’; turtle ‘‘
’’; frog ‘‘
’’; zebrafish ‘‘
’’; medaka ‘‘
’’; tilapia ‘‘
’’ and
stickleback ‘‘
’’.
usually considered to be crucial for human risk assessment of chemicals,
better understanding of similarities of these NRs sequences among
species will be useful to facilitate these extrapolations and better
understand the toxicities of environmental chemicals. In the present
study, pairwise alignments were constructed between sequences of
DBD/LBD of 48 human NRs and their corresponding orthologs in
the other eleven vertebrate species (Fig. 4). As expected, DBDs of the
orthologous proteins generally shared relatively great conservation with
sequences in human (Fig. 4, left), especially, for the mouse, rat and
dolphin, in which 94%–100% sequence similarities were observed for
most NRs, expect CAR (70%–89%), and almost 70% (31/46, 32/46 and
31/42, respectively) orthologous proteins showed 100% similarities with
sequences of the human. For bird, reptile, amphibian and teleost fishes,
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most NRs also displayed conservation of sequences (usually .90%),
especially for RORb (100% for all species). While there are also some
exceptions, such as PXR (61%–73%), CAR (64%–67%), and PPARa
and TR2 in teleost (87%–90% and 84%–87%, respectively), which
indicates potential alternations on target genes and signals for these
NRs among vertebrate species.
Compared to the more conserved sequences of DBD regions of
NRs among species, sequences of the LBD displayed more variation.
The greatest variation was observed for DAX1 (40%–81%), while the
least variation was observed for COUP-TFII (99%–100%) compared
with those in human (Fig. 4, right). To our best knowledge, this is the
first time all NRs LBD have been compared among vertebrates,
which showed a broader and novel insight to investigate the LBD
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Figure 3 | Characteristics of the 12 NRs families. (A) Phylogenetic tree for 12 NR3A1 (ERa) genes (B) The evolutionary relationships of NRs among
dolphin, rodents and human species. Left: the proportions of dolphin NRs with closer relationships with human compared to rodents are presented as
percent/number and blue colour. The proportions of rodents NRs with closer relationships with human are presented as percent/number and orange
colour. Green colour represents the NRs numbers with equivalent sequence similarities with human for dolphin and rodents. Right: phylogenetic tree for
NR2C1 and NR2A1 represents the different positions of NRs for dolphin. (C) Comparative searches for the ten lacked NRs in five bird species (D)
Paralogous gene copy numbers for each type of NRs (E) Comparative searches for NR1I genes (VDR, PXR and CAR) in 35 vertebrates, including 20
mammals, 5 birds, 2 reptiles, 1 amphibian and 7 teleost (details are described in Table S4). Phylogenetic tree was developed utilizing 35 full amino acid
sequences of VDR.
differences between species and between multiple NRs units. In the
present study, three groups were identified in general based on similarities in sequences of NRs. The first group contained 13 NRs
including THRa, THRb, RARa, RARb, RARc, RORa, RXRa,
RXRb, RXRc, COUP-TFII, ERRc, NURR1 and LRH1 (except some
orthologs for RARa, RORa, RXRb, RXRc and NURR1) with $90%
similarity of sequences of the LBDs for all eleven vertebrates compared with those of the human (Fig. 4, right). As observed for RXRa,
97–100% similarities in sequences, for the best alignment orthologs,
were observed from multiple sequence alignment (Fig. 5). Variations
in conservation of sequences, window averaged across 10 amino acid
residues, found that there were fewer than 5 variations in amino acid
residues among these 12 vertebrate species, and most of them were
observed in a-helix 3 to a-helix 6 of the LBD structures (Fig. 5).
RXRa commonly functions as a heterodimers with other NRs and
mainly mediates signaling of hormones derived from vitamin A
(retinol) such as 9-cis retinoic acid, and are involved in multiple
physiological functions of vertebrates such as embryonic patterning
and organogenesis, proliferation of cells and differentiation of tissues33. It has been reported that among vertebrates, such as mouse
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and human, LBDs of RXRa interacted with similar types of ligands
with similar binding affinities34,35. Sequence similarities of these 13
NRs among vertebrates suggested potential straightforward interspecies extrapolations when assessing toxicity of chemicals via these
NRs. Approximately 77% of NRs such as the well-known ERs, AR,
PR, PPARs and VDR can be sorted into the second group, exhibiting
60–100% similarities of sequences (for the best aligned orthologs)
compared with those of human. Similarities in sequences of these
NRs among four fishes were substantially the same and usually
$90% in mouse, rat and dolphin, showing apparent differences in
sequences of amino acids between teleosts and mammals.
Specifically, LBDs of NRs in the second group, such as ERa and
PPARc, always shared the same variations in amino acids within
four fishes, which were quite different from those of mammals
(Fig. 5 for ERa). ERa is a well-studied NR, activated by endogenous
and exogenous estrogens, and plays a variety of central physiological
roles, such as maintenance of reproductive, cardiovascular and central nervous systems in vertebrates36. Potencies of binding of ligands
to LBDs of ERa were different for fishes when compared to mammals. It has been reported that widespread chemicals like 4-t-octyl5
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Figure 4 | Pairwise alignments between DBD/LBD amino acid sequences of 48 human NRs and the corresponding orthologs in other eleven vertebrate
species. Left for the DBD sequence comparisons and right for the LBD. The sequence similarities are presented as the percentage (%) and relevant
color. NRs, with incomplete amino acid sequences of DBD/LBD, were not included in this comparison.
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Figure 5 | Variations in LBD sequence conservation across the sequence of RXRa, ERa and SHP. Left: LBD sequences for eleven vertebrates compared
to the related human nuclear receptors. All sequences were window averaged across 10 residues. Right: multiple sequence alignments among the 12
vertebrates. The sequence similarities are presented as the percentage (%) and relevant color. The LBD sequence of ERa in Dolphin was not included in
this comparison due to the incomplete amino acid sequences.
phenol and bisphenol A (BPA) bound with greater avidity to rainbow
trout ER than that of human or rat. Also, types of ligands were
various: of 34 chemicals tested, 29 can bind to ER of rainbow trout,
while only 20 of them can bind to ER of human/rat37. PPARc is also a
well-studied transcription factor, which could be activated by fatty
acids and is involved in lipid and glucose metabolism38. Reports on
binding strengths of LBDs for PPARc were rare, but interspecies
extrapolations on LBD binding activities can be likely to estimate,
due to the similar sequence characteristics between PPARc and ERa.
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In the third group, with less than 85% similarities in sequences of
eleven vertebrate species compared with those in human, four NRs
including PXR, CAR, DAX1 and SHP (Fig. 4) were classified as being
different from human. DAX1 and SHP, which belong to the subfamily NR0B, displayed the greatest variations among NRs and among
vertebrates (Fig. 4 and 5), a result which is consistent with those
reported previously that NRs in the NR0B group were a unique class
of NRs with among-species variability in sequences and lacking DBD
domains18. PXR and CAR were also assigned to this group, and
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exhibited apparent differences among vertebrates and even among
fishes. PXR and CAR can be activated by xenobiotics and have relatively broad abilities to bind ligands39. The unusually great diversity
in sequences of the LBD among species could be related to diversity
in binding activities among species. This is exemplified by the fact
that phenobarbital, a pharmaceutical that is generally detectable in
effluents of municipal waste water plants (WWTP), was a moderate
activator of the zebrafish PXR and exhibited greater binding affinity
with human PXR, while it did not bind to PXR of mouse39. These
differences among species might be due to the differences in diet and
physiology among vertebrates, and such largely differences of
sequences of PXR and CAR among vertebrates complicated the in
silico extrapolations.
Here, for the first time, genes that code for NRs and their relative
characteristics are provided for 12 vertebrate species used as model
animals in screening of toxic potencies of chemicals. These results
will help understanding of the NRs in vertebrates and will be useful
for clarifying mechanisms of toxic effects of environmental chemicals on these model species and also the extrapolations from the
effects on these surrogates to human.
Methods
Identification of NRs in 12 vertebrate genomics. Identification of sequences for
NRs was performed as described previously40,41 with slight modifications. In brief, the
putative NRs for each vertebrate were identified through a combination of BLASTn
and BLASTp searches of the genome and protein databases, which were obtained
from NCBI and Ensembl. The nucleotide and protein sequences of 165 described NRs
in three vertebrates (48 in human, 49 in mouse and 68 in Fugu rubripes) were
downloaded from GenBank and used as templates for interrogating the vertebrate
databases. Nucleotide homology searches were performed using the full nucleotide
sequences of each of the 165 NRs against these 12 genomic sequences database at
NCBI by use of nucleotide BLAST with a blastn algorithm and an e value cut off of 1e04. Protein sequences were then used to construct multiple sequence alignments by
ClustalX2 (http://www.clustal.org/clustal2/) and then the DNA-binding domain
(DBD) and the ligand-binding domain (LBD) amino acid sequences were
demonstrated. BLASTp searches were performed using the conserved DBD plus LBD
domains against the non-redundant vertebrate protein sequence database at NCBI by
use of protein BLAST with a blastp algorithm and an e value cut off of 1e-25. The e
cut-off values were set to be just loose enough to find all the Fugu NRs when using
human NRs as queries. Genes identified by BLASTn and BLASTp searches were then
combined and individual putative genes were sorted according their unique DNA and
amino acid sequences. All these putative genes were verified by online software
NRpred and iNR-PhysChem to remove the false-positive hits, and the NR0B1 and
NR0B2, which are known to lack the DBD region, were added to the final sets of NRs.
Details for the sequence searches were shown in Table S1. Finally, complete sequences
for each NR in each vertebrate species were loaded into Ensembl database. The
nomenclatures of NRs were based on Ensembl’s GeneTree and Orthology
annotations.
Genomic distributions. Genomic location for each nuclear receptor in seven
vertebrate genomes (human, mouse, rat, chicken, zebrafish, medaka and stickleback)
were retrieved via the Ensembl annotations, and then mapped onto complete
vertebrate karyograms.
Analyses of sequences of DBD and LBD. Sequences of peptides in the DBD and LBD
domains for each NR were identified by use of Pfam software (http://pfam.sanger.ac.
uk/, Pfam 27.0) and modified manually, based on characteristics of DBD and LBD
regions reported previously. The sequence of DBD, which is classified as a type-II zinc
finger motif, corresponds to a 75–80 amino acid residue segment, starting at the
location of two amino acid residues before the first conserved cysteine and
encompassing both C4 zinc fingers and the LBD, a flexible unit made of a-helices
containing of 170 to 210 amino acid residues, begin at the 12th residue of a-helix 3
and extended through a-helix 1042,43.
The pairwise alignments between sequences of the DBD and LBD of human
protein and corresponding orthologs in the other 11 vertebrates were constructed by
use of the NCBI BLASTp software with default parameters. Similarities in sequences
were calculated based on the numbers of identical residues over the total numbers of
aligned residues in human.
Phylogenetic analysis. Phylogenetic trees were constructed by use of amino acid
sequences of 48 types of NRs downloaded from Ensembl based on the set of
homologous NRs in the human. Only full- length molecules were included for the
analysis. Some genes without complete amino acid sequences in the Ensembl database
were retrieved from NCBI/EMBL/DDBJ databases (Table S3). They were also
included. The Ensembl ID of each NR used in the analyses is available in SI Table S2.
Conserved sequences of DBD and LBD for each NR were also isolated and used as a
supportive analysis. Sequences of DBD and LBD were combined and then aligned,
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except for NR0B1 and NR0B2. Multiple alignments of sequences of amino acids were
generated by use of ClustalX2 software with default parameters, and the results used
for construction of phylogenetic trees by implementation of the Neighbour-Joining
and Maximum-Likelihood algorithms with a Poisson model in MEGA6 software
(http://www.megasoftware.net/mega.php). Confidence for branching patterns was
assessed by bootstrap analysis (1000 replicates). For NR1I1 (VDR) analysis, the full
amino acid sequences of NR1I1 in 35 vertebrates, including 20 mammals, 5 birds, 2
reptiles, 1 amphibian and 7 teleost fishes (Table S4), were downloaded from the
Emsenbl database. These full amino acid sequences were then aligned and applied for
gene phylogenetic analysis by use of the same method described above.
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Acknowledgments
This study supported by the National Natural Science Foundation of China [41330637 and
41171385] and the 111 Project (B14001). Prof. Giesy was supported by the Canada Research
Chair program, a Visiting Distinguished Professorship in the Department of Biology and
Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong.
Author contributions
Y.B.Z. and J.Y.H. designed the experiments, Y.B.Z. and K.Z. performed the experiment and
analyzed the data, Y.B.Z., K.Z., J.P.G. and J.Y.H. wrote the manuscript. All authors
contributed to scientific discussions of the manuscript.
Additional information
Supplementary information accompanies this paper at http://www.nature.com/
scientificreports
Competing financial interests: The authors declare no competing financial interests.
How to cite this article: Zhao, Y., Zhang, K., Giesy, J.P. & Hu, J. Families of Nuclear
Receptors in Vertebrate Models: Characteristic and Comparative Toxicological Perspective.
Sci. Rep. 5, 8554; DOI:10.1038/srep08554 (2015).
This work is licensed under a Creative Commons Attribution 4.0 International
License. The images or other third party material in this article are included in the
article’s Creative Commons license, unless indicated otherwise in the credit line; if
the material is not included under the Creative Commons license, users will need
to obtain permission from the license holder in order to reproduce the material. To
view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
9
1
Supplementary information for:
2
Families of Nuclear Receptors in Vertebrate Models: Characteristic and Comparative
3
Toxicological Perspective
4
Yanbin Zhao1, Kun Zhang1, John P. Giesy2,3,4, and Jianying Hu1
5
1
6
Peking University, Beijing 100871, China
7
2
8
Saskatchewan, Saskatoon, Saskatchewan, Canada
9
3
MOE Laboratory for Earth Surface Processes, College of Urban and Environmental Sciences,
Department of Veterinary Biomedical Sciences and Toxicology Centre, University of
Department of Zoology, and Center for Integrative Toxicology, Michigan State University, East
10
Lansing, MI, USA
11
4
12
University of Hong Kong, Kowloon, Hong Kong, SAR, China
Department of Biology & Chemistry and State Key Laboratory in Marine Pollution, City
13
14
Address for Correspondence
15
Dr. Yanbin Zhao; Prof. Dr. Jianying Hu
16
College of Urban and Environmental Sciences
17
Peking University, Yi Fu Second Building
18
Beijing 100871 China
19
TEL & FAX: 86-10-62765520
20
Email: zhaoyb@pku.edu.cn; hujy@urban.pku.edu.cn
Figure S1. Phylogenetic analysis for 48 types of nuclear receptor genes in twelve vertebrate
species. Numbers at branches indicate the bootstrap probabilities (≥90%) with 1,000 replicates.
Neighbour-Joining trees of ClustalX-aligned full amino acid/DBD plus LBD sequences were
constructed and displayed for the majority of NRs. For some trees, which displayed better
topological structures in Maximum-Likelihood analysis, the ML trees were constructed instead.
21
22
23
24
25
26
Human_0B1
Human_0B2
90
Mouse_0B1
93
Mouse_0B2
100
Rat_0B1
Rat_0B2
100
Chicken_0B1
Zebrafish_1A1b
Medaka_1A1a
Zebrafish_0B2a
Tilapia_0B1a
Tilapia_1A1a
Tilapia_0B2
99
Zebrafish_0B1
Zebrafish_1A1a
Turtle_0B2
Turtle_0B1
Medaka_0B2
100
Stickleback_0B1
99
Tilapia_1A1b
Stickleback_0B2
Medaka_1A1b
Tilapia_0B1b
Dolphin_1B1
Human 1A2
96
Mouse_1B1
Mouse 1A2
90
Chicken_1B2
Xenopus_1B1
Duck_1B2
Zebrafish_1B1a
Chicken 1A2
Turtle_1B2
Medaka_1B1
Turtle 1A2
Rat_1B2
Mouse_1B2
99
Chicken_1B1
Rat 1A2
Duck 1A2
99
Human_1B2
98 Rat_1B1
Dolphin 1A2
99
Stickleback_1A1a
Human_1B1
100
Stickleback_1A1b
99
Medaka_0B1
96
Rat_1A1
Xenopus_1A1
Duck_0B2
100
Xenopus_0B1
100
95
98
Chicken_1A1
98
Chicken_0B2
Duck_0B1
99
Mouse_1A1
Dolphin_0B2
Dolphin_0B1
100
Human_1A1
100
Xenopus_1B2
Tilapia_1B1a
Xenopus 1A2
Medaka_1B2b
Zebrafish_1B1b
Zebrafish 1A2
Tilapia_1B1b
Medaka 1A2
Tilapia 1A2
Medaka_1B2a
Stickleback_1B1a
100
Tilapia_1B2b
100
Tilapia_1B2a
Stickleback 1A2
Stickleback_1B2
Human_1C1
Dolphin_1C1
100
Mouse_1C1
Dolphin_1B3
100 Human_1B3
Rat_1B3
100
100
Duck_1C1
100
Tilapia_1B3b
Zebrafish_1C1b
Stickleback_1B3a
Chicken_1C2
97
Medaka_1C1a
99 Duck_1C2
Zebrafish_1C2a
Tilapia_1C1a
Zebrafish_1B3a
Zebrafish_1C2b
Stickleback_1C1b
Zebrafish_1B3b
Stickleback_1C1a
Tilapia_1B3a
Stickleback_1B3b
Stickleback_1C2
Zebrafish_1C1a
Medaka_1B3a
94
100
Rat_1C2
Turtle_1C2
Xenopus_1C1
Medaka_1B3b
Dolphin_1C2
Mouse_1C2
90
Turtle_1C1
100
Xenopus_1B3
97
99
Chicken_1C1
100
Mouse_1B3
Human_1C2
Rat_1C1
100
Medaka_1C1b
Tilapia_1C1b
99
Medaka_1C2
Tilapia_1C2
Xenopus_1C2
27
Human_1C3
Human_1D1
Dolphin_1C3
Mouse_1D1
100
100
Mouse_1C3
100
Xenopus_1C3
Tilapia_1D1
Duck 1D2
99
Turtle 1D2
100
Xenopus 1D2
Zebrafish 1D2b
Medaka_1C3
Human 1F2
Tilapia_1C3
Mouse 1F2
Stickleback_1C3
Stickleback 1D2a
100
Zebrafish 1D2a
Medaka 1D2
Dolphin 1F2
Duck 1F2
Chicken_1F1
100
Duck_1F1
Tilapia 1D2b
100
Rat 1F2
98
97
Tilapia 1D2a
100
Stickleback 1D2b
98
Chicken 1F2
Turtle 1F2
Turtle_1F1
Xenopus 1F2b
Human_1F1
Human_1H2
Zebrafish 1F2
Dolphin_1F1
Dolphin_1H2
Medaka 1F2
Mouse_1F1
Rat_1F1
Mouse_1H2
Tilapia 1F2
100
Stickleback 1F2
Zebrafish_1F1a
97
Chicken 1D2
100
100
Stickleback_1D1
Zebrafish_1C3
93
100
Zebrafish_1D1
100
Rat 1D2
100
Xenopus_1D1
Turtle_1C3
99
Mouse 1D2
Dolphin_1D1
98 Chicken_1C3
Duck_1C3
100
Dolphin 1D2
Rat_1D1
100
98
100 Rat_1C3
Human 1D2
100
100
100
Rat_1H2
96
Human 1H4
Medaka_1F1
93
99 Tilapia_1F1a
Human 1H3
Xenopus_1F1
Dolphin 1H4
Dolphin 1H3
100
Tilapia_1F1b
Mouse 1H3
Zebrafish_1F1b
100
100
99
Duck 1H4
98
Mouse_1F3
Rat_1F3
99
100
Chicken_1F3a
Turtle 1H4
Turtle 1H3
Human_1F3
Chicken 1H4
100
Chicken 1H3
Xenopus 1H4
Xenopus 1H3
Dolphin_1F3
100
Rat 1H4
98
Rat 1H3
Duck 1H3
100
Mouse 1H4
100
Zebrafish 1H4
Stickleback 1H3
99
Zebrafish 1H3
100
Medaka 1H3
95
99
Duck_1F3a
Tilapia 1H4
100
Medaka 1H4
100
Stickleback 1H4
Tilapia 1H3
Turtle_1F3b
Zebrafish_1F3b
99
100
Medaka_1F3b
99
Dolphin 1I1
100
100
Zebrafish_1F3c
Medaka_1F3c
Rat 1I1
Rat 1I2
Xenopus 1I2
Zebrafish 1I2
Duck 1I1
100
Xenopus 1I1
Medaka_1F3a
Medaka 1I2
100
Turtle 1I1
Zebrafish_1F3a
100
Mouse 1I2
100
100
Tilapia_1F3a
100
Dolphin 1I2
Chicken 1I1
Stickleback_1F3a
100
Human 1I2
100
Mouse 1I1
Tilapia_1F3c
98
99
Human 1I1
Stickleback_1F3b
Tilapia 1I2
Zebrafish 1I1b
91
Tilapia_1F3b
Tilapia 1I1a
100
100
Medaka 1I1b
Human_1I3
Dolphin_1I3
100
Stickleback 1I1b
Zebrafish 1I1a
Medaka 1I1a
97
Tilapia 1I1b
Stickleback 1I1a
Mouse_1I3
100
Rat_1I3
Turtle_1I3
Xenopus_1I3
Human 2A1
28
Mouse 2A1
100
100
100
Dolphin 2A1
Mouse 2B1
Rat 2B1
Rat_2A2
Dolphin_2A2
Chicken 2A1
99
97
Mouse_2A2
100 Rat 2A1
100
Human 2B1
Human_2A2
90
100
Chicken_2A2
99
Duck 2A1
Chicken 2B1
Duck_2A2
100
Turtle 2A1
Turtle 2B1
Xenopus_2A2
Zebrafish 2A1
Xenopus 2B1
Medaka_2A2
Tilapia 2A1
99
Duck 2B1
95
100
Turtle_2A2
Xenopus 2A1
100
Dolphin 2B1
Tilapia_2A2
100
Stickleback 2A1
Zebrafish 2B1b
Zebrafish 2B1a
Stickleback_2A2
98
Medaka 2B1a
100
Tilapia 2B1
Human 2B2
99
Human 2B3
Dolphin 2B2
100
Mouse 2B2
Xenopus 2B2
Mouse 2C1
Chicken 2B3
100
Zebrafish 2B2a
100
Duck 2B3
Tilapia 2B2b
96
Turtle 2B3
Duck 2C1
Turtle 2C1
Zebrafish 2B3a
Zebrafish 2B2b
Medaka 2B2b
Xenopus 2C1
Zebrafish 2B3b
100
Tilapia 2B2a
100
100
Xenopus 2B3
Stickleback 2B2b
Stickleback 2C1
100
Tilapia 2B3
100
Stickleback 2B2a
Zebrafish 2C1
Medaka 2B1b
96
Rat 2C1
Chicken 2C1
97
Medaka 2B2a
100
Dolphin 2C1
100
Rat 2B3
99
Human 2C1
98
Mouse 2B3
Rat 2B2
100
Stickleback 2B1
Dolphin 2B3
100
Medaka 2C1
100
Tilapia 2C1
Stickleback 2B3
Human 2E3
Mouse 2E3
Human 2C2
100
Chicken 2E3
Turtle 2E3
Rat_2E1
Chicken 2C2
Xenopus 2E3
Chicken_2E1
Duck 2C2
100
100
Medaka 2E3a
Turtle_2E1
Xenopus 2C2
Rat_2F1
100
Dolphin_2F6
100
Mouse_2F6
98 Rat_2F6
Chicken_2F2
Turtle_2F2
99
Turtle_2F6
Xenopus_2F6
Xenopus_2F2
Zebrafish_2F6a
Duck_2F2
Zebrafish_2F1a
Zebrafish_2F6b
Tilapia_2F2b
Tilapia_2F1
Stickleback_2F1
Human_2F6
Mouse_2F2
100 Rat_2F2
Dolphin_2F1
90
Tilapia 2E3b
90
Human_2F2
Mouse_2F1
Chicken_2F1
Medaka 2E3b
100
93 Tilapia_2E1
Human_2F1
Xenopus_2F1
Stickleback 2E3
Medaka_2E1
99
Tilapia 2C2
99
Stickleback_2E1
100
Medaka 2C2
Tilapia 2E3a
91
Zebrafish_2E1
Stickleback 2C2
100
100
Xenopus_2E1
Zebrafish 2C2
100
Zebrafish 2E3
Duck_2E1
Turtle 2C2
Rat 2E3
Dolphin 2E3
Mouse_2E1
98 Rat 2C2
100
96
100
Dolphin_2E1
Mouse 2C2
94
99
Human_2E1
Dolphin 2C2
100
94
Stickleback_2F6b
Zebrafish_2F2
Medaka_2F2
100
Tilapia_2F6b
Stickleback_2F2b
Tilapia_2F6a
Tilapia_2F2a
100
Stickleback_2F2a
Medaka_2F6b
94
90
90
Medaka_2F6a
Stickleback_2F6a
29
Human 3A1
Human 3A2
Dolphin 3A1
Rat 3A1
100
92
100
Mouse 3A2
100
90
Chicken 3A1
100
99
100
99
Turtle 3B1
Xenopus 3B1
Medaka 3B1
Stickleback 3B1
Medaka 3A2b
97
100
Tilapia 3A2b
Stickleback 3A1
Zebrafish 3A2a
Human_3C1
Stickleback 3A2a
100
Dolphin_3C1
Medaka 3A2a
100
95
Mouse_3C1
Mouse_3B2
99
98
Rat_3B2
100
Dolphin_3B2
90
100 Chicken_3B2
100
99
Duck_3C1
Human_3B3
Turtle_3C1
Dolphin_3B3
Xenopus_3C1
100 Rat_3B3
Turtle_3B2
Zebrafish_3B2
100
Medaka_3C1b
Medaka_3B2a
100
100
Zebrafish_3C1
Duck_3B3
Tilapia_3C1a
92
Xenopus_3B3a
Stickleback_3B2a
Medaka_3C1a
Zebrafish_3B3a
Stickleback_3B2b
Stickleback_3C1a
Tilapia_3B3b
Medaka_3B2b
100
Stickleback_3C1b
Chicken_3B3
100
99
99
100
Tilapia_3B2b
Medaka_3B3a
Stickleback_3B3b
100
Medaka_3B3b
100
Mouse_3C4
100
Zebrafish_3B3b
100
Turtle_3C4
Mouse_3C2
100
100
100
Duck_3C2
99
100
99
Zebrafish_3C2
98
Stickleback_3C2
Stickleback_3C4b
100
Turtle_3C3
Medaka_3C4b
100
Tilapia_3C4b
Xenopus_3C3
Medaka_3C2
Tilapia_3C2
Zebrafish_3C4
Rat_3C3
Chicken_3C3
Xenopus_3C2
100
Xenopus_3C4
Mouse_3C3
100
Turtle_3C2
100
100 Duck_3C4
Dolphin_3C3
100
Chicken_3C2
Chicken_3C4
100
Human_3C3
Rat_3C2
100
Stickleback_3C4a
Stickleback_3C3
Zebrafish_3C3
Rat_3C4
Dolphin_3C4
Stickleback_3B3a
Dolphin_3C2
100
100
Xenopus_3B3b
Human_3C2
Human_3C4
95
Tilapia_3B3a
99
Tilapia_3C1b
Turtle_3B3
Tilapia_3B2a
100
Chicken_3C1
Mouse_3B3
Duck_3B2
100
Rat_3C1
Tilapia 3A2a
Human_3B2
100
Tilapia 3B1
100
Zebrafish 3A2b
Medaka 3A1
Tilapia 3A1
Zebrafish 3B1
Xenopus 3A2
Zebrafish 3A1
Mouse 3B1
99 Rat 3B1
Turtle 3A2
Xenopus 3A1
99
92
Rat 3A2
Duck 3A2
Turtle 3A1
100
100
Chicken 3A2
100 Duck 3A1
Human 3B1
Dolphin 3B1
Dolphin 3A2
Mouse 3A1
100
99
Medaka_3C4a
100
98
Tilapia_3C4a
30
Dolphin 4A1
100
Dolphin 4A3
100
Mouse 4A1
100
91
Rat_4A2
96
Mouse_4A2
100 Rat 4A3
95
Xenopus 4A1
Duck 4A3
Duck 4A1
Turtle 4A3
Zebrafish_4A2a
Zebrafish 4A3
Stickleback 4A1a
Medaka 4A1b
94
Chicken_4A2
Xenopus_4A2a
100
95
Zebrafish 4A1
100
Turtle_4A2
99
Chicken 4A3
100
Dolphin_4A2
100
Mouse 4A3
Rat 4A1
Turtle 4A1
100
91 Human_4A2
Human 4A3
Human 4A1
99
Medaka 4A3
100
99
Medaka 4A1a
Medaka_4A2a
99
Tilapia_4A2a
Tilapia 4A3
100
Tilapia 4A1a
Stickleback_4A2
Zebrafish_4A2b
Stickleback 4A3
Medaka_4A2b
Tilapia 4A1b
100
100
Stickleback 4A1b
Tilapia_4A2b
Xenopus_4A2b
Human_5A1
100
98 Human 5A2
Dolphin_5A1
Mouse_5A1
97
Dolphin 5A2
100
100
98
Xenopus_5A1
Zebrafish_5A1b
Zebrafish_5A1a
Stickleback_5A1a
99
99
Tilapia_5A1
Medaka_5A1b
Stickleback_5A1b
Human_6A1
Mouse_6A1
Rat_6A1
Chicken 5A2
Dolphin_6A1
96
Duck 5A2
100
Chicken_6A1
99 Turtle 5A2
Turtle_6A1
90
Xenopus 5A2
Zebrafish 5A2
Medaka_5A1a
100
100
Rat 5A2
Chicken_5A1
100
90
Mouse 5A2
100 Rat_5A1
Stickleback 5A2
100
99
Medaka 5A2
Tilapia 5A2
Xenopus_6A1
Zebrafish_6A1a
97
Tilapia_6A1
100 Stickleback_6A1
Zebrafish_6A1b
31
32
Figure S2. Schematic diagram depicts the evolution of PXR and CAR in vertebrates.
33
34
Table S1. Details for nuclear receptor sequence searches in 12 model vertebrates.
Human
Mouse
Rat
Dolphin
Chicken
Duck
Turtle
Xenopus
Zebrafish
Medaka
Tilapia
Stickleback
35
BLASTn
Hits
BLASTp
Hits
Sum
After
sortation
Verified by
software
NR0B
Subfamily
Final sets
of NRs.
33849
23014
8312
2834
3712
2381
2922
2289
9788
3601
7586
571
24967
12540
8896
2752
3761
4034
3421
3850
9230
4090
6630
268
58816
35554
17208
5586
7473
6415
6343
6139
19018
7691
14216
839
57
62
70
74
50
48
48
53
72
78
83
64
46
47
47
45
42
40
46
50
70
65
71
64
2
2
2
2
2
2
2
2
3
2
3
2
48
49
49
47
44
42
48
52
73
67
74
66
36
37
Table S2. Sequence ID. for each nuclear receptor gene in Ensembl database.
Human
Mouse
Rat
Dolphin
Chicken
Duck
Turtle
Xenopus
Zebrafish
Medaka
Tilapia
Stickleback
ENSG0000012
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
6351
000058756
000009066
00016893
000000270
00016001
0012754
00024399
000000151
00016941
00018247
00003766
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
000052654
00012005
00006456
00006540
NR1A1
ENSG0000015
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
1090
000021779
000006649
00001859
000011294
00006081
0008182
00003871
000021163
00008122
00010312
00007996
ENSG0000013
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
1759
000037992
000009972
00016901
000005629
00006377
0002372
00024390
000056783
00004373
00019915
00012955
ENSDARG00
ENSONIG000
ENSGACG000
000034893
00006314
00005297
NR1A2
NR1B1
ENSG0000007
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSORLG000
ENSONIG000
ENSGACG000
7092
000017491
000024061
00010874
000011298
00006432
0007930
00007272
00008502
00010320
00007999
ENSORLG000
ENSONIG000
00016394
00006493
NR1B2
ENSG0000017
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
2819
000001288
000012499
00002778
00012670
000034117
00015382
00012223
00009372
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
000054003
00007861
00019165
00000612
NR1B3
ENSG0000018
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
6951
000022383
000021463
00004136
000022985
00010641
0018221
00023454
000031777
00002413
00016715
00018958
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
000054323
00011091
00008831
00003703
NR1C1
ENSG0000011
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
2033
000002250
000000503
00009416
000002588
00004751
0005889
00015121
000044525
00006636
00011871
00008288
NR1C2
ENSDARG00
000009473
ENSG0000013
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
2170
000000440
000008839
00016565
000004974
00009031
0011100
00017422
000031848
00004432
00014331
00001665
ENSG0000012
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSONIG000
ENSGACG000
6368
000020889
000009329
00016894
0014806
00024397
000033160
00009283
00009356
ENSG0000017
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
4738
000021775
000046912
00010829
000011291
00005753
0008488
00003869
000003820
00016431
00008699
00012958
ENSDARG00
ENSONIG000
ENSGACG000
000009594
00010308
00007986
NR1C3
NR1D1
NR1D2
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
000031161
00007837
00012213
00000614
ENSDARG00
ENSORLG000
ENSONIG000
000059370
00015399
00019164
NR1D4
ENSG0000006
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
9667
000032238
000027145
00007718
000003759
00005866
0011314
00021123
000031768
00007645
00015289
NR1F1
ENSDARG00
ENSONIG000
000001910
00015603
ENSG0000019
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
8963
000036192
000013413
00008387
000015150
00007187
0005579
00031251
000033498
00012441
00010762
00011556
NR1F2
ENSXETG000
00008148
ENSG0000014
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
3365
000028150
000046831
00003151
000025988
00013051
0008995
00002131
000087195
00009486
00004686
00012280
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
000001035
00011493
0016262
000057231
00003765
00010247
00015341
ENSDARG00
ENSORLG000
ENSONIG000
000017780
00014886
00006222
NR1F3
ENSG0000002
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
5434
000002108
000013172
00014149
000008202
00010925
0010360
00000307
000043170
00001286
00005828
00017167
NR1H3
ENSG0000013
ENSMUSG00
ENSRNOG00
ENSTTRG000
1408
000060601
000019812
00002416
ENSMUSG00
ENSRNOG00
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSONIG000
ENSGACG000
000048938
000023073
000002170
00008338
0003828
00021443
000031046
00009252
00004938
ENSG0000001
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
2504
000047638
000007197
00016373
000011594
00013289
0005774
00030372
000057741
00011270
00014678
00011745
ENSG0000011
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
1424
000022479
000008574
00012578
000026166
00005087
0018108
00010658
000043059
00001063
00009200
00004763
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
000070721
00016402
00019378
00007975
NR1H2
NR1H5
NR1H4
NR1I1
ENSG0000014
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
4852
000022809
000002906
00016650
00018029
000029766
00017953
00014385
ENSG0000014
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSPSIG0000
ENSXETG000
3257
000005677
000003260
00009227
000028624
0004437
00031759
ENSG0000010
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
1076
000017950
000008895
00013004
000004285
00008950
0012689
00001775
000021494
00016380
00016515
00011485
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSONIG000
000015670
00011331
0017650
00016389
000012764
00005911
NR1I2
NR1I3
NR2A1
NR2A3
ENSG0000016
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
4749
000017688
000008971
00003691
000005708
00011794
0003756
00017845
000071565
00006996
00014490
00002422
ENSG0000018
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
6350
000015846
000009446
00009492
000002626
00013150
0011977
00012733
000057737
00012155
00013076
00018189
ENSDARG00
ENSORLG000
000035127
00016690
NR2A2
NR2B1
ENSG0000020
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
4231
000039656
000000464
00004291
00020416
000078954
00006476
00020007
00000096
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
000002006
00007020
00002873
00007982
NR2B2
ENSG0000014
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSONIG000
ENSGACG000
3171
000015843
000004537
00003653
000003406
00004831
0004871
00004750
000005593
00002143
00011685
NR2B3
ENSDARG00
000004697
ENSG0000012
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
0798
000005897
000006983
00016305
000011327
00006253
0017190
00023840
000045527
00004114
00008566
00010174
ENSG0000017
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
7463
000005893
000010536
00009876
000008519
00007538
0008928
00004817
000042477
00010877
00017240
00002941
ENSG0000011
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
2333
000019803
000050550
00008863
000015305
00010675
0006035
00014853
000017107
00013426
00013281
00008934
ENSG0000003
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
1544
000032292
000050690
00009410
000002093
0017480
00005219
000045904
00000011
00007109
00004739
ENSORLG000
ENSONIG000
00007175
00015396
NR2C1
NR2C2
NR2E1
NR2E3
ENSG0000017
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
5745
000069171
000014795
00001519
000027907
0009818
00011594
000052695
00010191
00011840
00010385
NR2F1
ENSPSIG0000
ENSDARG00
0010198
000017168
ENSG0000018
ENSMUSG00
ENSRNOG00
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
5551
000030551
000010308
000007000
00010629
0017164
00022346
000040926
00008429
00015133
00013235
ENSONIG000
ENSGACG000
00003070
00014846
NR2F2
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
00011046
000033172
00016315
00008594
00013191
NR2F5
ENSG0000016
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
0113
000002393
000016892
00003132
000027294
00003193
0013773
00013531
000003607
00008749
00010512
00007766
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
000003165
00008911
00010104
00015583
NR2F6
ENSG0000009
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
1831
000019768
000019358
00002996
000012973
00004585
0004166
00012364
000004111
00014514
00013354
00008711
ENSG0000014
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
0009
000021055
000005343
00000517
000011801
00011895
0018210
00007257
000016454
00017721
00005633
00007514
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
000034181
00018012
00001710
00000213
NR3A1
NR3A2
ENSG0000017
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
3153
000024955
000021139
00010296
0016751
00007211
000069266
00010624
00001778
00020287
ENSG0000011
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
9715
000021255
000010259
00001302
000010365
00012470
0017916
00013217
000040151
00016581
00015282
00010561
ENSORLG000
ENSONIG000
ENSGACG000
00009126
00020192
00007542
NR3B1
NR3B2
ENSG0000019
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
6482
000026610
000002593
00006004
000009645
00005309
0005595
00020932
000004861
00011528
00000573
00013426
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
00016948
000011696
00016819
00017162
00016275
ENSDARG00
ENSONIG000
ENSGACG000
000015064
00001134
00004898
NR3B3
NR3B4
ENSG0000011
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
3580
000024431
000014096
00003260
000007394
00007318
0015245
00001879
000025032
00006022
00017907
00018209
ENSORLG000
ENSONIG000
ENSGACG000
00001565
00008483
00020725
NR3C1
ENSG0000015
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
1623
000031618
000034007
00014440
000010035
00015146
0006383
00026061
000037025
00007530
00010029
00017193
ENSG0000008
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSGACG000
2175
000031870
000006831
00000030
000017195
00003887
0013654
00005482
000035966
00002651
00012162
ENSG0000016
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
9083
000046532
000005639
00004230
000004596
00006566
0010176
00005089
000067976
00008220
00012854
00018525
ENSORLG000
ENSONIG000
ENSGACG000
NR3C2
NR3C3
NR3C4
00009520
00017538
00020332
ENSG0000012
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
3358
000023034
000007607
00002817
00014123
0018018
00000579
000000796
00015557
00016717
00010788
ENSORLG000
ENSONIG000
ENSGACG000
00015279
00019260
00000045
NR4A1
ENSG0000015
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
3234
000026826
000005600
00005740
000012538
00012071
0008054
00031753
000017007
00016692
00008976
00005831
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
00024016
000044532
00000050
00012131
NR4A2
ENSG0000011
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
9508
000028341
000005964
00007458
000013568
00011263
0012281
000055854
00008732
00006026
00009027
ENSG0000013
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
6931
000026751
000012682
00017390
000001080
00004548
0006131
00011456
000017704
00016486
00020218
00003539
ENSDARG00
ENSORLG000
ENSGACG000
000023362
00013196
00018317
NR4A3
NR5A1
ENSG0000011
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
6833
000026398
000000653
00003256
000002182
00009302
0003632
00000314
000042556
00006933
00012517
00008896
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
000039116
00006019
00001686
00009952
NR5A2
NR5A5
ENSG0000014
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
8200
000063972
000013232
00017391
000001073
00004788
0006445
00008578
000018030
00016492
00020217
00003560
NR6A1
ENSDARG00
000014480
ENSG0000016
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
9297
000025056
000003765
00013272
000016287
00003894
0009740
00015374
000056541
00011824
00012111
00002817
NR0B1
ENSONIG000
00006662
ENSG0000013
ENSMUSG00
ENSRNOG00
ENSTTRG000
ENSGALG00
ENSAPLG000
ENSPSIG0000
ENSXETG000
ENSDARG00
ENSORLG000
ENSONIG000
ENSGACG000
1910
000037583
000007229
00016680
000000887
00010744
0017134
00011771
000044685
00004442
00006772
00007198
NR0B2
38
39
40
Table S3. Genes with incomplete/without DBD/LBD regions in the Ensembl database. Genes
marked in red means the full sequences were retrieved in NCBI/ EMBL/DDBJ databases.
Gene and related Ensembl ID.
Human
—
Mouse
—
Rat
Dolphin
NR2E3 (ENSRNOG00000050690); NR3A1 (ENSRNOG00000019358);
NR3C3 (ENSRNOG00000006831);
NR5A2 (ENSRNOG00000000653)
NR1A1 (ENSTTRG00000016893);
NR1B2 (ENSTTRG00000010874);
NR1C1 (ENSTTRG00000004136);
NR1F3 (ENSTTRG00000003151);
NR1I2 (ENSTTRG00000016650);
NR2A1 (ENSTTRG00000013004);
NR2B1 (ENSTTRG00000009492);
NR3A1 (ENSTTRG00000002996);
NR2B3 (ENSTTRG00000003653); NR2F6 (ENSTTRG00000003132);
NR3B2 (ENSTTRG00000001302);
NR4A1 (ENSTTRG00000002817);
NR4A3 (ENSTTRG00000007458)
Chicken
Duck
Turtle
NR1B1 (ENSGALG00000005629);
NR2F6 (ENSGALG00000027294);
NR1A1 (ENSAPLG00000016001);
NR1B1(ENSAPLG00000006377);
NR1F2 (ENSAPLG00000007187);
NR1F3b (ENSAPLG00000011493); NR1H4 (ENSAPLG00000013289);
NR1I1 (ENSAPLG00000005087);
NR2F6 (ENSAPLG00000003193);
NR3A1 (ENSAPLG00000004585);
NR3C3 (ENSAPLG00000003887);
NR4A1 (ENSAPLG00000014123);
NR4A2 (ENSAPLG00000012071);
NR5A1 (ENSAPLG00000004548);
NR6A1 (ENSAPLG00000004788);
NR0B1 (ENSAPLG00000003894)
NR1A1 (ENSPSIG00000012754);
NR1B1 (ENSPSIG00000002372);
NR1D1 (ENSPSIG00000014806);
NR2A2 (ENSPSIG00000003756);
NR2E3 (ENSPSIG00000017480);
NR2F1b (ENSPSIG00000010198);
NR3B1 (ENSPSIG00000016751);
NR4A2 (ENSPSIG00000008054);
NR5A1 (ENSPSIG00000006131);
NR1C3 (ENSXETG00000017422);
NR1F3 (ENSXETG00000002131);
NR6A1 (ENSPSIG00000006445)
Xenopus
Zebrafish
Medaka
Tilapia
Stickleback
NR1C2 (ENSXETG00000015121);
NR2A1 (ENSXETG00000001775);
NR4A2b (ENSXETG00000024016)
NR1C3 (ENSDARG00000031848);
NR1I1 (ENSDARG00000043059)
NR1B1 (ENSORLG00000004373);
NR2A1 (ENSORLG00000016380);
NR3C3 (ENSORLG00000002651);
NR6A1 (ENSORLG00000016492)
—
—
NR2F1 (ENSORLG00000010191);
41
42
43
Table S4. The vertebrate species used for NR1I1 (VDR) gene phylogenetic analysis.
Common name
Scientific name
Common name
Scientific name
Human
Gibbon
Gorilla
Macaque
Marmoset
Bushbaby
Cat
Dog
Ferret
Hedgehog
Rabbit
Dolphin
Pig
Opossum
Cow
Sheep
Mouse
Rat
Guinea Pig
Squirrel
Homo sapiens
Nomascus leucogenys
Gorilla gorilla gorilla
Macaca mulatta
Callithrix jacchus
Otolemur garnettii
Felis catus
Canis lupus familiaris
Mustela putorius furo
Erinaceus europaeus
Oryctolagus cuniculus
Tursiops truncatus
Sus scrofa
Monodelphis domestica
Bos taurus
Ovis aries
Mus musculus
Rattus norvegicus
Cavia porcellus
Ictidomys tridecemlineatus
Flycatcher
Zebra Finch
Duck
Chicken
Turkey
Ficedula albicollis
Taeniopygia guttata
Anas platyrhynchos
Gallus gallus
Meleagris gallopavo
Anole lizard
Chinese softshell turtle
Anolis carolinensis
Pelodiscus sinensis
Xenopus
Xenopus tropicalis
Coelacanth
Tilapia
Zebrafish
Tetraodon
Medaka
Platyfish
Stickleback
Latimeria chalumnae
Oreochromis niloticus
Danio rerio
Tetraodon nigroviridis
Oryzias latipes
Xiphophorus maculatus
Gasterosteus aculeatus