Trichoplax, the simplest known animal, contains an estrogen-related receptor but no estrogen
receptor: Implications for estrogen receptor evolution
Michael E. Baker*
Department of Medicine, 0693
University of California, San Diego
9500 Gilman Drive
La Jolla, CA 92093-0693
*Corresponding author
E-mail:
mbaker@ucsd.edu
Phone: 858-534-8317; Fax: 858-822-0873
Abstract. Although, as their names imply, estrogen receptors [ERs] and estrogen-related
receptors [ERRs] are related transcription factors, their evolutionary relationships to each other
are not fully understood. To elucidate the origins and evolution of ERs and ERRs, we searched
for their orthologs in the recently sequenced genome of Trichoplax, the simplest known animal,
and in the genomes of three lophotrochozoans: Capitella, an annelid worm, Helobdella robusta,
a leech, and Lottia gigantea, a snail. BLAST searches found an ERR in Trichoplax, but no ER.
BLAST searches also found ERRs in all three lophotrochozoans and invertebrate-like ERs in
Capitella and Lottia, but not in Helobdella. Unexpectedly we find that the Capitella ER
sequence is closest to ER
, unlike the other invertebrate ER sequences, which are closest to
ER
. Our database searches and phylogenetic analysis indicate that invertebrate ERs evolved in
a lophotrochozoan and steroid-binding ERs evolved in a deuterostome.
Key words: estrogen receptor evolution, invertebrate estrogen receptors; estrogen related
receptor, nuclear receptors, Trichoplax, lophotrochozoans,
Introduction.
From the beginning, when estrogen-related receptor
[ERR] and ERR were first
cloned [1], the ERR has been an enigma [2, 3]. As its name implies, ERR sequences are similar
to that of vertebrate estrogen receptors [ER]. The ligand-binding domain of human ER and
ERR
and ERR have about 35% sequence identity and 60% positive matches, when
conservative replacements such as arginine/lysine and glutamic acid/aspartic acid are considered.
.
2
Yet these ERRs do not bind estradiol or other steroids [1-3]. Subsequently, ERR
[4] was cloned
and also found to lack steroid-binding activity. Indeed, a bona fide biological ligand for an ERR
has not yet been identified. As a result, the ERR belongs to the orphan receptor group [5, 6] in
the nuclear receptor family of transcription factors [7-10].
An explanation for the absence of steroid binding by ERRs came from analysis of the
crystal structures of human ERR
[11, 12] and ERR [13], which showed that the ligand binding
site is too small to accommodate a steroid [11-14]. The crystal structures also showed that in
ERR
and ERR the activation function 2 (AF2) domain on -helix 12 is in a conformation for
productive interactions with coactivators [15, 16], which explains why the ERR does not require
a ligand to become transcriptionally active in cell assays [1-5]. In the last few years, there has
been progress in beginning to elucidate ERR functions, which include regulating bone formation
[2, 3, 17, 18] and mitochondrial biogenesis [18-20].
Complicating understanding of the evolution of ERRs and vertebrate ERs was the cloning
in the last five years of several invertebrate ERs from mollusks [21-25]. Invertebrate ERs, such
as octopus ER, have about 34% sequence identity and 58% positive matches with the estrogen-
binding domain in human ER
, and 29% identity and 56% positive matches with human ERR.
Similar to ERRs, invertebrate ERs do not bind estradiol with high affinity, in contrast to
vertebrate ERs, which are activated by 0.2 nM estradiol [26]. Also, similar to ERRs, invertebrate
ERs are constitutively active transcription factors in cell assays [21-25]. A biological function
for invertebrate ERs has not been reported.
The phylogenetic relationships of vertebrate and invertebrate ERs to each other and to
ERRs are still not fully understood [9, 17, 18, 21, 25]. When did the ancestral ER/ERR arise?
Was this ancestor more like an ERR or an ER? How did the estrogen-binding vertebrate ER and
the constitutively active invertebrate ERs evolve [8, 27-29]? That is, did vertebrate and
invertebrate ERs evolve from a gene duplication of an ancestral ER, or did the vertebrate and
invertebrate ERs evolve from separate ancestral genes? An opportunity to address these
questions comes from recent sequencing by the Joint Genome Initiative [http://genome.jgi-
psf.org] of genomes of Trichoplax, which is considered to be the simplest metazoan [30-32], and
of three lophotrochozoans: Capitella, a segmented worm, Helobdella, a leech and Lottia, a snail.
As reported here, BLAST [33] searches found an ERR, but no ER in Trichoplax,
indicating that ERRs are more ancient than ERs. BLAST searches of the three recently
.
3
sequenced lophotrochozoan genomes found ERRs in Capitella, Helodbdella and Lottia, and
invertebrate ERs in Capitella and Lottia. The current genome release of Helobdella does not
contain an invertebrate ER. To our surprise, a BLAST search of GenBank with the Capitella ER
sequence indicates that it is closest to ER
, in contrast to the other invertebrate ER sequences,
which are closest to ER
.
The evidence that invertebrate-like ERs are restricted to lophotrochozoans and our
phylogenetic analysis of protostome and deuterostome ERRs and ERs indicates that invertebrate
ERs share a common ancestor with protostome ERRs, and steroid-binding vertebrate ERs
evolved from an ancestor in a deuterostome [28].
Methods
BLAST [33] was used to collect ERR and ER sequences from the JGI server
[http://genome.jgi-psf.org] and GenBank. Two different methods, Clustal X 2.0 [34], which uses
a neighbor-joining algorithm [35], and PHYML [36], which uses a maximum likelihood
algorithm, were used to construct phylogenetic trees of various ERs, ERRs, human retinoid X
receptor-
(RXR) and amphioxus RXR.
For the Clustal X 2.0 phylogeny, the multiple alignment of ERs, ERRs and RXRs was
done using the iteration option for each alignment step in the multiple alignment. This alignment
was converted to a phylogenetic tree using the neighbor-joining algorithm [35] with a correction
of branch lengths for rate heterogeneity between sites.
For PhyML, the Muscle algorithm [37] was used to construct a multiple alignment.
PhyML was used with the WAG substitution model [38] and a gamma distribution of rates
between sites (four categories, parameter estimated by PhyML), and 100 bootstrap replicates.
The phylogenetic trees with Clustal X 2.0 and PhyML gave similar topologies and bootstrap
values.
Accession numbers are human ERR
[GenBank:AAQ93376], human ER
[GenBank:NP_000116], Aplysia californica (California sea hare) ER [GenBank:AAQ95045],
Capitella (polychaete worm) ER [jgi|Capca1|170275], Crassostrea gigas (Pacific oyster) ER
[GenBank:BAF45381], Lottia gigantea (Owl limpet) ER [jgi|Lotgi1|132166], Marisa
cornuarietis (giant rams horn) ER [GenBank:ABI97119], Nucella lapillus (Atlantic dogwhelk)
ER [GenBank: ABQ96884], Octopus vulgaris (Octopus) ER [GenBank:ABG00286], Thais
.
4
clavigera (rock shell) ER [GenBank:BAC66480], Human ER
[GenBank:6166154], Xenopus
tropicalis ER
[GenBank:NP_988866] and ER [GenBank:NP_001035101], Drosophila
melanogaster ERR [GenBank: NP_729340], Apis mellifera ERR [GenBank: 110756963],
Daphnia ERR [jgi|Dappu1|46682], Capitella ERR [jgi|Capca1|108381], Lottia ERR
[jgi|Lotgi1|168715], Helobdella ERR [jgi|Helro1|106750], human RXR
[GenBank:
NP_002948] and amphioxus RXR [GenBank: AAM46151]
Results and Discussion
Four Nuclear Receptors are present in a basal diploblast
The DNA and ligand-binding domains of human ERR
, human ER, octopus ER,
Aplysia ER, Thais ER and oyster ER were used as queries for BLAST searches for orthologs in
Trichoplax, Capitella, Helobdella, and Lottia on the JGI server. The BLAST search of
Trichoplex with human ERR
yielded four high scoring nuclear receptors. Searches with human
ER
and invertebrate ERs found the same genes in Trichoplax. To classify the four Trichoplax
genes, we used their sequences as queries for BLAST searches of GenBank. This identified
Trichoplex
jgi|Triad1|16711|gw1.23.179.1
as an ortholog of ERR; the other Trichoplax genes
appear to be orthologs of COUP, RXR or HNF4 [Table 1]. Thus, genes with similarity to
vertebrate ERR, COUP, RXR and HNF4 are found in a primitive multicellular animal belonging
to the phylum Placozoa [30-32].
Our analysis does not exclude the possibility of other nuclear receptors in Trichoplax
because our BLAST search focused on finding ancestors of ER and ERR. Also nuclear receptor
genes may have been lost in Trichoplax during its evolution from an ancestral metazoan.
Analyses of other simple metazoan genomes will provide a more definitive inventory of nuclear
receptors in basal metazoans.
We focused the rest of our analyses on the relationship of the Trichoplex ERR-like gene
to invertebrate and vertebrate ERRs and ERs. BLAST searches of the JGI server retrieved ERRs
from Capitella, Helobdella and Lottia, and an invertebrate ER from Capitella and Lottia.
BLAST did not find an invertebrate ER in Helobdella.
.
5
Table 1. Nuclear Receptor Genes in Trichoplax
Gene ID in JGI Databank
Homolog in
GenBank
BLAST
score
% Identity and %Positives, and
Gaps
jgi|Triad1|16711|gw1.23.179.1
ligand-binding domain
Human ERR
pdb|1KV6|A
6e-55
Identities: 99/222 (44%)
Positives:155/222 (69%)
Gaps: 0/222 (0%)
>
jgi|Triad1|49897|fgeneshTA2_pm
.C_scaffold_2000050
Human RXR
AAH63827.1|
1e-124 Identities:216/330
(65%)
Positives:260/330 (78%)
Gaps:17/330 (5%)
jgi|Triad1|50786|fgeneshTA2_pm.
C_scaffold_12000032
HNF4
NP_849180.1
1e-125
Identities: 223/324 (68%)
Positives: 265/324 (81%)
Gaps: 7/324 (2%)
>
jgi|Triad1|9010|gw1.23.150.1
DNA-binding domain
Human ERR
AAH64700.1
1e-28
Identities: 53/85 (62%)
Positives: 67/85 (78%)
Gaps: 0/85 (0%)
>
jgi|Triad1|21656|e_gw1.2.1246.1
Human
COUP
ref|NP_005645.1
3e-73
Identities: 143/322 (44%)
Positives; 205/322 (63%)
Gaps; 12/322 (3%)
The Trichoplax genome at JGI was searched with the amino acid sequence for human ER
.
Column 1 lists the five entries that were retrieved, two of which correspond to the ligand-binding
and DNA-binding domains of human ERR
.
Columns 2 and 3 show the highest scoring entry in GenBank and its BLAST score.
Column 4 shows the % identities, positives, which include identities and conservative
replacements, and the gaps in the BLAST alignment.
A BLAST search of the JGI server retrieved an ERR from Daphnia, a water flea.
BLAST did not find an invertebrate ER in Daphnia. We also used BLAST to retrieve
invertebrate ER sequences from A. californica, O. vulgaris, C. gigas, and two snails: T. clavigera
[23] and M. cornuarietis [22] and ERR sequences from M. cornuarietis, A. mellifera, and D.
melanogaster.
Divergence of Capitella ER from other invertebrate ERs.
To better understand the relationship of invertebrate ERs to steroid-binding ERs, we used
BLAST to search GenBank with the domain on each invertebrate ER which corresponds to the
steroid binding domain on vertebrate ERs, with the goal of determining how similar each
invertebrate ER is to the steroid-binding vertebrate ERs. To our surprise, Capitella ER is closest
to vertebrate ER
, unlike the other invertebrate ER sequences, which are closest to ER. For
example, BLAST found minnow ER
[GenBank: ABS84945] as the closest vertebrate protein to
Capitella ER. Following in the BLAST output were eleven ER
entries and then amphioxus
ERR [GenBank: AAU88063]. Much later in the BLAST output was mouse ER
[GenBank:
.
6
P19785]. BLAST searches showed that the other invertebrate ERs were closest to vertebrate
ER
, and then closest to vertebrate ER and then closest to vertebrate ERR.
To follow-up these BLAST analyses, we did pairwise BLAST comparisons of each
invertebrate ER with human ER
, ER and ERR. As shown in Table 2, pairwise BLAST
analyses show that Capitella ER is closer to human ER
than to human ER. Interestingly,
octopus ER has about equal sequence similarity to human ER
and human ER. However, a
BLAST search of GenBank found that octopus ER clearly was closest to ER
. BLAST found
the closest vertebrate sequence to octopus ER was golden hamster ER
[GenBank: AAD53956],
which was followed by over twenty ER
sequences. The pairwise BLAST analyses in Table 2
show that the other invertebrate ERs are closer to ER
than to ER.
Table 2. Invertebrate ERs and ERRs in JGI and GenBank
Animal Characteristics
ER
ERR
Trichoplax
Diploblast
Placozoan
No Yes
Fruit Fly
Drosophila
Ecdysozoan
Arthropod
No Yes
Water Flea
Daphnia
Ecdysozoan
Arthropod
No Yes
Sea Slug
Aplysia **
Lophotrochozoan
Mollusk
Yes, *
None in GenBank
Snail
Thais **
Lophotrochozoan
Mollusk
Yes, *
None in GenBank
Snail
Marissa **
Lophotrochozoan
Mollusk
Yes, *
Yes
Oyster **
Crassostrea
Lophotrochozoan
Mollusk
Yes, *
None in GenBank
Octopus **
Lophotrochozoan
Mollusk
Yes, *
None in GenBank
Snail
Lottia
Lophotrochozoan
Mollusk
Yes No
Bristle Worm
Capitella
Lophotrochozoan
Annelid
No Yes
Leech
Helobdella
Lophotrochozoan
Annelid
No Yes
*Constitutive transcriptional activity. Receptor does not bind estradiol. **Complete genome has
not been sequenced.
.
7
Invertebrate ERs evolved in lophotrochozoans
To clarify further the evolutionary relationships of various invertebrate and vertebrate
ERRs and ERs, we constructed a phylogenetic tree of their ligand-binding domains as shown in
Figure 1. The vertebrate ER and ERR part of the phylogeny is in agreement with previous
analyses [8, 29, 39]. The phylogeny indicates that vertebrate ERs and invertebrate ERs diverged
from a common ancestor at node A before the evolution of deuterostomes.
Human RXR
Amphioxus RXR
Trichoplax ERR
Human ERR
Amphioxus ERR
Helobdella ERR
Capitella ERR
Lottia ERR
Marisa ERR
69
100
Apis ERR
Fly ERR
Daphnia
ERR
69
89
100
66
95
Capitella ER
Oyster ER
Octopus ER
Marisa ER
Nucella ER
Thais ER
100
Aplysia ER
Lottia ER
80
61
81
59
Lamprey ER
Human ER
Chick ER
55
Human ER
Chick ER
98
100
100
53
100
99
43
74
98
94
100
0.2
0.4
0.9
1.3
0.5
0.3
0
.
5
0.3
0.6
1.3
0.4
A
1.1
X. tropicalis ER
X. tropicalis ER
Figure 1. Phylogenetic analysis of invertebrate and vertebrate ERs and ERRs.
The phylogenetic tree was constructed using PhyML [36] under the WAG substitution model
[38], with a gamma distribution of rates between sites (four categories, parameter estimated by
PhyML), and 100 bootstrap replicates. Shown at the nodes are bootstrap values for each branch
of the tree, which is the percent this cluster was found in the 100 bootstrap trials. Branches with
bootstrap values that are greater than fifty percent are significant. Branch lengths are
proportional to the distance between proteins. Due to space limitations, we show values for
selected branches. Human RXR
and amphioxus RXR were used as outgroups for the
phylogenetic tree.
.
8
Like our BLAST analyses [Table 2], the phylogeny shows that Capitella ER has diverged
substantially from the other invertebrate ERs, which cluster together.
The phylogeny and the absence of invertebrate ERs in ecdyzoa suggests that invertebrate
ERs arose in a lophotrochozoan from an ERR-like ancestor. A practical application of this
phylogeny is to suggest that invertebrate ERs are likely to have function(s) that resemble ERR
functions [2, 17-20].
The evolution of invertebrate ERs from an ERR-like ancestor is consistent with
functional similarities between invertebrate ERs and vertebrate ERRs. Both vertebrate ERRs
and invertebrate ERs are constitutively active and do not bind estradiol. The crystal structures of
human ERRs [11-14] and a 3D model of octopus ER [40] indicates that their ligand-binding
domains are too small to accommodate estradiol.
Steroid-regulated vertebrate ERs evolved in a deuterostome
The absence of an invertebrate ER outside of lophotrochozoans and the absence of an
invertebrate ER in the recently completed sea urchin genome [41] suggests that a steroid-binding
vertebrate ER evolved in a deuterostome [27, 28, 42, 43], in which case, vertebrate and
invertebrate ERs evolved from different ancestors.
Which mutations led to the evolution of estrogen-dependent activation in vertebrate ERs?
Analyses of the 3D structures of human ERR
[11] and ERR [13] reveals that the volume of
their ligand-binding pockets are about 100 Å
3
and 220 Å
3
, respectively, which is much less than
369 Å
3
found in human ER
[14]. The more compact ligand-binding pocket in ERRs [13,14] is
thought to explain why ERRs do not bind estradiol, which has a van der Waals volume of 251
Å
3
[44] and, thus, easily fits into human ER
. The 3D structures of ERR and ERR also reveal
that the AF2 domain is in a position to have productive interactions with coactivators and
regulate gene transcription.
Information clarifying the basis for the transcriptional properties of ERR
comes from
Greschik et al. [13], who modeled estradiol in ERR
and compared it with estradiol in ER [45].
They identified two residues, Leu-345 and Phe-435 in ERR
that had steric clashes with the D
ring of estradiol. Mutation of these residues to Ile and Leu, respectively, as found in ER
,
reduced steric interference with estradiol. As a result, the mutant ERR
bound estradiol,
although with low affinity. However, as Greschik et al. [13] noted, unexpectedly, there was no
.
9
change in transcriptional activity of the ERR
mutant, which suggests that in the estradiol-ERR
mutant complex, the AF2 domain is in the proper configuration to bind coactivators. This
contrasts with binding of estradiol to vertebrate ERs, which causes a conformational change in
AF2 on
-helix 12, so that the ER can bind co-activators [45]. A similar conformational change
occurs in other steroid receptors upon binding of their cognate steroid [15, 16].
If the ligand-activated vertebrate ER evolved from an ERR, then Greschik et al.'s studies
indicate that additional mutation(s) in the ERR ancestor had to occur to increase the affinity for
estradiol and also alter the configuration of AF2 in order for binding of a ligand to be required
for transcriptional activity. Alignment ERR
with human ER [13] [40] reveals an insertion of a
total of twelve amino acids distributed among three sites in the steroid binding domain of ER
compared to ERR
. These insertions map to loops between -helices in human ER. One or
more of these insertions may be important in altering the ligand-binding pocket and/or
conformation of AF2 to yield a steroid-dependent ER.
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