The Journal of Immunology
Evolution of the Ikaros Gene Family: Implications for the
Origins of Adaptive Immunity1
Liza B. John,*† Simon Yoong,* and Alister C. Ward2†
Members of the Ikaros family of transcription factors are important for immune system development. Analysis of Ikaros-related
genes from a range of species suggests the Ikaros family derived from a primordial gene, possibly related to the present-day
protostome Hunchback genes. This duplicated before the divergence of urochordates to produce two distinct lineages: one that
generated the Ikaros factor-like (IFL) 2 genes of urochordates/lower vertebrates and the Pegasus genes of higher vertebrates, and
one that generated the IFL1 genes of urochordates/lower vertebrates, the IKFL1 and IKFL2 genes of agnathans and the remaining
four Ikaros members of higher vertebrates. Expansion of the IFL1 lineage most likely occurred via the two intervening rounds of
whole genome duplication. A proposed third whole genome duplication in teleost fish produced a further increase in complexity
of the gene family with additional Pegasus and Eos members. These findings question the use of IFL sequences as evidence for the
existence of adaptive immunity in early chordates and vertebrates. Instead, this study is consistent with a later emergence of
adaptive immunity coincident with the appearance of the definitive lymphoid markers Ikaros, Aiolos, and Helios. The Journal
of Immunology, 2009, 182: 4792– 4799.
T
ranscription factors are often called “master regulators” of
development as they function in mediating the complex
changes in gene expression required to produce the diverse array of cells needed in a multicellular organism (1). A common motif found in transcription factors is the zinc finger motif,
consisting of a zinc atom in specific coordination with four cysteine and/or histidine residues. Such motifs can be involved in
either protein-nucleic acid or protein-protein interactions, with
more than one zinc finger commonly found in the same protein,
often in the form of tandem arrays (2, 3). Zinc finger transcription
factors exhibit broad evolutionary conservation, including hematopoiesis. For example, GATA-related proteins regulate blood cell
development from Drosophila to man (4, 5).
The Ikaros family of zinc finger transcription factors are important regulators of immune system development (6). This family is
characterized by two sets of highly conserved zinc finger motifs: a
set of usually four zinc fingers located at the N terminus involved
in DNA binding, and a set of two zinc fingers at the C terminus,
which enable the Ikaros proteins to dimerize with themselves or
other family members (7). The mammalian Ikaros family consists
of Ikaros, Helios, Aiolos, Eos, and Pegasus. Ikaros and Aiolos are
highly conserved and expressed in lymphoid tissues, such as the
spleen and thymus, as well as PBL (6, 8, 9). Loss or mutation of
Ikaros results in dramatic decreases in T cells, B cells, NK, and
lymphoid-derived dendritic cells, as well as effects on myeloid cell
*Center for Cellular and Molecular Biology, School of Life and Environmental Sciences, Deakin University, Burwood, Victoria, Australia; and †School of Medicine,
Deakin University, Waurn Ponds, Victoria, Australia
Received for publication July 21, 2008. Accepted for publication February 4, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This project was supported by a Deakin Central Research Grant. L.B.J. received an
Australian Postgraduate Award.
2
Address correspondence and reprint requests to Prof. Alister C. Ward, School of
Medicine, Deakin University, Waurn Ponds, Victoria, Australia. E-mail address:
[email protected]
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.0802372
lineages (9 –11). Similarly, loss of Aiolos results in severe depletion of peritoneal and recirculating B cells and, in contrast, the
spontaneous production of autoantibodies (12). Deregulation of
Ikaros and Aiolos, particularly their splice variants, can result in
cellular hyperproliferation and the development of lymphomas and
leukemias (7, 9, 10, 13). Helios is also involved in T cell differentiation, at least partly via heterodimerization with Ikaros (7).
Constitutive expression of mutant forms of Helios leads to aggressive T cell lymphoma (14, 15). Eos is more broadly expressed,
being found in skeletal muscle, heart, and liver in addition to hematopoietic tissues such as thymus and spleen (16, 17). However,
its function remains relatively poorly understood. Pegasus is distinct from the other Ikaros members, as it contains only three Nterminal zinc fingers, with divergent sequence and DNA binding
specificity. It is also widely expressed but its function remains to
be determined (17).
Ikaros family homologues and related genes have been identified in a diverse range of species (17–26), the presence of
which has been used as evidence of lymphocyte-like gene regulatory programs in these organisms (19, 20, 22, 23, 27). However, despite this, a comprehensive perspective on the origins of
this important family has remained elusive. To further our evolutionary understanding, we have explored the repertoire of
Ikaros family of transcription factors in amphioxus, sea squirt,
lamprey, teleost fish, and other species. Detailed bioinformatics
analysis has allowed the formulation of an evolutionary model
for the genesis of the Ikaros gene family. This suggests that
Ikaros-related genes were derived from a primordial gene, possibly related to the present-day Hunchback genes of protostomes, which underwent duplication before the divergence of
urochordates/lower vertebrates from higher vertebrates. This
yielded a distinct Pegasus-related lineage that has remained
largely intact since that time, and another lineage that ultimately produced Ikaros, Aiolos, Helios, and Eos genes by the
time of divergence of the cartilaginous fish, likely via the two
intervening rounds of whole genome duplication that occurred
during this time. A further increase in complexity of the family
has occurred in teleost fish following a proposed third whole
The Journal of Immunology
4793
FIGURE 1. Sequence alignment of zinc finger motifs of Ikaros-related proteins. N-terminal (upper panel) and C-terminal (lower panel) zinc finger (ZF)
motifs from human (Hs; Homo sapiens), medaka (Ol; Oryzias latipes), lamprey (Pm; Petromyzon marinus and Lf; Lampetra fluviatilis), sea squirt (Ci;
Ciona intestinalis) and amphioxus (Bf; Branchiostoma floridae and Bb; Branchiostoma belcheri tsingtaunese) Ikaros-related proteins (Ikaros, Aiolos,
Helios, Eos, Pegasus, and Ikaros factor-like (IFL/IKFL) 1 and 2) and fruit fly (Dm; Drosophila melanogaster) Hunchback (Hb) were aligned using the
ClustalX program, as indicated. Asterisks (ⴱ) represent identical residues, colons (:) represent highly related residues and dots (.) represent moderately
related residues. Conserved essential cysteine (C) and histidine (H) residues, presumably involved in zinc coordination in each finger, are indicated in gray
with an alternate cysteine in ZF4 and ZF5 bolded. Note the absence of ZF1 in medaka Helios and Ikaros, and ZF4 in Pegasus and IFL2 sequences. The
absence of ZF1 in Ciona is likely due to limits in sequence availability.
genome duplication in this lineage. These findings have
prompted a reassessment of the origins of adaptive immunity.
Materials and Methods
Identification of new Ikaros-related sequences
Whole genomic sequence, high throughput genomic, and expressed sequence tag (EST)3 databases of amphioxus, sea squirt, lamprey, and teleost
fish were searched using known human orthologues of the Ikaros family to
identify Ikaros-related sequences. In addition, cDNA clones for ESTs representing zebrafish genes encoding Pegasus (AY394931-full-length), Eos
(CK397774 and AL927666-partial), and Helios (DN896784-partial) were
obtained and sequenced in full. Raw sequence analysis, contig assembly,
and basic sequence manipulation and were performed using Sequencher
4.1.4 (GeneCodes) or the web-based basic local alignment search tool
(BLAST) 2Sequences (http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.
cgi). GenomeScan (http://genes.mit.edu/genomescan.html) was used to
predict coding exons from sequences derived solely from whole genomic
sequence or genomic scaffolds. The following new sequences were identified; Xenopus tropicalis (western clawed frog) Aiolos (ENSX
ETP00000033186), Danio rerio (zebrafish) Pegasus (BN001214), Eos
(BN001212) and Helios (BN001213); Tetraodon nigroviridis (spotted
green pufferfish) Ikaros (BN001216), Aiolos (BN001289), Pegasus1
(BN001218), Pegasus2 (BN001290), Eos1 (BN001215), Eos2
(BN001291), and Helios (BN001217); Takifugu rubripes (fugu pufferfish)
Eos1 (BN001285), Eos2 (BN001286), Helios (BN001287), and Aiolos
(BN001288); Oryzias latipes (medaka) Eos1 (BN001294), Eos2
(BN001282), Helios (BN001283), and Aiolos (BN001284) and Petromyzon marinus (sea lamprey) Pegasus (Ikaros factor-like (IFL) 2)
(GENSCAN00000129115 and GENSCAN00000133626). The latter was
identified using pre-ensembl contigs (http://pre.ensembl.org/Petromyzon_
marinus/index.html) and the UCSC lamprey genome browser database
3
Abbreviations used in this paper: EST, expressed sequence tag; IFL/IKFL, Ikaros
factor-like; ZF, zinc finger; ISH, in situ hybridization; BLAST, basic local alignment
search tool.
(http://genome.ucsc.edu/cgi-bin/hgGateway?clade ⫽ other&org ⫽
Lamprey&db ⫽). Also retrieved were Ciona intestinalis (sea squirt) IFL1
(BN001219) and IFL2 (BN001220), which have been assigned model identity numbers (144428 and 131901, respectively) in the Ciona intestinalis
database version 1.0 (20). In addition, Branchiostoma floridae (amphioxus)
IFL1 N terminus (BN001292) and C terminus (BN001293), and IFL2 C
terminus (BN001281) were identified from GenBank and JGI Branchiostoma floridae v1.0 genome browser database (http://genome.jgi-psf.org/
Brafl1/Brafl1.home.html) searches. Predicted sequences of zinc finger domains were also retrieved for Ikaros members from Callorhinchus milii
(elephant shark).
Database interrogation and assembly of known sequences
BLASTN (nucleotide query vs nucleotide database), BLASTX (translated
query-protein), and TBLASTN (nucleotide-protein) search algorithms
were used on the National Center for Biotechnology Information (NCBI)
(www.ncbi.nlm.nih.gov/BLAST/) and Ensembl (www.ensembl.org) websites to identify Ikaros-related sequences from representative organisms.
Sequences retrieved from GenBank (http://www.ncbi.nlm.nih.gov) or Ensembl were: Homo sapiens (human) Ikaros AAP88838, Aiolos AAF13493,
Helios AAF09441, Eos AAG39221, and Pegasus AAG39220, Mus musculus (mouse) Ikaros NP_033604, Aiolos XP_283022, Helios NP_035900,
Eos NP_035902 and Pegasus NP_780324, Gallus gallus (chicken) Ikaros
NP_990419, Aiolos NP_990151, Helios NP_989938 and Pegasus
NP_001026766, Xenopus tropicalis (western clawed frog) Ikaros
NP_001015698, Helios NP_001116930, Eos ENSXETP00000020847 and
Pegasus NP_001017355, Danio rerio (zebrafish) Ikaros AAC61763,
Takifugu rubripes (fugu pufferfish) Ikaros ENSTRUG00000017779, Pegasus1 ENSTRUG00000001038 and Pegasus2 ENSTRUG00000010762,
Oryzias latipes (medaka) Ikaros ENSORLT00000025472, Pegasus1
ENSORLT00000007007 and Pegasus2 ENSORLT00000011938, Raja eglanteria (skate) Ikaros AF163848, Aiolos AF163850, Helios AF163847,
Eos AF163849, Petromyzon marinus (sea lamprey) IKFL1 AAF82350,
Lampetra fluviatilis (river lamprey) IKFL2 AAL67304, Myxine glutinosa
(hagfish) composite IFL (renamed IKFL1) AAP84653, Oikopleura dioica
(urochodate) IFL AAP84655, Strongylocentrotus purpuratus (purple sea
4794
IKAROS FAMILY EVOLUTION
FIGURE 2. Phylogenetic analysis of Ikaros-related proteins. The sequences of a comprehensive set of Ikaros-related proteins were aligned using
ClustalX, and a phylogenetic tree generated using the Neighbor-Joining (N-J) algorithm and visualized with the TreeView program. Branch labels
indicate the robustness of the tree over 100 bootstrap replicates, while the scale bar indicates an evolutionary distance of 0.05 amino acid
substitutions per site. Shading indicates respective Ikaros, Aiolos, Helios, Eos, and Pegasus clusters, while red, blue, and yellow rectangles indicate
potential groupings derived from successive duplication events during evolution. Newly identified proteins are shown in red. Abbreviations: Bf;
Branchiostoma floridae, amphioxus, Bb; Branchiostoma belcheri tsingtaunese, lancelet, Ci; Ciona intestinalis, sea squirt, Dr; Danio rerio, zebrafish,
Gg; Gallus gallus, chicken, Hs; Homo sapiens, human, Lf; Lampetra fluviatilis, river lamprey, Mg; Myxine glutinosa, hagfish, Mm; Mus musculus,
mouse, Od; Oikopleura dioica, tunicate, Ol; Oryzias latipes, medaka, Pm; Petromyzon marinus, sea lamprey, Re; Raja eglanteria, skate, Sp;
Strongylocentrotus purpuratus, purple sea urchin, Tn; Tetraodon nigroviridis, spotted green pufferfish, Tr; Takifugu rubripes, fugu pufferfish, Xt;
Xenopus tropicalis, western clawed frog.
The Journal of Immunology
4795
FIGURE 3. Microsynteny analysis of Ikaros-related gene loci. Schematic representation of sea squirt (Ci; Ciona intestinalis), lamprey (Pm; Petromyzon
marinus and Lf; Lampetra fluviatilis), human (Hs; Homo sapiens), frog (Xt; Xenopus tropicalis), and medaka (Ol; Oryzias latipes) Ikaros-related gene loci.
Orthologous genes are grouped within shaded boxes with species and individual chromosome (C), scaffold (S), and contig derivation indicated. Arrows
represent the relative orientation and position of each gene, color-coded on the basis of gene relatedness with Ikaros-related genes in red. A line with two
dashes as indicated for sea squirt IFL1 represents a large distance in the chromosome, while the zig-zag lines represent different scaffolds. Note that no other
genes are found on the contigs that lamprey IKFL genes are located. Gene abbreviations: ABCA12: ATP-binding cassette, subfamily A, member 12;
ACAD-SB/L: Acyl-Coenzyme A dehydrogenase, short/long branched chain; BARD1: BRCA1 associated RING domain 1; CHAT: choline acetyltransferase; CUZD1: CUB and zona pellucida domain 1; ERBB2/3/4: v-erb-a erythroblastic leukemia viral oncogene homologue 2/3/4; FIGNL1: Fidgetin-like
1; GPR26: G protein-coupled receptor 26; GRB7/10: Growth receptor bound 7/10; HMX3: Homeo box (H6 family) 3; IF-A: Intermediate filament protein
A; IGFBP4: insulin-like growth factor binding protein 4; MLLT6: myeloid/lymphoid or mixed-lineage leukemia translocated to 6; PARG: Poly(ADPribose) glycohydrolase; PA2G4: proliferation-associated 2G4; PSMB3: proteasome subunit, ␤ type 3; PSMD3: proteasome 26S subunit, non-ATPase 3;
RGR: retinal G protein coupled receptor; RPS26: ribosomal protein S26; SPAG16: Sperm associated Ag 16; STAT2: Signal transducer and activator of
transcription protein 2; SUOX: Sulfite oxidase; ZPBP (2): zona pellucida binding protein (2).
urchin) IFL1 CX559654 and Branchiostoma belcheri tsingtaunese (amphioxus) IFL1 ABH05923, together with the Hunchback genes Caenorhabditis elegans (nematode) Hunchback-like AAD16170, Drosophila
melanogaster (fruit fly) Hunchback NP_731267, Helobdella robusta
(leech) Hunchback-like AAY43810, Helobdella triserialis (leech) hbl1
CAA62741 and hbl2 X91395, and Platynereis dumerilii (Dumeril’s clam
worm) Hunchback-like CAJ78430.
Sequence analysis and nomenclature
For phylogenetic analysis, all known and novel protein sequences were
aligned using the ClustalX version 1.83 algorithm and GONNET matrix
model (Freeware) (28). This matrix was used to calculate the alignment
with penalties applied for opening and extending gaps in the alignment. A
phylogenetic tree was created using the bootstrapped Neighbor-Joining algorithm of 100 replicates in PAUP (Freeware). Trees were formatted using
njplot (http://pbil.univ-lyon1.fr/software/njplot.html) and viewed in TreeView (Freeware: http://taxonomy.zoology.gla.ac.uk/rod/treeview.html)
(29). Additional analysis using maximum parsimony (30) and maximum
likelihood (31) algorithms was performed using the PHYLIP version 3.68
(http://evolution.genetics.washington.edu/phylip.html) software package to
confirm phylogenetic topologies. Synteny analysis was performed using
Ensembl (http://www.ensembl.org) and NCBI Map Viewer (http://www.
ncbi.nlm.nih.gov/mapview) using the Ciona intestinalis (JGI v2), Petromyzon marinus (PMAR3), Homo sapiens (NCBI 36/Build 36.2), Xenopus
tropicalis (JGI v4.1) and Oryzias latipes (NIG MEDAKA1) databases.
Analysis of splicing was performed by comparing the genomic scaffold to
the relevant cDNA sequences, applying the GT-AG rule (32).
Results
Identification of Ikaros-related sequences in teleost fish,
lamprey, sea squirt, and amphioxus
Exhaustive bioinformatic analysis of zebrafish genomic databases
revealed the presence of single homologues for Helios, Eos, and
Pegasus, as well as the previously described Ikaros (33), but not
the related Aiolos (Fig. 2 and data not shown). The presence of
corresponding ESTs verified that all were expressed. A similar
analysis of the medaka, fugu, and green spotted pufferfish genomes
revealed an identical complement of Ikaros and Helios genes (Figs.
1 and 2), but identified duplications of Pegasus and Eos genes
(Figs. 1 and 2 and supplemental Fig. 2).4 Divergent Aiolos genes
were also identified in these species (Fig. 1 and 2). Predicted
genomic sequences retrieved from elephant shark also showed
high level of identity to zinc finger domains of Ikaros members
(supplementary Fig. 1). Bioinformatic analysis of the lamprey genome identified, in addition to the previously discovered IKFL1
and IKFL2 genes, a Pegasus-like gene, which grouped with the
IFL2 subset (Fig. 1 and 2). In sea squirt, two Ikaros-related (IFL)
4
The online version of this article contains supplemental material.
4796
IKAROS FAMILY EVOLUTION
FIGURE 4. Comparison of splicing patterns for the Ikaros-related
genes. Schematic representation of
the splicing pattern for Ikaros-related
genes; Ikaros (I), Aiolos (A), Helios
(H), Eos (E), and Pegasus (P) of the
indicated organisms. Splice sites are
indicated by open rounded triangles
and exons indicated by black boxes.
Zinc fingers are numbered and denoted by gray boxes. The dotted line
indicates limited sequence data available for amphioxus IFL1 and IFL2,
and Ciona and lamprey IFL2. Note
that medaka/fugu Aiolos and zebrafish Eos has an extra splice site
following ZF4, and medaka and zebrafish Helios, together with
medaka Ikaros, lack ZF1.
proteins, designated IFL1 and IFL2, with homology with IFL sequences identified in other urochordates and other early vertebrates
were discovered (Fig. 1 and data not shown). In addition, partial
sequences for the N terminus and C terminus zinc finger motifs of
IFL1 and IFL2 genes in two amphioxus species were also identified (Figs. 1 and 2).
Zinc fingers of Ikaros-related sequences show high conservation
and an alteration to the common C2H2 motif
Alignment of the N-terminal and C-terminal arrays of zinc finger
(ZF) motifs revealed high level of identity and conservation of
each motif between species and especially among the human and
medaka orthologues (Fig. 1). In general Ikaros, Aiolos, Helios, and
Eos proteins aligned more closely with amphioxus and Ciona
IFL1, whereas Pegasus proteins aligned more closely with IFL2
(Fig. 1). Pegasus and IFL2 both lack one of the N-terminal zinc
fingers, which the alignment suggests is ZF4, rather than ZF1 as
suggested by others (17), an assertion supported by phylogenetic
analysis of individual ZF3 and ZF4 sequences (data not shown).
Medaka Ikaros and Helios, in contrast, were missing ZF1. Hunchback showed consistent homology to the Ikaros-related proteins,
particularly in ZF2, ZF3, ZF5, and ZF6 (Fig. 1). Finally, the C2H2
motif was observed in all zinc fingers, except for ZF4 of IFL1 and
Aiolos, as well as Eos1 (medaka), which all possessed a variant
C2HC motif. However, many of the Ikaros sequences have a cysteine residue immediately before the last histidine in the ZF4 and
ZF5 motif, suggesting the IFL1 and Aiolos variants do not necessarily change the motif dramatically.
Phylogenic analysis of Ikaros-related sequences
To investigate potential evolutionary relationships, Ikaros-related
sequences were assembled from a range of different species, along
with the most closely related zinc finger protein from invertebrate
species, Hunchback. Phylogenetic trees were constructed using the
Neighborhood-Joining algorithm from ClustalX alignments of
these sequences (Fig. 2 and supplementary Fig. 2). The robustness
of these trees was confirmed by the relatively high bootstrapping
values for each branch, and by using the parsimony maximum
The Journal of Immunology
4797
FIGURE 5. Evolutionary model for the Ikaros-related gene family. Schematic representation of the evolution of the Ikaros-related gene family. Upper
panel, “X” represents the primordial gene (possibly related to protostome Hunchback indicated by “?”), from which all the Ikaros family members were
derived. Likely duplication events are denoted by red boxes, with those corresponding to known whole genome duplication events, numbered as shown.
The duplication of X produced two precursor lineages before cephalochordate and urochordate divergence; one related to IFL1 and the other related to IFL2,
denoted by blue boxes. Duplication 1 occurred before agnathan divergence producing IKLF1 and IKLF2 precursors in the IFL1 lineage. Duplication 2 gave
rise to the individual Ikaros members from the IKFL precursors; IKFL1 produced Ikaros and Aiolos, and IKFL2 produced Helios and Eos, in cartilaginous
fish and tetrapods. In contrast, Pegasus genes have evolved from the IFL2-related precursor. Duplication 3 was a fish-specific event that produced duplicated
members in medaka and pufferfish, as indicated. Absent genes in zebrafish are denoted by “x”. The zinc finger motifs for each member are indicated as
follows: zinc finger (ZF) 1, blue; ZF2, brown; ZF3, green; ZF4, yellow; ZF5, purple; and ZF6, orange. Lower panel, An evolutionary tree is indicated, along
with proposed whole genome duplications (1/2/3R in red with gray shading). This is not drawn to scale.
likelihood PHYLIP 3.68 algorithm, which produced a similar
branching structure (data not shown).
The trees indicated two distinct clades within the Ikaros family
(red boxes). The first clade consisted of IFL2 sequences from amphioxus and Ciona along with Pegasus sequences from various
higher vertebrate species, with IFL2 being most divergent. The
second clade consisted of IFL/IFL1 sequences from sea urchin,
amphioxus, tunicates, and Ciona, IKFL sequences from agnathans,
as well as Helios, Eos, Ikaros, and Aiolos sequences from a variety
of higher vertebrates. The IFL/IFL1 sequences were clearly divergent from the other sequences, which subsequently split into two
branches, one consisting of Helios and Eos along with IKLF2 sequences and the other of all Ikaros and Aiolos along with IKLF1
sequences (blue boxes). Each of these branches split into individual Ikaros, Aiolos, Helios, and Eos clusters (yellow boxes). Finally, the Hunchback proteins grouped closest to the Ikaros-related
sequences, a consistent result regardless of the outgroup or algorithm used (supplementary Fig. 2 and data not shown).
Synteny analysis of Ikaros family genes in higher vertebrates
Synteny analysis was performed on Ikaros family members from
sea squirt, lamprey, and three representative higher vertebrates:
human, frog, and medaka. Genes with conserved synteny were
observed for each Ikaros member: HMX3, ACAD-SB and CUZD1related genes for Pegasus; ZPBP, FIGNL1-related, GRB10-related
and ERBB1-related genes for Ikaros; IGFBP4, PSMD3-related,
ZPBP2, GRB7-related, ERBB2-related, and MLLT6-related genes
for Aiolos; STAT2-related, SUOX, RSP26-related, ERBB3-related,
and PA2G4-related for Eos, as well as ABCA12-related, BARD1related, ERBB4-related, ACAD-L, and SPAG16-related for Helios
(Fig. 3), confirming the orthologous assignment of frog Aiolos and
Eos, and medaka Ikaros, Aiolos, Helios, and the duplicated Eos
member. However, conserved syntenic relationships were also observed between different Ikaros genes: Ikaros and Aiolos shared
synteny with GRB and ZPBP homologues (Ikaros: GRB10 and
ZPBP; Aiolos: GRB7 and ZPBP2), while Helios and Eos share
synteny with ERBB homologues (Eos: ERBB3; Helios: ERBB4),
confirming close evolutionary derivation of each gene pair. Ikaros
and Aiolos also shares synteny with an ERBB homologue (Ikaros:
ERBB1; Aiolos: ERBB2) highlighting a likely common evolutionary ancestry between the gene pairs and also to Ciona IFL1 which
contains an ERBB gene on its loci. Similarly, Pegasus and Helios
also shared conserved synteny with ACAD homologues, again suggesting a common evolutionary derivation. Synteny analysis of the
duplicated teleost eos and pegasus genes confirmed differential
gene loss between species (supplementary Fig. 3).
Preservation of splice sites within Ikaros-related genes
Comparison of splicing patterns reinforced the presumed evolutionary relationships (Fig. 4). Ikaros, Aiolos, Helios, and Eos have
an identical splicing pattern except for an extra exon in zebrafish
Eos and medaka Aiolos, together with the absence of zinc finger
4798
sequences in teleost Helios. IFL1 and lamprey IKFL genes showed
a similar pattern, except for three additional exons in Ciona. In
contrast, while Pegasus genes in lamprey and higher vertebrates
had their first ZF on a separate exon like the other Ikaros genes, the
remainder lie on a single exon like Hunchback. IFL2 showed similarities to both Pegasus and other Ikaros genes.
Discussion
The Ikaros family of zinc finger proteins represent an important
class of transcriptional regulators, with key roles identified for
some members in adaptive immunity (7). To further our understanding of the evolution of this family, we identified and characterized related genes in teleost fish, agnathans, cephalochordates,
and other species. Detailed bioinformatics analysis of these and
other Ikaros-related sequences has shed new light on the evolutionary origins of this family with implications for understanding
the emergence of adaptive immunity.
Phylogenetic analysis of full-length protein sequences (Fig. 2
and supplemental Fig. 2) highlighted the likely evolutionary relationships between specific IFL genes of urochordates and lower
vertebrates and Ikaros family members of higher verterbrates:
IFL1 genes clustering with Ikaros, Aiolos, Helios, and Eos, and
IFL2 with Pegasus. Within the former cluster, distinct Ikaros/Aiolos and Helios/Eos subclusters were present, typical of a gene family expanded through successive rounds of duplication, with the
agnathan IKFL1 and IKFL2 likely representing derivatives of the
respective intermediates. These relationships were confirmed and
further emphasized by similarities in the sequences of individual
zinc fingers (Fig. 1 and supplementary Fig. 1), arrangements of
respective gene loci (Fig. 3 and supplementary Fig. 3), and splicing
patterns (Fig. 4).
On the basis of these data, we propose a model describing the
evolution of Ikaros-related genes (Fig. 5). This suggests that Ikaros
members evolved from a primordial gene (“X”). This duplicated
before the divergence of cephalochordates and urochordates (34),
producing a lineage represented by present-day IFL2 in urochordates and Pegasus in higher vertebrates, and another lineage represented by present-day IFL/IFL1 in urochordates and lower vertebrates. The latter lineage underwent successive duplications to
produce intermediate precursors, related to present day agnathan
IKFL1 and IKFL2, and from these the Ikaros/Aiolos and Helios/
Eos gene pairs, respectively, by the time of the divergence of cartilaginous fish. Two rounds of whole genome duplications occurred during this time (35, 36), providing a convenient
mechanism for how this occurred. A third round of whole genome
duplications resulted in the further expansion of the Ikaros gene
family by duplications of Pegasus and Eos members in the teleost
lineage, although these appear to have been lost in zebrafish where
gene loss appears to have occurred. Collectively, these data suggest that Pegasus likely retains a more ancient function compared
with other members of the higher vertebrate Ikaros family
member.
The proposed model of Ikaros gene family evolution has implications for understanding the evolution of the immune system. For
example, the presence of IFL/IKFL sequences has been used as
evidence for the existence of a lymphocyte-like gene regulatory
apparatus in urochordates and early vertebrates (19, 20, 22), by
analogy with Ikaros (as well as Aiolos and Helios), which are
largely expressed in a lymphoid-specific manner with lymphoidspecific gene regulatory functions (8, 9, 11, 37). However, the
evolutionary relationships delineated in this study would suggest
that IFL/IKFL genes may not necessarily have immune functions.
Thus, IFL2 is likely to reflect a functional homologue of the more
divergent and broadly expressed Pegasus, for which expression has
IKAROS FAMILY EVOLUTION
been observed in the adult mammalian brain, heart, skeletal muscle, kidney, and liver by Northern blot analysis (17), as well as
specifically in the forebrain, hindbrain, eye, and gut during zebrafish embryogenesis as judged by in situ hybridization (ISH)
(L. B. John and A. C. Ward, unpublished data). As for IFL1, the
amphioxus equivalent was found to be expressed in ovary and gills
by ISH (22), while urochordate IFL1 was strongly expressed in the
oocyte and filter house as judged by ISH (20) and, significantly,
failed to bind the consensus Ikaros site (20). Similarly, lamprey
IKFL genes were found to be expressed in intestinal epithelium
and strongly in the ovary using ISH, with further expression in
liver and gills detected by RT-PCR (19). This collectively suggests
that ascribing an immune-related function to the IFL genes may be
a dangerous assumption, although general expression patterns do
not themselves rule out such a role. With this important caveat, our
model places the appearance of the definitively lymphoid Ikaros,
Aiolos, and Helios proteins coincidentally with the timing of the
“emergence” of adaptive immunity at around 450 million years
ago, as argued by others (35), although this remains controversial.
The protein Hunchback, found in various invertebrate species,
has been previously described as having sequence similarity with
Ikaros, particularly at the C terminus (38, 39). Despite this, a direct
evolutionary relationship has not been considered, in part due to
the divergent function of Hunchback, a “gap” gene playing a crucial role in controlling pattern formation and segmentation during
development (40 – 44), compared with the lymphopoietic function
of Ikaros, Helios, and Aiolos (7). However, because Hunchback
showed the highest homology of any protostome sequence to
Ikaros family members (data not shown), consistently grouping
with them in phylogenetic analysis (supplementary Fig. 2 and data
not shown) with the same topology of C2H2 type zinc finger motifs
(45, 46) that showed reasonable alignment with those of Ikaros
family members (Fig. 1), the possibility of shared evolutionary
origins from primordial gene X should be entertained.
Acknowledgments
We thank Clifford Liongue for very helpful discussions regarding phylogenetic analysis and sequence alignments.
Disclosures
The authors have no financial conflict of interest.
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