J Mol Evol (2010) 71:427–436
DOI 10.1007/s00239-010-9396-1

Evolution of Conserved Non-Coding Sequences Within
the Vertebrate Hox Clusters Through the Two-Round Whole
Genome Duplications Revealed by Phylogenetic Footprinting
Analysis
Masatoshi Matsunami • Kenta Sumiyama •Naruya Saitou

Received: 27 July 2010 / Accepted: 17 September 2010 / Published online: 28 October 2010
Ó Springer Science+Business Media, LLC 2010

Abstract As a result of two-round whole genome duplications, four or more paralogous Hox clusters exist invertebrate genomes. The paralogous genes in the Hox
clusters show similar expression patterns, implying shared
regulatory mechanisms for expression of these genes.
Previous studies partly revealed the expression mechanisms of Hox genes. However, cis-regulatory elements that
control these paralogous gene expression are still poorly
understood. Toward solving this problem, the authors
searched conserved non-coding sequences (CNSs), which
are candidates of cis-regulatory elements. When comparing
orthologous Hox clusters of 19 vertebrate species, 208intergenic conserved regions were found. The authors thensearched for CNSs that were conserved not only between
orthologous clusters but also among the four paralogous
Hox clusters. The authors found three regions that areconserved among all the four clusters and eight regions thatare conserved between intergenic regions of two paralogous Hox clusters. In total, 28 CNSs were identified in theparalogous Hox clusters, and nine of them were newly
found in this study. One of these novel regions bears a
RARE motif. These CNSs are candidates for gene
expression regulatory regions among paralogous Hox
Electronic supplementary material The online version of thisarticle (doi:10.1007/s00239-010-9396-1) contains supplementarymaterial, which is available to authorized users.
M. Matsunami  K. Sumiyama  N. Saitou
Department of Genetics, School of Life Science, The Graduate
University for Advanced Studies (SOKENDAI),
Mishima 411-8540, Japan
M. Matsunami  K. Sumiyama  N. Saitou (&)
Division of Population Genetics, National Institute of Genetics,
Yata 1111, Mishima 411-8540, Japane-mail: saitounr@lab.nig.ac.jp

clusters. The authors also compared vertebrate CNSs withamphioxus CNSs within the Hox cluster, and found that
two CNSs in the HoxA and HoxB clusters retain homology
with amphioxus CNSs through the two-round whole genome duplications.
Keywords Hox cluster  Two-round whole genome
duplications  Conserved non-coding sequences 
Phylogenetic footprinting  Retinoic acid response
element  Amphioxus

Introduction
Vertebrate genomes show evidence of widespread gene
duplications compared to invertebrate genomes. Ohno
(1970) proposed the existence of two-round whole genome
duplications (2R WGD) during the early vertebrate evolution, now known as the 2R hypothesis. Before theamphioxus genome was reported by Putnam et al. (2008),
this hypothesis was extensively debated (e.g., Holland et al.
1994; Gibson and Spring 2000; Hughes et al. 2001).
Genome duplications generated paralogous genes andcomplex gene regulatory mechanisms in vertebrate evolution (e.g., Dehal and Boore 2005). These paralogous genes
often share the same expression patterns, but some may
acquire new expression patterns. The changes of gene
expression are mainly because of changes in cis-regulatory
elements (Carroll 2001).
Identifying the cis-regulatory sequences that control
spatial and temporal gene expression is a challenging issue.
Because gene regulatory elements are expected to be conserved because of their functional importance, searching forevolutionarily conserved non-coding sequences (CNSs)
would be an effective strategy for finding candidates of

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functional elements. Let us note that the gene regulatory
elements which are not conserved are very rare (Werauchand Hughes 2010). Previous studies have already shownthat CNSs are abundant in vertebrate genomes (Bejerano
et al. 2004; Woolfe et al. 2005). Genome-wide comparative
approaches have also reported the existence of paralogous
CNSs (Bejerano et al. 2004; Woolfe et al. 2005; McEwen
et al. 2006), and most of them are located in paralogous
gene clusters that code for transcriptional factors. These
results imply that paralogous CNSs contribute to clusterorganization and/or their neighboring gene expression patterns. The authors therefore focused on the vertebrate Hox
clusters because they contain abundant CNSs.
The Hox genes orchestrate the development of animal
body plans. They consist of more than four physicallylinked clusters in different chromosomes in vertebrate
genomes (Pearson et al. 2005; Lemons and McGinnis2006). Hox genes of each cluster are expressed along theanterior–posterior body axis in the same order as lining up
on the chromosome, a feature called ‘‘colinearity’’ (GarciaFernàndez 2005). Paralogous genes of the Hox clusters
show the similar expression pattern, which suggests that
there might be shared gene expression regulatory mechanisms among paralogous Hox clusters.
The duplication of Hox clusters influences clusterarchitecture and patterns of non-coding sequence evolution. The duplicated non-coding regions within the Hox
clusters are mainly studied for teleost fish (e.g., Chiu
et al. 2002; Santini et al. 2003; Prohaska et al. 2004).
The third round whole genome duplication occurred after
the 2R WGD in the teleost lineage. Chiu et al. (2002)
and Prohaska et al. (2004) found massive loss ofsequence conservation in teleost HoxA cluster non-coding regions after the 3R WGD. Therefore, teleosts are
not suitable for analyzing duplicated Hox cluster noncoding sequences.
In the case of 2R WGD, Kim et al. (2000) described one
paralogous CNS within the four Hox clusters. However,analysis of non-coding sequences of the Hox clusters
within vertebrates, especially mammalian species, is not
sufficient. There are probably two reasons for this. First,
the functional paralogous conservation cannot be detectedeasily. This is because the 2R WGD were very ancient
events which occurred approximately half a billion years
ago and the non-coding sequences experienced higher
evolutionary rates compared to protein coding sequences.
This is probably because cis-regulatory elements are
redundant and may be changed by binding site turnover(Hancock et al. 1999). Second, only a few invertebrate
sequences that are more closely related to vertebrates andthat still retain cluster structure are available. With therecent abundance in vertebrate genomes sequences, theauthors can now analyze the evolution of non-coding

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sequences within the Hox clusters after 2R WGD. However, identifying CNSs within Hox clusters before 2R
WGD remains a challenge.
Recently, Hox cluster sequences of two different
amphioxus species, Branchiostoma floridae and B. lanceolatum were reported by Amemiya et al. (2008) andPascual-Anaya et al. (2008), respectively. Because
amphioxus is the chordate bearing a syntenic Hox cluster
which is most closely related to vertebrates, these data
would be very informative for inferring the evolution of
non-coding regions within Hox clusters before 2R WGD.
Detection of the functional turnover of transcription
factor binding site (TFBS) is one interesting problem. In
the Drosophila genome, the TFBS turnover frequently
occurred (Ludwig et al. 2005). Ray et al. (2008) developed
a program to find the functional turnover motifs using
experimental results as training data. Some cis-regulatory
regions showed the TFBS turnovers also in vertebrates(Werauch and Hughes 2010). However, these data are
difficult to utilize for finding other functional turnover
events for various reasons such as insufficient experimental
data, short alignment length, and low mutation rate.
Therefore, the authors did not examine the functional
turnover of the TFBS in this study.
In this study, the authors identified orthologous CNSs
within the vertebrate Hox clusters, and found conserved
loci among paralogous Hox clusters. The authors comparedthese CNSs with amphioxus–human CNSs reported by
Pascual-Anaya et al. (2008) using phylogenetic footprinting to find CNSs that can be dated back to amphioxus. This
study identified and mapped vertebrate CNSs within thefour vertebrate Hox clusters using comprehensive genome
comparisons.

Materials and Methods
Identification of Vertebrate Hox CNSs
Genomic sequences of Hox clusters were obtained for thefollowing 18 vertebrate species from UCSC Genome Bioinformatics (http://genome.ucsc.edu/): Human (Homo sapiens), mouse (Mus musculus), chimpanzee (Pan troglodytes),orangutan (Pongo pygmaeus abelii), rhesus macaque(Macaca mulatta), marmoset (Callithrix jacchus), rat (Rattus norvegicus), guinea pig (Cavia porcellus), cat (Felis
catus), dog (Canis familiaris), horse (Equus caballus), cow(Bos taurus), opossum (Monodelphis domestica), platypus(Ornithorhynchus anatinus), chicken (Gallus gallus), zebra
finch (Taeniopygia guttata), lizard (Anolis carolinensis), andfrog (Xenopus tropicalis). Partial sequences of the horn shark(Heterodontus francisci) that included Hox clusters (DDBJ/
EMBL/GenBank accession numbers are AF224262 andJ Mol Evol (2010) 71:427–436

AF224263) were also used for this study. The authors
excluded teleost fishes, which have undergone the additional
genome duplication in their lineages. Protein coding regions
were filtered based on the RefSeq project (http://www.ncbi.
nlm.nih.gov/RefSeq/) annotation. Alternative exons were
not considered in this analysis. BLAST homology search(Altschul et al. 1997) was performed on this data set withdefault parameter setting and cutoff scores of[200.
Orthologous CNSs were systematically named based on
their genomic locations and BLAST scores. For example,
the CNS that is located at the intergenic region between
HoxA7 and HoxA6 with the highest BLAST score wasnamed ‘‘A76-1.’’
These CNSs were aligned using CLASTALW(Thomopson et al. 1994), and divided into three categories toinvestigate the depth of conservation: placental mammals,amniotes, and vertebrates. The authors then searched forconserved sequences that were conserved not only between
orthologous clusters but also among paralogous four Hox
clusters using BLAST search with the cutoff score ofless than 30. Annotations of TFBS motifs were mainly
based on the TRANSFAC database (http://www.biobaseinternational.com/pages/index.php?id=transfac).
Analysis of Paralogous CNSs
To investigate the non-coding transcribed regions of the
Hox clusters, transcriptional information of mRNAs and
ESTs within the human and mouse Hox clusters wereobtained from the UCSC Genome Bioinformatics database,and these transcripts were mapped on the region.
Phylogenetic footprinting analysis was carried out foreach orthologous CNSs that also have paralogous conservation. Each vertebrate CNS was aligned using CLASTALW. The substitution number of each aligned site wasestimated parsimoniously using Fitch’s (1971) algorithm.
The guide phylogenetic tree (Supplementary Fig. 1) necessary for this analysis was taken from Murphy et al.
(2004). In parallel, the likelihood estimation of ancestral
sequence of each vertebrate CNS was inferred using PAML
4 (Yang 2007).
Comparison with Amphioxus Hox CNSs
Pascual-Anaya et al. (2008) compared Hox clusters of two
different amphioxus species (Branchistoma floridae and B.
lanceolatum) to each human Hox cluster and defined 75
human–amphioxus CNSs (amphiCNS). These amphiCNSs
were obtained and were sorted by identity. The authors
named amphiCNSs by the order of their identity. The
amphiCNSs were compared with vertebrate CNSs to
identify significant conserved region among chordates.

429

Results
Orthologous CNSs Within Vertebrate Hox Clusters
The authors defined 208 CNSs in total: 64, 43, 60, and 41
for HoxA, B, C, and D clusters, respectively. Genomic
locations of these CNSs are graphically shown in Fig. 1,
and detailed information of all these CNSs is shown in
Supplementary Table 1. Many of these orthologous CNSs
overlap microRNAs and cis-regulatory elements whichhave previously been described (Mainguy et al. 2003;
Yekta et al. 2004). Because sequence information is notcomplete or homologous sequence is lacking, some CNSs
were not found in several species (see Supplementary
Table 2). As an example of a cis-regulatory element, C98-1
corresponds to the HoxC8 early enhancer which is necessary for proper HoxC8 expression (Juan and Ruddle 2003).
Other CNSs might bear similar enhancer functions.
The findings of this study are consistent with previous
observations (Prohaska et al. 2004; Chiu et al. 2002),
confirming that orthologous CNSs were detected effectively. Moreover, using our criteria, the authors also
detected 160 new CNSs (see Fig. 1 and Supplementary
Table 1).
The authors detected a larger number of CNSs in Hox5Hox3 (corresponding to Drosophila Antp and Ubx/abdA)
intergenic sequences than in other intergenic sequences(Fig. 1). This region has abundant alternatively spliced
coding RNAs and long non-coding RNAs (Mainguy et al.
2007). This observation suggests that functionally
unknown CNSs in this region contribute to these alternative splicing events. In contrast, posterior regions of Hox
clusters have poor conservation except for upstream
regions of Evx1 and Evx2.
The 208 CNSs were divided into six categories: placental
mammals, placental mammals ? marsupials, placental
mammals ? marsupials ? monotremes, amniotes, tetrapods, and vertebrates, based on the depth of conservation(Table 1). The level of conservation of orthologous CNSs
varies among the four Hox clusters; HoxA has the highest
number (64) of CNSs in total, while HoxD has the smallest
number (41) of CNSs due to the small numbers of CNSs
conserved among amniotes and tetrapods. The HoxC cluster
has the highest number (33) of CNSs conserved only amongplacental mammals, while the HoxB cluster has the highest
number (11) of CNSs in placental mammals ? marsupials.
This result, however, does not mean that the HoxC cluster isthe least conserved (see Discussion).
Paralogous CNSs Among Hox Clusters
The authors found 28 paralogous conserved elements intotal (8, 6, 6, and 8 for Hox A, B, C, and D clusters,

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EVX1

A13

A11

A10

A9

A7

A6 A5

A4

A3

A2 A1

HoxA

B13

B9

DP1

DP2

DP8

DP7

DP4

HoxB

B8 B7

B6 B5

B4

TP1 (FCS)

C13

C12

C11

C10

C9

C8

B3 B2

TP3 (RARE2)

TP2 (RARE1)
C6 C5

B1

C4

HoxC
DP3
EVX2

D13 D12 D11DP5
D10 D9

D8

DP6
D4

D3

D1

HoxD
10 kbpFig. 1 The schematic diagram of orthologous CNSs and paralogous
CNSs among human Hox clusters. Exons of protein coding genes are
represented by light green boxes. The orange ovals are orthologous
CNSs. The blue and red ovals indicate locations of paralogous CNSs
conserved also among the two clusters and the four clusters,
respectively. The blue dotted lines show either microRNA (DP4) or

Table 1 Conservation depth of each CNS
HoxA

HoxB

HoxC

HoxD

Placental mammals

4

5

33

2

Above ? Marsupials

2

11

0

1

Above ? Monotremes

8

6

3

6

Amniotes

17

7

8

7

Tetrapods

17

14

16

9

Vertebrates

16

–

–

16

Total

64 (48)43 (43)60 (60)41 (25)

Note: There is no genomic sequence data for horn shark HoxB and
HoxC clusters, so ‘‘Vertebrates’’ depth CNSs are not determined, as
shown with hyphens. As a result of this, values in parentheses in‘‘Total’’ are those excluding CNSs shared in all vertebrates

respectively). Three quartets of CNSs are conserved among
all the four Hox clusters, and the authors named them TP(tetra-paralogous), as shown in Fig. 1. The authors carriedout the phylogenetic footprinting analysis to infer significantly conserved motifs among these three TPs. The authors
found the highly conserved region in each CNS, and theseoverlap with paralogous conserved regions (see Supplementary Fig. 2). Multiple sequence alignments of three TP
CNSs are shown in Fig. 2. It should be noted that thesesequences are reconstructed ancestral ones. TP2 and TP3
contain retinoic acid response elements (RAREs). Intergenic
regions of upstream or downstream of Hox4 genes areabundant with functional RAREs (Mainguy et al. 2003).
Despite this, RAREs located downstream of HoxC4 has notbeen reported before. The authors found a new evolutionarily
highly conserved sequence containing RARE in this region.

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non-syntenic DP (DP6). The paralogous CNSs whose name enclosedby blue rectangle is newly detected. Especially the newly detected
HoxC CNS of TP3 is highlighted by blue circle. The light blue circled
HoxA CNSs were not identified by Prohaska et al. (2004). TP tetraparalog, DP di-paralog (Color figure online)

These motifs might maintain gene expression pattern ofclusters cooperatively.
The remaining TP1 was discovered by Kim et al. (2000),
and they named it four cluster sequence (FCS). Though theauthors found conserved motifs in the FCS (Fig. 2), thesemotifs have no experimental corroboration. Then theauthors mapped transcripts within Hox clusters. As a result,
136 CNSs overlap with transcribed regions (see Supplementary Table 1). FCS corresponds to the bidirectionaltranscript start sites (TSS) which encode alternative spliced
RNAs of Hox genes and antisense non-coding RNAs
(Fig. 3a). These CNSs might play important roles in thecolinear expression pattern of the Hox cluster. Anotherparalogous CNS between Hox5 and Hox4 overlapped theregion of TSS and alternative exons (Fig. 3b), suggestingthat CNSs function as cis and trans regulatory elements.
Eight pairs of CNSs are conserved between two paralogous Hox clusters; and the authors named them DP(di-paralogous), as shown in Fig. 1. Results of phylogenetic
footprinting analysis and pairwise sequence alignment are
shown in Supplementary Figs. 2 and 3, respectively. The
DP6 CNS is not located at syntenic region and the conservation is poor. Other DP CNSs are located at the syntenic
region of each cluster and include functional elements(Table 2). DP1 and DP2 which are located at the upstreamof Evx1 and Evx2 have cis-regulatory functions (Lehoczky
et al. 2004). The region called ‘‘distal limb enhancer’’ in the
HoxD cluster is essential for the posterior HoxD gene
expression of appendicle (Spitz et al. 2001). The DP4 paircorresponds to microRNAs mir-196b and mir-196a-2. They
belong to the mir-196 family. This family is composed of

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431

Fig. 2 Multiple alignments ofthree TP CNS sequences.
a–c are the results of multiplealignments of paralogous
conserved regions derived fromeach TP CNS. Aligned
sequences are ancestral
sequences estimated from each
CNS using PAML4 program(Yang 2007). Alignments are
generated using CLUSTALW(Thomopson et al. 1994). The
putative TFBS are highlighted
by orange (Color figure online)

Fig. 3 The scheme ofparalogous conserved
bidirectional promoters. The
authors mapped the paralogous
CNSs on bidirectional transcript
start sites which code alternative
splicing RNAs of Hox genes
and antisense RNAs. Paralogous
CNSs are a TP1 and b DP7. The
blue and red lines are sense
RNAs and antisense RNAs,
respectively. DDBJ/EMBL/
GenBank accession numbers ofthese RNA sequences are also
shown (Color figure online)(B) DP7(A) TP1 (FCS)
DB289031

DA758599

HoxA
A7

A6

DA750122
A5

AK292200

A4

AK291182

HoxB
B7

B6

DA741273

AK314829

B5

BC034922
X07495

B4

1 kbp

HoxC
C6

C8

C5

C4

BC047605

HoxD
D8

BC034000

three members, which are mapped between Hox10 andHox9 of HoxA, HoxB and HoxC clusters (Yekta et al.
2004). However, another member, mir-196a-1, was difficult
to detect because of poor conservation. The authors thus
defined only two microRNA members as CNS.
Comparison Between Vertebrate CNSs and Amphioxus
CNSs Within Hox Clusters
Phylogenetic footprinting can be used to detect significantly
conserved sequences between vertebrates and the amphioxus

D4

Hox cluster. Because the conservation of non-coding region
between amphioxus and vertebrates is poor, Pascual-Anaya
et al. (2008) defined CNS in the case of human–amphioxus
comparison as approximately 60% identity and 50-bp
length region. They reported 75 amphiCNSs. However,this might include CNSs which are not conserved among allvertebrates, but conserved only between human andamphioxus.
To remove these CNSs and to identify CNSs conserved among all vertebrates, the authors collected
multiple orthologous vertebrate sequences and carried out

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Table 2 Possible functions of Tetra (TP) and Di (DP) paralogous CNSs
Name

ID

Function

Putative TFBS

References

A76-1

Anterior Hox promotera

Homeobox, E-box

Kim et al. (2000), This study

B76-1

Anterior Hox promotera

Homeobox, E-box

Kim et al. (2000), This study

C86-1

Anterior Hox promoteraHomeobox, E-box

Kim et al. (2000), This study

D84-2

Anterior Hox promotera

Homeobox, E-box

Kim et al. (2000), This study

A54-2

Hox4 Enhancer

RARE

Mainguy et al. (2003)(A) TP CNSs
TP1

TP2

TP3

B54-3

Hox4 Enhancer

RARE

Mainguy et al. (2003)

C54-3

Hox4 Enhancer

RARE

Mainguy et al. (2003)

D84-3

Hox4 Enhancer

RARE

Mainguy et al. (2003)

A43-7

Hox3 Enhancer

RARE

Mainguy et al. (2003)

B43-3
C4-3

Hox3 Enhancer
Hox4 Enhancera

RARE
RARE

Mainguy et al. (2003)
This study

D43-9

Hox3 Enhancer

RARE

Mainguy et al. (2003)(B) DP CNSs
DP1
DP2
DP3
DP4
DP5
DP6
DP7
DP8
a

E1-1

Hox13 Enhancer (distal limb enhancer)

PPAR-a, GATA-1, POU1F1a, Homeobox

Lehoczky et al. (2004)

E2-1

Hox13 Enhancer (distal limb enhancer)

PPAR-a, GATA-1, POU1F1a, Homeobox

Spitz et al. (2001)

E1-2

Hox13 Enhancer (distal limb enhancer)

C-Myb, Homeobox, YY1

Lehoczky et al. (2004)

E2-3

Hox13 Enhancer (distal limb enhancer)

C-Myb, Homeobox, YY1

Spitz et al. (2001)

C1110-2

Hox10 enhancera

SF1, CP1, Homeobox

This study

D1110-3

Hox10 enhancera

SF1, CP1, Homeobox

This study

A109-2

MicroRNA (mir-196 family)–

Yekta et al. (2004)

C109-2

MicroRNA (mir-196 family)–

Yekta et al. (2004)

C109-4

Hox9 enhancera

GR, E-box, CAT-box

This study

D109-1
B43-1

aHox9 enhancer
Hox3 enhancera

GR, E-box, CAT-box
GR

This study
This study

D84-1

Hox3 enhancera

GR

This study

A54-1

Bidirectional promotera

E-box, NF-1, E-box, CAT-box, TATA-box

This study

B54-1

Bidirectional promoter

E-box, NF-1, E-box, CAT-box, TATA-box

Dinger et al. (2008)

A43-12

Hox3 enhancera

USF, Homeobox

This study

B43-5

Hox3 enhancera

USF, Homeobox

This study

Putative function

phylogenetic footprinting analysis. The authors then identified the highly conserved ‘‘core region’’ of each vertebrate CNS. By comparing amphiCNSs with the vertebrate
CNSs, the authors found that only 16 out of 75 amphiCNSs
overlap with the vertebrate CNSs. Eight of them show deep
conservation; they are conserved among all the vertebrates
used in this study(see vertebrate CNSs information shownin Supplementary Table 2). Two of eight amphiCNSs werealigned with the ‘‘core region’’ of the vertebrate CNSs;
they are conserved among all the chordates used in thisstudy (Fig. 4a). These are located at the HoxA and HoxB
anterior regions, and supported a previous observation thatthe posterior region is more divergent than the anterior
region (Ferrier et al. 2000).
The remaining six amphiCNSs did not correspond to the‘‘core region’’ of the vertebrate CNSs (Fig. 4b and123

Supplementary Fig. 4). Interestingly, the ‘‘core region’’ ofthe vertebrate CNSs is often adjacent to the amphioxus–
human conserved regions. At last, only 2 out of 75 amphiCNSs are significantly highly conserved among
chordates.

Discussion
The authors defined 208 CNSs within the vertebrate Hox
clusters. To infer the depth of sequence conservation, theauthors investigated the existence of orthologous CNSs fromvertebrate species. The depth of conservation is different
with each cluster. The HoxC cluster shows the shallowest
conservation. Despite this result, the HoxC cluster retains
some paralogous CNSs. Shallow conservation of the HoxC

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433

Fig. 4 The phylogenetic footprinting analysis within chordates. The
authors compared the vertebrate CNSs with amphioxus–human CNSs
(amphiCNSs). The results of phylogenetic footprinting are described.
Each orange box corresponds to amphiCNS. a CNSs conserved

among all the vertebrates. b CNSs not conserved among all thevertebrates. Each green box represents highly conserved region
among vertebrates identified by phylogenetic footprinting. Each axis
and color is the same as Supplementary Fig. 2 (Color figure online)

cluster could be an artifact. Because the intergenic sequence
data of HoxC cluster is the poorest, the authors cannot detect
the intergenic conservation from several species accurately.
If the sequence data of Hox clusters are complete, then theabundance of CNS of each Hox cluster may not be sodifferent.
The number of the CNSs located at anterior region ishigher than that of CNSs located at posterior region. The
divergence of posterior paralogous Hox genes are more
rapid compared with other paralogous Hox genes, called‘‘posterior flexibility’’ (Ferrier et al. 2000). For example,
because posterior genes of the HoxD cluster are regulated
not only by each gene regulatory element but also by the

global control regulatory element located 240 kb upstreamof the cluster (Spitz et al. 2003), the intergenic region of
the posterior HoxD cluster might have poor conservation.
The posterior HoxA genes show similar expression patternwith the posterior HoxD genes. Therefore, this tendency
applies to the HoxA cluster. The HoxA cluster also haveglobal control enhancers located at upstream of the cluster(Lehoczky and Innis 2008).
The DP CNSs have many putative TFBSs (Table 2 andSupplementary Fig. 3). The homeobox binding motifs areespecially abundant. This suggests that DP CNSs areimportant for the auto regulatory mechanism of the four
vertebrate Hox clusters. Each Hox protein may bind to

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cis-regulatory regions of other Hox genes and controls theexpression patterns. E-box is the motif related to the HLH(helix-loop-helix) transcription factor. HLH and homeobox
proteins mainly regulate the expression pattern of Hox
genes. The DP7 CNSs bear the conserved TATA-box. Thissuggests that the DP7 CNSs have promoter function as theauthors described.
The authors identified three paralogous regions conserved among the four Hox clusters. One of them, FCS,
was previously reported (Kim et al. 2000). Surprisingly,
many RNAs are transcribed in this area. Different directional transcripts are started in the HoxA cluster. FCS of
the HoxB cluster corresponds with TSS of the HoxB6 gene.
In the HoxC cluster, FCS is the TSS of HoxC6, HoxC5 andHoxC4 coding transcripts. In the HoxD cluster, FCS might
control different directional transcripts. Not only FCS but
also other paralogous CNSs (DP7) between HoxA andHoxB clusters overlap with TSS and alternative exons(Fig. 3). Experimental approach revealed long non-coding
antisense RNA started from this HoxB cluster region(Dinger et al. 2008). Because RNA data are insufficient todetect all the cluster transcripts, some of these transcripts
are partial and were found only in human and/or mouse. It
is probable that these paralogous CNSs play importantroles in alternative transcription in other tetrapod species.
The other two TP CNSs (TP2 and TP3) include the
RARE (Mainguy et al. 2003). Their functions are experimentally confirmed (Morrison et al. 1997). Retinoids arethought to exert their activities at the transcriptional level,
acting as ligands to activate nuclear receptors. These
nuclear receptors recognize DNA sequences closely related
to 50 -(A/G)G(G/T)TCA-30 . Previous studies suggested that
retinoic acids contribute to the expressions of Hox genes(Dubrulle and Pourquié 2004). TP2 and TP3 have type11
and type3 RAREs, respectively. A conserved sequence,
TP3, downstream of HoxC4 gene was newly detected inthis study. This sequence is located more than 20 kb awayof the HoxC4 gene and corresponds to type3 RAREs.
Amphioxus also has RARE in this intergenic region (Wadaet al. 2006). However, the authors could not detect thiselement in this study. Only one motif conservation is difficult to detect using this method. Other motifs of thoseparalogous CNSs might function as cis-regulatory elementthat cooperates with RAREs.
It is possible that these TP CNSs are key components ofcluster organization. The motifs within them might havealready existed in the ancestor of vertebrates who had only
one Hox cluster. Because other motifs are not conserved
within the orthologous region of invertebrates but conserved in the paralogous region of vertebrates, they wereacquired after the emergence of vertebrates.
Pascual-Anaya et al. (2008) reported 75 amphiCNSs
which might include CNSs that are not conserved among

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J Mol Evol (2010) 71:427–436

Gain of ancestral TP/DP CNSs

11

2R WGD

Loss of some DP CNSs
Gain of orthologous CNSs

amphiHox

HoxA

3

5

56

37

HoxB

5

3
54

33

HoxC

HoxD

Tetrapods

Fig. 5 The loss and gain of Hox CNSs during the chordate evolution.
The Numbers of gain and loss of CNSs, shown in red and blue colors,
respectively, are apportioned to the known Hox gene tree (Color figure
online) (Color figure online) (Color figure online) (Color figure online)

all the vertebrates but conserved only between human andamphioxus. To remove these CNSs and to increase statistical significance, the authors compared multiple orthologous vertebrate sequences. The authors found that two
amphiCNSs are overlapped and conserved in vertebrate
CNSs. Ancestral DNA sequences of these CNSs haveprobably been under strong selective constraint throughout
the chordate evolution, though their conservation isdetected in only one Hox cluster. Other amphiCNSs might
not be conserved among all the vertebrates. However, theauthors should deal with this problem carefully, for only
two amphioxus genomes were used to detect CNSs conserved among chordates. More information of the Hox
cluster from non-vertebrate chordate genome is necessary
to obtain the complete picture of chordate CNSs.
The loss and gain of Hox CNSs are shown in the Fig. 5.
After the 2R WGD, the massive gains of CNSs were
occurred. In contrast, the conservation of non-coding
regions in the invertebrate genomes is low. This difference
on the Hox clusters may be related with the evolution ofvarious unique features of vertebrates. When vertebrates
acquired the more complex morphogenesis, the Hox clusters may become more conservative. To solve why thesehighly conserved CNS were appeared, it is necessary toconsider the relationship between the non-coding functions
and evolutionary conservations.
In summary, the authors efficiently detected orthologous
CNSs of vertebrates. The authors identified three paralogous CNSs, and one of them bears a newly detected RARE
motif. These CNSs are conserved among all the paralogous
Hox clusters, and might contribute to Hox cluster organization and gene expression patterns.
Acknowledgments The authors thank Drs. Kiyoshi Ezawa, Hiroki
Kokubo, Kazuho Ikeo, Toshihiko Shiroishi, and Hiroyuki Sasaki for
their many helpful discussions, suggestions, and comments. The

J Mol Evol (2010) 71:427–436
authors appreciate English polishing by Ms Rumiko Suzuki, Ms
Mahoko Takahashi, and Mr. Tim Jinam. This study was supported
partly by The Graduate University for Advanced Studies (Sokendai) to
M.M. and by Grant-in-Aid for scientific research from the Ministry of
Education, Culture, Sports, Science, and Technology of Japan to N.S.

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