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Vertebrate Paralogous Conserved Noncoding Sequences
May Be Related to Gene Expressions in Brain
Masatoshi Matsunami1,2,4, and Naruya Saitou1,2,3,*
1

Department of Genetics, School of Life Science, Graduate University for Advanced Studies (SOKENDAI), Mishima, Japan

2

Division of Population Genetics, National Institute of Genetics, Mishima, Japan3

Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan

4

Present address: Laboratory of Ecology and Genetics, Graduate School of Environmental Science, Hokkaido University, Sapporo, Japan*Corresponding author: E-mail: saitounr@lab.nig.ac.jp.
Accepted: December 18, 2012

Abstract
Vertebrate genomes include gene regulatory elements in protein-noncoding regions. A part of gene regulatory elements are expected to be conserved according to their functional importance, so that evolutionarily conserved noncoding sequences (CNSs) might
be good candidates for those elements. In addition, paralogous CNSs, which are highly conserved among both orthologous loci andparalogous loci, have the possibility of controlling overlapping expression patterns of their adjacent paralogous protein-coding genes.
The two-round whole-genome duplications (2R WGDs), which most probably occurred in the vertebrate common ancestors,
generated large numbers of paralogous protein-coding genes and their regulatory elements. These events could contribute to theemergence of vertebrate features. However, the evolutionary history and influences of the 2R WGDs are still unclear, especially innoncoding regions. To address this issue, we identified paralogous CNSs. Region-focused Basic Local Alignment Search Tool (BLAST)
search of each synteny block revealed 7,924 orthologous CNSs and 309 paralogous CNSs conserved among eight high-quality
vertebrate genomes. Paralogous CNSs we found contained 115 previously reported ones and newly detected 194 ones. Throughcomparisons with VISTA Enhancer Browser and available ChIP-seq data, one-third (103) of paralogous CNSs detected in this study
showed gene regulatory activity in the brain at several developmental stages. Their genomic locations are highly enriched near thetranscription factor-coding regions, which are expressed in brain and neural systems. These results suggest that paralogous CNSs areconserved mainly because of maintaining gene expression in the vertebrate brain.
Key words: noncoding sequences, genome duplication, vertebrates, gene regulation.

Introduction
Regulation of gene expression in a spatial and temporalmanner is crucial during the vertebrate development. Suchcomplex transcriptional regulations are thought to be
mediated by the co-ordinated binding of transcription factors
known as cis-regulatory elements. They allow integration ofmultiple signals to regulate the expression of specific genes.
These elements may not act on the physically closest gene but
can act across intervening genes (Spitz et al. 2003). It was
shown that certain genomic regions contain arrays of conserved noncoding sequences (CNSs), which are candidatesof cis-regulatory elements, clustered around developmental
regulatory genes (Bejerano et al. 2004; Woolfe et al. 2005;
McEwen et al. 2006; Hufton et al. 2009; Lee et al. 2011;

Takahashi and Saitou 2012). Some of them already tested
have been shown to act as enhancers in transgenic reporter
assays (Pennacchio et al. 2006; McEwen et al. 2006; Hufton
et al. 2009; Lee et al. 2011). These genomic regions also showconserved synteny that is a prominent feature of vertebrate
genomes. Moreover, these regions are conserved not only
orthologously but also paralogously.
These paralogous synteny are most probably derived fromancient genome duplications. Ohno (1970) proposed that the
two-round whole-genome duplications (2R WGDs) happenedat the intersection of early vertebrate evolution, now called as
the 2R hypothesis. It is clear that the 2R WGDs occurred earlyin the vertebrate evolution from phylogenetic and syntenic
analysis of vertebrates and invertebrates (Dehal and Booreß The Author(s) 2012. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which
permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Paralogous Conserved Noncoding Sequences in Vertebrates

2005; Nakatani et al. 2007; Putnam et al. 2008; Kuraku et al.
2009). These conserved synteny blocks are bearing paralogous
genes and CNSs (Kikuta et al. 2007). They are under thestrong evolutionary constraints and must have played important roles in the vertebrate evolution. However, the CNSs generated by the 2R WGDs and conserved among paralogous
synteny blocks are still not completely documented. These
sequences have a vertebrate-specific conservation and might
be related to vertebrate morphological features such as neuralcrest cells and complex brains. Thus, detecting paralogous
CNSs and inferring their characteristics are important in understanding the evolution of vertebrate genomes after the 2R
WGDs.
The vertebrate Hox clusters are one of the most wellknown examples of highly conserved paralogous syntenic regions (e.g., Garcia-Fernández 2005; Matsunami et al. 2010).
Vertebrates were shown to possess at least four Hox clusters,
whose genes are intimately involved in axial patterning and astrict relationship exists between respective genes and theirexpression limits in somitic and neural tissues. As a result of
their intimate involvement in early development, a change ofHox gene expression often triggers a vertebrate morphologicalchange (Cohn and Tickle 1999). The paralogous genes of the
Hox clusters show the similar expression patterns, which suggest that there might be shared gene regulatory mechanisms
among paralogous Hox clusters. Previously, we carried out the
search of CNSs within these clusters, not only among orthologous clusters but also among paralogous clusters, and identified three paralogous CNSs conserved within all four Hox
clusters of vertebrate species, which experienced no further
genome duplication (Matsunami et al. 2010). These CNSs
should contribute to the Hox cluster organization and gene
expression patterns.
We used a region-focused homology search to detect
weak paralogous conservations in this study. The method ofparalogous CNS identification is critical for the result. Previous
studies were mainly based on MegaBLAST (Zhang et al. 2000)
search of whole-genome sequences to detect the paralogous
CNSs (Bejerano et al. 2004; McEwen et al. 2006) on wholevertebrate species. This method is faster than conventional
Basic Local Alignment Search Tool (BLAST) search (Altschul
et al. 1997) and is effective to identify the paralogous CNSs
showing prominent high conservation among vertebrate
genomes. However, it is difficult to detect weak conservationof paralogous noncoding regions by using this strategy.
The orthologous conservation of noncoding region is statistically highly significant and easy to detect. In contrast, theparalogous conservation of noncoding region is usually
weaker than orthologous conservation of noncoding regions(e.g., Matsunami et al. 2010). To overcome this problem, aregion-focused BLAST search of each synteny block is useful.
This improved method allowed us to identify much weakerparalogous conservations derived from the 2R WGDs.

In this study, we characterized paralogous synteny blocks
derived from the 2R WGDs by using vertebrate genome dataand identified both orthologous and paralogous CNSs derived
from the 2R WGDs. These paralogous CNSs are frequently
located near the protein-coding regions functioned as transcription factors expressed in brain and neural systemand have potential to control similar expression patterns ofparalogs.

Materials and Methods
Identification of Conserved Synteny Blocks Afterthe 2R WGDs
Nakatani et al. (2007) reported 118 conserved vertebrate linkage (CVL) regions within the human genome through comparison with medaka fish genome sequences. A total of
10,618 protein-coding genes exist in these CVL regions.
Each of these regions retain four, three, or two paralogous
gene sets or there is only one gene without any paralogous
counterparts. These CVL regions were used as synteny block
data. The Ensembl BioMart interface (http://www.ensembl.
org/biomart/martview/, last accessed January 7, 2013) wasused to download gene information. Paralogous genes(four, three, or two gene retentions) were used as markers
of paralogous conservation to identify paralogous synteny
blocks. When paralogous genes show the same combinationof CVL groups, these paralogous gene sets are regarded asparalogous synteny blocks.

Identification of Paralogous CNSs
We identified paralogous CNSs within vertebrate genomes byusing the paralogous synteny block data. First, the humangenome sequences (Homo sapiens; NCBI36) were downloaded from the Ensembl database (http://www.ensembl.
org/, last accessed January 7, 2013), and they were divided
into the CVL regions following Nakatani et al. (2007). Repeat
and coding regions of each block were masked based on the
annotations of Ensembl database. BLAST (Altschul et al. 1997)
searches were carried out between human and mouse (Musmusculus; NCBI m37) orthologous blocks to detect orthologous CNSs. The default parameter settings of BLAST searchwere used. The cutoff value of BLAST search directly influences the result of orthologous CNS detection. Because weeffectively detected the orthologous CNSs from the Hox genecluster regions in our previous study (Matsunami et al. 2010),
the same cutoff bit score (200) was used for the human–
mouse comparison. To evaluate the conservation, human–
mouse CNSs were compared with six other vertebrate genomes. Species used were dog (Canis familiaris; CanFam 2.0),
cow (Bos taurus; Btau_4.0), opossum (Monodelphis domestica; monDom5), chicken (Gallus gallus; WASHUC2), lizard(Anolis carolinensis; AnoCar1.0), and frog (Xenopus tropicalis;
JGI 4.1). Teleosts were excluded because they underwent an

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additional genome duplication in their common ancestor. The
cutoff e value to identify orthologous CNSs was 105. Fromthese orthologous CNSs, we determined CNSs conserved
among all the eight vertebrate species (see supplementary
fig. S1, Supplementary Material online, for their phylogenetic
relationship). Those orthologous CNSs were compared witheach other with the threshold of 103 e value to detect
paralogous CNSs. We also searched the amphioxus
genome (Branchiostoma floridae v1.0; Putnam et al. 2008)
to confirm whether the detected paralogous CNSs wereconserved for invertebrate genomes using default BLAST
settings.

Functional Validation of Paralogous CNSs
The detected paralogous CNSs were compared withpreviously reported sequences, which were already tested
for the enhancer activities. The 1,619 human and mousenoncoding elements tested in transgenic mice at 11.5 days
postcoitum (dpc) were downloaded from VISTA Enhancer
Browser (http://enhancer.lbl.gov/, last accessed January 7,
2013; Visel et al. 2007). These sequences were comparedwith our paralogous CNSs through BLAST searches.
We also used murine ChIP-seq data produced by Shen et al.
(2012) to validate gene regulatory function of paralogous
CNSs. Enrichment of H3K4me3 or polII-binding signals is
indicative of an active promoter, whereas the presence of
H3K27ac outside promoter regions can be used as marks
for enhancers (Kim et al. 2005; Heintzman et al. 2007,2009; Creyghton et al. 2010; Rada-Iglesias et al. 2011).
CTCF binding is considered as a mark for potential insulator
elements (Kim et al. 2007). We downloaded the murine
ChIP-seq peak data (CTCF, H3K4me3, H3K27ac, and polII)and predicted enhancer data at E14.5 brain, 8-week-old
adult cortex, and 8-week-old adult cerebellum from murine
ENCODE HP (http://chromosome.sdsc.edu/mouse/, lastaccessed January 7, 2013). Overlaps between these peakdata and our paralogous CNSs were investigated. It should
be noted that Shen et al. (2012) carefully examined levels ofconservations of mouse genomic sequences they found. Thisis a clear contrast to the article of the ENCODE Project
Consortium (2012) who puzzlingly assigned newly defined"biochemical functions" for 80% of the human genome.
Other possible functions of paralogous CNSs are noncoding RNA (ncRNA), such as microRNA (miRNA) or long intergenic ncRNA (lincRNA). The human ncRNA sequences(H. sapiens; NCBI37) were downloaded from the Ensembl
database (http://www.ensembl.org/, last accessed January 7,2013) and compared against paralogous CNSs to evaluate thepossibility that paralogous CNSs function as ncRNA.

Ontology Analysis of Paralogous CNS-Harboring Genes
The paralogous CNS-harboring genes were defined, and theirfeatures were inferred in the following manner. The closest

paralogs derived from the 2R WGDs, which are conserved
among both paralogous loci, were defined as paralogous
CNS-harboring genes (supplementary fig. S2, Supplementary
Material online). We then conducted statistical analysis byusing Gene Ontology database (http://www.geneontology.
org/, last accessed January 7, 2013) to find significantly enriched paralogous CNS-harboring genes. Analysis of genefunction enrichment was performed using Fatigo+ web
server (Al-Shahrour et al. 2007). The paralogous CNSharboring genes were compared with the entire humangenes in the Gene Ontology database to detect the overrepresented paralogous CNS-harboring genes. The expression
regions and timings of paralogous CNS-harboring genes werealso analyzed. The eGenetics (http://www.nhmrc.gov.au/
your_health/egenetics/index.htm, last accessed January 7,2013) database was used to investigate gene expression ofparalogous CNS-harboring genes. Human anatomical systemdata, which give information about gene expression regions
and expressed timings, were obtained from the eGenetics
database by using the Ensembl Biomart (Kelso et al. 2003).
We counted numbers of each gene category (paralogs derived
from the 2R WGDs and paralogous CNS-harboring genes expressed in each organ and timing) and obtained their frequencies by dividing the numbers by the number of all genes.

Results
Identification of Orthologous CNSs
To identify orthologous CNSs shared among vertebrate species, we carried out comprehensive BLAST searches. We discovered 67,052 CNSs from human and mouse genome
comparison under the following settings: >100 bp lengthand >78% similarity. Their average length was 318 bp. We
compared these human–mouse CNSs with other vertebrate
species. The genomes of dog, cow, opossum, chicken, lizard,
and frog shared 62,611, 65,878, 44,726, 24,549, 19,724,
and 10,664 CNSs with human and mouse, respectively. The
number of orthologous CNSs is gradually decreased following
the order of the evolutionary divergence from human andmouse. Interestingly, we identified more than 10,000 noncoding conservations from the genome of frog (X. tropicalis) in
spite of the large divergence from mammals. Among the67,052 human–mouse orthologous CNSs, 7,650 CNSs wereconserved in all the eight species employed in this study; they
are listed in supplementary table S1, Supplementary Material
online. These CNSs may be recognized among all vertebrate
species, which did not experience further genome duplications. Each synteny block contains 65 orthologous CNSs on
average. The synteny blocks are scattered across the wholegenome of each species except for the mammalian Y chromosome. The lack of conservation might be related to the characteristic feature of this sex chromosome. We could not findany correlations between orthologous gene density andorthologous CNS density, although important development

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genes such as Hox or Sox have many CNSs, other CNSs areequally distributed in the genome.

Highly Conserved Synteny Blocks
We then identified pairs of paralogous block sets (diparalogous sets), trios of paralogous block sets (triparalogous sets),and quartets of paralogous block sets (tetraparalogous sets)(tables 1 and 2). The genomic locations of paralogous synteny
blocks are shown in figure 1. Lundin et al. (2003) reported thatthe chromosomes bearing the Hox clusters frequently include
paralogous genes derived from the 2R WGDs and are organized large synteny blocks. These blocks have been considered
as hallmarks of the 2R WGDs and include not only the Hox
clusters but also other important genes such as Dlx, Gbx, Gli,and collagen genes. Our study also showed that Hox-linked
paralogous synteny blocks had prominent paralogous conservation of not only coding regions but also noncoding regions.
These Hox-linked paralogous synteny blocks were oneof highly conserved synteny blocks including abundant

Table 1
Number of Paralogous CNS Harboring Genes: Gene and CNS Loss
Pattern of Duplicated Regions
Conservation
Level
4
3
2
1
Total

No. of Paralogous
Gene Group(No. of Genes)

No. of Paralogous
CNS Group(No. of CNSs)

50 (50  4 ¼ 200)
220 (220  3 ¼ 660)
861 (861  2 ¼ 1,722)
8,036 (8,036  1 ¼ 8,036)
9,167 (10,618)0
3 (3  3 ¼ 9)150 (150  2 ¼ 300)
7,341 (7,341  1 ¼ 7,341)
7,494 (7,650)

Table 2
Number of Paralogous CNS Harboring Genes
No. of Paralogous CNSs
Quartets of paralogous genes
4
3
2
0
Total
Trios of paralogous genes
3
2
0
Total
Pairs of paralogous genes
2
0
Total

No. of CNS Harboring Genes
0
2
13
36
50
1
29
190
220
31
830
861

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FIG. 1.—Paralogous synteny blocks within the human genome. Genomic distribution of paralogous synteny blocks is shown. (A) Di-, (B) tri-, and (C) tetraparalogous blocks are identified by the geneorder and homology.

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FIG. 2.—Scheme of Hox-linked paralogous block. Paralogous gene conservations and paralogous CNSs conservations are shown. Hox-linked paralogous
synteny blocks also show prominent conservation of not only coding regions but also noncoding regions. Tetraparalogous, triparalogous, and diparalogous
genes are represented as red, blue, and green thin lines, respectively, and diparalogous CNSs represent green thick lines. We could not identify tetraparalogous or triparalogous CNSs in these regions.

paralogous genes. These blocks also have the high numbers ofparalogous CNSs (shown with thick green lines in the fig. 2)
and protein-coding genes. The paralogous gene-dense
regions correspond to the dense paralogous CNS regions.
Because each paralogous Hox gene showing similar expression pattern controls the vertebrate early development, CNSs
in the Hox cluster might function as cis-regulatory elements
such as already known paralogous conserved elements(Lehoczky et al. 2004) and control the similar expression ofparalogous Hox or neighboring genes.

Paralogous CNSs
From the vertebrate-specific orthologous CNSs, 309 paralogous CNSs were identified, and they are listed in supplementary table S2, Supplementary Material online. Only threeparalogous CNSs (SB2CNS1101, SB78CNS1119, andSB59CNS435) showed weak sequence similarities withthose in the amphioxus genome. Because we could notdetect clear sequence similarities between sequences of
the amphioxus genome and vertebrate paralogous CNS
groups, vertebrate paralogous CNSs might have emerged
after amphioxus and the common vertebrate ancestor
diverged. We thus focus on these vertebrate-specific CNSs
in the later analyses.
We found diparalogous and triparalogous CNSs; however,tetraparalogous CNSs were not detected, probably because
conservation of noncoding regions was lower than that ofcoding regions. Each paralogous synteny block bears several
conservation levels of genes and CNSs, such as trios or pairs.
We compared paralogous CNSs detected in this study withalready described CNSs. Among the 309 paralogous CNSs,
194 (63%) were newly determined, whereas confirming
115 previously reported paralogous CNSs (McEwen et al.
2006). There were 15 paralogous CNSs corresponding to

ncRNA (5 pairs, 1 trio, and 2 members of pair). ThesencRNA included 12 miRNA and 3 lincRNA (supplementarytable S3, Supplementary Material online). The paralogous
CNS trio was located near the MEF family genes (MEF2A,
MEF2C, and MEF2D). These paralogous CNSs function as
ncRNA and are assumed to regulate the expression of neighboring genes.
According to VISTA Enhancer Browser database (Visel et al.
2007), 83 (27%) paralogous CNSs were already validated fortheir enhancer function through transgenic mice experiments.
The 61% (51 of 83) of CNSs had positive enhancer functions,when mice were at 11.5 days postcoitum. Although the remaining 22 (39%) CNSs were not detected to have an enhancer activity, they have a possibility of silencer function. Among
CNSs having positive enhancer functions, the 82% (42 of 51)
of CNSs showed the prominent expression at the developmental brain region. Moreover, we used the available
ChIP-seq data to confirm the enhancer function of paralogous
CNSs in the brain region. From the read mapping data of
ChIP-seq, 55, 16, and 17 paralogous CNSs corresponded toat least one of ChIP-seq peaks at E14.5 brain, 8-week-old
adult cortex, and 8-week-old adult cerebellum, respectively(supplementary table S2, Supplementary Material online).
In summary, 72 paralogous CNSs were overlapped with
ChIP-seq peaks. Although these experiments covered only
three time points of developmental stages, 103 paralogous
CNSs (42 CNSs are positive at enhancer database and 72 CNSs
are positive at ChIP-seq data) showed enhancer function in
the brain region. These results suggest that paralogous CNSs
may frequently regulate genes, which express brain regions atseveral developmental stages.
Figure 3 illustrates one of paralogous CNS pairs located in
CVL11 and CVL35. These CVLs contain paralogous transcription factor genes (POU3F2 and POU3F3), and they are

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FIG. 3.—Paralogous CNSs shared between POU3F2 and POU3F3 genes. Genomic locations of each orthologous CNS in the human genome and thealignment of paralogous CNS are shown. This paralogous CNS pair is located nearby POU3 paralogs, POU3F2 (BRN2), and POU3F3 (BRN1), that is derived
from the 2R WGDs. These are strong candidates of gene regulatory sequences of these paralogs.

assigned as "paralogous CNS-harboring genes" for these CVL
regions. In the multiple alignment, orthologous CNSs are obviously highly conserved. However, the conservation of paralogous CNSs is weaker than that of orthologous CNSs, sothat previous studies could not detect these paralogous CNSs.
These newly detected paralogous CNSs have a possibility towork as a distal enhancer for POU3F paralogous genes.

Location of CNSs and Paralogous CNS-Harboring Genes
We searched paralogous CNS-harboring genes of each paralogous CNS (tables 1 and 2). We then inferred the functional
bias of paralogous CNS-harboring genes based on the Gene
Ontology database and the gene expression database.
Tables 3 and 4 present paralogous CNS-harboring genes
with particularly abundant paralogous CNSs. The majority ofparalogous CNSs are located at intron, upstream region or
downstream region of the genes encoding transcription
factor. We identified three gene families each of which retained paralogous CNS trios (fig. 4A–C) and five gene families
each of which retained more than three paralogous CNS pairs(fig. 4D–H). The functions of paralogous CNSs located nearwell-studied transcription factors such as FoxP1/P2, Sox14/21,
and the Irx cluster are already known (de la Calle-Mustienes

et al. 2005; McEwen et al. 2006). These paralogous CNSs
function as distal enhancers and partially share their gene
expression regions between paralogous pairs. Among otherparalogous CNSs, some paralogous CNSs also showed experimentally validated distal enhancer functions in the databaseor function as ncRNA (supplementary tables S2 and S3,
Supplementary Material online). This result strongly suggeststhat the remaining paralogous CNS pairs whose function is
not yet tested have also enhancer or ncRNA function.
Table 5 is the result of the gene ontology analysis. The
paralogous CNS-harboring genes were compared with theentire human genes in the Gene Ontology database. We
found that paralogous CNSs were frequently located neargenes which function as sequence-specific DNA binding(i.e., transcription factors). These results are consistent withprevious studies and suggest that, after the 2R WGDs,
genes which function as gene regulation were more conservative than genes having other functions.
The expression regions and stages of paralogous
CNS-harboring genes were investigated by examining theeGenetics database. We found that paralogous CNSharboring genes frequently include genes expressed in thebrain at early developmental stages (supplementary fig. S3,
Supplementary Material online). The proportions ofGenome Biol. Evol. 5(1):140–150. doi:10.1093/gbe/evs128 Advance Access publication December 24, 2012

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Table 3
List of Paralogous CNSs Harboring Genes: Pair of Paralogous CNSs
Number of Pairs
6
5
4
3
2

1

Pair of Harboring Gene
FOXP1&FOXP2, ZNF503&ZNF703
IRX1&IRX3
PBX1&PBX3, SALL1&SALL3
EBF1&EBF3, EVX1&EVX2, NR2F1&NR2F2, POU4F1&POU4F2, SOX5&SOX6
ESRP1&ESRP2, FOXB1&FOXB2, HOXA5&HOXB5, LMO1&LMO3, LRBA&NBEA, LRP3&LRP12, NEUROD1&NEUROD2,
NRXN1&NRXN3, OTX1&OTX2, POU3F1&POU3F2, POU3F2&POU3F3, PRDM16&MECOM, SLIT2&SLIT3, SOX14&SOX21,
TCF4&TCF12, TFAP2A&TFAP2B, TOX&TOX3, TSHZ1&TSHZ2, VRK1&VRK2
ACTL6A&ACTL6B, ARL5A&ARL5C, ARSB&ARSI, ARSJ&ARSB, BACE1&BACE2, BMP3&GDF10, CCNL1&CCNL2, CPA1&CPA2,
CUX1&CUX2, DNM1&DNM3, ENPP2&ENPP3, FOXO1&FOXO3, FOXP2&FOXP4, GNB2&GNB4, GPC2&GPC6, GPC3&GPC5,GPM6A&PLP1, GRIA2&GRIA3, HMGB1&HMGB3, HOXA4&HOXD4, HSF2&HSF4, ING1&ING2, INPP5D&SH2D1A, IRX2&IRX5,
KANK1&KANK4, KCNK9&KCNK15, KHDRBS2&KHDRBS3, LASS3&LASS6, MACF1&DST, MBNL1&MBNL2, MCTP1&MCTP2,
MEF2A&MEF2C, MEIS1&MEIS2, NFIA&NFIB, NPNT&EGFL6, ODZ2&ODZ3, P4HA1&P4HA2, PDE4B&PDE4C, PIK3C2A&PIK3C2B,
PLS1&PLS3, PTCH1&PTCH2, QSOX1&QSOX2, R3HCC1&c10orf28, RALA&RALB, RHAG&RHCG, RNF38&RNF44, SALL1&SALL4,
SEC24C&SEC24D, SEPT6&SEPT10, SGMS1&SGMS2, SH3RF3&SORBS2, SHH&IHH, SLC12A1&SLC12A3, SLC12A2&SLC12A3,
SLC4A4&SLC4A10, SLC6A15&SLC6A18, SLC9A2&SLC9A3, SLIT1&SLIT2, SMAD2&SMAD3, SOX1&SOX2, SOX2&SOX3,
ST8SIA3&ST8SIA4, SULF1&GNS, TFAP2A&TFAP2C, ZEB2&KIAA0087, ZFHX3&ZFHX4, ZIC2&ZIC3, ZNF423&ZNF521

Table 4
List of Paralogous CNSs Harboring Genes: Trio of Paralogous CNSs
Number of Trio

Trio of Harboring Gene

1

MEF2A&MEF2C&MEF2D, NFIA&NFIB&NFIX, and GRIA1&GRIA2&GRIA4

paralogous CNSs enhancer activities were compared with theproportions of the entire enhancer database (table 6 and supplementary table S2, Supplementary Material online). Majorityof paralogous CNSs in the enhancer database show expression in the brain at an early developmental stage (P < 0.01, 2test). These results imply that existing paralogous CNSs may
contribute to the vertebrate-specific complex brain morphology at their early developmental stages.

Because we used genome sequences of many species, otherconserved elements missing in some species were notincluded in our results. These missed conserved elements
may be overlapped with long gap regions. Because some
CNSs are missed because of low genome quality, our results
may underestimate the number of orthologous CNSs.
However, it is clear that orthologous CNSs we detected are significantly conserved throughout the vertebrate evolution.
Why the paralogous synteny block is conserved remains
elusive. The genomic regulatory block hypothesis is proposed
to explain this enigmatic synteny blocks (Becker and Lenhard
2007; Kikuta et al. 2007). This hypothesis suggests that CNSs
scattered across each synteny block prevent each block frombreakage of the synteny. Under this hypothesis, paralogous
CNSs maintain paralogous conserved synteny. We inferred therelationship between the distribution of paralogous CNSs andthe distribution of paralogous genes. As the result, paralogous
gene orders (synteny) and paralogous CNS conservations are
weakly correlated, especially for tetraparalogous synteny
blocks (Pearson’s product–moment correlation coefficient ¼ 0.485; fig. 5). This means that paralogous synteny
blocks bearing many paralogs also include abundant paralogous CNSs. In other words, highly conserved syntenic regions
have more paralogous CNSs. This implies that these paralogous CNSs may constrain the synteny blocks from the breakage and play a key role in the genomic regulatory block
hypothesis.

Discussion
In this study, we identified vertebrate-specific CNSs, and they
may be related to the vertebrate-specific features. Previously,
CNSs were described by several criteria. Bejerano et al. (2004)
defined 483 ultra-conserved elements (UCEs). These included
coding regions and 200 bp length with 100% identity
among human, rat, and mouse. Woolfe et al. (2007) defined
6,957 CNEs that were 40 bp length and 65% identity between human and fugu. These genome-wide studies usedvery stringent criteria and often missed rather weak paralogous conservations. It is difficult to compare those results with
our present study, partly because compared genome sequences are different. Our results covered 308 human–rat–
mouse UCEs and 3,388 human–fugu CNEs, whereas otherconserved elements previously detected are not overlappedwith our CNSs. We defined vertebrate-specific CNSs as conserved among all the eight vertebrate species in this study.

146 Genome Biol. Evol. 5(1):140–150. doi:10.1093/gbe/evs128 Advance Access publication December 24, 2012

Genome Biol. Evol. 5(1):140–150. doi:10.1093/gbe/evs128 Advance Access publication December 24, 2012250 kbp

CVL. 78 (Chr. 16)
SALL1

SALL3

FOXP2

FOXP1

GRIA4

GRIA2

GRIA1

0

100kbp

CVL. 56 (Chr. 9)

CVL. 5 (Chr. 1)

100 kbp

CVL
CVL. 46 (Chr. 8)

H

0

CVL. 89 (Chr.19)

CVL. 51 (Chr. 9)

10 kbp

CVL. 2 (Chr. 1)

CVL. 59 (Chr. 10)

E

0

B

PBX3

PBX1

ZNF703

ZNF503

NFIX

NFIB

NFIA

0

500 kbp

CVL. 78 (Chr. 16)

CVL. 26 (Chr. 5)

50kbp

CVL
CVL. 4 (Chr.
(Ch 1)F

0

CVL. 27 (Chr. 5)

CVL. 76 (Chr. 15)

C

MEF2D

MEF2A

MEF2C

IRX3

IRX1

FIG. 4.—The locations of paralogous CNS. The triparalogous CNSs were identified only near (A) GRIA gene family, (B) NFI gene family, and (C) MEF2 gene family. The paralogous CNS-harboring genes
having more than three pair-paralogous CNS pairs are only five gene families. These are (D) FOXP1/P2, (E) ZNF503/703, (F) IRX1/3, (G) SALL1/3, and (H) PBX1/3. Protein-coding regions of paralogous
CNS-harboring genes are represented by black boxes. The orange ellipse is paralogous CNSs. The connected lines show paralogous conservation of each CNS.

0

50 kbp

CVL. 87 (Chr. 18)

G

0

CVL. 44 (Chr. 7)

10 kbp

CVL. 63 ((Chr. 11))

CVL. 25 (Chr. 4)

CVL. 28 (Chr. 5)

CVL. 18 (Chr. 3)

D

0

A

Paralogous Conserved Noncoding Sequences in Vertebrates

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The majority of vertebrate CNSs may have been generated
before the divergence of the major extant vertebrate lineages.
The sequencing of the genome of a cephalochordate, amphioxus (Branchiostoma ﬂoridae), has uncovered traces of theorigins of very small number of vertebrate CNSs (Holland
et al. 2008; Putnam et al. 2008). Although we also foundTable 5Overrepresented Gene Functions of Host Genes
GO Term

P

Sequence-speciﬁc DNA binding (GO:0043565)
Ionotropic glutamate receptor activity (GO:0004970)
Phosphoinositide binding (GO:0035091)
Lipid kinase activity (GO:0001727)
1-Phosphatidylinositol-3-kinase activity (GO:0016303)
Follicle-stimulating hormone receptor activity (GO:0004963)
Low-density lipoprotein receptor activity (GO:0005041)

3.39E–15
7.69E–05
6.05E–05
5.33E–04
8.92E–06
1.77E–04
3.41E–04

NOTE.—Adjusted P values are calculated by comparing the distribution of thehost genes with that of human genes.

Table 6
Proportion of Enhancer Activities
Expression
No expression
At brain region
At other region
Total

Paralogous CNSs

All Sequences in Database22 (26.51%)
42 (50.60%)
19 (22.89%)
83815 (50.34%)
416 (25.69%)
388 (23.97%)
1,619

few weak conservations in noncoding regions between theamphioxus genome and vertebrate orthologous CNSs, we
could not detect conservations that were shared among
each vertebrate paralogous loci and amphioxus noncoding
regions. Invertebrate groups have been found to possess
their own sets of CNSs (Glazov et al. 2005; Vavouri et al.
2007), and interestingly, there are similarities between thefunctions of genes around with both vertebrate and invertebrate CNSs cluster. This suggests parallel evolution of CNS
networks (Vavouri et al. 2007). Consequently, although theslow evolution of coding sequences can be charted readily
across the invertebrate/vertebrate boundary, the CNSs changed very quickly during the vertebrate evolution. Recently,
noncoding sequences that are conserved from several basal
vertebrates were reported. The elephant shark (Callorhinchus
milii) is a cartilaginous fish and a basal jawed vertebrate. Its
genome contains a few thousand vertebrate CNSs in spite of
their low-coverage genome information (Lee et al. 2011).
However, the number of CNSs retained in the sea lamprey(Petromyzon marinus), one of extant jawless vertebrates, ismuch smaller than that of other vertebrates (McEwen et al.
2009). The lamprey CNSs show remarkably shorter lengths
than those of vertebrates and low homology with vertebrate
CNSs. Whether the jawless vertebrate genomes experiencethe 2R WGDs is still unclear, so that the orthologies ofsequences are difficult to assign (Kuraku et al. 2009).
Nevertheless, these observations suggest that vertebrate
CNSs have not constantly evolved. We can interpret that lamprey noncoding sequences are extremely changed such as

FIG. 5.—Relationship between numbers of paralogs and paralogous CNSs derived from the 2R WGDs. Paralogs and paralogous CNSs within the synteny
blocks were counted and were plot to a scatter plot. The horizontal axis is the number of conserved paralogous genes. Vertical axis is the number ofconserved paralogs CNSs. The red line is an approximate linear regression of tetraparalogous block points. There is a clear positive correlation.

148 Genome Biol. Evol. 5(1):140–150. doi:10.1093/gbe/evs128 Advance Access publication December 24, 2012

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their coding regions (Qiu et al. 2011), or the evolutionary rateof jawed vertebrate noncoding region has become slower
after the jawless vertebrate lineages branched off.
To answer these issues, we should analyze the high-quality
basal vertebrate genomes. In either case, the existence ofmassive CNSs is not an usual situation compared with otherinvertebrate species closer to vertebrates. These CNSs might
contribute to vertebrate-specific features.
The existences of paralogous CNSs detected in thisstudy are difficult to explain by previous duplication models.
Classical models predicted that the most likely fate of duplicated genes is the degeneration of one of the pair to a pseudogene (or completely lost from the genome) or less frequently
the acquisition of novel gene functions as a result of alterations in coding or regulatory sequences in a process known
as neo-functionalization. Force et al. (1999) proposed the
duplication–degeneration–complementation (DDC) model inwhich duplicated genes undergo complementary deleterious
mutations in independent subfunctions, so that both genes
are required to share the functions of the ancestral gene.
These models are difficult to explain the existence of paralogous CNSs. An alternative model is the gene balance hypothesis proposed by Papp et al. (2003). It postulates that selection
against gene dosage imbalances will promote the retention ofparticular types of genes, though this model has not explicitly
been applied to evolutionary fates of noncoding sequences(Papp et al. 2003). Immediately after a WGD event,genome-wide relative gene dosage is maintained, but subsequent step-wise mutation or deletion of duplicated genes canlead to deleterious dosage imbalances. Genes whose proteins
have many interaction partners may be more sensitive to thesedosage changes, possibly leading to an over-retention ofhighly connected gene functions, such as transcriptional regulators and signaling complexes. Conversely, small-scale duplications immediately disrupt relative dosage, so highly
connected genes should avoid this type of duplication
during evolution. Duplicated genes derived from the 2R
WGDs are more sensitive to dosage change than other duplicated genes and frequently associated with disease in humangenome (Makino and McLysaght 2010). This different correlation between gene retention after WGD and small-scale duplication is a key distinction between the gene balance
hypothesis and the DDC model; DDC model should promote
the same patterns of gene retention for all types of gene duplication. In support of the gene balance hypothesis, vertebrate genes that function in transcription regulation orsignal transduction are over-retained after the 2R WGDevents but not after small-scale duplications (Blomme et al.
2007). We also found that the paralogous CNSs are frequently
retained near the transcription factors. The transcription anddevelopmental genes have more complex function than othergenes, such as pleiotropic expressions, highly connected protein networks, and dosage sensitivity. These characters may
allow greater subfunctionalization. They often share gene

expression regions and have similar functions among paralogous genes. However, the existence of paralogous CNSs isdifficult to be explained by the DDC model, because thismodel does not assume same enhancer functions among paralogous loci. The one possible explanation of the existence ofparalogous CNSs is the gene balance hypothesis (Papp et al.
2003). These paralogous CNSs have possibility to control similar expression patterns of paralogs and dosage compensationof paralogs through the highly conserved sequences.
The alternative possible function of paralogous CNSs isncRNA. Rinn et al. (2007) reported interchromosomal interactions between paralogous regions through ncRNA. Someenhancers act not only cis but also trans via ncRNA transcription (Ørom et al. 2010). Paralogous CNSs may be related tothese interchromosomal interactions of duplicated genome
regions.

Supplementary Material
Supplementary figures S1–S3 and table S1–S3 are availableat Genome Biology and Evolution online (http://www.gbe.
oxfordjournals.org/).

Acknowledgments
The authors thank Drs. Shigehiro Kuraku, Akatsuki Kimura,Toshiyuki Takano, Hiroshi Akashi, and Toshihiko Shiroishi for
their many helpful discussions, suggestions, and comments.
This work was supported partly by the Graduate University for
Advanced Studies (SOKENDAI) and SOKENDAI Short-term
Study-abroad Program to M.M. and by a Grant-in-Aid forscientific research from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan to M.M. and N.S.

Literature Cited
Al-Shahrour F, et al. 2007. FatiGO+: a functional profiling tool for genomic
data. Integration of functional annotation, regulatory motifs and interaction data with microarray experiments. Nucleic Acids Res. 35:
W91–W96.
Altschul SF, et al. 1997. Gapped BLAST and PSI-BLAST: a new generationof protein database search programs. Nucleic Acids Res. 25:
3389–3402.
Becker TS, Lenhard B. 2007. The random versus fragile breakage
models of chromosome evolution: a matter of resolution.
Mol Genet Genomics. 278:487–491.
Bejerano G, et al. 2004. Ultraconserved elements in the human genome.
Science 304:1321–1325.
Blomme T, et al. 2007. The gain and loss of genes during 600 million yearsof vertebrate evolution. Genome Biol. 7:R43.
Cohn MJ, Tickle C. 1999. Developmental basis of limblessness and axial
patterning in snakes. Nature 399:474–479.
Creyghton MP, et al. 2010. Histone H3K27ac separates active from poisedenhancers and predicts developmental state. Proc Natl Acad Sci U S A.
107:21931–21936.
Dehal P, Boore JL. 2005. Two rounds of whole genome duplication in theancestral vertebrate. PLoS Biol. 3:1700–1708.

Genome Biol. Evol. 5(1):140–150. doi:10.1093/gbe/evs128 Advance Access publication December 24, 2012

149

GBE

Matsunami and Saitou

de la Calle-Mustienes E, et al. 2005. A functional survey of the enhancer
activity of conserved non-coding sequences from vertebrate Iroquois
cluster gene deserts. Genome Res. 15:1061–1072.
ENCODE Project Consortium. 2012. An integrated encyclopedia of DNA
elements in the human genome. Nature 489:57–74.
Force A, Lynch M, Pickett FB, Amores A, Yan YL. 1999. Preservation of
duplicate genes by complementary, degenerative mutations. Genetics
151:1531–1545.
Garcia-Fernández J. 2005. The genesis and evolution of homeobox geneclusters. Nat Rev Genet. 6:881–892.
Glazov EA, Pheasant M, McGraw EA, Bejerano G, Mattick JS. 2005.
Ultraconserved elements in insect genomes: a highly conserved
intronic sequence implicated in the control of homothorax mRNA
splicing. Genome Res. 15:800–808.
Heintzman ND, et al. 2007. Distinct and predictive chromatin signaturesof transcriptional promoters and enhancers in the human genome.
Nat Genet. 39:311–318.
Heintzman ND, et al. 2009. Histone modifications at human enhancers
reflect global cell-type-specific gene expression. Nature 459:108–112.
Holland LZ, et al. 2008. The amphioxus genome illuminates vertebrate
origins and cephalochordate biology. Genome Res. 18:1100–1111.
Hufton AL, et al. 2009. Deeply conserved chordate noncoding sequences
preserve genome synteny but not drive gene duplication retention.
Genome Res. 19:2036–2051.
Kelso J, et al. 2003. eVOC: a controlled vocabulary for unifying gene
expression data. Genome Res. 13:1222–1230.
Kikuta H, Fredman D, Rinkwitz S, Lenhard B, Becker TS. 2007. Retroviralenhancer detection insertions in zebrafish combined with comparative
genomics reveal genomic regulatory blocks—a fundamental featureof vertebrate genomes. Genome Biol. 8(1 Suppl):S4.
Kim TH, et al. 2005. A high-resolution map of active promoters in thehuman genome. Nature 436:876–880.
Kim TH, et al. 2007. Analysis of the vertebrate insulator protein
CTCF-binding sites in the human genome. Cell 128:1231–1245.
Kuraku S, Meyer A, Kuratani S. 2009. Timing of genome duplications
relative to the origin of the vertebrates: did cyclostomes diverge
before or after? Mol Biol Evol. 26:47–59.
Lee AP, Kerk SY, Tan YY, Brenner S, Venkatesh B. 2011. Ancient vertebrate conserved noncoding elements have been evolving rapidly inteleost fishes. Mol Biol Evol. 28:1205–1215.
Lehoczky JA, Williams ME, Innis JW. 2004. Conserved expression domains
for genes upstream and within the HoxA and HoxD clusters suggests
a long-range enhancer existed before cluster duplication. Evol Dev. 6:
423–430.
Lundin LG, Larhammar D, Hallböök F. 2003. Numerous groups of chromosomal regional paralogies strongly indicate two genome doublings at
the root of the vertebrates. J Struct Funct Genomics. 3:53–63.
Makino T, McLysaght A. 2010. Ohnologs in the human genome aredosage balanced and frequently associated with disease. Proc Natl
Acad Sci U S A. 107:9270–9274.
Matsunami M, Sumiyama K, Saitou N. 2010. Evolution of conserved
non-coding sequences within the vertebrate Hox clusters throughthe two-round whole duplications revealed by phylogenetic footprinting analysis. J Mol Evol. 71:427–436.
McEwen GK, et al. 2006. Ancient duplicated conserved noncoding
elements in vertebrates: a genomic and functional analysis. Genome
Res. 16:451–465.
McEwen GK, Goode DK, Parker HJ, Woolfe A, Callaway H, et al. 2009.
Early evolution of conserved regulatory sequences associated with
development in vertebrates. PLoS Genet. 12:e1000762.
Nakatani Y, Takeda H, Kohara Y, Morishita S. 2007. Reconstruction of thevertebrate ancestral genome reveals dynamic genome reorganizationin early vertebrates. Genome Res. 17:1254–1265.
Ohno S. 1970. Evolution by gene duplication. New York: Springer-Verlag.
Ørom UA, et al. 2010. Long noncoding RNAs with enhancer-like function
in human cells. Cell 43:46–58.
Papp B, Pál C, Hurst LD. 2003. Dosage sensitivity and the evolution of genefamilies in yeast. Nature 424:194–197.
Pennacchio LA, et al. 2006. In vivo enhancer analysis of human conserved
non-coding sequences. Nature 444:499–502.
Putnam NH, et al. 2008. The amphioxus genome and the evolution of thechordate karyotype. Nature 453:1064–1071.
Qiu H, Hildebrand F, Kuraku S, Meyer A. 2011. Unresolved orthology andpeculiar coding sequence properties of lamprey genes: the KCNA
gene family as test case. BMC Genomics 12:325.
Rada-Iglesias A, et al. 2011. A unique chromatin signature uncovers early
developmental enhancers in humans. Nature 470:279–283.
Rinn JL, et al. 2007. Functional demarcation of active and silent
chromatin domains in human HOX loci by noncoding RNAs. Cell
129:1311–1323.
Shen Y, et al. 2012. A map of the cis-regulatory sequences in the mouse
genome. Nature 488:116–20.
Spitz F, Gonzalez F, Duboule D. 2003. A global control region defines achromosomal regulatory landscape containing the HoxD cluster. Cell
113:405–417.
Takahashi M, Saitou N. 2012. Identification and characterization of
lineage-specific highly conserved noncoding sequences in mammalian
genomes. Genome Biol Evol. 4:641–657.
Vavouri T, Walter K, Gilks WR, Lehner B, Elgar G. 2007. Parallel evolutionof conserved non-coding elements that target a common set ofdevelopmental regulatory genes from worms to humans. Genome
Biol. 8:R15.
Visel A, Minovitsky S, Dubchak I, Pennacchio LA. 2007. VISTA Enhancer
Browser—a database of tissue-specific human enhancers. Nucleic
Acids Res. 35:D88–D92.
Woolfe A, et al. 2005. Highly conserved non-coding sequences areassociated with vertebrate development. PLoS Biol. 3:116–130.
Woolfe A, et al. 2007. CONDOR: a database resource of developmentally-associated conserved non-coding elements. BMC Dev Biol. 7:
100.
Zhang Z, Schwartz S, Wagner L, Miller W. 2000. A greedy algorithm foraligning DNA sequences. J Comput Biol. 7:203–214.
Associate editor: Wen-Hsiung Li

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