Genome Biology and Evolution Advance Access published April 13, 2012 doi:10.1093/gbe/evs035

Identification and characterization of lineagespecific highly conserved noncoding sequences inmammalian genomes

1) Department of Genetics, School of Life Science, Graduate University for Advanced Studies,
Mishima 411-8540, Japan
2) Division of Population Genetics, National Institute of Genetics, Mishima 411-8540, Japan
3) Department of Genetics, Stanford University, Stanford, California 94305, U.S.A.

Key words: lineage specific evolution, conserved noncoding sequence, mammals
Running title: Lineage-specific HCNSs in mammals

Corresponding author:
Naruya Saitou
Division of Population Genetics
National Institute of Genetics
Mishima, 411-8540, Japan
Phone: +81-559-81-6790
Fax: +81-559-81-6789
Email: saitounr@lab.nig.ac.jp
Web: http://sayer.lab.nig.ac.jp/~saitou/ª 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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Mahoko Takahashi1,2,3) and Naruya Saitou2,1)

ABSTRACT

Vertebrate genome comparisons revealed that there are highly conserved noncoding
sequences (HCNSs) among a wide range of species, and many of which contain regulatory
elements. However, recently emerged sequences conserved in specific lineages have not been wellstudied. Toward this end, we identified 8,198 primate and 21,128 specific HCNSs as
representative ones among mammals from human-marmoset and mouse-rat comparisons,

HCNSs were under purifying selection, indicating that they may harbor important functions. We
selected the top 1,000 largest HCNSs and compared the lineage-specific HCNS-flanking genes(LHF genes) with UCE (ultraconserved element)-flanking genes. Interestingly, the majority of
LHF genes were different from UCE-flanking genes. This lineage-specific set of LHF genes wasmore enriched in protein binding function. Conversely, the number of LHF genes which were also
shared by UCEs was small but significantly larger than random expectation, and many of thesegenes were involved in anatomical development as transcriptional regulators, suggesting thatcertain groups of genes preferentially recruit new HCNSs in addition to old HCNSs which areconserved among vertebrates. This group of LHF genes might be involved in the various levels of
lineage-specific evolution among vertebrates, mammals, primates, and rodents. If so, theemergence of HCNSs in and around these two groups of LHF genes developed lineage-specificcharacteristics. Our results provide new insight into lineage-specific evolution through interactions
between HCNSs and their LHF genes.

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respectively. Derived allele frequency analysis of primate-specific HCNSs showed that theseINTRODUCTION
From the inception of molecular evolutionary studies, protein non-coding regions weresuspected to be involved in gene regulation (Zuckerkandl and Pauling 1965; Britten and Davidson
1971; King and Wilson 1975). Now it is widely accepted that some non-coding regions play
important roles in gene regulation (e.g., Carroll 2005). The functional elements are expected to
evolve more slowly than surrounding nonfunctional DNA, as they are under purifying selection

important from the functional point of view. In fact, 5% of the human genome is conserved(Mouse Genome Sequencing Consortium 2002), a considerably higher proportion than that (2%)
of the protein coding regions (International Human Genome Sequencing Consortium, 2004).
Many highly conserved noncoding sequences (HCNSs) among vertebrates have now been
identified (Ahituv et al. 2004; Bejerano et al. 2004; Siepel et al. 2005), and some of which arereported to function as distal enhancers for neighboring genes (e.g. Woolfe et al. 2005;
Pennacchio et al. 2006).
The regions conserved in only one restricted lineage such as primates and rodents are
considered to be recently emerged HCNSs. These HCNSs may have gained new functions todevelop the lineage-specific characteristics after diverging from the ancestral species. However,the commonly accepted strategy for detecting regulatory regions is to identify HCNSs among awide range of species such as vertebrates. This approach only identifies the regions conserved
among diverged species, and does not detect sequences conserved in just one particular smalllineage. Indeed, such comparisons among vertebrate genomes are known to miss a large numberof highly constrained mammalian-specific functional elements despite the fact that these elements
are all under similarly intense levels of purifying selection in mammals (Aparicio et al. 2002;!

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(Kimura 1983; Nei 1987). Therefore, sequences that are more highly conserved are likely to beHillier et al. 2004; Cooper et al. 2005).
Challenges and limitations exist for studies seeking to identify the evolution of regulatory
regions by detecting changes that were accelerated as a result of lineage-specific positive selection(Pollard et al. 2006; Prabhakar et al. 2006, 2008). These studies focused only on human-specificchanges. However, positive selection is not the only possible explanation for these lineage-specificaccelerated sequences. Biased gene conversion (BGC) is a neutral mutation process associatedwith meiotic recombination, which favors a special kind of mutation pattern (Marais 2003). BGC(Galtier and Duret 2007; Dreszer et al. 2007; Duret and Galtier 2009; Sumiyama and Saitou 2011).
In addition, there are reports that the selective pressure affecting the evolution of regulatory
elements in the hominid lineage is significantly relaxed compared to that of the rodent lineage(Kryukov et al. 2005), and that regulatory elements in hominids may be diverging at a neutral rate
(Keightley et al. 2005). All of these elements point to the difficulty in detecting evidence of
positive selection in one lineage.
Another challenge for finding the lineage-specific regulatory regions was to identify
HCNSs found only in one lineage comprised of very closely related species. The primates is oneof the lineages of closely related species compared to the mammals and vertebrates. Sequencecomparisons only among primates are likely to capture functional components of the lineage due
to shared biological processes (Boffelli et al. 2003). However, to date, this strategy of comparing
genomes among closely related species has been applied only to the very limited regions.
Furthermore, the goal of this method was to identify all sequences conserved among species atvarious levels of divergence, such as vertebrates, mammals, and primates, but not primate-specific
HCNSs. In contrast to the lineage-specific phenotypic changes, the HCNSs which are conserved!

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can create strong substitution hotspots, thereby leading to spurious signatures of positive selection

only in one particular lineage have not been well studied. Thus, to expand our understanding of
lineage-specific evolution, we identified HCNSs that were conserved in a particular lineage (either
in primates or in rodents), and compared characteristics of the lineage-specific HCNSs with those
conserved among mammals and vertebrates. We used human and marmoset genomes for detecting
primate-specific HCNSs, while mouse and rat genomes were used for detecting rodent-specific
HCNSs (fig. 1). The lineage-specific HCNSs identified in this study are expected to provide new
insight into how one lineage evolved from a common ancestor.

Genomes used in this study
We used a total of 13 vertebrate genomes with over 6X coverage and high quality sincealignments containing low-coverage genomes cause misalignment. All genomes were obtained
from the UCSC Genome Bioinformatics database (http://genome.ucsc.edu/). They are medaka(Oryzias latipes; oryLat2), sticleback (Gasterosteus aculeatus; gasAcu1), fugu (Takifugu rubripes;fr2), tetradon (Tetraodon nigroviridis; tetNig2), zebrafish (Danio rerio; danRer6), frog (Xenopus
tropicalis; xenTro2), lizard (Anolis carolinensis; anoCar1), chicken (Gallus gallus; galGal3), dog(Canis familiaris; canFam2), horse (Equus caballus; equCab1), cow (Bos taurus; bosTau4), rat(Rattus norvegicus; rn4), mouse (Mus musculus; mm9), marmoset (Callithrix jacchus; calJac3),and human (Homo sapiens; hg19). Genomic alignments between human and marmoset andbetween mouse and rat were also retrieved from the UCSC Genome Bioinformatics database.
Genome sequences of rhesus macaque (Macaca mulatta; rheMac2), orangutan (Pongo pygmaeus
abelii; ponAbe2), and chimpanzee (Pan troglodytes; panTro3) were also used for extraction ofprimate-specific HCNSs.
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MATERIALS AND METHODSFiltering repeats and coding sequences
Repetitive sequences (chrN_rmsk tables) in the human and mouse genomes were obtained fromthe UCSC database. All repetitive sequences were excluded from the analysis. Filtering of coding
regions was performed based on the annotation (CCDS.20080902) of NCBI CCDS project

database (http://www.ensembl.org/) (Hubbard et al 2002).

Extraction of primate shared and rodent shared HCNSs
We applied a sliding window analysis to UCSC pairwise noncoding alignments of humanmarmoset and mouse-rat (http://genome.ucsc.edu/). We first extracted the repeat-masked noncoding regions and performed sliding window analysis. The window and step size were set to be
100bp and 25bp, respectively. When making sliding window sequences, we kept only thesequences that had no gap in a window. To estimate p-values for lineage-specific HCNSs, we
calculated the divergence of the non-gapped non-coding regions between human-marmoset andmouse-rat pairwise alignments (~10% and ~14% for autosomes and ~9% and ~14% forchromosome X, respectively). We assumed that these average genome divergences are neutral
substitution rates and obtained statistical significance of the lineage-specific HCNSs by using abinomial distribution.

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Downloaded from http://gbe.oxfordjournals.org/ at Idenkagaku Kenkyujo on April 15, 2012(http://www.ncbi.nlm.nih.gov/projects/CCDS/) (Pruitt et al. 2009) and the Ensembl humanIdentification of lineage-specific HCNSs
Discontiguous MegaBLAST homology search (Zhang et al. 2000) was performed to extract
primate-specific HCNSs against the non-primate vertebrate genomes. Similarly, rodent-specific
HCNSs were extracted by performing MegaBLAST search against the non-rodent vertebrate
genomes. Parameters for MegaBLAST were discontiguous word template size 16bp, word
matches 12bp and mismatch penalty -2. Alignable sequences may be homologous regions. We
therefore removed the MegaBLAST hits with !30% identity and !30 bp in length from primate

elements (McGaughey et al 2008). The homologous sequences among mammals (e.g. human anddog) with " 30 bp length and " 30% identity can be found throughout the genome and areassumed to be neutral when assessing average genome identity. However, the homologous
sequences among diverged vertebrates (e.g. human and fish) are considered to be functional
elements. We removed these alignable sequences among vertebrate genomes (birds, lizard, frog,
and fish) from the lineage-specific HCNSs using UCSC multiway alignments. In addition, since
there is no closely related species available for rodent lineage, we applied further filtering only for
extraction of primate-specific HCNSs and removed the HCNSs that were not found or showed low
identity (<98%) in the rhesus macaque, orangutan, and chimpanzee genomes.
To make analyses of these lineage-specific HCNSs easier, we extracted the top 1,000 largest
HCNSs, as longer sequences were considered to be under stronger constraint. We assumed that theconstraints on the HCNSs in the same bin (class of length) were equal. HCNSs were chosen fromthe first bin to the n-th bin until the total number approached 1,000. Additional HCNSs werechosen by random from the (n+1)-th bin to reach the total HCNS numbers to be 1,000.

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shared and rodent shared HCNSs since the sequences with !40% identity may contain functional

SNP detection
We downloaded human SNP data from the Hapmap database (http:// hapmap.ncbi.nlm.nih.gov/)
and mouse SNP data from NCBI dbSNP build 128 (http://www.ncbi.nlm.nih.gov/SNP). We
extracted only the SNP with minor allele frequencies of at least 0.01 in one of four populations(YRI, CEU, JPT, and CHB) in humans, and one of all strains in mice. The densities of the SNPs in
the repeat masked noncoding sequences in the human and mouse genomes were used to estimate
the expected SNP numbers in HCNSs, and these were compared with the observed SNP numbers

Derived allele frequency estimation
To estimate derived allele frequency (DAF), we converted the coordinates of primate-specific
HCNSs into those of hg18 to obtain allele frequencies in human populations provided by HapMap
release 27. We determined the ancestral allele by using chimpanzee alleles defined by UCSC
snp126OrthoPt2Pa2Rm2. An SNP locus was discarded whenever the allele of its orthologous
chimpanzee locus did not match either human allele. We used 2 $ 2 contingency tables to compare
DAF distribution for SNPs within primate-specific HCNSs with all non-repetitive humannoncoding genomes.

Gene Ontology analysis
We looked for significantly enriched gene categories in primate and rodent LHF genes in the Gene
Ontology (GO) database (http://www.geneontology.org/) (Ashburner et al. 2000). The assignment!

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of GO terms and the test for statistical enrichment of those terms were performed with GOstat
using goa_human and mgi GO gene association database (http://gostat.wehi.edu.au/) (Beissbarthand Speed 2004). The enrichment of InterPro domains (http://www.ebi.ac.uk/interpro/) of humanand mouse genes associated with HCNSs was determined by Fisher's exact test. The correction for
multiple comparisons was performed by using the false discovery rate (FDR) option in GOstat.

Analysis of dN and dS levels for LHF genes

macaque, and mouse-rat pairs (Hubbard et al 2002), and extracted only the LHF genes (and LHF
orthologs) that were located within 1 Mb of HCNSs in all genomes. dN and dS values were also
downloaded from Ensembl (Vilella et al 2008). These values were estimated by using codeml in
the PAML package (model=0, NSsites=0) (Yang et al. 1997). With dN and dS values of one-toone pair orthologs in Ensembl homolog lists, we calculated the means of dN and dS of LHF genes
and all genes in the human and mouse genomes. Statistical analysis (one-sample t-test, two tailed)was conducted using the R package (http://www.r-project.org). For UCE-flanking genes, we usedgenes that were located within 1 Mb of UCEs in both human and mouse genomes. With these
extracted genes, the same procedure was used for estimation of dN and dS for UCE-flanking genes.

Expected number of genes shared by lineage-specific HCNSs and UCEs
The expected number of overlapping genes among lineage-specific HCNSs and UCEs wascalculated by random sampling simulation. This random sampling weights the chance of choosing
a gene by! +,-! length of the chromosome where the gene is located.! We randomly selected thesame number of genes as the primate LHF genes from the human genome, those as the rodent!

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We obtained ortholog lists from Ensembl through biomart for human-marmoset, human-rhesus

LHF genes from the mouse genome, and those as the noncoding UCE-flanking genes from bothhuman and mouse genomes, in each 10,000 replicates. Using these data sets, we counted thenumber of shared genes between primate LHF and UCE-flanking genes, rodent LHF and UCEflanking genes, and primate and rodent LHF genes, and obtained the expected numbers foroverlapping genes. Chi-squared tests were conducted for observed and expected numbers using
the R package.

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RESULTS
Determination of parameters to extract lineage-specific HCNSs
One important parameter when identifying highly conserved sequences among closely
related species is the window size used to compare sequences. Although larger windows havemore statistical power to detect significantly conserved sequences among closely related species,

possible window size which is still large enough to detect conserved sequences. Another importantparameter for identifying conserved sequences among closely related species is the threshold forextraction of conserved sequences. Particularly for closely related species, this substitution
number must not be too small because the effect of sequencing errors on the determination ofsignificantly conserved sequences may not be negligible. Taking this into consideration, athreshold of 100% identity increases the number of false negative identification of HCNSs and isthus too strict. For these reasons, the window size for sequence comparison among closely related
species should be determined by considering the substitution numbers within a given window.
By way of preliminary analysis, we examined which window size was most appropriate for
identifying HCNSs in closely related species by assuming a simple model in which thesubstitution rate within a given window follows a binomial distribution. The window size setting
is a more sensitive process in the human and marmoset comparison than that in the mouse and rat
comparison because the average genomic divergence between human and marmoset (non-gapped
non-coding region: ~10%) is smaller than that between mouse and rat (~14%). We thus estimatedthe number of substitutions in HCNSs between human and marmoset. First, we defined that the
HCNSs reside in the lowest 5% of the left tail of the distribution, and obtained the expected!

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smaller windows provide better resolution for the analysis. Thus, it is important to set the smallest

number of substitutions of HCNSs in 50, 100, 150 and 200bp windows using the average genome
identity of non-gapped noncoding region as a neutral rate. We then chose to use 100 bp since thelength was relatively small but the range of expected substitution numbers in the HCNS was
between 0 and 5. This window size also has adequate statistical power for rodents whose genetic
divergence was larger than primates. For the simplicity of the analysis, we therefore used the same
window length to extract rodent HCNSs for the simplicity of the analysis.
The first step involved extraction of 100bp primate/rodent shared conserved sequences from2). The pairwise alignments were obtained from the UCSC Genome Bioinformatics database. We
first limited the region used in this analysis to repeat masked noncoding sequences (350 Mbp inhuman-marmoset and 210 Mbp in mouse-rat pairwise alignments). By using this repeat masked
noncoding human-marmoset and mouse-rat pairwise alignments, we created 100bp sliding
windows, which have no gaps from non-repetitive alignments, with a step size of 25bp. The total
numbers of repeat-masked and non-gapped 100bp sliding windows in human-marmoset andmouse-rat were 13,618,548 and 8,187,889, respectively.
From these windows, we extracted HCNSs using empirical conservation cutoffs andobtained only the sequences which have no substitution (0.78% of the total 100 bp fragments), one
substitution or fewer (2.2%), and two substitutions or fewer (4.3%) in the human and marmoset
pair, and those with no substitution (0.94%), one or fewer (2.5%), and two or fewer (4.5%) in themouse and rat pair. When we extracted sequences that had three or fewer substitutions, thepercentage of the total fragments exceeded 5% in both comparisons. Thus, we determined that asubstitution number of two was the appropriate threshold for extraction of HCNSs of primates androdents (P < 2.1x10-3 and P < 1.5x10-5, respectively, the binomial model). These numbers!

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determined nucleotide identity thresholds for highly conserved regions in primates and rodents tobe !98% ([100-2]/100). Note that we determined thresholds specifically for chromosome X
because the mutation rate on the X chromosome of mammalian genomes is known to be lower
than that of autosomes (Miyata et al. 1987; Takahata et al. 1995; Makova and Li 2002). To
estimate the appropriate calibration parameter for chromosome X, we examined the number ofsubstitutions in autosome and chromosome X separately in non-gapped noncoding pairwisealignments of human-marmoset and mouse-rat pairs. In the human-marmoset comparison, we didchromosome X and found 2 substitutions in a window (!98% identity, P < 4.8x10-3, the binomial
model). However, in the mouse-rate comparison, we observed difference between the two andfound only one substitution in a window (!99% identities, P < 1.2x10-5, the binomial model). We
calibrated the threshold for chromosome X only for mouse-rat sequences as 99 % ([100-1]/100)
and obtained a total of 590,678 and 356,529 conserved 100bp sequences from the humanmarmoset pair and mouse-rat pair, respectively. These extracted sequences account for less than
2% of the human and mouse genomes. The extraction of conserved sequences is, however, only astarting point for the extraction of lineage-specific HCNSs, which were filtered further in the next
step.

Extraction of lineage-specific HCNSs
The second step is an extraction of lineage-specific HCNSs. We performed MegaBLAST
search against vertebrate genomes with extracted HCNSs (590,678 and 162,304 from primates androdents, respectively). The primate- and rodent-specific HCNSs are conserved sequences that haveemerged after the divergence of these lineages from their ancestors (fig. 1), such that they are!

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Downloaded from http://gbe.oxfordjournals.org/ at Idenkagaku Kenkyujo on April 15, 2012not observe any difference in the number of substitution in the HCNSs between autosome andfound only in primates or rodents. We thus removed all HCNS homologous sequences that were
found in non-primate and non-rodent vertebrates. For extraction of primate-specific HCNSs, anadditional filtering criterion was applied, and we limited to the HCNSs that were also conserved in
the rhesus macaque, orangutan and chimpanzee genomes. The remainders were 8,198 and 21,128
for primate- and rodent-specific HCNSs, respectively. For simplicity, we used the top 1,000
largest lineage-specific HCNSs for both rodents and primates when comparing theircharacteristics. The primate- and rodent-specific HCNSs range in size from 125 to 375bp and 175(approximated with p-distance) in those 1,000 primate- and rodent-specific HCNSs and their±10,000bp flanking regions, respectively. The patterns indicate that only the HCNSs are under thestrong constraints, relative to their flanking regions. The ±500bp flanking regions shown in theinsets showed a smaller number of differences compared to those of genome averages. However,the number of substitutions of lineage-specific HCNSs is clearly much lower even when comparedto that of ±500 bp flanking regions.

SNP analysis
A single nucleotide polymorphism (SNP) is a good indicator for detection of theselective constraint on the sequence in question. We investigated the number of SNPs overlaid on
the lineage-specific HCNSs in humans and mice (HapMap Consortium 2005; Sherry et al. 2001),
and found that less than 10% of lineage-specific HCNSs had SNPs (MAF < 0.01). The majority oflineage-specific HCNSs have no SNPs and the numbers of SNPs in both primate (SNP density persite: 9.74x10-4) and rodent (1.96x10-4) lineages were significantly smaller than genome wide
averages (1.6x10-3 and 5.0x10-4 for human and mouse non-repetitive genomes, respectively). (P!

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to 425bp, respectively. Figs. 3A and 3B show the average numbers of substitutions per site<< 0.01 for primate and rodent specific HCNSs, Chi- squared test).
To measure the relative level of purifying selection acting on HCNSs, we analyzed derived
allele frequency (DAF) distributions of primate-specific HCNSs and compared these distributions
with those of the human genome (fig. 4). Purifying selection is likely the main evolutionary force
preventing the vertebrate HCNS from accumulating mutations (Katzman et al. 2007).
Quantitatively, the signature of the purifying selection can be observed as a shift in the allele
frequency toward ancestral alleles. (Drake et al. 2006). We observed the levels of DAF "0.1 andJapanese (ASN), and American of European Ancestor (CEU). At the level of DAF "0.1, only the
YRI and ASN populations showed a significant excess of rare derived alleles of SNPs within
primate-specific HCNSs compared with the genome average (P < 0.05, Chi-squared test).
However, at the level of DAF "0.2, all populations showed a significant excess of rare allele of the
SNPs (P < 0.006, Chi-squared test). This is consistent with previously published results on nonlineage-specific HCNSs (Drake et al. 2006; Ovcharenko 2008, Katzman et al 2007), suggestingthat purifying selection is acting on the primate-specific HCNSs.

General features of the lineag- specific HCNSs
Genic category
To determine if there are any general trends in the distribution of the lineage-specific
HCNSs, we compared three annotation categories (intron, intergenic, and UTR) of the lineagespecific HCNSs. The fractions of lineage-specific HCNSs and their genic categories are shown infig. 5. The fractions of HCNSs residing in UTRs and introns in both primates and rodents weremuch higher than those of the whole human and mouse genomes, respectively (P < 10-15, Chi-!

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0.2 within primate-specific HCNSs in three human populations: Yoruba (YRI), Han Chinese +squared test). The most striking difference was found in the UTR category, where the fractions of
UTR in the primate and rodent-specific HCNSs were 3 times and 5.5 times higher than those ofhuman and mouse genomes, respectively. This increased fraction of UTRs is consistent with thetendency of HCNSs in vertebrates (e.g., Siepel et al. 2005; Woolfe et al. 2006). However, thefractions of UTR category in lineage-specific HCNSs were lower than those in non-lineage
specific vertebrates in UTRs (~6%). In addition, the fractions of intergenic+UTR and intronic
categories differed between primate- and rodent-specific HCNSs (P = 1.04 x 10-4, Chi-squared

GO analysis of the lineage specific HCNS-flanking genes
The function of genes that are located near lineage-specific HCNSs may provide important
information for understanding the lineage-specific evolution. We therefore examined thestatistically overrepresented functions of the lineage-specific HCNS-flanking genes (LHF genes).
We first obtained the distance between the lineage-specific HCNSs and their LHF genes, andfound that 96.2% and 97.5% of intergenic HCNSs are located within 1 Mb of the transcription
start site of LHF genes in human and mouse, respectively. The longest distance between a target
developmental gene and its experimentally verified enhancer is ~1 Mb; the reported genes are
SHH (Lettice et al. 2003; Sagai et al. 2005), SOX9 (Bishop et al. 2000), and SHOX (Sabherwal etal. 2007). These findings indicate that at least some HCNSs may be associated with LHF genes as
distal regulatory elements.
Next, we looked for functional categories of LHF genes as defined by the Gene Ontology(GO) database (Beissbarth and Speed 2004), and obtained significantly enriched functions of LHF
genes. The top 30 over-represented gene functions are shown in table 1. In primate and rodent!

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LHF genes, statistically over-represented functions were developmental process, protein binding,and regulation of transcription. In developmental process, anatomical structure (P = 3.1x10-66 andP = 3.1x10-66 for primate and rodent LHF genes, respectively) and nervous system development(P = 6.1x10-54 and P = 1.9x10-13) were enriched. In transcriptional regulation, positive regulationof transcription (P = 7.1x10-23 for primate LHF genes) and regulation of transcription (P = 5.0x107

for rodent LHF genes) were overrepresented. This tendency of over-represented gene functions

is consistent with previous studies of highly conserved noncoding sequences among vertebrates

rodent-specific LHF genes showed significant over-representation of protein binding (P = 9.7x1036

and P = 3.3x10-16) compared to vertebrate HCNSs (e.g. Bejerano et al. 2004a; Siepel A. et al.

2005; Woolfe et al. 2005). We further examined the LHF genes in protein binding category andfound that they were enriched in nervous system development (P = 2.93x10-37), positive regulationof transcription (P = 8.27x10-13), and transcription cofactor binding (P = 2.3x10-9) which are also
important for developmental process and transcriptional regulation.

Selective constraints on LHF genes
In order to identify evolutionary constraints on LHF genes, we examined the nonsynonymous (dN) and synonymous (dS) substitution rates in LHF genes. First, dN and dS values
for human-marmoset and mouse-rat pairs were obtained from Ensembl (Vilella et al. 2008). Using
primate (or rodent) LHF genes that had annotated orthologs within 1Mbp of the HCNS in humanand marmoset (mouse and rat) genomes, we calculated the means of dN and dS of genes in eachpair. We then compared dN and dS of LHF genes with those of the genome average. LHF genes
are expected to have important functions, and in fact, means of dN/dS ratios in all pairs were!

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(e.g. Bejerano et al. 2004a; Siepel A. et al. 2005; Woolfe et al. 2005). However, both primate- andsignificantly smaller than those of genome averages (P < 0.001 in all pairs, one-sample t-test).
However, dS values of primate and rodent LHF genes were significantly smaller than those of
genome averages as well as dN values (P < 0.001 in human-marmoset and mouse-rodent pairs;
table 2), indicating stronger constraint on flanking genes not only at amino acid sequence level but
also at nucleotide level.
The main advantage of studying HCNSs that are found in only one particular lineage is that
we can compare evolutionary constraints on the LHF genes with those of orthologs which have notheir orthologs that have no lineage-specific HCNS, we compared dN and dS levels of orthologs ofprimate (rodent) LHF genes with genome averages in rodent (primate) pair. We defined orthologs
of primate LHF genes in rodents and those of rodent LHF genes in primates as primate LHF
orthologs and rodent LHF orthologs, respectively (see fig. 6). As in the LHF genes, the primate
and rodent LHF orthologs had significantly smaller dN/dS ratios as well as dN levels, whencompared with those of genome averages in mouse-rat (human- marmoset) pair (P < 0.001, one
sample t-test; table 2(A) and table 2(B)).
On the other hand, there was no significant difference between dS values of primate androdent LHF orthologs and those of genome wide genes (P > 0.05, table 2(A) and table 2(B)). Thisfinding indicates that the evolutionary constraint on LHF genes is stronger than the constraint on
genes that have no lineage-specific HCNSs. We also analyzed levels of constraints using dN anddS values in genes flanking noncoding ultraconserved elements (UCEs) which are an extreme caseof HCNSs among vertebrates (Bejerano et al. 2004a). All mean values (dN/dS ratio, dN, and dSvalues) of UCE-flanking genes were significantly smaller than genome averages (table 2(C)). This
result further supports the finding lower dS is an important signature of the genes that are!

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HCNS in another lineage. To investigate differences in selective constraints on LHF genes andassociated with HCNSs.

Comparison of lineage-specific HCNS-flanking genes with vertebrate HCNS-flanking genes! ! ! GO analysis of the LHF genes showed that the most statistically over-represented
functions were developmental process and transcriptional regulation, which were quite similar tothe over-represented functions of HCNSs conserved among vertebrates (e.g. Bejerano et al. 2004a;

found near the same genes as those of vertebrate HCNSs whose origin are older than primate- androdent-specific HCNSs.
To address this question, we first examined the number of flanking genes which were shared
among primate- and rodent-specific HCNSs and noncoding UCEs (fig. 7). The majority ofprimate- and rodent-specific HCNSs (980 and 985, respectively) had LHF genes within 1 Mbp.
They were often clustered near a small subset of genes, and the numbers of LHF genes forprimate- and rodent-specific HCNSs were 820 and 516, respectively. This is consistent withprevious studies of vertebrate HCNSs including UCEs. Furthermore, a total of 11 LHF genes wereshared among lineage-specific HCNS- and UCE-flanking genes (fig. 7A). The number of genes
shared by primate-specific HCNSs and UCEs, and rodent-specific HCNSs and UCEs were 31 and41, respectively (fig. 7, B and C in the Venn diagram). Interestingly, we found that the numbers ofgenes shared by lineage-specific HCNSs and UCEs were significantly larger than random
expectation (P < 10-4). Over-represented functions of GO categories for the LHF genes
overlapping with UCE-flanking genes were mainly involved in regulation of transcription, DNAbinding, anatomical structure development and intracellular membrane-bound organelle (fig. 7,
scatter plots A through C). Note that rodent LHF genes overlapping with UCE-flanking genes!

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Woolfe et al. 2005). This observation raised the question whether the lineage-specific HCNSs arewere mildly enriched in anatomical structure development (P < 0.056). Many of these genes
shared by lineage-specific HCNSs and UCEs are well studied and known to play an important roleas transcriptional regulators during vertebrate development.
Similarly, we examined LHF genes shared by primate- and rodent-specific HCNSs andfound the numbers of overlapping LHF genes were also significantly larger than random
expectation (P < 10-4) (fig. 7, D in the Venn diagram). In contrast, the proportion of primate androdent LHF genes that were not shared by any gene set were 82.4 % and 74.6%, respectively (fig.

lineage-specific. The main over-represented gene functions were regulation of transcription,
protein binding, anatomical structure development and intracellular membrane-bound organelle(fig. 7 scatter plots D through F). Taking into consideration that the UCE-flanking genes that do
not include LHF genes were enriched in both DNA binding and protein binding, while genes
involved in protein binding were less significant compared to those of DNA binding, thedifference between the gene functions of data sets A through F was whether or not DNA binding
was over-represented (fig. 7, scatter plot G).
Examples of these overlapping LHF genes are shown in fig. 8. Lineage-specific HCNSs as
well as UCEs were found in and around the PBX genes. A primate-specific HCNS and UCEs
were located within PBX1, and rodent-specific HCNSs and UCEs were located in and around
PBX3 (figs. 8A and 8B). PBX genes act as co-factors for various transcription factors such as
HOX genes (e.g. Rauskolb and Wieschaus 1994; Mann, 1995, Mann and Chan, 1996, Mann and
Affolter, 1998), and are involved in chromatin modification (e.g. Cirillo et al., 2002; Berkes et al.
2004). The lineage-specific HCNSs were also associated with SOX genes. Both primate- androdent-specific HCNSs were found in and around SOX13 and SOX6 (Figs. 8C and 8D),!

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7, E and F in the Venn diagram). This finding demonstrates that the majority of the LHF genes arerespectively. In addition, a primate-specific HCNS and a rodent-specific HCNS were also locatednear SOX9 and SOX11, respectively. SOX genes are transcriptional activators that are required
for normal development of the central nervous, chondrogenesis and maintenance of cardiac andskeletal muscle cells (Wegner, 1999; Wegner and Stolt 1995).
MEF2C is an interesting example of primate and rodent HCNSs and UCE shared LHF genes.
The genomic positions of the lineage-specific HCNSs and UCEs for MEF2C are shown in fig. 8E.
MEF2C belongs to the evolutionarily ancient MADS family of transcription factors which play

genetic programs that control cell differentiation, proliferation, morphogenesis, survival andapoptosis of a wide range of cell types (Shore and Sharrocks, 1995; Potthoff and Olson, 2007).
TLE genes also recruited lineage-specific HCNSs. Another LHF gene that was shared among
primate- and rodent-specific HCNSs and UCEs is TLE4 (fig. 8F). This is a transcriptional corepressor that binds to a number of transcription factors and inhibits the transcriptional activation
mediated by PAX5, and by CTNNB1 and TCF family members in Wnt signaling (Molenaar et al.
1998; Eberhard, 2000; Yaklichkin et al, 2007). An interesting exceptional example of the LHF
genes is NPAS3 (fig. 8G). This gene is a brain-enriched transcription factor belonging to the basic
helix-loop-helix-PAS superfamily, the members of which carry out diverse functions, including
circadian oscillations, neurogenesis, toxin metabolism, hypoxia, and tracheal development(Kamnasaran et al., 2003), shown as HAR in fig. 8G. NPAS3 has not only vertebrate HCNSs but
also a human accelerated region (Pollard et al. 2006). Three primate-specific HCNSs, one rodentspecific HCNS and three UCEs are located in NPAS3. Another example of an LHF gene that isthought to have a critical impact on lineage-specific evolution is FOXP1. Primate-specific HCNSs,
a rodent-specific HCNS and 3 UCEs were found in and around FOXP1 (fig. 8H). FOXP1 is amember of the FOX family of transcription factor, and plays important roles in the regulation of!

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central roles in the transmission of extracellular signals to the genome and in the activation of thetissue- and cell type-specific gene transcription (e.g. Kaufmann and Knochel 1996; Carlsson and
Mahlapuu 2002).

DISCUSSION

found that the lineage-specific HCNSs showed the signature of purifying selection at the SNPlevel as well as the nucleotide level (figs. 3 and 4). We found that the LHF genes as a whole wereenriched in gene functions similar to those UCE-flanking genes (table 2). However, the majorityof LHF genes and UCE-flanking genes are independent sets (fig. 7, E through G in the Venn
diagram). This suggests that a particular group of genes are preferentially associated with lineagespecific HCNSs instead of vertebrate HCNSs (fig. 7, scatter plots E through G). Thus, this groupof genes may be regulated through HCNSs in a lineage-specific manner. Similarly, we found that
there are UCE-flanking genes that have no lineage-specific HCNSs. These genes are thought to bea core set that may have contributed to the development of fundamental characteristics ofvertebrates. On the other hand, we found that the number of LHF genes that were also UCEflanking genes was significantly larger than random expectation (fig. 7, B and C in the Venn
diagram). This suggests that certain groups of genes tend to recruit new HCNSs in addition to thevertebrate (old) HCNSs such as UCEs. The genes at the intersection of all lineages were the mostextreme example (fig. 7, A in the Venn diagram). These genes may have contributed to theevolution of different levels of organisms, e.g. vertebrates, primates, and rodents. A particularly
noteworthy feature of LHF genes is that even genes that are highly conserved among vertebrates,
and which play an important role in the developmental process (e.g. FOXP1 and PBX genes),!

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In this study, we identified a total of 8,198 primate- and 21,128 rodent-specific HCNSs, andhave lineage-specific HCNSs. This is so because regulatory regions for the conservative genes arealso conserved among a wide range of species.
Although the primate- and rodent-specific HCNSs are found only in primates and rodents,
respectively (fig. 1), there may be HCNSs that have been lost only in these lineages. Comparisonsof ancient vertebrate conserved noncoding elements (aCNEs) which were present in the common
ancestor of jawed vertebrates in Hox cluster loci showed that many of aCNEs have diverged
beyond recognition in teleost fish (Lee et al. 2010). However, another study of HCNSs among 4(Mclean and Bejerano 2008). It is also known that there is a slowdown in substitution rates of
UCEs in tetrapods (Stephen et al 2008). These observations indicate that the loss rate of
mammalian and tetrapod HCNSs was smaller than expected. Therefore, if there are HCNSs thathave been lost only in primate and rodent lineages, many of them are expected to be derived fromaCNEs. The evolutionary process by which new regulatory networks are created may be driven by
the addition and loss of HCNSs to genes that play an important role in development.
It is also possible that new lineage-specific HCNSs and old vertebrate or mammalian
HCNSs have the same function even if their sequences are not homologous. This suggests that the
new-lineage specific HCNSs were created during functional turnover, and that gaining the new
HCNSs did not contribute to the lineage-specific evolution. In such cases, the gain and loss of
HCNSs frequently occur within and around these vertebrate developmental genes and their gene
expressions are maintained among a wide range of species. However, it is impossible to knowwhether or not non-homologous long sequences have the same function by performing
computational analysis alone.
In TF binding sites, it is known that there is turnover, including gain and loss of DNA!

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mammals showed that the loss rate of ultraconserved-like HCNSs in rodents was only 0.086%

sequence motifs (Cliften et al. 2003; Kellis et al. 2003; Gasch et al. 2004; Stark et al. 2007) as
well as alterations in motif spacing relative to the start of transcription, or to other motifs (Ihmels
et al. 2005; Tanay et al. 2005). Recently, a number of genome-scale studies using
immunoprecipitation were performed to compare TF-binding patterns (Borneman et al. 2007;
Tuch et al. 2008; Bradley et al. 2010; Lavoie et al. 2010; Schmidt et al. 2010) or mRNA
expression profiles across species (Ihmels et al. 2005; Tanay et al. 2005; Hogues et al. 2008; Field
et al. 2009; Wapinski et al. 2010). Many of these studies have identified transcriptional programs(2007) found that the TF Tec1 binds only 20% of the same target genes in comparisons between
Saccharomyces cerevisiae and the closely related S. bayanus and S. mikatae, and that thisdifference is due to the gain and loss of canonical Tec1 cis-regulatory motifs.
Kim et al. (2010) found abundant transcription at neuronal enhancers that are evolutionary
conserved. However, the lineage-specific HCNSs have only a few partial matches with ESTs andknown RNA genes. This suggests that the majority of HCNS functions are cis-regulatory elements,
and in fact many studies reported that vertebrate HCNSs showed enhancer activities (e.g. Poulin etal. 2005; Woolfe et al. 2005; Pennacchio et al. 2006; Lareau et al. 2007; Ni et al. 2007). In
addition, we compared lineage-specific HCNSs and CNEs of other known vertebrate HCNSs(Woolfe et al. 2005, 2007) to determine whether there was any difference in the results ofcomparisons with UCEs. The CNEs are over 1400 HCNSs which were identified by human-fugu
comparison and which include sequences overlapping with UCEs. As expected, we obtained
similar results between noncoding UCEs and lineage-specific HCNSs in dN and dS and in sharedgenes analyses.
We also found that there were differences between dS values of LHF genes and those of!

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genome wide genes (table 2(A) and table 2(B)). It is known that the neutral mutation rate hasregional biases in mammalian genomes (Mouse Genome sequencing Consortium 2002; Hardison
et al. 2003), and low dS genes tend to have similar GO functional categories such astranscriptional regulation and development (Chuang and Li 2004). However, the low mutation rateof LHF genes does not affect substitution numbers in HCNSs, because there is no correlation
between dS values and distances between HCNSs and their LHF genes (Pearson’s r=0.04 to 0.05).
This raises the question as to why there is correlation between HCNSs and low dS genes.

when it affects splicing and/or mRNA stability (Chamary et al. 2006). To investigate this, we
examined the number of splicing variants in LHF genes. Both the primate and rodent LHF genes
have significantly higher numbers of splicing variants than genome averages (one sample t-test, P< 2.50$10-2 and P < 1.87$10-7, respectively.). However, LHF orthologs also showed relatively
higher number of splicing variants (P < 3.0$10-2 and P < 2.8$10-2, respectively). This does not
explain the difference in dS values between LHF genes and the LHF orthologs. A second
possibility is the change in chromatin structure. Genes that display a strong constraint atsynonymous sites are preferentially located in closed regions of the genome because they require
tight transcriptional regulation (Prendergast et al. 2007). Moreover, this strong constraint on LHF
genes at the nucleotide level suggests that many regulatory proteins may bind to the genes andinteract with HCNSs for tight regulation of the gene expression.
We carefully chose the species used in this study by considering their coverage and quality.
Nevertheless, there are some limitations in these analyses of lineage-specific HCNSs due to thesmall number of genomes of closely related species. We were able to use only two species
genomes for the rodent lineage. We found small differences in genic categories overlapping
primate- and rodent-specific HCNSs (figs. 5A and 5B). The number of rodent-specific HCNSs!

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One possible explanation is that a lower dS gene may be constrained at nucleotide leveloverlapping intergenic and UTR was significantly larger than the comparable number in primates(P < 10-4, Chi-squared test). We found another small difference in over-represented gene functions.
However, we cannot determine whether these differences are derived from lineage-specificcharacteristics, from the different number of species used for extraction of primate and rodent
HCNSs, or from annotation problems. Further studies of lineage-specific HCNSs are necessary toobtain clear pictures of lineage-specific evolution.
! ! In spite of the small differences, similar tendencies were found in the constraints on thelineage-specific HCNSs were created in and around two categories of genes. In the first category,lineage-specific HCNSs are created near protein coding genes which had no HCNSs before. This
expands the set of LHF genes that differ from those of ancestral (mammalian and vertebrate)
HCNSs. These genes are more enriched in protein binding, many of which are involved in nervous
development, compared to ancestral HCNS flanking genes, suggesting that the lineage-specificevolution may be driven by changes in the regulation of protein interaction during nervous systemdevelopment. In the second category, lineage-specific HCNSs are newly added to particulargroups of genes which already have vertebrate HCNSs. One of the major gene groups codestranscriptional regulators involved in anatomical development, and may be involved in the various
levels of lineage-specific evolution such as vertebrates, mammals, primates, and rodents. Many ofthe lineage-specific HCNSs and vertebrate HCNSs are likely to be associated with different genesets. The lineage-specific evolution through HCNSs thus occurred by obtaining both new HCNS
and LHF gene sets that differ from the “core” sets of vertebrate HCNS and its associated gene.

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primate and rodent LHF genes and their functions. Our analyses of LHF genes imply that theACKNOWLEDGMENTS
We thank the Genome Sequencing Center at WUSTL for providing marmoset genome sequences;Drs. Jaume Bertranpetit, Arcadi Navarro, Hafid Laayouni, Kenta Sumiyama, and Kirill Kryukov
for useful discussion. MT received Research Fellowship of the Japan Society for the Promotion of
Science for Young Scientists. This study was supported partly by Grant-in-Aid for scientific

N.S, and by SOKENDAI Short-term Study-abroad Program to M.T.

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research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to

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$)!

Table 1 Top 30 overrepresented functions of LHF genes.
!

(A) Primate LHF genes
GO

GO:0045935
GO:0050794
GO:0031325
GO:0009893
GO:0009887
GO:0007165
GO:0000902
GO:0032989
GO:0022008
GO:0008134
GO:0048468
GO:0003712
GO:0007267
GO:0048522
GO:0048699

P -value

Anatomical structure development
System development
Multicellular organismal development
Developmental process
Nervous system development
Multicellular organismal process
Protein binding
Anatomical structure morphogenesis
Cellular developmental process
Cell differentiation
Organ development
Cell communication
Regulation of biological process
Biological regulation
Positive regulation of transcription
Positive regulation of nucleobase, nucleoside, nucleotide and nucleic acidmetabolic process
Regulation of cellular process
Positive regulation of cellular metabolic process
Positive regulation of metabolic process
Organ morphogenesis
Signal transduction
Cell morphogenesis
Cellular structure morphogenesis
Neurogenesis
Transcription factor binding
Cell development
Transcription cofactor activity
Cell-cell signaling
Positive regulation of cellular process
Generation of neurons

3.10E-66
2.10E-64
2.32E-58
3.18E-55
6.11E-54
9.41E-51
9.72E-36
2.66E-32
6.39E-29
6.39E-29
8.93E-29
5.11E-24
2.08E-23
7.14E-23
1.39E-22
1.98E-21
5.97E-21
1.49E-20
7.10E-20
2.53E-19
8.86E-19
1.60E-16
1.60E-16
6.43E-16
1.09E-15
1.49E-15
1.57E-15
6.28E-15
7.14E-15
1.09E-14(B) Rodent LHF genes
GO
GO:0005515
GO:0007275
GO:0048731
GO:0032502
GO:0048856
GO:0007399
GO:0009653
GO:0050789
GO:0065007
GO:0032990
GO:0030030
GO:0048858
GO:0009887
GO:0048666
GO:0050794
GO:0031175
GO:0007167
GO:0048869
GO:0030154
GO:0048513
GO:0030182!

Gene function

P -value

Protein binding
Multicellular organismal development
System development
Developmental process
Anatomical structure development
Nervous system development
Anatomical structure morphogenesis
Regulation of biological process
Biological regulation
Cell part morphogenesis
Cell projection organization and biogenesis
Cell projection morphogenesis
Organ morphogenesis
Neuron development
Regulation of cellular process
Neurite development
Enzyme linked receptor protein signaling pathway
Cellular developmental process
Cell differentiation
Organ development
Neuron differentiation

3.03E-17
4.43E-15
7.66E-14
1.89E-13
1.92E-13
1.92E-13
3.25E-12
4.09E-12
2.32E-11
1.00E-10
1.00E-10
1.00E-10
1.02E-09
1.02E-09
1.40E-09
3.86E-09
1.18E-08
3.45E-08
3.45E-08
3.72E-08
4.58E-08$*!

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GO:0048856
GO:0048731
GO:0007275
GO:0032502
GO:0007399
GO:0032501
GO:0005515
GO:0009653
GO:0048869
GO:0030154
GO:0048513
GO:0007154
GO:0050789
GO:0065007
GO:0045941

Gene function

GO:0022610
GO:0007155
GO:0000904
GO:0007267
GO:0005216
GO:0016477
GO:0010468
GO:0022838
GO:0045449

Biological adhesion
Cell adhesion
Cellular morphogenesis during differentiation
Cell-cell signaling
Ion channel activity
Cell migration
Regulation of gene expression
Substrate specific channel activity
Regulation of transcription

4.74E-08
4.74E-08
1.26E-07
1.68E-07
1.81E-07
2.00E-07
3.90E-07
4.53E-07
4.99E-07!
Notes. The P value was determined by Fisher's exact test, and corrected with FDR method. Only the gene funcions
belonging to “Biological process” and “Molecular function” of GO category are shown (Ashburner et al. 2000). A
total of 980 and 985 LHF genes that locate within 1 Mbp of HCNSs were used for primate and rodent specific HCNSs,
respectively.

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%/!

Table 2 dN and dS values!

(A) dN and dS of LHF genes in the human-marmoset pair!

Number of genes

All genes1

Primate LHF genes

15,011

462

Orthologs ofrodent LHF genes
319

Average dN

0.0407 (0.0494)

0.0250** (0.0362)

0.0255** (0.0287)

Average dS

0.1684 (0.1140)

0.1306** (0.0834)

0.1740 (0.1279)

Average dN/dS3

0.2495 (0.2599)

0.1852** (0.2123)

0.1585**(0.1710)(B) dN and dS of LHF genes in the mouse-rat pair

Number of geneAll genes2

Rodent LHF genes

16,104

517

Orthologs ofprimate LHF genes
306

Average dN

0.0408 (0.0509)

0.0285**(0.0434)

0.0262** (0.0352)

Average dS

0.2091 (0.0877)

0.1943* (0.0948)

0.2002 (0.0815)

Average dN/dS3

0.1910 (0.2360)

0.1330** (0.1731)

0.1241** (0.1248)!
"#$!dN and dS of UCE-flanking genes

Primates
Number of gene

Rodents

141

122

Average dN

0.0245* (0.0378)

0.0208* (0.0244)

Average dS

0.1225** (0.0844)

0.1705** (0.0748)

Average dN/dS3

0.1857* (0.2381)

0.1075** (0.1078)!!

Note: Numbers in parenthesis represent standard deviation. Values with asterisks represent significant diffeences (P<0.001, one sample t-test) from the genome averages. ** and * represent P <10-9 and 10-3, respectively. 1 All genes in3
the human genome. 2 All genes in the mouse genome. dN/dS ratio was calculated only for genes with dS > 0.

!

%"!

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FIGURE LEGENDS
Fig. 1 Phylogenetic relationship of species mainly used in this study
The blue, yellow and purple circles represent primate-specific, rodent-specific, and vertebrateshared HCNSs, respectively. The approximate divergence times for ancestral species of eachlineage are shown on the tree (Hedges and Kumar 2003; Mouse Genome Sequencing Consortium
2002; Gibbs et al. 2004; She et al. 2006).

Pairwise alignment represent human- marmoset and mouse-rat alignments for extraction ofprimate and rodent-specific HCNSs, respectively. After the step “remove vertebrate homologous
regions”, another filtering was applied for primate comparison, and the sequences that were notconserved in other primate species (rhesus macaque, orangutan and chimpanzee) were removed.

Fig. 3 Substitution rates in lineage-specific intergenic HCNSs and their flanking regions
The average substitution number per site within 100 bp window in the range of ±10,000bp of the
top largest 1,000 primate-specific HCNSs (A), and rodent-specific HCNSs (B). The insets show
enlarged distributions in the range of ±1,500bp. The red lines represent average substitution
numbers per site of non-gapped non-coding regions in the human and mouse genomes,
respectively. The error bars are 95% confidence intervals of substitution rate in each window.
Fig. 4 DAF distribution in primate-specific HCNS!

%#!

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Fig. 2 The procedure of extraction of primate and rodent-specific HCNSs

DAF distribution of Yoruba from Nigeria (YRI) (A), Han Chinese from Beijing combined withJapanese from Tokyo (ASN) (B), and American of European ancestry (CEU) (C). Light gray andblue bars represent data for SNPs in the non-repetitive human genome and SNPs within primatespecific HCNSs. Error bars were estimated using binominal distribution as %2 = (pq)/n, where p
represented the fraction of SNPs in a particular bin, q represented 1-p, and n represented the total
number of SNPs. All primate-specific HCNSs (8,198) were used for this analysis.

The pie charts show percentages of genic categories in the human genome (left) and primatespecific HCNSs (right)(A), in the mouse genome (left) and rodent-specific HCNSs (right)(B). The
percentages of UTRs become markedly elevated in the lineage-specific HCNSs. The distributionof genic categories between genomes and lineage-specific HCNSs showed significant difference(P < 10-15, Chi-squared test).

Fig. 6 Definition of LHF orthologs
The primate and rodent LHF orthologs are defined as the ortholog of primate LHF gene in rodents(Gene A in rodents), and the ortholog of rodent LHF gene in primates (Gene B in primates).
Although primate and rodent LHF genes recruited lineage-specific HCNSs after the divergence ofeach lineage from the common ancestor, the majority of primate and rodent LHF orthologs did not.

Fig. 7 Comparison of genes among lineage-specific HCNSs and UCEs!

%$!

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Fig. 5 Fractions of genic categories in whole genomes and lineage-specific HCNSs

Upper 7 panels show the scatter plots of the number of over-represented gene functions and theirp-values obtained by GO analysis. The letters A through G in the scatter plots are corresponding
to the letters in the Venn diagram which shows the number of overlapping LHF genes among
primate and rodent-specific HCNSs and UCEs (numbers in brackets).

Fig. 8 Examples of lineage-specific HCNS and UCE distributions

HCNSs, respectively. Examples of PBX1 (A), Pbx3 (B), SOX13 (C), Sox6 (D), MEF2C (E),
TLE4 (F), NPAS3 (G) and FOXP1 (H) are shown in the figure. When LHF genes are of primatespecific HCNSs, the distribution of HCNSs and UCEs are always shown on the human genes. All
genes but NPAS3 are highly conserved in vertebrates. For NPAS3, both human and mouse genes
are shown since there is no intronic region corresponding to rodent-specific HCNSs in the humangene. As an additional information, the human accelerated region (HAR) is shown.

!

%%!

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Purple, light blue, and yellow circles represent the position of UCE, primate, and rodent-specific

!!"#$#% Myr!

Mouse!

!&'$&(!)*+!

Rat!
"!,"'!)*+!

Dog!
Cow!
Horse!
Chicken!
Lizard!
Frog!
Fish!

Vertebrate shared !

Marmoset!

Rodent specific! Primate specific! 35-40 Myr!

Chimpanzee!
Orangutan!
Macaque!

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Human!

Pairwise alignments!

Mask coding and!!repeat regions!

Extract HCNSs "!
in each lineage

Remove vertebrate "!
homologous regions"! 100bp HCNSs!
Merge overlapping

Lineage specific HCNSs!

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Create 100bp non-gapped sequences"!
with 25bp step size

Distance from HCNS (bp)!

B!

Distance from HCNS (bp)!

Substitution rate per site

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Substitution rate per siteA!

Fraction of SNPs!
Fraction of SNPs!

C!

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YRI!

ASN!

CEU!

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DAF!

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B!

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Fraction of SNPs!

A!

UTRs! 0.4%!
25%!
Intronic!

75%!

Intergenic!

B!

UTRs!0.6%!
24%!
Intronic!

UTRs!1.2%!

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A!

47%!

51.8%!

Intronic! Intergenic!

UTRs! 3%!

36%!
75%!

Intergenic!

Intronic!

61%!

Intergenic!

HCNS

Gene B

Human
HCNS
Gene A
Gene B≈ 40 Myr

Gene A

Marmoset≈ 75 Myr

Gene B≈ 24 Myr

Mouse
Gene A

Gene BPrimate LHF ortholog

HCNS

Gene A

Rat
Rodent LHF gene
Gene B
HCNS

Rodents

Rodent LHF ortholog

Primates

Gene A

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Primate LHF gene!

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DNA binding!
Anatomical structure development!
Protein binding!
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Intracellular membrane-bound organelle!

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8696!95:;6<E!

F!
Rodent LHF genes!

(385)

D!
(100)

Primate LHF genes!

(676)

A!
C!

(20)

B!
G!

(33)!

(82)!
UCE-flanking
genes!

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A!
chr1: 164000000

164100000

164200000

200 kb164300000

hg19
164500000
164600000

164400000

164700000

164800000

PBX1

B!
100 kbchr2:

34050000

34100000

mm934200000

34150000

Pbx3

C!
chr1:

50 kb
204020000 204030000

204010000

204040000

204050000

204060000

204070000

hg19
204080000

204090000

SOX13

chr7: 122500000

200 kb122700000

122600000

mm9122800000

122900000

123000000

123100000

Sox6

E!

200 kbchr5:

88100000

88200000

88300000

hg19
88500000

88400000

88600000

MEF2C

F!
hg19

500 kbchr9:

82000000

82100000

82200000

82300000

82400000

82500000

82600000

82700000

82800000

82900000

83000000

83100000

83200000

TLE4

G!

200 kbchr12:

54400000

54450000

54500000

54550000

54600000

54650000mm954700000

54750000

54800000

54850000

54900000

54950000

Npas3

55000000

55050000

55100000

55150000

Mouse!
Human!

chr14:

33500000

33600000

33700000

200 kb33800000

33900000

hg19
34000000

34100000

34200000

NPAS3
HAR!

H!
hg19

500 kbchr3:
70500000

70600000

70700000

70800000

70900000

71000000

71100000

71200000

71300000

71400000

71500000

71600000

FOXP1

UCE!

Primate specific HCNS!

Rodent specific HCNS!

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