MBE Advance Access originally published online on December 20, 2006
Molecular Biology and Evolution 2007 24(3):784-791; doi:10.1093/molbev/msl205
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Research Articles |
Comparison of Pax1/9 Locus Reveals 500-Myr-Old Syntenic Block and Evolutionary Conserved Noncoding Regions
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* Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen, China
Key Laboratory of Cellular and Molecular Evolution, Kunming Institute of Zoology, The Chinese Academy of Sciences, Kunming, China
Kunming Primate Research Center, The Chinese Academy of Sciences, Kunming, China
Chinese Human Genome Center at Shanghai, Shanghai, China
E-mail: wangyq{at}xmu.edu.cn.
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Abstract |
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Identification of conserved genomic regions within and between different genomes is crucial when studying genome evolution. Here, we described regions of strong synteny conservation between vertebrate deuterostomes (tetrapods and teleosts) and invertebrate deuterostomes (amphioxus and sea urchin). The shared gene contents across phylogenetically distant species demonstrate that the conservation of the regions stemmed from an ancestral segment instead of a series of independent convergent events. Comparison of the syntenic regions allows us to postulate the primitive gene organization in the last common ancestor of deuterostomes and the evolutionary events that occurred to the 3 distinct lineages of sea urchin, amphioxus, and vertebrates after their separation. In addition, alignment of the syntenic regions led to the identification of 8 noncoding evolutionarily conserved regions shared between amphioxus and vertebrates. To our knowledge, this is the first report of conserved noncoding sequences shared by vertebrates and nonvertebrates. These noncoding sequences have high possibility of being elements that regulate neighboring genes. They are likely to be a factor in the maintenance of conserved synteny over long phylogenetic distance in different deuterostome lineages.
Key Words: amphioxus • conserved synteny • ncECR • duplication • evolution
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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The nature of the last common ancestor of vertebrates is of enormous research interest. This area of interest has been rigorously researched in the past and present. Knowledge in this area has increased steadily, which, in the not too distant time, we hope to understand in the evolution process. It has been recognized that the origin of vertebrates is significantly related to the profusion of gene duplication events (Pennisi 2001
). The correlation between the increase of gene number and dramatic jumps in morphological complexity has led to a widely accepted notion that the duplication of existing genes was the source of raw materials for the great bursts of innovations seen in the vertebrate lineage. However, the extent and nature of the events (2 successive polyploidizations, single polyploidization, or limited segmental duplications) remained a subject of vigorous debate (Larhammar et al. 2002; McLysaght et al. 2002; Friedman and Hughes 2003). Comparisons of vertebrate species with one another and with invertebrate outgroups could bring further knowledge to the debate. Prior studies have shown that cephalochordate amphioxus is the closest living invertebrate relative of vertebrates (Wada and Satoh 1994), and it has evolved into its present form almost instantly preceding the vertebrate gene duplication (Panopoulou et al. 2003). This knowledge of cephalochordate amphioxus offers us a compelling understanding of the ancestral state of preduplicated genome (Castro and Holland 2003; Horton and Gibson-Brown 2002; Luke et al. 2003; Panopoulou et al. 2003; Castro et al. 2004).
To better comprehend the duplication pattern at the origin of vertebrates, it is important to research the evolutionary history of given chromosomal paralogy regions. Such research will provide evidences for different duplication models as well as offer new insights into the dynamics of genome evolution and the emergence of new functions. In 2003, Santagati et al. identified a pair of paralogy regions containing 5 genes from 4 distinct gene families (Pax1/9, Nkx2, Slc, and FoxA) in human chromosome 20p and 14q. These regions are hypothesized to have formed either through segmental or whole-genome duplication in early vertebrate evolution. The availability of genomic sequences from invertebrate species would greatly increase the reliability of comparative genomic analysis to delineate the ancestry and divergence of the paralogy region. In this study, we have sequenced and annotated the Pax1/9 containing bacterial artificial chromosome (BAC) clone from Chinese amphioxus Branchiostoma belcheri. Through comparison of syntenic regions from humans, amphioxi, and a variety of other species, we are able to illustrate the stigmata of conservation among different deuterostome lineages that exist in spite of more than 500 Myr of divergence and evolution from their last common ancestor. Moreover, cross-species alignment of the contiguous sequences corresponding to the conserved syntenic blocks enables the identification of noncoding evolutionary conserved regions (ncECRs; Shin et al. 2005) that are shared among the divergent groups of vertebrate and invertebrates. To our knowledge, the conservation over such a great evolutionary distance has not been reported before. These ncECRs represent potential regulatory elements associated with the presence of genes in the conserved syntenic blocks.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Sequencing of the Pax1/9 Locus and the Surrounding Genes in B. belcheri
We have documented the isolation of BAC clone 71P5 from B. belcheri containing Pax1/9 gene located on a 140-kb genomic insert in the BAC vector pBACe3.6 in our previous work (Wang et al. 2005
). This clone was sonicated, and the resulting fragments (2 kb) were subcloned and sequenced to cover the BAC clone with 8x overlap. The sequences were then analyzed and assembled with the PHRED/PHRAP/CONSED package (University of Washington, Seattle, WA; http://www.phred.org). For the annotation, we adopted the gene finding program GenScan to predict potential genes (http://bioweb.pasteur.fr/seqanal/interfaces/genscan.html). The entire sequence and the predicted genes were investigated by homology searching against National Center for Biotechnology Information databases (http://ncbi.nlm.nih.gov/BLAST/) with BlastN, BlastX, and TBlastX algorithms (Altschul et al. 1990).
Synteny Conservation of Pax1/9 Loci among Deuterostomes
Scaffold bearing amphioxus Pax1/9 gene was retrieved from the Joint Genome Institute (JGI) v.1.0 Branchiostoma floridae draft genome assembly (http://genome.jgi-psf.org/) using TBlastN search with the correspondent full-length protein sequence (BfPax1/9, GenBank accession number U20167) as query. The adjacent genes of BfPax1/9 were investigated to determine whether the orthologues could be mapped to the vicinity of Pax1 or Pax9 loci in human genome.
The loci conservation of syntenic genes shared between amphioxi and humans were determined via database searching (JGI, UCSC, and Ensembl) against the genome assemblages of 15 species including 6 mammals (chimpanzee, Pan troglodytes; dog, Canis familiaris; cow, Bos taurus; mouse, Mus musculus; rat, Rattus norvegicus; opossum, Monodelphis domestica), 1 bird (chicken, Gallus gallus), 1 amphibian (western clawed frog, Xenopus tropicalis), 3 teleost fishes (zebra fish, Danio rerio; pufferfish, Tetraodon nigroviridis; and fugu, Takifugu rubripes), 1 urochordate (ascidian, Ciona intestinalis), 1 echinoderms (sea urchin, Strongylocentrotus purpuratus), 1 insect (fruit fly, Drosophila melanogaster), and 1 worm (nematode, Caenorhabditis elegans).
Phylogenetic Reconstruction
Phylogenetic reconstruction was employed to access the orthology and paralogy relationships of genes residing in the Pax1/9 loci. The putative protein sequences were either retrieved by database searching (accession numbers provided in supplementary table 1, Supplementary Material online) or predicted through alignment of genomic sequences to known cDNA sequences by Genewise (http://www.ebi.ac.uk/Wise2/). Amino acid sequences were aligned using the ClustalX program (Thompson et al. 1994). The alignments were visually verified and corrected where necessary to improve accuracy. The phylogenetic trees were constructed using Neighbor-Joining (P distance) method from version 3.1 of MEGA program (Kumar et al. 2004). Confidence on each node was assessed by 1,000 bootstrap replicates.
Identification of ncECR
Syntenic genomic regions were retrieved from genome assemblies. Each gene and exon in the regions was annotated from start to end. Repetitive sequences were detected and removed by the program RepeatMasker (http://www.repeatmasker.org/) on slow setting. Local multiple alignments of the resulting masked sequences were generated and visualized by using MULAN free Web server (Ovcharenko et al. 2005; http://mulan.dcode.org/) with the "increase alignments sensitivity" option. To detect ncECR presence across vertebrates and invertebrates, a high-resolution threshold of 50% identity in a 50-bp window was adopted. All identified ncECRs were tested individually by using BlastN to exclude their presence in other positions of the genomes. Statistical analyses were conducted with paired t-test (2-tailed) to determine whether ncECRs were significantly more conserved than their adjacent segments. Potential binding sites for transcription factors were analyzed using MatInspector (http://www.genomatrix.de; Cartharius et al. 2005).
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Pax1/9 Gene Environment in Amphioxus
From B. belcheri BAC clone 71P1, we obtained 134 kb of sequence with 4 gaps organized into 5 contigs. Combining GenScan prediction and homology search, a total of 5 genes are identified. They are dehydrogenase/reductase family member 7 (Dhrs7), Pax1/9, solute carrier family 25 member 21 (Slc25A21), egg laying defective 9 homolog 3 (Egln3), and hepatocyte nuclear factor 3 (HNF3) family member HNF3-1. The clone 71P1 contains a complete coding sequence of the former 4 genes and a 649-bp segment at the sequence end, which is identical to the 3' untranslated regions of B. floridae HNF3-1 mRNA (GenBank accession number X96519), indicating that this gene is present immediately beside the genomic stretch. Because the nomenclature of the HNF3 family has been revised to FoxA genes (Kaestner 2000
), we refer to amphioxus HNF3-1 as FoxA1/2A hereafter. To determine if the gene loci are conserved in amphioxus lineage, the genome assembly of another amphioxus species B. floridae is examined, and the result shows that the corresponding sequence of clone 71P5 is localized in scaffold 42. Comparison of the orthologous regions between 2 amphioxus species reveals identical gene contents and spatial organization (fig. 1). In addition, 2 more genes are found in the downstream region of B. floridae. One gene encoding HNF3-2 (GenBank accession number Y09236) is present in close tail-to-tail arrangement to FoxA1/2A. We refer to it as FoxA1/2B hereafter. The second one is mirror-image polydactyly gene 1 (Mipol1) residing downstream of FoxA1/2B.
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Region of Conserved Synteny Maintained throughout 500 Myr of Evolution
As indicated in figure 1, synteny between the segment in amphioxus and 2 regions in human genome is found through genomic comparison of Pax1/9 gene environments. Among 7 genes in the amphioxus segment, 4 (Pax1/9, Slc25A21, FoxA1/2A, and FoxA1/2B) can be mapped to the pair of human 20p–14q paralogy regions (Santagati et al. 2003
) and the other 3 genes (Mipol1, Egln3, and Dhrs7) have orthologues on 14q but not 20p. Egln3 and Dhrs7 are distantly associated with the physically linked 4 genes of Pax9, Slc25A21, Mipol1, and FoxA1. To better understand the evolution of ancient syntenic region shared between amphioxi and humans, the conservation of orthologous gene loci of the available complete genome sequences from diverse species were investigated (fig. 1). Comparison of vertebrate orthologous regions showed that the organization of syntenic gene group Egln3-Pax9-Slc25A21-Mipol1-FoxA1-Dhrs7 is highly conserved in diverse tetrapod species. However, in teleost lineage only the linkage between Pax9 and Slc25A21 is observed. The paralogous cluster of Pax9-containing syntenic group includes 3 genes of Pax1, Slc25A5l, and FoxA2. The association between Pax1 and FoxA2 is conserved throughout vertebrate. The computer-predicted pseudogene Slc25A5l is only encountered in the syntenic region of humans and chimpanzees, suggesting that the remnant of ancient duplicated copy had been erased from nonprimate vertebrates.
In invertebrate species, the strongest synteny conservation is observed in the sea urchin genome. The close linkage of 4 genes, including Slc25A21, Pax1/9, Mipol1, and FoxA1/2 is identified in scaffold 37. In the genome draft of C. intestinalis, a genomic segment of 25 kb long (scaffold 764) comprising Slc25A21 and Pax1/9 is found, but no Mipol1- or FoxA1/2-coding sequence is identified, suggesting that these 2 genes are either discarded or positioned in the gap of the Ciona genome assembly. In the genome of C. elegans, the genes of Pax1/9, Egln3, and FoxA1/2 are mapped to the same chromosome albeit the large intervening genomic space between them. In D. melanogaster, all identifiable orthologues are not syntenic. This result is not surprising because the lineage of protostomes diverged to a great extent from that of deuterostomes.
Phylogenetic Reconstruction
We constructed Neighbor-Joining trees to access the phylogenetic relationship of 6 gene families localized in the surveyed region of syntenic conservation (supplementary fig. 1, Supplementary Material online). Genes used in this analysis are listed in supplementary table 1 (Supplementary Material online). In general, the internal topology of each tree agrees fairly well with the accepted evolutionary relationship of the organisms and is supported by high bootstrap values. The invertebrate genes are placed at the base of the trees. The 2 gene families of Pax1/9 and FoxA1/2 both have 2 vertebrate members and exhibited similar topology. The 2 vertebrate genes show greater similarity to each other than their invertebrate counterparts. In the gene family FoxA1/2, the 2 B. floridae genes (BfFoxA1/2A and BfFoxA1/2B) are clustered together, suggesting gene duplication specific to the amphioxus lineage.
Identification of ncECR
To detect putative regulatory elements conserved across vertebrates and invertebrates, we searched for ncECRs in the syntenic region conserved in diverse deuterostomes species using multiple sequence alignment tool, Mulan. A total of 8 conserved elements shared by amphioxus and vertebrates were identified in highly variable sequence background. These elements are 50–124 bp in amphioxus genome and can be divided into 2 groups. The first group includes 4 elements (A1–A4) shared with vertebrate Pax1-FoxA2 genomic segments and the second comprises another 4 elements (B1–B4) shared with vertebrate Pax9-Slc25A21-Mipol1-FoxA1 intervals. The spatial arrangement and alignment are shown in figure 2 and supplementary figure 2 (Supplementary Material online). The sequence similarities range from 52.8% to 74% between the 2 amphioxus species, from 68.9% to 95.2% between the vertebrate species, and from 53.2% to 73.2% between the 2 clades of amphioxus and vertebrates. Statistical analysis reveals that with the exception of element A1, the ncECRs identified in our study are significantly more conserved than the adjacent segments (paired t-test, 2-tailed P < 0.05). In addition, element A1 does not widely deviate from the significance (P = 0.056). The result indicates that negative selection has acted on these elements.
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The order, position, and orientation of those identified elements are strictly conserved within each individual lineage of amphioxus or vertebrate, but highly variant between the 2 lineages (fig. 2). One exception is element A4 residing downstream of FoxA2 (FoxA1/2B) with relative conserved orientation in both lineages. Additionally, this element also displayed the highest level of conservation as suggested by wider evolutionary spectrum (including teleosts) and higher overall sequence identity (90% vs. 55–80%) than to the other 7 elements. These evidences strongly indicate that A4 is involved in fundamental regulating function of FoxA.
We employed the MatInspector program to examine whether any known transcription factor–binding sites were contained in the conserved elements. The results show that a putative FoxA2-binding site exists in the element A3 of both amphioxus and vertebrates. Previous studies demonstrated that both cross-regulation (by FoxA2 and FoxA3) and autoregulation (by FoxA1 itself) featured in the regulation of FoxA1 gene (Kaestner et al. 1999). The binding site of FoxA2 in element A3 is probably required to mediate the regulation of Fox genes.
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Phylogenetic Analysis
Of the 7 genes corresponding to 6 gene families predicted from the amphioxus genomic sequences, 3 (Pax1/9, FoxA1/2A, and FoxA1/2B) were cloned previously and their phylogenetic relationships analyzed (Holland et al. 1995
; Shimeld 1997). Here, we performed phylogenetic analysis on all 6 gene families by incorporating sequence of taxa examined in our study (supplementary fig. 1 and table 1, Supplementary Material online). The topology of Pax1/9 is consistent with previous studies (Holland et al. 1995), indicating that the vertebrate Pax1 and Pax9 originated by duplication after the divergence of amphioxus. The phylogenetic analysis of FoxA1/2 gene family confirms the earlier hypothesis that the ancient FoxA1/2 had independently duplicated in the 2 lineages of amphioxus and vertebrate (Shimeld 1997). Furthermore, the identification of physically linked FoxA1/2A and FoxA1/2B in B. floridae genome revealed that these 2 genes were formed through tandem duplication. The 4 newly predicted amphioxus genes (Dhrs7, Egln3, Slc25A21, and Mipol1) displayed an orthology relationship toward vertebrate sequences.
Evolution of Syntenic Block
Evidences from genomic loci and evolutionary reconstruction strongly suggest that the region surrounding Pax1/9 genes are evolutionarily related between humans and amphioxi. Such homology can be explained by 2 alternative hypotheses. The first hypothesis is the conservation of an ancestral state and the other is the convergence of evolutionary events. In order to test the hypotheses, we investigated whether any conservation could be found in the genomes of other species. This investigation reveals strong synteny conservation over wide evolutionary spectrum of deuterostome, supporting the hypothesis of a conserved ancestral state.
Identification of homologous genomic regions is an essential prerequisite to study the evolution of genomes, both within and between organisms. Identifying intergenomic homology allows researchers to assess the impact of rearrangement events, whereas intragenomic homology gives insights into the duplication history of a genome (Simillion et al. 2004). Analysis of the conserved synteny between vertebrate paralogy regions and nonvertebrate deuterostome chromosomal segments supports the dynamics that shaped the region hosting Pax1/9 gene members. We propose that a linked array of genes comprising Pax1/9, Slc25A21/5l, Egln3, Mipol1, and FoxA1/2 existed in the genome of early deuterostomes, which was then passed to sea urchin, amphioxus, and vertebrates. Lineage-specific rearrangement of the major deuterostome clades can be inferred through the comparison of positional relationship of orthologous genes. A putative model for the evolution of the syntenic region harboring Pax1/9 is given in figure 3.
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In amphioxus, vertebrates, and C. intestinalis, Slc25A21 lies downstream of Pax1/9 (Pax9). However, this gene is located upstream of Pax1/9 in sea urchin. We also noticed that a gap of 36 kb long is present immediately upstream of Slc25A21 in the sea urchin genome assembly. This observation allows us to speculate that during the evolution of sea urchin, the local rearrangement had translocated the genomic segment encompassing Slc25A21 and Egln3 (block 1 in fig. 3) upstream of Pax1/9 and the Egln3 gene localized in the gap of the region.
Compared with sea urchin and tetrapods, the amphioxus region features 2 peculiarities. One is the reversed order and orientation of the block containing Mipol1 and the neighboring FoxA1/2B gene and the second is that 2 FoxA family members (FoxA1/2A and FoxA1/2B) are closely linked in tail-to-tail fashion. Phylogenetic analysis shows that FoxA1/2A and FoxA1/2B from amphioxi are coorthologous to the FoxA1 and FoxA2 from humans (Shimeld 1997). Therefore, we hypothesize that in amphioxus lineage, the genomic region harboring Mipol1 and FoxA1/2 (block 2 in fig. 3) underwent segmental inverse duplication followed by the disposal of 1 Mipol1 copy.
In the genome of diverse vertebrate species, there are 2 chromosomal segments showing similar gene contents and organization to the single invertebrate region. Thus, we infer that during the early stages of vertebrate evolution, the chromosomal segment containing a cluster of at least 5 genes were duplicated. The congruent timing of vertebrate gene expansion and the duplication of 2 paralogous gene pairs of Pax1/9 and FoxA1/2 (Holland et al. 1995; Shimeld 1997) imply that the sister segments could have formed from whole-genome duplication at the early stage of the vertebrate formation. Subsequently, the 2 sets of paralogous genes diverged independently up to the present time, acquiring distinct functions. In the region containing Pax1, the paralogues of Mipol1 and Egln3 were discarded, and the Slc25A5l was pseudogenized after the genome duplication. In the region harboring Pax9, all single copies of ancestral genes were retained albeit an interchromosal translocation that moved Egln3 to a distant position.
The evolutionary scenario we present here supports the idea that the ancestral segment containing Pax1/9, Slc25A21, and FoxA1/2 had duplicated in vertebrate progenitors as proposed by Santagati et al. (2003). Our finding that the 2 additional genes of Egln and Mipol1 are also part of the duplicated segment extends further the characterization of the syntenic region. Furthermore, by incorporating genomic information of invertebrate species, we are able to deduce that the evolutionary events are specific occurrences to the lineages of cephalochordates (amphioxus) and echinoderms (sea urchin). Another marked difference is that our scenario does not include the tandem pair of nature killer class 2 (NK2) genes (Nkx2-1/4 and Nkx2-2/9), which are previously regarded as part of the syntenic region (Santagati et al. 2003). Our genome assembly search reveals that there is a pair of tightly linked NK2 class genes in the genome of amphioxus and sea urchin; however, none resides in the scaffold harboring Pax1/9 gene (data not shown). With separate evolution paths, the correlation diminishes to an insignificant level between natural killer (NK) class genes and the syntenic regions.
Conserved Noncoding Sequences in the Region
The existence of conserved syntenies between vertebrates and invertebrates has been studied previously. Such studies have focused on 2 areas: the clustering of families of related genes and the relative positions of genes physically associated in one lineage whose orthologues are linked to the genomes of another lineage. The former is characterized by the clustering of Hox (Garcia-Fernandez and Holland 1994), Parahox (Brooke et al. 1998), and Nkx genes (Luke et al. 2003), whereas the later is represented by the major histocompatibility cells (Castro and Holland 2003) and Insulin–Relaxin gene families (Olinski et al. 2006). The case of highly conserved genomic region encompassing unrelated genes in vertebrates and invertebrates as deciphered in our study has not been addressed before. The degree of conservation in the gene content in the proximity of Pax1/9 may suggest that the regulatory elements of the neighboring genes are largely conserved across great evolutionary distances. One of the selective forces that keep the genes closely linked may stem from the fact that adjacent genes share common cis-regulatory elements (Peifer et al. 1987). Dispersing of these genes would deprive one of them of its cis-regulatory elements and lead to deleterious mutation. In the study of Santagati et al. (2003), 2 conserved noncoding sequences that function as tissue-specific enhancers of neighboring genes are identified through the comparison of the syntenic regions spanning Nkx2-9, Pax9, and Slc25a21 from humans, mice, and fugus. Interestingly, one of the elements residing in the intron of Slc25A21 was found to be the cis-regulatory sequences of neighboring Pax9 gene (Santagati et al. 2003); suggesting the fixation of regulatory elements inside the territory of neighboring genes might constitute a functional bond to limit the gene rearrangement, and as a result, the linked organization is retained in the phylogenetically distant species.
In this study, we provided a novel expansion to earlier genomic comparison by including species from both the invertebrate and vertebrate with double conserved synteny blocks surrounding Pax1 and Pax9 loci. The analysis allows the identification of 8 noncoding regions conserved between amphioxus and vertebrates. The sequence similarity among vertebrate species is higher than that between vertebrates and invertebrates, as can be expected from the evolutionary distance of the compared species. Interestingly, in the 5 elements (A1, A2, B1, B3, and B4) where the sequences of both amphioxus species are available, the amphioxus sequences exhibited low level of conservation as suggested by nucleotide identity ranging from 52.8% to 74%, the degree comparable to that between amphioxus and vertebrates (53.2–73.2%). The sister taxa B. floridae and B. belcheri diverged about 112 MYA (Nohara et al. 2004), whereas the ancestor they share with vertebrates existed for approximately 500 Myr. Taking the variation level and the divergence time into account, the amphioxus elements appeared to have experienced lower selective constraint than their vertebrate counterparts. It is possible that the observed changes are generally neutral and do not alter their regulatory role extensively. It is known that stabilizing selection on transcriptional output allows slightly deleterious mutations to persist, compensated for by adaptive changes elsewhere in the promoter and resulting in continuous binding-site turnover (Ludwig et al. 2000, 2005; Balhoff and Wray 2005; Gompel et al. 2005). The insufficient sensitivity of comparative genomic analysis could leave many of the functional elements undetected. Although phylogenetic footprinting is a widely accepted and a powerful approach, we still need to be cautious of its limitation.
To our knowledge, the noncoding sequence similarity between vertebrates and invertebrates has never been described before. In 2005, Woolfe et al. reported sequences that are highly conserved between humans and fugus, but failed to find similarity in the invertebrate sequence databases, including the whole-genome sequences of C. intestinalis, D. melanogaster, and C. elegans. The noncoding sequence homology between amphioxus and vertebrates identified here indicates the presence of conserved regulatory modules between the 2 lineages. In vivo reporter constructs using amphioxus genomic DNA in transgenic mice (Manzanares et al. 2000) and chicken (Wada et al. 2006) support the hypothesis. Therefore, we can infer that for the purpose of better understanding the mechanism and evolution of gene regulation, amphioxus is preferred to other invertebrate models.
As a preliminary attempt to exploit the extent of noncoding conservation between vertebrates and invertebrates, we have also inspected 3 individual loci of Pax2/5/8, Pax3/7, and Pax6, but no ncECR could be identified. The enrichment of ncECR in the Pax1/9 locus is congruent with the recent knowledge that highly conserved noncoding sequences are located in and around genes involved in the regulation of transcription and development (Bejerano et al. 2004; Shin et al. 2005; Siepel et al. 2005; Woolfe et al. 2005). In vertebrates and invertebrates, there are 2 developmental regulators (Pax1/9 and FoxA1/2) residing in the regions of synteny being researched in this paper. The expression profiles of Pax1/9 and FoxA1/2 further supports the hypothesis that the functional relationship between ncECRs with several associated genes may be one of the selective forces that maintain the associated genes tightly through 500 Myr evolution. In amphioxus, the Pax1/9, FoxA1/2A, and FoxA1/2B expressed in the developing pharyngeal (Holland et al. 1995; Shimeld 1997), whereas the vertebrate FoxA1, FoxA2, Pax1, and Pax9 all expressed in thymus (Dooley et al. 2005), strongly indicating that the linked genes might share common regulatory elements.
Based on the estimation that the common ancestors of amphioxus and vertebrates diverged 500 MYA (Holland et al. 1992), the noncoding sequences identified in this study are conserved over half a billion years of parallel evolution, and thus, they are valid candidates for functional significance. Although the transient transgenic method developed by Yu et al. (2004) for amphioxus embryos is generally the accepted method, the system is still not well established yet due to the limitation of the embryo resource. Other well-developed systems, such as mice or zebra fish, probably offer the experimental evaluation for these cis-regulatory modules. Furthermore, the completion of amphioxus genome project provides a valuable resource to promote our understanding of the relationships between conserved sequences and the biological functions they confer and shed light on the evolutionary forces that have shaped chordate genomes.
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Supplementary Material |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Supplementary table 1 and figures 1 and 2 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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The authors are grateful to 2 anonymous reviewers for their helpful suggestions and comments and Mr Bang Yoke Leong for his linguistical help. This work is supported by National Nature Science Foundation of China (No. 30470938 & No. 30570208), Natural Science Foundation of Fujian Province, China (No. D0510002), and grant from the Science and Technology Bureau of Xiamen (No. 3502Z20042015).
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Footnotes |
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Jianzhi Zhang, Associate Editor
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References |
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