MBE Advance Access originally published online on May 9, 2008
Molecular Biology and Evolution 2008 25(8):1521-1525; doi:10.1093/molbev/msn109
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Letters |
Convergent Evolution of Clustering of Iroquois Homeobox Genes across Metazoans
Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain
E-mail: jordigarcia{at}ub.edu.
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Abstract |
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 TOP Abstract Supplementary MaterialAcknowledgementsReferences |
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Vertebrate and Drosophila Iroquois genes are organized in clusters of 3 genes sharing blocks of conserved regulatory sequences. Here, we report a 3-gene cluster in the basal, preduplicative chordate amphioxus. Surprisingly, however, the origin of the amphioxus cluster is independent of those in vertebrates and drosophilids. Investigation of genomic organization of Iroquois genes in other 17 metazoan genomes revealed a fourth independent 3-gene cluster organization in polychaetes, as well as additional 2- and 4-gene clusters in other clades, in one of the most striking examples of convergence in genomic organization described so far. The recurrent independent evolution of Iroquois clusters suggests a functional importance of this organization for these genes, perhaps related to the sharing of regulatory elements. Consistent with this, comparative analysis of genomic regions flanking the 3 amphioxus Irx genes revealed several blocks of sequences, conserved for at least 100 Myr. Finally, we discuss the possible causes and implications of the convergent evolution of this genomic and regulatory organization throughout metazoans.
Key Words: amphioxus • Iroquois • genome evolution • gene cluster • convergent evolution • synteny conservation
Restructuring and shuffling of genome architecture are a major source for evolutionary change; however, little is known about the mechanisms underlying these changes. With the completion of several metazoan genome sequencing projects, many key features of genome structure have been discovered; the finding of gene regulatory blocks, conserved synteny, and gene deserts have given rise to stimulating hypothesis about the origin, conservation, and evolution of genomic structure and function. In this regard, the presence of conserved cis-regulatory elements has been shown to be crucial both in maintaining gene clustering and in promoting extensive gene-free regions (Nobrega et al. 2003
; Ovcharenko et al. 2005; Engstrom et al. 2007; Kikuta et al. 2007).
Iroquois genes represent a paradigmatic example of the relationship between gene regulation and genome organization (de la Calle-Mustienes et al. 2005). Iroquois genes are homeobox transcription factors of the TALE superclass implicated in key developmental processes during animal development. In vertebrates, Irx genes regulate proneural genes (Bellefroid et al. 1998; Gómez-Skarmeta et al. 1998) and are involved in several other processes during gastrulation, nervous system regionalization, and organ patterning (Briscoe et al. 2000; Gómez-Skarmeta et al. 2001; Kobayashi et al. 2002). Perhaps, the most prominent feature of Irx genes is their functional genomic organization, consisting of complexes of 3 clustered genes. In tetrapods, due to the whole-genome duplications occurred at the origin of the vertebrate lineage (Dehal and Boore 2005), there are 6 Irx genes grouped into 2 paralogous genomic clusters: IrxA, containing Irx1, Irx2, and Irx4, and IrxB, containing Irx3, Irx5, and Irx6 (Peters et al. 2000). Interestingly, the developmental expression patterns of Irx1 and Irx2 and of Irx3 and Irx5, respectively, are almost identical, whereas the expression of the third gene of each cluster, Irx4 or Irx6, is generally more divergent (Bellefroid et al. 1998; Gómez-Skarmeta et al. 1998; Garriock et al. 2001; Houweling et al. 2001). These expression patterns can be explained by the differential distribution of several highly conserved noncoding regions (HCNRs [Sandelin et al. 2004; Woolfe et al. 2005]) within the clusters, which drive expression in the common territories where several Irx genes are expressed (de la Calle-Mustienes et al. 2005). These HCNRs act as enhancers shared by more than one gene and provide an explanation as to why vertebrate Irx genes have remained in cluster (de la Calle-Mustienes et al. 2005). Interestingly, a parallel situation is observed in Drosophila, where the 3 Iroquois genes also share expression domains and specific regulatory elements (Gomez-Skarmeta and Modolell 2002), although the vertebrate and fruit fly clusters originated independently (Peters et al. 2000; de la Calle-Mustienes et al. 2005).
Here, we study the genomic organization of Iroquois genes in the basal, preduplicative chordate amphioxus Branchiostoma floridae. As in vertebrates, we found a cluster of 3 genes (BfIrxA, BfIrxB, and BfIrxC). To explore the evolution of these clusters, we performed gene tree analyses using phylogenetic methods. Interestingly, the 3 B. floridae genes form a clade (supplementary fig. SM1, Supplementary Material online), indicating that these 3 genes represent duplications of a single ancestral gene after the divergence of cephalochordates from other chordates. The clusters' independent origins are also underscored by the different strand orientation of the genes (2 genes 3'-to-3' in Branchiostoma, 2 genes 5'-to-5' in vertebrates, fig. 1). In addition, a search using reverse transcriptase–polymerase chain reaction (RT-PCR) for Irx genes in a sister amphioxus species, Branchiostoma lanceolatum, split from B. floridae around 100 MYA (Cañestro et al. 2002; Nohara et al. 2005; Kon et al. 2007), yield orthologs for the 3 B. floridae Irx genes. All 3 amphioxus genes are expressed in a similar temporal manner during whole embryonic development, with a peak of expression from neurula to 1-gill-slit larva stages, as indicated by RT-PCR analysis (data not shown).
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Genomic analysis of other 17 available metazoan genomes, including species from placozoans, cnidarians, and the 3 main bilaterian clades (Ecdysozoans, Lophotrochozoans, and Deuterostomes), revealed further surprises. Strikingly, we found a fourth independent 3-gene cluster of Iroquois genes in the polychaete Capitella capitata. Another cluster, of 4 genes, was found in the mollusk Lottia gigantea, and 2 other independent clusters, consisting of 2 genes each, were found in each of the 2 tunicates species examined (Ciona savigni and Ciona intestinalis, as previously reported [Wada et al. 2003
]) (fig. 1). In each case, genes within a lineage show greater similarity to each other than to genes from other lineages, indicating recurrent gene duplication leading to these clusters (supplementary fig. SM1, Supplementary Material online). Considering that the basal metazoan groups (placozoans and cnidarians) and the nonchordate deuterostomes harbor a single Iroquois gene (fig. 1), the observed pattern implies at least 13 tandem gene duplications of Iroquois genes in 6 independent lineages, with further convergence on exactly 3-gene clusters in 4 deeply diverged metazoan groups. This is, to our knowledge, the most remarkable example of convergence in genomic organization described so far.
What explains this recurrent convergent evolution? In vertebrate models, the presence of shared HCNRs acting as regulatory elements, particularly the ultraconserved regions (UCRs), may act as a constraint to maintain the cluster integrity (de la Calle-Mustienes et al. 2005). Does the amphioxus cluster show similar organization? We searched for the elements described within the vertebrate Irx cluster in amphioxus but failed to find a clear match, consistent with independent evolutionary origins of the Branchiostoma and vertebrate clusters. However, comparison of the genomic regions surrounding the 3 B. floridae Irx genes revealed lineage-specific conserved regions. We divided the amphioxus cluster into 3 regions, each containing one of the genes and surrounding noncoding sequences (see supplementary methods, Supplementary Material online). Crossed VISTA (Frazer et al. 2004) analysis of these regions revealed several repeated blocks with high sequence similarity (fig. 2A). Most blocks are present in 2 copies, but some are present in 3 copies. Three-copy sequences lie nearby the coding sequences (one for each gene; fig. 2B, yellow boxes), consistent with being part of minimal promoters, untranslated regions (UTRs), or other proximal cis elements. Two-copy blocks are present in opposite orientation, in agreement with the opposite transcriptional orientations of the genes, and are located further away from the coding sequences and may thus be able to function over longer distances (and so to influence expression of all 3 genes; fig. 2B, turquoise and red boxes). These 2-copy elements likely were initially doubly duplicated along with the coding sequences but have since been differentially lost (fig. 2C). Importantly, we have also identified and cloned these blocks in B. lanceolatum, which indicates that they have been conserved for around 100 MYA (Cañestro et al. 2002; Nohara et al. 2005; Kon et al. 2007), strongly suggesting a functional role for these elements.
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The finding of potential regulatory elements that are present in fewer than 3 copies is consistent with a role for these elements in stabilizing the Irx cluster in Branchiostoma as in other metazoans (Gomez-Skarmeta and Modolell 2002
; de la Calle-Mustienes et al. 2005). Whereas the initial duplicated genomic region might have contained all the necessary regulatory sequences, loss of some of these elements might have rendered the genes dependent on proximity to the other copies, ensuring the continued linkage of the paralogs in a cluster. At the same time, the presence of multiple Irx genes would allow for the emergence of copy-specific regulatory elements and patterns, aiding the emergence of new developmental programs.
Related to the presence of noncoding regulatory sequences, genes with complex regulation usually show large surrounding genomic regions devoid of genes (gene deserts) (Nelson et al. 2004). Similarly, clusters of highly regulated genes usually have large intergenic regions between cluster members, as is the case of the Irx clusters in vertebrates (de la Calle-Mustienes et al. 2005). The amphioxus Irx cluster also shows very large intergenic regions within the cluster, much larger than the distances between the genes in the proximity and than the average genomic intergenic distance (table 1). A similar pattern is observed for clusters of all species and in the case of the surrounding regions of the species with a single Irx gene, with the exception of the placozoans and cnidarians (table 1 and supplementary file 1, Supplementary Material online).
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Interestingly, the study of the Irx surrounding regions across metazoans yielded another surprising observation. Amphioxus Irx cluster is flanked directly upstream by an ortholog of the Drosophila CG10632 locus and downstream by the carbonic anhydraseVIII (CAVIII) gene. This synteny is conserved to sea urchin (where only a single Irx is present), making sea urchin's organization highly resembling of the hypothesized ancestral Irx organization in the amphioxus lineage (fig. 2C) (Unfortunately, we were not able to identify any clearly conserved noncoding sequence between amphioxus and sea urchin using VISTA analysis). Strikingly, CG10632 orthologs are also immediately 5' of the Irx cluster in all studied insects and in the crustacean Daphnia pulex (black arrowheads in fig. 1). Moreover, in lophotrochozoans, the CG10632 ortholog is within the Irx cluster, shedding light on the events leading to lophotrochozoan's Irx cluster formation and further supporting the independent evolution of this cluster. In the case of CAVIII, although the synteny is not conserved to protostomes, this gene is flanking upstream the single Irx gene in placozoans (white arrowhead in fig. 1).
These data strongly support that the linkage between CG10632, CAVIII, and Iroquois genes, which has been retained in amphioxus and sea urchin, is ancestral at least to bilaterians. In the case of CG10632, this linkage has been maintained in members of all major metazoans groups, encompassing more than 600 million years of evolution, in one of the most striking examples of conserved synteny between 2 phylogenetically unrelated genes. For this reason, we propose for this gene the name Sosondowah (sowah), after the mythological hunter tied to her doorpost by the Iroquois sky's goddess.
In summary, the results presented here show that the organization of Iroquois genes in gene complexes has evolved independently several times in metazoan evolution, especially generating clusters of 3 genes. The number of convergent clusterization events thus seems to exceed what would be expected merely by chance. If so, it is tempting to speculate that tandem duplication and neighborhood organization for Iroquois genes are fixed in evolution at a high rate, maybe related to the functional importance of shared regulatory elements. Intriguingly, our results would suggest that gene tandem duplication is more widespread than previously thought, being only fixed at high rates in cases in which a cluster organization bear a functional relevance, as it seems the case for the Iroquois complexes. An alternative explanation is that some genomic regions are more prone to undergo tandem duplication; however, there is currently no evidence that specific groups of genes or types of sequences are more likely to duplicate than others. Whatever the case, the study of Iroquois gene organization will help to understand how and why functional architectures evolve in metazoans genomes. Understanding the causes and mechanisms involved in the evolution of these genes will help to understand the flexibility, constraints, and evolvability of genome organization.
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Supplementary Material |
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Supplementary file 1, methods, and figure SM1 are available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/). Accession numbers EU754744 [GenBank] -EU754750.
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Acknowledgements |
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TOPAbstractSupplementary MaterialAcknowledgementsReferences |
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We thank Scott W. Roy for critical reading of the manuscript and extremely helpful comments and suggestions and Jim Langeland for kindly providing the B. floridae cDNA library. We particularly thank an anonymous referee for insightful suggestions. J.G-F. thanks Laura for inspiration. This work was funded by grant BFU2005-00252 from the Ministerio de Educación y Ciencia (MEC), Spain. M.I. holds FPI and I.M. FPU fellowships (MEC).
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Footnotes |
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1 These authors contributed equally to this work.
Billie Swalla, Associate Editor
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References |
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