- Email: [email protected]
The Urbilaterian Super-Hox cluster
Update 2 Pastinen, T. et al. (2004) A survey of genetic and epigenetic variation affecting human gene expression. Physiol. Genomics 16, 184–193 3 Gimelbrant, A. et al. (2007) Widespread monoallelic expression on human autosomes. Science 318, 1136–1140 4 Wang, J. et al. (2007) Monoallelic expression of multiple genes in the CNS. PLoS ONE 2, e1293 5 Pollard, K.S. et al. (2008) A genome-wide approach to identifying novelimprinted genes. Hum. Genet. 122, 625–634 6 Serizawa, S. et al. (2004) One neuron-one receptor rule in the mouse olfactory system. Trends Genet. 20, 648–653 7 Alexander, M.K. et al. (2007) Differences between homologous alleles of olfactory receptor genes require the Polycomb Group protein Eed. J. Cell Biol. 179, 269–276 8 Mlynarczyk-Evans, S. et al. (2006) X chromosomes alternate between two states prior to random X-inactivation. PLoS Biol. 4, e159 9 Bacher, C.P. et al. (2006) Transient colocalization of X-inactivation centres accompanies the initiation of X inactivation. Nat. Cell Biol. 8, 293–299
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10 Xu, N. et al. (2006) Transient homologous chromosome pairing marks the onset of X inactivation. Science 311, 1149–1152 11 Zhao, Z. et al. (2006) Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions. Nat. Genet. 38, 1341–1347 12 Gimelbrant, A.A. et al. (2005) Monoallelic expression and asynchronous replication of p120 catenin in mouse and human cells. J. Biol. Chem. 280, 1354–1359 13 Mostoslavsky, R. et al. (2001) Asynchronous replication and allelic exclusion in the immune system. Nature 414, 221–225 14 Ensminger, A.W. and Chess, A. (2004) Coordinated replication timing of monoallelically expressed genes along human autosomes. Hum. Mol. Genet. 13, 651–658 15 Singh, N. et al. (2003) Coordination of the random asynchronous replication of autosomal loci. Nat. Genet. 33, 339–341 0168-9525/$ – see front matter . ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2008.03.005 Available online 29 April 2008
Genome Analysis
The Urbilaterian Super-Hox cluster Thomas Butts1, Peter W.H. Holland1 and David E.K. Ferrier1,2 1 2
Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK Present address: The Gatty Marine Laboratory, University of St Andrews, St Andrews, Fife, KY16 8LB, UK
Comparison of whole genome sequences of representative animals enables reconstruction of the ancestral bilaterian genome: the starting point from which most extant animal lineages evolved. The Hox gene cluster patterns the anterior–posterior axis of bilaterians. Here we show that this cluster was embedded within a larger homeobox gene cluster, the Super-Hox cluster, in the ancestral bilaterian. This Super-Hox cluster contained at least eight genes alongside the core Hox genes (‘EuHox’ genes).
Ancient homeobox gene neighbours The Bilateria represent 99% of all described living animal species, and as such, reconstructing the biology of their last common ancestor, the Urbilateria, is of fundamental importance for understanding animal evolution. In the postgenomic era, this reconstruction has begun to be extended to the urbilaterian genome (e.g. [1]). The homeobox genes are particularly well suited for reconstruction of ancestral genomes because of their widespread conservation and robust family level phylogeny. Their diversity arose predominantly by tandem duplication, which can create functional clusters of genes, such as the Hox cluster, or can survive as relics of the ancestral organization simply because of a lack of extensive genomic rearrangement. Here we take the existence of such evolutionarily related genes that are also close neighbours on the chromosome, such as the Antennapedia-class (ANTP-class) homeobox genes, to represent such relics of the ancestral genome organization, assuming this to be much more likely than a secondary Corresponding author: Ferrier, D.E.K. ([email protected]).
reassociation of these genes on independent evolutionary lineages. The ANTP-class is the largest class of the homeobox superfamily, and is central to the evolution of animal development. In addition to the Hox genes, it comprises several other gene families whose members are sometimes found in small gene clusters in animal genomes. This propensity for gene clustering, and the organization of gene cluster members in vertebrate genomes, have led to the hypothesis of an ancestral ‘Mega-cluster’ deep in animal evolutionary history [2,3]. The ANTP-class originated before the radiation of the Metazoa [4]; recent data from the genome of the starlet sea anemone Nematostella vectensis have shown that most extant families within this class were present, by inference in a Mega-cluster, before the divergence of the cnidarian and bilaterian lineages [5,6]. Chromosomal rearrangement and breakage subsequently began to break up this Mega-cluster and disperse its constituent genes. By contrast, the Hox and NK cluster genes have been conserved in tight genomic clusters in some extant genomes. It has been suggested that temporal colinearity – a process whereby the progressive activation of the genes along the anterior–posterior axis during development is mirrored by their genomic position within the cluster – has been the major constraining force on the organization of the Hox gene cluster [7,8], because these clusters have disintegrated in animals with rapid or highly determinative development (e.g. [9]). The function of the NK cluster is less well studied than its Hox counterpart, although all of the constituent genes are involved in mesoderm patterning and differentiation [10]. Nevertheless, a clusterwide phenomenon that explains their conservation in 259
Update Glossary Bilaterian: An animal that has a single plane of symmetry along its anteriorposterior axis (bilateral symmetry). Deuterostome: An animal in the group Deuterostomia, in which the mouth/ stomodaeum arises by a secondary invagination during embryogenesis rather than from the blastopore. Includes Echinodermata, Hemichordata, Xenoturbellida and Chordata [16]. Homeobox: A DNA motif, usually of 180 bp, that encodes a distinctive sequence-specific DNA binding domain (the homeodomain) with three a helices, two of which are in a Helix-turn-Helix conformation. Hox gene: A gene that is orthologous to the homeobox genes of the Drosophila and mammalian Hox gene clusters, which pattern the anterior– posterior axes of bilaterian animals. Metazoa: Synonymous with the kingdom Animalia (multicellular, heterotrophic animals). Protostome: An animal in the group Protostomia, the second great bilaterian clade, in which generally the mouth/stomodaeum forms from the blastopore. Urbilateria: The last common ancestor of the bilaterians [27], originally taken to be the last common ancestor of the Protostomia and Deuterostomia. However, its precise meaning is dependent on the phylogenetic position of the acoelomorph flatworms, which have been suggested to be basal bilaterians [28], although this is still debated [29]. In the present context, because the genes of the Mega-cluster existed well before the last common ancestor of all bilaterians, the precise position of the acoelomorph flatworms is irrelevant and hence we retain the use of the term Urbilateria. The acoelomorph problem does mean, however, that the number of genes in the Hox cluster of Urbilateria is not completely resolved, and the seven used here comes from protostomedeuterostome comparisons [26], but may have been fewer if the low number of Hox genes presently isolated from Acoelomorpha [28,30] is an accurate representation of the ancestral bilaterian state.
insects and their breakage in chordates [11] is elusive at present. In addition to these extant gene clusters, the remains of the ancestral Mega-cluster organization have been revealed in genomic comparisons of human, mouse and Drosophila [2,12]; however, the extent of clustering that remained in the last common ancestor of the Bilateria (the Urbilaterian) is not clear. We have analysed the content and organization of the ANTP-class homeobox genes of the Florida lancelet and red flour beetle. These species have both retained a greater extent of ancient genomic organization than is present in other sequenced bilaterian genomes, enabling us to reconstruct the homeobox gene cluster that existed around the Hox cluster in the Urbilaterian. Amphioxus clustering When reconstructing genomic evolutionary history, animals that exhibit low levels of genomic rearrangement will be of primary importance. From our analysis presented here and in other studies [13–15], it seems that the Florida lancelet, Branchiostoma floridae (also known as amphioxus), and the red flour beetle, Tribolium castaneum, will both prove fruitful reference points in this endeavour. Amphioxus has for a long time cast light on the events that surrounded the invertebrate–vertebrate transition. Despite its recent repositioning as the basal chordate lineage rather than as the sister taxon to vertebrates [16], it retains many aspects of ancestral chordate genomic organization that have been lost in the urochordates, the true invertebrate sister group of the vertebrates, such as intact Hox [9,14,17,18] and ParaHox [19,20] clusters. In addition, the remains of the NK cluster in amphioxus resemble the vertebrate pre–whole genome duplication condition [11], but with the additional retention of NK6 and NK7 in a cluster next to the Lbx–Tlx gene pair. 260
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In addition to the prototypical Hox and ParaHox clusters and NK cluster remnants, our analysis shows amphioxus possesses two additional examples of clustering (Figure 1). First, the amphioxus genes Mnxa and Rough are separated by an intergenic distance of only 20 kb. Second, a novel cluster of homeobox genes is present that consists of four genes, Engrailed, Nedxa, Nedxb and Dlx, within 135 kb [13]. The Nedx (Next to Distalless) family is widespread across bilaterians (its Drosophila ortholog is CG13424) but has been lost from vertebrates. It is notable that these novel examples of clustering are both present on the same chromosome as the Hox cluster, which, as in Cnidaria, is flanked by Evx [21,22] (although, in amphioxus, there are two Evx genes [23]). In contrast to the situation in vertebrate genomes, and contrary to previous models [24], Mox is present further upstream from the posterior end of the Hox cluster (220 kb from AmphiEvxB). Tribolium clustering In the recently sequenced Tribolium genome [15], further evidence of homeobox gene clustering was found in addition to the intact Hox [25] and NK clusters in this animal. On the X chromosome, the single copy genes Btn (the ortholog of chordate Mox genes), Rough and Hex are clustered within 80 kb. In addition, the genome of Tribolium contains a further uncharacterized homeobox gene cluster consisting of the genes Exex (the ortholog of Mnx), Nedx, Dll and Eve. This cluster spans 190 kb on chromosome 7. The Dlx–Nedx gene pair is distinctive because it is also present in amphioxus, suggesting a selective pressure for its retention. Such a pressure is most likely to have arisen because of enhancer sharing, whereby a nearby cis element regulates both genes. Rebuilding Urbilateria Combining the genomic data on novel homeobox gene clusters from both amphioxus and the red flour beetle, it was possible for us to reconstruct the extent of homeobox gene clustering at the Hox locus in the Urbilaterian (Figure 1). From our reconstruction, it can be seen that, as well as the canonical Hox genes (the ‘EuHox’ genes), eight other ANTP-class genes were clustered at the Urbilaterian Hox locus. This is in addition to the minimum number of seven EuHox genes in the Urbilaterian [26] (however, see ‘Urbilateria’ in the Glossary). Together these genes comprised the ‘Super-Hox’ cluster, which contained at least 15 genes in the last common ancestor of the bilaterians. Given the frequency of genome rearrangements and especially inversions, even in slowly evolving genomes, it is currently impossible to predict the ancestral order of the genes at this locus, other than in reference to the canonical Hox genes, where selection acts to maintain gene order and constrain the overall clustered organization. With the possible exception of the Dlx–Nedx gene pair, the remnants of the SuperHox cluster in amphioxus and beetles are likely to be simply the products of evolutionary inertia dating from the origin of these families >550 million years ago (Mya).
Update
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Figure 1. The Urbilaterian Super-Hox cluster and its remnants. The gene order shown is not necessarily indicative of ancestral arrangement, which is impossible to infer at present because of the lack of functional selection for gene order, coupled with the frequency of inversions compared with other types of genomic rearrangement. In amphioxus, the gene encoding Nedx has been duplicated; in beetles, engrailed has duplicated. The Hox cluster (11 genes in beetles and 15 in amphioxus) is shown as a single hatched box that represents multiple genes (the ‘EuHox’ genes). LG , linkage group.; *, linked to the remains of the NK cluster. Note the linkage is not drawn to scale. All intergenic distances are <80 kb. The bold diagonal lines represent intergenic gaps >1 Mb; standard lines represent a gap of <250 kb.
Likewise, with evidence from beetle and amphioxus to supplement genomic information from other model organisms, the NK cluster of Urbilateria can be inferred to have contained nine genes: Msx, NK4, NK3, Lbx, Tlx, NK1, NK5, NK6 and NK7 [3,11,13]. Concluding remarks The ancestral Antennapedia (ANTP) Mega-cluster, which existed before the Cnidarian-Bilaterian Ancestor, did not break up as quickly as has previously been imagined, with only selectively maintained gene clusters (the Hox and ParaHox clusters and possibly the NK cluster) being retained. Instead, the break-up of the Mega-cluster occurred gradually and independently among extant animal lineages. Certain genomes (e.g. amphioxus and the red flour beetle) are less derived from the urbilaterian condition than others. Comparison of the organization of ANTP-class genes in these two genomes reveals the evolutionary history of ANTP-class gene clustering in unprecedented detail. Of the ancestral homeobox Mega-cluster, at least 24 genes existed as two clusters in the urbilaterian: the 9-gene NK cluster (see above) and the 15+ gene Super-Hox cluster (Figure 1). It is not clear from the beetle and amphioxus comparison whether the SuperHox and NK clusters were linked in the urbilaterian, but further genome sequences from other less derived organisms could resolve this. Also, at this point, it must be stressed that 15 actually represents a lower limit for the number of genes in the Super-Hox cluster and is based on the falsifiable assumption that no further instances of novel clus-
tering will be found in as yet unsequenced bilaterian genomes. Acknowledgements The authors thank Dan Rokhsar and the Joint Genome Institute for coordinating the amphioxus genome project and Stephen Richards and the sequencing centre at the Baylor College of Medicine for coordinating the Tribolium genome project. Work in the authors’ laboratories is funded by the BBSRC and the Wellcome Trust.
References 1 Raible, F. et al. (2005) Vertebrate-type intron-rich genes in the marine annelid Platynereis dumerilii. Science 310, 1325–1326 2 Pollard, S.L. and Holland, P.W.H. (2000) Evidence for 14 homeobox gene clusters in human genome ancestry. Curr. Biol. 10, 1059–1062 3 Garcia-Ferna`ndez, J. (2005) The genesis and evolution of homeobox gene clusters. Nat. Rev. Genet. 6, 881–892 4 Larroux, C. et al. (2007) The NK homeobox gene cluster predates the origin of Hox genes. Curr. Biol. 17, 706–710 5 Ryan, J.F. et al. (2006) The cnidarian-bilaterian ancestor possessed at least 56 homeoboxes: evidence from the starlet sea anemone, Nematostella vectensis. Genome Biol. 7, R64 6 Kamm, K. and Schierwater, B. (2006) Ancient complexity of the nonHox ANTP gene complement in the anthozoan Nematostella vectensis. Implications for the evolution of the ANTP superclass. J. Exp. Zool. B Mol. Dev. Evol. 306, 589–596 7 Ferrier, D.E.K. and Minguillo´n, C. (2003) Evolution of the Hox/ ParaHox gene clusters. Int. J. Dev. Biol. 47, 605–611 8 Monteiro, A.S. and Ferrier, D.E.K. (2006) Hox genes are not always collinear. Int. J. Biol. Sci. 2, 95–103 9 Seo, H.C. et al. (2004) Hox cluster disintegration with persistent anteroposterior order of expression in Oikopleura dioica. Nature 431, 67–71 10 Jagla, K. et al. (2001) A cluster of Drosophila homeobox genes involved in mesoderm differentiation programs. Bioessays 23, 125–133 261
Update 11 Luke, G.N. et al. (2003) Dispersal of NK homeobox gene clusters in amphioxus and humans. Proc. Natl. Acad. Sci. U. S. A. 100, 5292–5295 12 Coulier, F. et al. (2000) MetaHox gene clusters. J. Exp. Zool. B Mol. Dev. Evol. 288, 345–351 13 Putnam, N.H. et al. The amphioxus genome and the evolution of the chordate karyotype. (in press) 14 Holland, L.Z. et al. The amphioxus genome illuminates vertebrate origins and cephalochordate biology. (in press) 15 Richards, S. et al. The first genome sequence of a beetle, Tribolium castaneum, a model for insect development and pest biology. Nature (in press) 16 Bourlat, S.J. et al. (2006) Deuterostome phylogeny reveals monophyletic chordates and the new phylum Xenoturbellida. Nature 444, 85–88 17 Garcia-Ferna`ndez, J. and Holland, P.W.H. (1994) Archetypal organisation of the amphioxus Hox gene cluster. Nature 370, 563–566 18 Ferrier, D.E.K. et al. (2000) The amphioxus Hox cluster: deuterostome posterior flexibility and Hox14. Evol. Dev. 2, 284–293 19 Brooke, N.M. et al. (1998) The ParaHox gene cluster is an evolutionary sister of the Hox gene cluster. Nature 392, 920–922 20 Ferrier, D.E.K. and Holland, P.W.H. (2002) Ciona intestinalis ParaHox genes: evolution of Hox/ParaHox cluster integrity, developmental mode, and temporal colinearity. Mol. Phylogenet. Evol. 24, 412– 417 21 Chourrout, D. et al. (2006) Minimal ProtoHox cluster inferred from bilaterian and cnidarian Hox complements. Nature 442, 684–687
Trends in Genetics Vol.24 No.6 22 Castro, L.F.C. and Holland, P.W.H. (2003) Chromosomal mapping of ANTP class homeobox genes in amphioxus: piecing together ancestral genomes. Evol. Dev. 5, 459–465 23 Ferrier, D.E.K. et al. (2001) Amphioxus Evx genes: implications for the evolution of the midbrain-hindbrain boundary and the chordate tailbud. Dev. Biol. 237, 270–281 24 Minguillo´n, C. and Garcia-Ferna`ndez, J. (2003) Genesis and evolution of the Evx and Mox genes and the extended Hox and ParaHox gene clusters. Genome Biol. 4, R12 25 Brown, S.J. et al. (2002) Sequence of the Tribolium castaneum homeotic complex: the region corresponding to the Drosophila melanogaster antennapedia complex. Genetics 160, 1067–1074 26 Balavoine, G. et al. (2002) Hox clusters and bilaterian phylogeny. Mol. Phylogenet. Evol. 24, 366–373 27 De Robertis, E.M. and Sasai, Y. (1996) A common plan for dorsoventral patterning in Bilateria. Nature 380, 37–40 28 Bagun˜a, J. and Riutort, M. (2004) The dawn of bilaterian animals: the case of acoelomorph flatworms. Bioessays 26, 1046–1057 29 Philippe, H. et al. (2007) Acoel flatworms are not Platyhelminthes: evidence from phylogenomics. PLoS ONE 8, e717 30 Jime´nez-Guri, E. et al. (2006) Hox and ParaHox genes in Nemertodermatida, a basal bilaterian clade. Int. J. Dev. Biol. 50, 675–679 0168-9525/$ – see front matter . ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2007.09.006 Available online 9 May 2008
Genome Analysis
Mutation of miRNA target sequences during human evolution Paul P. Gardner1 and Jeppe Vinther2 1
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton CB10 1SA, UK Molecular Evolution Group, Department of Biology, University of Copenhagen, Copenhagen Biocenter, DK-2200 Copenhagen N, Denmark
2
It has long-been hypothesized that changes in non–protein-coding genes and the regulatory sequences controlling expression could undergo positive selection. Here we identify 402 putative microRNA (miRNA) target sequences that have been mutated specifically in the human lineage and show that genes containing such deletions are more highly expressed than their mouse orthologs. Our findings indicate that some miRNA target mutations are fixed by positive selection and might have been involved in the evolution of human-specific traits.
Selection on microRNA target sequences The microRNA (miRNA) gene family is a highly conserved and important part of gene regulation in animals and plants [1]. The miRNAs are short (22 bases long) RNA molecules, which are incorporated into a regulatory protein complex. Here, the miRNAs determine the binding specificity of the complex by hybridisation to partially complementary sequences, primarily in the 30 untranslated region (UTR) of mRNAs. The majority of functional animal miRNA target sequence requires perfect base pairCorresponding author: Vinther, J. ([email protected]).
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ing between bases 2–7 of the miRNA and the target sequence, accompanied by an adenine in position 1 of the target or an additional base pair between the target and position 8 of the miRNA [2–4]. When recruited to a 30 UTR, the miRNA complex destabilizes the mRNA and reduces translation [5,6] by mechanisms that are still not completely understood [7,8]. In humans (Homo sapiens), about one third of all protein-coding genes contain conserved target sequences for the 163 miRNA families that are conserved between humans and dogs [2,9,10]. These miRNA target sequences have been under strong purifying selection and are, on average, 3.5 times more conserved than control sequences [2] and depleted in single nucleotide polymorphisms (SNPs) [11]. Moreover, the 30 UTRs of mRNAs co-expressed with tissue-specific miRNAs are depleted in target sequences of those miRNAs, indicating that a significant proportion of the miRNA target sequences resulting from mutations in the 30 UTRs are selected against [12,13]. In contrast to transcription factors, which have degenerate binding specificity, miRNA target sequences have strict sequence requirements to be functional (i.e. presence of a specific 7mer sequence). It therefore requires only a single mutation to inactivate a miRNA target sequence. In
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10 Xu, N. et al. (2006) Transient homologous chromosome pairing marks the onset of X inactivation. Science 311, 1149–1152 11 Zhao, Z. et al. (2006) Circular chromosome conformation capture (4C) uncovers extensive networks of epigenetically regulated intra- and interchromosomal interactions. Nat. Genet. 38, 1341–1347 12 Gimelbrant, A.A. et al. (2005) Monoallelic expression and asynchronous replication of p120 catenin in mouse and human cells. J. Biol. Chem. 280, 1354–1359 13 Mostoslavsky, R. et al. (2001) Asynchronous replication and allelic exclusion in the immune system. Nature 414, 221–225 14 Ensminger, A.W. and Chess, A. (2004) Coordinated replication timing of monoallelically expressed genes along human autosomes. Hum. Mol. Genet. 13, 651–658 15 Singh, N. et al. (2003) Coordination of the random asynchronous replication of autosomal loci. Nat. Genet. 33, 339–341 0168-9525/$ – see front matter . ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tig.2008.03.005 Available online 29 April 2008
Genome Analysis
The Urbilaterian Super-Hox cluster Thomas Butts1, Peter W.H. Holland1 and David E.K. Ferrier1,2 1 2
Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK Present address: The Gatty Marine Laboratory, University of St Andrews, St Andrews, Fife, KY16 8LB, UK
Comparison of whole genome sequences of representative animals enables reconstruction of the ancestral bilaterian genome: the starting point from which most extant animal lineages evolved. The Hox gene cluster patterns the anterior–posterior axis of bilaterians. Here we show that this cluster was embedded within a larger homeobox gene cluster, the Super-Hox cluster, in the ancestral bilaterian. This Super-Hox cluster contained at least eight genes alongside the core Hox genes (‘EuHox’ genes).
Ancient homeobox gene neighbours The Bilateria represent 99% of all described living animal species, and as such, reconstructing the biology of their last common ancestor, the Urbilateria, is of fundamental importance for understanding animal evolution. In the postgenomic era, this reconstruction has begun to be extended to the urbilaterian genome (e.g. [1]). The homeobox genes are particularly well suited for reconstruction of ancestral genomes because of their widespread conservation and robust family level phylogeny. Their diversity arose predominantly by tandem duplication, which can create functional clusters of genes, such as the Hox cluster, or can survive as relics of the ancestral organization simply because of a lack of extensive genomic rearrangement. Here we take the existence of such evolutionarily related genes that are also close neighbours on the chromosome, such as the Antennapedia-class (ANTP-class) homeobox genes, to represent such relics of the ancestral genome organization, assuming this to be much more likely than a secondary Corresponding author: Ferrier, D.E.K. ([email protected]).
reassociation of these genes on independent evolutionary lineages. The ANTP-class is the largest class of the homeobox superfamily, and is central to the evolution of animal development. In addition to the Hox genes, it comprises several other gene families whose members are sometimes found in small gene clusters in animal genomes. This propensity for gene clustering, and the organization of gene cluster members in vertebrate genomes, have led to the hypothesis of an ancestral ‘Mega-cluster’ deep in animal evolutionary history [2,3]. The ANTP-class originated before the radiation of the Metazoa [4]; recent data from the genome of the starlet sea anemone Nematostella vectensis have shown that most extant families within this class were present, by inference in a Mega-cluster, before the divergence of the cnidarian and bilaterian lineages [5,6]. Chromosomal rearrangement and breakage subsequently began to break up this Mega-cluster and disperse its constituent genes. By contrast, the Hox and NK cluster genes have been conserved in tight genomic clusters in some extant genomes. It has been suggested that temporal colinearity – a process whereby the progressive activation of the genes along the anterior–posterior axis during development is mirrored by their genomic position within the cluster – has been the major constraining force on the organization of the Hox gene cluster [7,8], because these clusters have disintegrated in animals with rapid or highly determinative development (e.g. [9]). The function of the NK cluster is less well studied than its Hox counterpart, although all of the constituent genes are involved in mesoderm patterning and differentiation [10]. Nevertheless, a clusterwide phenomenon that explains their conservation in 259
Update Glossary Bilaterian: An animal that has a single plane of symmetry along its anteriorposterior axis (bilateral symmetry). Deuterostome: An animal in the group Deuterostomia, in which the mouth/ stomodaeum arises by a secondary invagination during embryogenesis rather than from the blastopore. Includes Echinodermata, Hemichordata, Xenoturbellida and Chordata [16]. Homeobox: A DNA motif, usually of 180 bp, that encodes a distinctive sequence-specific DNA binding domain (the homeodomain) with three a helices, two of which are in a Helix-turn-Helix conformation. Hox gene: A gene that is orthologous to the homeobox genes of the Drosophila and mammalian Hox gene clusters, which pattern the anterior– posterior axes of bilaterian animals. Metazoa: Synonymous with the kingdom Animalia (multicellular, heterotrophic animals). Protostome: An animal in the group Protostomia, the second great bilaterian clade, in which generally the mouth/stomodaeum forms from the blastopore. Urbilateria: The last common ancestor of the bilaterians [27], originally taken to be the last common ancestor of the Protostomia and Deuterostomia. However, its precise meaning is dependent on the phylogenetic position of the acoelomorph flatworms, which have been suggested to be basal bilaterians [28], although this is still debated [29]. In the present context, because the genes of the Mega-cluster existed well before the last common ancestor of all bilaterians, the precise position of the acoelomorph flatworms is irrelevant and hence we retain the use of the term Urbilateria. The acoelomorph problem does mean, however, that the number of genes in the Hox cluster of Urbilateria is not completely resolved, and the seven used here comes from protostomedeuterostome comparisons [26], but may have been fewer if the low number of Hox genes presently isolated from Acoelomorpha [28,30] is an accurate representation of the ancestral bilaterian state.
insects and their breakage in chordates [11] is elusive at present. In addition to these extant gene clusters, the remains of the ancestral Mega-cluster organization have been revealed in genomic comparisons of human, mouse and Drosophila [2,12]; however, the extent of clustering that remained in the last common ancestor of the Bilateria (the Urbilaterian) is not clear. We have analysed the content and organization of the ANTP-class homeobox genes of the Florida lancelet and red flour beetle. These species have both retained a greater extent of ancient genomic organization than is present in other sequenced bilaterian genomes, enabling us to reconstruct the homeobox gene cluster that existed around the Hox cluster in the Urbilaterian. Amphioxus clustering When reconstructing genomic evolutionary history, animals that exhibit low levels of genomic rearrangement will be of primary importance. From our analysis presented here and in other studies [13–15], it seems that the Florida lancelet, Branchiostoma floridae (also known as amphioxus), and the red flour beetle, Tribolium castaneum, will both prove fruitful reference points in this endeavour. Amphioxus has for a long time cast light on the events that surrounded the invertebrate–vertebrate transition. Despite its recent repositioning as the basal chordate lineage rather than as the sister taxon to vertebrates [16], it retains many aspects of ancestral chordate genomic organization that have been lost in the urochordates, the true invertebrate sister group of the vertebrates, such as intact Hox [9,14,17,18] and ParaHox [19,20] clusters. In addition, the remains of the NK cluster in amphioxus resemble the vertebrate pre–whole genome duplication condition [11], but with the additional retention of NK6 and NK7 in a cluster next to the Lbx–Tlx gene pair. 260
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In addition to the prototypical Hox and ParaHox clusters and NK cluster remnants, our analysis shows amphioxus possesses two additional examples of clustering (Figure 1). First, the amphioxus genes Mnxa and Rough are separated by an intergenic distance of only 20 kb. Second, a novel cluster of homeobox genes is present that consists of four genes, Engrailed, Nedxa, Nedxb and Dlx, within 135 kb [13]. The Nedx (Next to Distalless) family is widespread across bilaterians (its Drosophila ortholog is CG13424) but has been lost from vertebrates. It is notable that these novel examples of clustering are both present on the same chromosome as the Hox cluster, which, as in Cnidaria, is flanked by Evx [21,22] (although, in amphioxus, there are two Evx genes [23]). In contrast to the situation in vertebrate genomes, and contrary to previous models [24], Mox is present further upstream from the posterior end of the Hox cluster (220 kb from AmphiEvxB). Tribolium clustering In the recently sequenced Tribolium genome [15], further evidence of homeobox gene clustering was found in addition to the intact Hox [25] and NK clusters in this animal. On the X chromosome, the single copy genes Btn (the ortholog of chordate Mox genes), Rough and Hex are clustered within 80 kb. In addition, the genome of Tribolium contains a further uncharacterized homeobox gene cluster consisting of the genes Exex (the ortholog of Mnx), Nedx, Dll and Eve. This cluster spans 190 kb on chromosome 7. The Dlx–Nedx gene pair is distinctive because it is also present in amphioxus, suggesting a selective pressure for its retention. Such a pressure is most likely to have arisen because of enhancer sharing, whereby a nearby cis element regulates both genes. Rebuilding Urbilateria Combining the genomic data on novel homeobox gene clusters from both amphioxus and the red flour beetle, it was possible for us to reconstruct the extent of homeobox gene clustering at the Hox locus in the Urbilaterian (Figure 1). From our reconstruction, it can be seen that, as well as the canonical Hox genes (the ‘EuHox’ genes), eight other ANTP-class genes were clustered at the Urbilaterian Hox locus. This is in addition to the minimum number of seven EuHox genes in the Urbilaterian [26] (however, see ‘Urbilateria’ in the Glossary). Together these genes comprised the ‘Super-Hox’ cluster, which contained at least 15 genes in the last common ancestor of the bilaterians. Given the frequency of genome rearrangements and especially inversions, even in slowly evolving genomes, it is currently impossible to predict the ancestral order of the genes at this locus, other than in reference to the canonical Hox genes, where selection acts to maintain gene order and constrain the overall clustered organization. With the possible exception of the Dlx–Nedx gene pair, the remnants of the SuperHox cluster in amphioxus and beetles are likely to be simply the products of evolutionary inertia dating from the origin of these families >550 million years ago (Mya).
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Figure 1. The Urbilaterian Super-Hox cluster and its remnants. The gene order shown is not necessarily indicative of ancestral arrangement, which is impossible to infer at present because of the lack of functional selection for gene order, coupled with the frequency of inversions compared with other types of genomic rearrangement. In amphioxus, the gene encoding Nedx has been duplicated; in beetles, engrailed has duplicated. The Hox cluster (11 genes in beetles and 15 in amphioxus) is shown as a single hatched box that represents multiple genes (the ‘EuHox’ genes). LG , linkage group.; *, linked to the remains of the NK cluster. Note the linkage is not drawn to scale. All intergenic distances are <80 kb. The bold diagonal lines represent intergenic gaps >1 Mb; standard lines represent a gap of <250 kb.
Likewise, with evidence from beetle and amphioxus to supplement genomic information from other model organisms, the NK cluster of Urbilateria can be inferred to have contained nine genes: Msx, NK4, NK3, Lbx, Tlx, NK1, NK5, NK6 and NK7 [3,11,13]. Concluding remarks The ancestral Antennapedia (ANTP) Mega-cluster, which existed before the Cnidarian-Bilaterian Ancestor, did not break up as quickly as has previously been imagined, with only selectively maintained gene clusters (the Hox and ParaHox clusters and possibly the NK cluster) being retained. Instead, the break-up of the Mega-cluster occurred gradually and independently among extant animal lineages. Certain genomes (e.g. amphioxus and the red flour beetle) are less derived from the urbilaterian condition than others. Comparison of the organization of ANTP-class genes in these two genomes reveals the evolutionary history of ANTP-class gene clustering in unprecedented detail. Of the ancestral homeobox Mega-cluster, at least 24 genes existed as two clusters in the urbilaterian: the 9-gene NK cluster (see above) and the 15+ gene Super-Hox cluster (Figure 1). It is not clear from the beetle and amphioxus comparison whether the SuperHox and NK clusters were linked in the urbilaterian, but further genome sequences from other less derived organisms could resolve this. Also, at this point, it must be stressed that 15 actually represents a lower limit for the number of genes in the Super-Hox cluster and is based on the falsifiable assumption that no further instances of novel clus-
tering will be found in as yet unsequenced bilaterian genomes. Acknowledgements The authors thank Dan Rokhsar and the Joint Genome Institute for coordinating the amphioxus genome project and Stephen Richards and the sequencing centre at the Baylor College of Medicine for coordinating the Tribolium genome project. Work in the authors’ laboratories is funded by the BBSRC and the Wellcome Trust.
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Genome Analysis
Mutation of miRNA target sequences during human evolution Paul P. Gardner1 and Jeppe Vinther2 1
Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton CB10 1SA, UK Molecular Evolution Group, Department of Biology, University of Copenhagen, Copenhagen Biocenter, DK-2200 Copenhagen N, Denmark
2
It has long-been hypothesized that changes in non–protein-coding genes and the regulatory sequences controlling expression could undergo positive selection. Here we identify 402 putative microRNA (miRNA) target sequences that have been mutated specifically in the human lineage and show that genes containing such deletions are more highly expressed than their mouse orthologs. Our findings indicate that some miRNA target mutations are fixed by positive selection and might have been involved in the evolution of human-specific traits.
Selection on microRNA target sequences The microRNA (miRNA) gene family is a highly conserved and important part of gene regulation in animals and plants [1]. The miRNAs are short (22 bases long) RNA molecules, which are incorporated into a regulatory protein complex. Here, the miRNAs determine the binding specificity of the complex by hybridisation to partially complementary sequences, primarily in the 30 untranslated region (UTR) of mRNAs. The majority of functional animal miRNA target sequence requires perfect base pairCorresponding author: Vinther, J. ([email protected]).
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ing between bases 2–7 of the miRNA and the target sequence, accompanied by an adenine in position 1 of the target or an additional base pair between the target and position 8 of the miRNA [2–4]. When recruited to a 30 UTR, the miRNA complex destabilizes the mRNA and reduces translation [5,6] by mechanisms that are still not completely understood [7,8]. In humans (Homo sapiens), about one third of all protein-coding genes contain conserved target sequences for the 163 miRNA families that are conserved between humans and dogs [2,9,10]. These miRNA target sequences have been under strong purifying selection and are, on average, 3.5 times more conserved than control sequences [2] and depleted in single nucleotide polymorphisms (SNPs) [11]. Moreover, the 30 UTRs of mRNAs co-expressed with tissue-specific miRNAs are depleted in target sequences of those miRNAs, indicating that a significant proportion of the miRNA target sequences resulting from mutations in the 30 UTRs are selected against [12,13]. In contrast to transcription factors, which have degenerate binding specificity, miRNA target sequences have strict sequence requirements to be functional (i.e. presence of a specific 7mer sequence). It therefore requires only a single mutation to inactivate a miRNA target sequence. In