Cephalochordates, the sister group of vertebrates + tunicates, are evolving particularly slowly. Therefore, genome comparisons between two congeners of Branchiostoma revealed so many conserved noncoding elements (CNEs), that it was not clear how many are functional regulatory elements. To more effectively identify CNEs with potential regulatory functions, we compared noncoding sequences of genomes of the most phylogenetically distant cephalochordate genera, Asymmetron and Branchiostoma, which diverged approximately 120-160 million years ago. We found 113,070 noncoding elements conserved between the two species, amounting to 3.3% of the genome. The genomic distribution, target gene ontology, and enriched motifs of these CNEs all suggest that many of them are probably cis-regulatory elements. More than 90% of previously verified amphioxus regulatory elements were re-captured in this study. A search of the cephalochordate CNEs around 50 developmental genes in several vertebrate genomes revealed eight CNEs conserved between cephalochordates and vertebrates, indicating sequence conservation over >500 million years of divergence. The function of five CNEs was tested in reporter assays in zebrafish, and one was also tested in amphioxus. All five CNEs proved to be tissue-specific enhancers. Taken together, these findings indicate that even though Branchiostoma and Asymmetron are distantly related, as they are evolving slowly, comparisons between them are likely optimal for identifying most of their tissue-specific cis-regulatory elements laying the foundation for functional characterizations and a better understanding of the evolution of developmental regulation in cephalochordates.

Biosciences at Rice Rice University Houston Texas Present address Dovetail Genomics Santa Cruz CA 95060

Biosciences at Rice Rice University Houston Texas Present address Gene by Gene Ltd Houston TX 77008

Biosciences at Rice Rice University Houston Texas Present address Institute for Research on Cancer and Aging Nice CNRS UMR 7284 INSERM U1081 Nice 06107 France

Department of Transcriptional Regulation Institute of Molecular Genetics Prague 14220 Czech Republic

Institute of Cellular and Organismic Biology Academia Sinica Taipei Taiwan

Marine Biology Research Division Scripps Institution of Oceanography UC San Diego La Jolla California

See more in PubMed

Acemel RD, et al. 2016. A single three-dimensional chromatin compartment in amphioxus indicates a stepwise evolution of vertebrate Hox bimodal regulation. Nat Genet. 48:336–341. PubMed

Akimenko MA, Johnson SL, Westerfield M, Ekker M. 1995. Differential induction of four msx homeobox genes during fin development and regeneration in zebrafish. Development 121:347–357. PubMed

Anwar S, et al. 2015. Identification and functional characterization of novel transcriptional enhancers involved in regulating human GLI3 expression during early development. Dev Growth Differ. 57:570–580. PubMed PMC

Beaster-Jones L, Schubert M, Holland LZ. 2007. Cis-regulation of the amphioxus engrailed gene: insights into evolution of a muscle-specific enhancer. Mech Dev. 124:532–542. PubMed

Bejerano G, et al. 2004. Ultraconserved elements in the human genome. Science 304:1321–1325. PubMed

Bendall AJ, Ding J, Hu G, Shen MM, Abate-Shen C. 1999. Msx1 antagonizes the myogenic activity of Pax3 in migrating limb muscle precursors. Development 126:4965–4976. PubMed

Benjamini Y, Hochberg Y. 1995. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B. 57:289–300.

Bessa J, et al. 2009. Zebrafish enhancer detection (ZED) vector: a new tool to facilitate transgenesis and the functional analysis of cis-regulatory regions in zebrafish. Dev Dyn. 238:2409–2417. PubMed

Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. PubMed PMC

Braasch I, et al. 2016. The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons. Nat Genet. 48:427–437. PubMed PMC

Bradley RK, et al. 2009. Fast statistical alignment. PLoS Comput Biol. 5:e1000392.. PubMed PMC

Britten RJ. 1997. Mobile elements inserted in the distant past have taken on important functions. Gene 205:177–182. PubMed

Buenrostro JD, Giresi PG, Zaba LC, Chang HY, Greenleaf WJ. 2013. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods 10:1213–1218. PubMed PMC

Candia AF, Wright CV. 1995. The expression pattern of Xenopus Mox-2 implies a role in initial mesodermal differentiation. Mech Dev. 52:27–36. PubMed

Clarke SL, et al. 2012. Human Developmental enhancers conserved between deuterostomes and protostomes. PLoS Genet. 8:e1002852.. PubMed PMC

Conesa A, et al. 2005. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676. PubMed

Corbo JC, Levine M, Zeller RW. 1997. Characterization of a notochord-specific enhancer from the Brachyury promoter region of the ascidian, Ciona intestinalis. Development 124:589–602. PubMed

Davidson EH. 2011. Evolutionary bioscience as regulatory systems biology. Dev Biol. 357:35–40. PubMed PMC

Dayal S, et al. 2004. Creation of cis-regulatory elements during sea urchin evolution by co-option and optimization of a repetitive sequence adjacent to the spec2a gene. Dev Biol. 273:436–453. PubMed

Dickmeis T, et al. 2001. Expression of the anti-dorsalizing morphogenetic protein gene in the zebrafish embryo. Dev Genes Evol. 211:568–572. PubMed

Dogan N, et al. 2015. Occupancy by key transcription factors is a more accurate predictor of enhancer activity than histone modifications or chromatin accessibility. Epigenetics Chromatin 8:16.. PubMed PMC

Doglio L, et al. 2013. Parallel evolution of chordate cis-regulatory code for development. PLoS Genet. 9:e1003904.. PubMed PMC

Fatica A, Bozzoni I. 2014. Long non-coding RNAs: new players in cell differentiation and development. Nat Rev Genet. 15:7–21. PubMed

Fidler AL, et al. 2014. A unique covalent bond in basement membrane is a primordial innovation for tissue evolution. Proc Natl Acad Sci U S A. 111:331–336. PubMed PMC

Fisher R. 1922. On the interpretation of χ2 from contingency tables, and the calculation of P. J R Stat Soc. 85:87–94.

Frazer KA, Pachter L, Poliakov A, Rubin EM, Dubchak I. 2004. VISTA: computational tools for comparative genomics. Nucleic Acids Res. 32:W273–W279. PubMed PMC

Furlong RF. 2005. Insights into vertebrate evolution from the chicken genome sequence. Genome Biol. 6:207.. PubMed PMC

Gardner PP, et al. 2011. Rfam: Wikipedia, clans and the “decimal” release. Nucleic Acids Res. 39:D141–D145. PubMed PMC

Grabherr MG, et al. 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 29:644–652. PubMed PMC

Guil S, Esteller M. 2012. Cis-acting noncoding RNAs: friends and foes. Nat Struct Mol Biol. 19:1068–1075. PubMed

Harris RS. 2007. Improved pairwise alignment of genomic DNA. Ph.D. dissertation, Pennsylvania State University.

Hausser J, Zavolan M. 2014. Identification and consequences of miRNA-target interactions - beyond repression of gene expression. Nat Rev Genet. 15:599–612. PubMed

Heinz S, et al. 2010. Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol Cell. 38:576–589. PubMed PMC

Hemberg M, et al. 2012. Integrated genome analysis suggests that most conserved non-coding sequences are regulatory factor binding sites. Nucleic Acids Res. 40:7858–7869. PubMed PMC

Hiller M, Schaar BT, Bejerano G. 2012. Hundreds of conserved non-coding genomic regions are independently lost in mammals. Nucleic Acids Res. 40:11463–11476. PubMed PMC

Holland LZ, et al. 2008. The amphioxus genome illuminates vertebrate origins and cephalochordate biology. Genome Res. 18:1100–1111. PubMed PMC

Holland LZ, Yu JK. 2004. Cephalochordate (amphioxus) embryos: procurement, culture, and basic methods. Methods Cell Biol. 74:195–215. PubMed

Holland ND, Holland LZ, Heimberg A. 2015. Hybrids between the Florida amphioxus (Branchiostoma floridae) and the Bahamas lancelet (Asymmetron lucayanum): developmental morphology and chromosome counts. Biol Bull. 228:13–24. PubMed

Holland PW, Koschorz B, Holland LZ, Herrmann BG. 1995. Conservation of Brachyury (T) genes in amphioxus and vertebrates: developmental and evolutionary implications. Development 121:4283–4291. PubMed

Huang S, et al. 2014. Decelerated genome evolution in modern vertebrates revealed by analysis of multiple lancelet genomes. Nat Commun. 5:5896.. PubMed PMC

Hufton AL, et al. 2009. Deeply conserved chordate noncoding sequences preserve genome synteny but do not drive gene duplicate retention. Genome Res. 19:2036–2051. PubMed PMC

Irimia M, et al. 2012a. Comparative genomics of the Hedgehog loci in chordates and the origins of Shh regulatory novelties. Sci Rep. 2:433.. PubMed PMC

Irimia M, et al. 2012b. Extensive conservation of ancient microsynteny across metazoans due to cis-regulatory constraints. Genome Res. 22:2356–2367. PubMed PMC

Irvine SQ. 2013. Study of cis-regulatory elements in the ascidian Ciona intestinalis. Curr Genomics. 14:56–67. PubMed PMC

Ishibashi M, Noda AO, Sakate R, Imanishi T. 2012. Evolutionary growth process of highly conserved sequences in vertebrate genomes. Gene 504:1–5. PubMed

Jagla T, et al. 1999. Plasticity within the lateral somatic mesoderm of Drosophila embryos. Int J Dev Biol. 43:571–573. PubMed

Kajitani R, et al. 2014. Efficient de novo assembly of highly heterozygous genomes from whole-genome shotgun short reads. Genome Res. 24:1384–1395. PubMed PMC

Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 30:772–780. PubMed PMC

Kawakami K, et al. 2004. A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev Cell 7:133–144. PubMed

Kon T, et al. 2007. Phylogenetic position of a whale-fall lancelet (Cephalochordata) inferred from whole mitochondrial genome sequences. BMC Evol Biol. 7:127.. PubMed PMC

Kozomara A, Griffiths-Jones S. 2013. miRBase: annotating high confidence microRNAs using deep sequencing data. Nucleic Acids Res. 42:D68–D73. PubMed PMC

Lang M, et al. 2010. Conservation of shh cis-regulatory architecture of the coelacanth is consistent with its ancestral phylogenetic position. Evodevo 1:11.. PubMed PMC

Li H, et al. 2009. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25:2078–2079. PubMed PMC

Lunter G, Goodson M. 2011. Stampy: a statistical algorithm for sensitive and fast mapping of Illumina sequence reads. Genome Res. 21:936–939. PubMed PMC

Maeso I, Irimia M, Tena JJ, Casares F, Gómez-Skarmeta JL. 2013. Deep conservation of cis-regulatory elements in metazoans. Philos Trans R Soc Lond B Biol Sci. 368:20130020.. PubMed PMC

Maeso I, et al. 2012. An ancient genomic regulatory block conserved across bilaterians and its dismantling in tetrapods by retrogene replacement. Genome Res. 22:642–655. PubMed PMC

Maeso I, Tena JJ. 2016. Favorable genomic environments for cis-regulatory evolution: a novel theoretical framework. Semin Cell Dev Biol. 57:2–10. PubMed

Makunin IV, Kolesnikova TD, Andreyenkova NG. 2014. Underreplicated regions in D. melanogaster are enriched with fast evolving genes and highly conserved noncoding sequences. Genome Biol Evol. 6:2050–2060. PubMed PMC

Manzanares M, et al. 2000. Conservation and elaboration of Hox gene regulation during evolution of the vertebrate head. Nature 408:854–857. PubMed

Marçais G, Kingsford C. 2011. A fast, lock-free approach for efficient parallel counting of occurrences of k-mers. Bioinformatics 27:764–770. PubMed PMC

Martinez-Morales JR. 2015. Toward understanding the evolution of vertebrate gene regulatory networks: comparative genomics and epigenomic approaches. Brief Funct Genomics 15(4):315–321. PubMed

McEwen GK, et al. 2009. Early evolution of conserved regulatory sequences associated with development in vertebrates. PLoS Genet. 5:e1000762.. PubMed PMC

McKenna A, et al. 2010. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20:1297–1303. PubMed PMC

Meader S, Ponting CP, Lunter G. 2010. Massive turnover of functional sequence in human and other mammalian genomes. Genome Res. 20:1335–1343. PubMed PMC

Minguillón C, Garcia-Fernàndez J. 2002. The single amphioxus Mox gene: insights into the functional evolution of Mox genes, somites, and the asymmetry of amphioxus somitogenesis. Dev. Biol. 246:455–465. PubMed

Nawrocki EP, Kolbe DL, Eddy SR. 2009. Infernal 1.0: inference of RNA alignments. Bioinformatics 25:1335–1337. PubMed PMC

Nelson AC, Wardle FC. 2013. Conserved non-coding elements and cis regulation: actions speak louder than words. Development 140:1385–1395. PubMed

Nguyen PD, et al. 2014. Haematopoietic stem cell induction by somite-derived endothelial cells controlled by meox1. Nature 512:314–318. PubMed

Nohara M, Nishida M, Miya M, Nishikawa T. 2005. Evolution of the mitochondrial genome in cephalochordata as inferred from complete nucleotide sequences from two epigonichthys species. J Mol Evol. 60:526–537. PubMed

Ono H, Kozmik Z, Yu JK, Wada H. 2014. A novel N-terminal motif is responsible for the evolution of neural crest-specific gene-regulatory activity in vertebrate FoxD3. Dev Biol. 385:396–404. PubMed

Van Otterloo E, et al. 2012. Novel Tfap2-mediated control of soxE expression facilitated the evolutionary emergence of the neural crest. Development 139:720–730. PubMed PMC

Parker HJ, Sauka-Spengler T, Bronner M, Elgar G. 2014. A reporter assay in lamprey embryos reveals both functional conservation and elaboration of vertebrate enhancers. PLoS One 9:e85492.. PubMed PMC

Pascual-Anaya J, D’Aniello S, Garcia-Fernàndez J. 2008. Unexpectedly large number of conserved noncoding regions within the ancestral chordate Hox cluster. Dev Genes Evol. 218:591–597. PubMed

Patel RK, Jain M. 2012. NGS QC toolkit: a toolkit for quality control of next generation sequencing data. PLoS One 7:e30619.. PubMed PMC

Paten B, et al. 2011. Cactus: algorithms for genome multiple sequence alignment. Genome Res. 21:1512–1528. PubMed PMC

Pennacchio L, et al. 2006. In vivo enhancer analysis of human conserved non-coding sequences. Nature 444:499–502. PubMed

Putnam NH, et al. 2008. The amphioxus genome and the evolution of the chordate karyotype. Nature 453:1064–1071. PubMed

Quinlan AR, Hall IM. 2010. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26:841–842. PubMed PMC

Ramos C, Robert B. 2005. msh/Msx gene family in neural development. Trends Genet. 21:624–632. PubMed

Royo JL, et al. 2011. Transphyletic conservation of developmental regulatory state in animal evolution. Proc Natl Acad Sci U S A. 108:14186–14191. PubMed PMC

Russo MT, et al. 2004. Regulatory elements controlling Ci-msxb tissue-specific expression during Ciona intestinalis embryonic development. Dev Biol. 267:517–528. PubMed

Sanges R, et al. 2013. Highly conserved elements discovered in vertebrates are present in non-syntenic loci of tunicates, act as enhancers and can be transcribed during development. Nucleic Acids Res. 41:3600–3618. PubMed PMC

Saudemont A, et al. 2008. Complementary striped expression patterns of NK homeobox genes during segment formation in the annelid Platynereis. Dev Biol. 317:430–443. PubMed

Schmid KJ, Tautz D. 1997. A screen for fast evolving genes from Drosophila. Proc Natl Acad Sci U S A. 94:9746–9750. PubMed PMC

Schmieder R, Edwards R. 2011a. Fast identification and removal of sequence contamination from genomic and metagenomic datasets. PLoS One 6:e17288.. PubMed PMC

Schmieder R, Edwards R. 2011b. Quality control and preprocessing of metagenomic datasets. Bioinformatics 27:863–864. PubMed PMC

Schulte-Merker S, van Eeden FJ, Halpern ME, Kimmel CB, Nüsslein-Volhard C. 1994. no tail (ntl) is the zebrafish homologue of the mouse T (Brachyury) gene. Development 120:1009–1015. PubMed

Sharman AC, Shimeld SM, Holland PWH. 1999. An amphioxus Msx gene expressed predominantly in the dorsal neural tube. Dev Genes Evol. 209:260–263. PubMed

Simakov O, et al. 2015. Hemichordate genomes and deuterostome origins. Nature 527:459–465. PubMed PMC

Slater GSC, Birney E. 2005. Automated generation of heuristics for biological sequence comparison. BMC Bioinformatics 6:31.. PubMed PMC

Stanke M, Steinkamp R, Waack S, Morgenstern B. 2004. AUGUSTUS: a web server for gene finding in eukaryotes. Nucleic Acids Res. 32:W309–W312. PubMed PMC

Stergachis AB, et al. 2014. Conservation of trans-acting circuitry during mammalian regulatory evolution. Nature 515:365–370. PubMed PMC

The ENCODE Project Consortium 2007. Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project. Nature 447:799–816. PubMed PMC

Tsagkogeorga G, Turon X, Galtier N, Douzery EJP, Delsuc F. 2010. Accelerated evolutionary rate of housekeeping genes in tunicates. J Mol Evol. 71:153–167. PubMed

Valouev A, et al. 2008. Genome-wide analysis of transcription factor binding sites based on ChIP-Seq data. Nat Methods 5:829–834. PubMed PMC

Vavouri T, McEwen GK, Woolfe A, Gilks WR, Elgar G. 2006. Defining a genomic radius for long-range enhancer action: duplicated conserved non-coding elements hold the key. Trends Genet. 22:5–10. PubMed

Vavouri T, Walter K, Gilks WR, Lehner B, Elgar G. 2007. Parallel evolution of conserved non-coding elements that target a common set of developmental regulatory genes from worms to humans. Genome Biol. 8:R15.. PubMed PMC

Venkatesh B, et al. 2014. Elephant shark genome provides unique insights into gnathostome evolution. Nature 505:174–179. PubMed PMC

Villar D, et al. 2015. Enhancer evolution across 20 mammalian species. Cell 160:554–566. PubMed PMC

Wada H, Escriva H, Zhang S, Laudet V. 2006. Conserved RARE localization in amphioxus Hox clusters and implications for Hox code evolution in the vertebrate neural crest. Dev Dyn. 235:1522–1531. PubMed

Waterhouse AM, Procter JB, Martin DMA, Clamp M, Barton GJ. 2009. Jalview Version 2-A multiple sequence alignment editor and analysis workbench. Bioinformatics 25:1189–1191. PubMed PMC

Woolfe A, et al. 2005. Highly conserved non-coding sequences are associated with vertebrate development. PLoS Biol. 3:e7.. PubMed PMC

Yousaf A, Sohail Raza M, Ali Abbasi A. 2015. The evolution of bony vertebrate enhancers at odds with their coding sequence landscape. Genome Biol Evol. 7:2333–2343. PubMed PMC

Yu JK, Holland ND, Holland LZ. 2004. Tissue-specific expression of FoxD reporter constructs in amphioxus embryos. Dev Biol. 274:452–461. PubMed

Yue JX, Yu JK, Putnam NH, Holland LZ. 2014. The transcriptome of an amphioxus, Asymmetron lucayanum, from the Bahamas: a window into chordate evolution. Genome Biol Evol. 6:2681–2696. PubMed PMC

Molecular Fingerprint of Amphioxus Frontal Eye Illuminates the Evolution of Homologous Cell Types in the Chordate Retina