Alice Tay

Survey Sequencing and Comparative Analysis of the Elephant Shark (Callorhinchus milii) Genome

Owing to their phylogenetic position, cartilaginous fishes (sharks, rays, skates, and chimaeras) provide a critical reference for our understanding of vertebrate genome evolution. The relatively small genome of the elephant shark, Callorhinchus milii, a chimaera, makes it an attractive model cartilaginous fish genome for whole-genome sequencing and comparative analysis. Here, the authors describe survey sequencing (1.4× coverage) and comparative analysis of the elephant shark genome, one of the first cartilaginous fish genomes to be sequenced to this depth. Repetitive sequences, represented mainly by a novel family of short interspersed element–like and long interspersed element–like sequences, account for about 28% of the elephant shark genome. Fragments of approximately 15,000 elephant shark genes reveal specific examples of genes that have been lost differentially during the evolution of tetrapod and teleost fish lineages. Interestingly, the degree of conserved synteny and conserved sequences between the human and elephant shark genomes are higher than that between human and teleost fish genomes. Elephant shark contains putative four Hox clusters indicating that, unlike teleost fish genomes, the elephant shark genome has not experienced an additional whole-genome duplication. These findings underscore the importance of the elephant shark as a critical reference vertebrate genome for comparative analysis of the human and other vertebrate genomes. This study also demonstrates that a survey-sequencing approach can be applied productively for comparative analysis of distantly related vertebrate genomes.

Evidence for at least six Hox clusters in the Japanese lamprey (Lethenteron japonicum).

Significance Lampreys and hagfishes (cyclostomes) are the only living group of jawless vertebrates and therefore are important for the study of vertebrate evolution. We have characterized Hox clusters in the Japanese lamprey (Lethenteron japonicum), and shown that it contains at least six Hox clusters as compared with four Hox clusters in tetrapods. This suggests that the lamprey lineage has undergone an additional round of genome duplication compared with tetrapods. Several conserved noncoding elements (CNEs) were predicted in the Hox clusters of lamprey, elephant shark, and human. Transgenic assay of CNEs demonstrated their potential to function as cis-regulatory elements. Thus, these CNEs may represent part of the core set of cis-regulatory elements that were present in the common ancestor of vertebrates. Cyclostomes, comprising jawless vertebrates such as lampreys and hagfishes, are the sister group of living jawed vertebrates (gnathostomes) and hence an important group for understanding the origin and diversity of vertebrates. In vertebrates and other metazoans, Hox genes determine cell fate along the anteroposterior axis of embryos and are implicated in driving morphological diversity. Invertebrates contain a single Hox cluster (either intact or fragmented), whereas elephant shark, coelacanth, and tetrapods contain four Hox clusters owing to two rounds of whole-genome duplication (“1R” and “2R”) during early vertebrate evolution. By contrast, most teleost fishes contain up to eight Hox clusters because of an additional “teleost-specific” genome duplication event. By sequencing bacterial artificial chromosome (BAC) clones and the whole genome, here we provide evidence for at least six Hox clusters in the Japanese lamprey (Lethenteron japonicum). This suggests that the lamprey lineage has experienced an additional genome duplication after 1R and 2R. The relative age of lamprey and human paralogs supports this hypothesis. Compared with gnathostome Hox clusters, lamprey Hox clusters are unusually large. Several conserved noncoding elements (CNEs) were predicted in the Hox clusters of lamprey, elephant shark, and human. Transgenic zebrafish assay indicated the potential of CNEs to function as enhancers. Interestingly, CNEs in individual lamprey Hox clusters are frequently conserved in multiple Hox clusters in elephant shark and human, implying a many-to-many orthology relationship between lamprey and gnathostome Hox clusters. Such a relationship suggests that the first two rounds of genome duplication may have occurred independently in the lamprey and gnathostome lineages.

Ancient noncoding elements conserved in the human genome.

Cartilaginous fishes represent the living group of jawed vertebrates that diverged from the common ancestor of human and teleost fish lineages about 530 million years ago. We generated ~1.4× genome sequence coverage for a cartilaginous fish, the elephant shark (Callorhinchus milii), and compared this genome with the human genome to identify conserved noncoding elements (CNEs). The elephant shark sequence revealed twice as many CNEs as were identified by whole-genome comparisons between teleost fishes and human. The ancient vertebrate-specific CNEs in the elephant shark and human genomes are likely to play key regulatory roles in vertebrate gene expression.

Integration of the Genetic Map and Genome Assembly of Fugu Facilitates Insights into Distinct Features of Genome Evolution in Teleosts and Mammals

Abstract The compact genome of fugu (Takifugu rubripes) has been used widely as a reference genome for understanding the evolution of vertebrate genomes. However, the fragmented nature of the fugu genome assembly has restricted its use for comparisons of genome architecture in vertebrates. To extend the contiguity of the assembly to the chromosomal level, we have generated a comprehensive genetic map of fugu and anchored the scaffolds of the assembly to the 22 chromosomes of fugu. The map consists of 1,220 microsatellite markers that provide anchor points to 697 scaffolds covering 86% of the genome assembly (http://www.fugu-sg.org/). The integrated genome map revealed a higher recombination rate in fugu compared with other vertebrates and a wide variation in the recombination rate between sexes and across chromosomes of fugu. We used the extended assembly to explore recent rearrangement events in the lineages of fugu, Tetraodon, and medaka and compared them with rearrangements in three mammalian (human, mouse, and opossum) lineages. Between the two pufferfishes, fugu has experienced fewer chromosomal rearrangements than Tetraodon. The gene order is more highly conserved in the three teleosts than in mammals largely due to a lower rate of interchromosomal rearrangements in the teleosts. These results provide new insights into the distinct patterns of genome evolution between teleosts and mammals. The consolidated genome map and the genetic map of fugu are valuable resources for comparative genomics of vertebrates and for elucidating the genetic basis of the phenotypic diversity of ∼25 species of Takifugu that evolved within the last 5 My.

Elephant shark (Callorhinchus milii) provides insights into the evolution of Hox gene clusters in gnathostomes

We have sequenced and analyzed Hox gene clusters from elephant shark, a holocephalian cartilaginous fish. Elephant shark possesses 4 Hox clusters with 45 Hox genes that include orthologs for a higher number of ancient gnathostome Hox genes than the 4 clusters in tetrapods and the supernumerary clusters in teleost fishes. Phylogenetic analysis of elephant shark Hox genes from 7 paralogous groups that contain all of the 4 members indicated an ((AB)(CD)) topology for the order of Hox cluster duplication, providing support for the 2R hypothesis (i.e., 2 rounds of whole-genome duplication during the early evolution of vertebrates). Comparisons of noncoding sequences of the elephant shark and human Hox clusters have identified a large number of conserved noncoding elements (CNEs), which represent putative cis-regulatory elements that may be involved in the regulation of Hox genes. Interestingly, in fugu more than 50% of these ancient CNEs have diverged beyond recognition in the duplicated (HoxA, HoxB, and HoxD) as well as the singleton (HoxC) Hox clusters. Furthermore, the b-paralogs of the duplicated fugu Hox clusters are virtually devoid of unique ancient CNEs. In contrast to fugu Hox clusters, elephant shark and human Hox clusters have lost fewer ancient CNEs. If these ancient CNEs are indeed enhancers directing tissue-specific expression of Hox genes, divergence of their sequences in vertebrate lineages might have led to altered expression patterns and presumably the functions of their associated Hox genes.

Conserved regulation of the lymphocyte-specific expression of lck in the Fugu and mammals

The lck gene encodes a lymphocyte-specific protein-tyrosine kinase that is implicated in T cell maturation and signaling. In mammals, the transcription of the lck gene is regulated by two independent promoters, the proximal promoter, which is active in thymocytes, and the distal promoter, which dominates in mature T cells. In the human and mouse lck gene loci, the two promoter elements are separated by at least 40 kb and 10 kb, respectively. In this study, we have cloned and sequenced 60 kb from the pufferfish (Fugu rubripes) lck locus. The promoter region of the Fugu lck spans only 4.2 kb and contains a proximal and a distal promoter in the 2.3-kb region adjacent to the coding sequence. By generating transgenic mice, we have demonstrated that the compact promoter of the Fugu lck contains regulatory elements that direct expression to lymphoid organs of mice. We were able to localize the regulatory elements to a short region of 830 bp without losing specificity to cultured human T cell line. These results show that the basic mechanisms that mediate lymphocyte-specific expression are conserved between teleosts and mammals. The short promoter of the Fugu lck isolated by us offers a powerful tool for labeling T cells, targeting expression, and manipulating T cell activity in fishes as well as in mammals.

STAT4 is a target of the hematopoietic zinc-finger transcription factor Ikaros in T cells

STAT4 is a transcription factor activated in response to IL‐12, and is involved in Th1 cell development. The molecular mechanisms controlling the transcription of the STAT4 gene are however, unclear. Sequence comparison of the 5′ flanking regions of human, mouse and pufferfish (Fugu rubripes) Stat4 genes revealed a high frequency of Ikaros (Ik) binding elements in all three species. We then investigated the role of Ik binding elements in the human STAT4 promoter using Jurkat T cells. Transactivation, electrophoretic mobility shift assay and RNA interference‐mediated gene knockdown experiments revealed that Ik is involved in the regulation of STAT4 in human T cells.

Sequencing of Pax6 loci from the elephant shark reveals a family of Pax6 genes in vertebrate genomes, forged by ancient duplications and divergences

Pax6 is a developmental control gene essential for eye development throughout the animal kingdom. In addition, Pax6 plays key roles in other parts of the CNS, olfactory system, and pancreas. In mammals a single Pax6 gene encoding multiple isoforms delivers these pleiotropic functions. Here we provide evidence that the genomes of many other vertebrate species contain multiple Pax6 loci. We sequenced Pax6-containing BACs from the cartilaginous elephant shark (Callorhinchus milii) and found two distinct Pax6 loci. Pax6.1 is highly similar to mammalian Pax6, while Pax6.2 encodes a paired-less Pax6. Using synteny relationships, we identify homologs of this novel paired-less Pax6.2 gene in lizard and in frog, as well as in zebrafish and in other teleosts. In zebrafish two full-length Pax6 duplicates were known previously, originating from the fish-specific genome duplication (FSGD) and expressed in divergent patterns due to paralog-specific loss of cis-elements. We show that teleosts other than zebrafish also maintain duplicate full-length Pax6 loci, but differences in gene and regulatory domain structure suggest that these Pax6 paralogs originate from a more ancient duplication event and are hence renamed as Pax6.3. Sequence comparisons between mammalian and elephant shark Pax6.1 loci highlight the presence of short- and long-range conserved noncoding elements (CNEs). Functional analysis demonstrates the ancient role of long-range enhancers for Pax6 transcription. We show that the paired-less Pax6.2 ortholog in zebrafish is expressed specifically in the developing retina. Transgenic analysis of elephant shark and zebrafish Pax6.2 CNEs with homology to the mouse NRE/Pα internal promoter revealed highly specific retinal expression. Finally, morpholino depletion of zebrafish Pax6.2 resulted in a “small eye” phenotype, supporting a role in retinal development. In summary, our study reveals that the pleiotropic functions of Pax6 in vertebrates are served by a divergent family of Pax6 genes, forged by ancient duplication events and by independent, lineage-specific gene losses.

Conserved synteny between the Fugu and human PTEN locus and the evolutionary conservation of vertebrate PTEN function.

Mutations of PTEN, which encodes a protein-tyrosine and lipid phosphatase, are prevalent in a variety of human cancers. The human genome ‘draft’ sequence still lacks organization and much of the PTEN and adjacent loci remain undefined. The pufferfish, Fugu rubripes, by virtue of having a compact genome represents an excellent template for rapid vertebrate gene discovery. Sequencing of 56 kb from the Fugu pten (fpten) locus identified four complete genes and one partial gene homologous to human genes. Genes neighboring fpten include a PAPS synthase (fpapss2) differentially expressed between non-metastatic/metastatic human carcinoma cell lines, an inositol phosphatase (fminpp1) and an omega class glutathione-S-transferase (fgsto). We have determined the order of human BAC clones at the hPTEN locus and that the locus contains hPAPSS2 and hMINPP1 genes oriented as are their Fugu orthologs. Although the human genes span 500 kb, the Fugu genes lie within only 22 kb due to the compressed intronic and intergenic regions that typify this genome. Interestingly, and providing striking evidence of regulatory element conservation between widely divergent vertebrate species, the compact 2.1 kb fpten promoter is active in human cells. Also, like hPTEN, fpten has a growth and tumor suppressor activity in human glioblastoma cells, demonstrating conservation of protein function.

Sequencing and Analysis of Full-Length cDNAs, 5′-ESTs and 3′-ESTs from a Cartilaginous Fish, the Elephant Shark (Callorhinchus milii)

Cartilaginous fishes are the most ancient group of living jawed vertebrates (gnathostomes) and are, therefore, an important reference group for understanding the evolution of vertebrates. The elephant shark (Callorhinchus milii), a holocephalan cartilaginous fish, has been identified as a model cartilaginous fish genome because of its compact genome (∼910 Mb) and a genome project has been initiated to obtain its whole genome sequence. In this study, we have generated and sequenced full-length enriched cDNA libraries of the elephant shark using the ‘oligo-capping’ method and Sanger sequencing. A total of 6,778 full-length protein-coding cDNA and 10,701 full-length noncoding cDNA were sequenced from six tissues (gills, intestine, kidney, liver, spleen, and testis) of the elephant shark. Analysis of their polyadenylation signals showed that polyadenylation usage in elephant shark is similar to that in mammals. Furthermore, both coding and noncoding transcripts of the elephant shark use the same proportion of canonical polyadenylation sites. Besides BLASTX searches, protein-coding transcripts were annotated by Gene Ontology, InterPro domain, and KEGG pathway analyses. By comparing elephant shark genes to bony vertebrate genes, we identified several ancient genes present in elephant shark but differentially lost in tetrapods or teleosts. Only ∼6% of elephant shark noncoding cDNA showed similarity to known noncoding RNAs (ncRNAs). The rest are either highly divergent ncRNAs or novel ncRNAs. In addition to full-length transcripts, 30,375 5′-ESTs and 41,317 3′-ESTs were sequenced and annotated. The clones and transcripts generated in this study are valuable resources for annotating transcription start sites, exon-intron boundaries, and UTRs of genes in the elephant shark genome, and for the functional characterization of protein sequences. These resources will also be useful for annotating genes in other cartilaginous fishes whose genomes have been targeted for whole genome sequencing.