Elephant shark genome provides new insights into bone formation and adaptive immunity in humans

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Elephant shark genome provides new insights into bone formation and adaptive immunity in humans A*STAR-led international consortium completely decodes the first shark-family member genome 1. Singapore, Jan, 09, 2014 - Scientists at A*STAR's Institute of Molecular and Cell Biology (IMCB) led an international team of researchers that sequenced and analysed the genome of the elephant shark. A comparison of the elephant shark genome with human and other vertebrate genomes revealed why the skeleton of sharks consists entirely of cartilage instead of bones. The findings carry potential implications for human bone disease treatment. The analysis also sheds new light on the origin of the adaptive immune system. The findings of this study are published in the 9 January, 2014 issue of the prestigious scientific journal, Nature.

2. The collaboration with 12 international institutions was headed by IMCB's Prof Byrappa Venkatesh, who is also a chairperson of the "Genome10K" Project. The project was largely funded by the National Institutes of Health (NIH), USA, the world's foremost centre for the support of biomedical research. Analysis of the elephant shark genome

3. Elephant shark is a member of cartilaginous fishes, which are the oldest living group of jawed vertebrates that diverged from bony vertebrates about 450 million years ago. Cartilaginous fishes include sharks, rays, skates and chimaeras. Unlike humans and other bony vertebrates, cartilaginous fishes are unable to replace cartilage with bone. Among over 1,200 species of cartilaginous fishes, the elephant shark was sequenced due to its compact genome size. Analysis of the elephant shark genome identified a family of genes absent in elephant shark but present in all bony vertebrates. A significant reduction in bone formation was observed when a member of this gene family was knocked out in zebrafish, thereby indicating the importance of this gene family in bone formation. The finding has important implications towards our understanding of bone-related diseases such as osteoporosis and hence the development of effective therapeutic strategies for them.

4. An unexpected finding was that elephant shark appears to lack special types of immune cells previously considered essential for defence against viral/bacterial infections and preventing autoimmune reactions such as diabetes and rheumatoid arthritis . Despite this seemingly primitive organization of the immune system, sharks exhibit robust immune defences and are long-lived. By challenging long-held notions, this discovery has opened up a new avenue towards the development of non-intuitive strategies to shape the immune functions of humans. 5. The study also found that the elephant shark genome is the slowest evolving among all vertebrates, including the coelacanth, popularly known as a "living fossil", whose genes were recently shown to be evolving slower than those of other bony vertebrates . Furthermore, large blocks of elephant shark and human chromosomes were found to be highly similar. The markedly slow evolution of the elephant shark genome and its similarity to the human genome, further underscores its importance as a reference genome for comparative genomic studies aimed at better understanding of the human genome.

6. Prof Byrappa Venkatesh, Research Director at IMCB, and lead author of the Nature paper added, "The slow evolving genome of the elephant shark is probably the best proxy for the ancestor of all jawed-vertebrates that became extinct a long time ago. It is a cornerstone for improving our understanding of the development and physiology of human and other vertebrates as illustrated by our analysis of the skeletal system and immune system genes."

7. Dr Wes Warren, senior author of the Nature paper commented, "Although cartilaginous vertebrates and bony vertebrates diverged about 450 million years ago, with the elephant shark genome in hand, we can now begin to identify key genetic adaptations in the evolutionary tree."

8. Prof Hong Wanjin, Executive Director of IMCB, said, "Over the years, IMCB has carried out several remarkable genomic projects and we are excited to showcase yet another milestone. Not only has our research team sequenced the first shark-family member genome, they have also uncovered many insights significant to the field of genomic and medical research through the genome analysis."

9. Prof Sir David Lane, Chief Scientist at A*STAR, said, "We are delighted that a Singapore laboratory conceived and led a major international genome project. The international research grant from NIH, USA is a testimony of Singapore's world-class standard in genomics research."

### Notes for Editor: The research findings described in this media release can be found in the 9 January, 2014 issue of Nature Journal, under the title, "Elephant shark genome provides unique insights into gnathostome evolution" by Byrappa Venkatesh1,2, Alison P. Lee1, Vydianathan Ravi1, Ashish K. Maurya3, Michelle M. Lian1, Jeremy B. Swann4, Yuko Ohta5, Martin F. Flajnik5, Yoichi Sutoh6, Masanori Kasahara6, Shawn Hoon7, Vamshidhar Gangu7, Scott W. Roy8, Manuel Irimia9, Vladimir Korzh10, Igor Kondrychyn10, Zhi Wei Lim1, Boon-Hui Tay1, Sumanty Tohari1, Kiat Whye Kong7, Shufen Ho7, Belen Lorente-Galdos11,12, Javier Quilez11,12, Tomas Marques-Bonet11,12, Brian J. Raney13, Philip W. Ingham3, Alice Tay1, LaDeana W. Hillier14, Patrick Minx14, Thomas Boehm4, Richard K. Wilson14, Sydney Brenner1 & Wesley C. Warren14

1Comparative Genomics Laboratory, Institute of Molecular and Cell Biology, A*STAR, Biopolis, Singapore 138673.
2Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 119228.
3Developmental and Biomedical Genetics Laboratory, Institute of Molecular and Cell Biology, A*STAR, Biopolis, Singapore 138673.
4Department of Developmental Immunology, Max-Planck-Institute of Immunobiology and Epigenetics, Stuebeweg 51, 79108 Freiburg, Germany
5Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201 USA
6Department of Pathology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan
7Molecular Engineering Laboratory, Biomedical Sciences Institutes, A*STAR, Biopolis, Singapore 138673.
8Department of Biology, San Francisco State University, San Francisco, CA 94132, USA
9 Banting and Best Department of Medical Research and Donnelly Centre, University of Toronto, Toronto, M5S 3E1, Canada
10 Fish Developmental Biology Laboratory, Institute of Molecular and Cell Biology, A*STAR, Biopolis, Singapore 138673.
11Institut de Biologia Evolutiva, (UPF-CSIC), PRBB, 08003 Barcelona, Spain.
12Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Catalonia, Spain
13Center for Biomolecular Science and Engineering, School of Engineering, University of California Santa Cruz, Santa Cruz, CA 95064, USA
14 The Genome Institute at Washington University, St. Louis, MO 63108, USA

Correspondence should be addressed to Byrappa Venkatesh

Full text of the article can be accessed from http://www.nature.com/nature/journal/v505/n7482/index.html.

For more information about the elephant shark genome, please visit: http://esharkgenome.imcb.a-star.edu.sg.

AGENCY FOR SCIENCE, TECHNOLOGY AND RESEARCH (A*STAR)

Enclosed: Annex A – Key Findings of "Elephant Shark Genome Provides Unique Insights into Gnathostome Evolution" Annex B – About Professor Byrappa Venkatesh and his Research Group

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About Institute of Molecular and Cell Biology (IMCB) The Institute of Molecular and Cell Biology (IMCB) was established in 1987 at the National University of Singapore (NUS) before becoming an autonomous research institute (RI) of A*STAR and moving to Biopolis in 2004. IMCB strives to maintain the scientific excellence of PI-driven research and at the same time aims to promote collaborative team-based projects of medical and industrial relevance. Funded primarily by the Biomedical Research Council (BMRC) of A*STAR, IMCB's research activities focus on four major fields: Animal Models of Development and Disease, Cancer Genetics and Therapeutics, Cell Biology in Health and Disease, and Structural Biology and Drug Discovery. For more information about IMCB, please visit http://www.imcb.a-star.edu.sg.

About the Agency for Science, Technology and Research (A*STAR) The Agency for Science, Technology and Research (A*STAR) is Singapore's lead public sector agency that fosters world-class scientific research and talent to drive economic growth and transform Singapore into a vibrant knowledge-based and innovation driven economy.

In line with its mission-oriented mandate, A*STAR spearheads research and development in fields that are essential to growing Singapore's manufacturing sector and catalysing new growth industries. A*STAR supports these economic clusters by providing intellectual, human and industrial capital to its partners in industry.

A*STAR oversees 18 biomedical sciences and physical sciences and engineering research entities, located in Biopolis and Fusionopolis, as well as their vicinity. These two R&D hubs house a bustling and diverse community of local and international research scientists and engineers from A*STAR's research entities as well as a growing number of corporate laboratories. For more information about A*STAR, please visit http://www.a-star.edu.sg.

Annex A

Key Findings of "Elephant Shark Genome Provides Unique Insights into Gnathostome Evolution"

Species: Elephant shark (Callorhinchus milii) aka ghost shark, inhabits temperate waters of the continental shelves off southern Australia and New Zealand, at depths of 200 to 500 meters. From approximately 1,200 species of cartilaginous fishes which include sharks, rays, skates and chimaeras, elephant shark was chosen as a model because of its compact genome (1 billion bases) which is one third the size of the human genome (3 billion bases).

Summary of Scientific Paper: The paper reports (1) the sequencing and analysis of the whole-genome of the first cartilaginous fish, the elephant shark. (2) The elephant shark genome is the slowest evolving of all known vertebrates, including the "living fossil" coelacanth, and features extensive conservation with tetrapod genomes, making it a good model for comparative analyses of jawed vertebrate (gnathostome) genomes. (3) Functional studies suggest that the lack of genes encoding secreted calcium-binding proteins (SCPPs) in cartilaginous fishes explains the absence of bones in their endoskeleton. (4) The adaptive immune system of cartilaginous fishes was found to be unusual; it lacks the canonical CD4 co-receptor and most transcription factors, cytokines and cytokine receptors related to the CD4 lineage despite the presence of polymorphic major histocompatibility complex class II molecules. It thus presents a new model for understanding the origin of adaptive immunity.

(1) The First Whole-Genome Sequence of a Cartilaginous Fish: The project was initiated and led by Singapore scientist, Prof Byrappa Venkatesh and involved 12 institutions from Singapore, USA, Germany, Japan, Canada and Spain. The project was supported mainly by a grant from the National Human Genome Research Institute (NHGRI) of the National Institutes of Health (NIH), USA.

The research team has successfully sequenced the whole-genome of elephant shark. Analysis of the genome provided useful insights into how we see the human genome and its evolution.

(2) The Elephant Shark Genome is the Slowest Evolving among all Vertebrates: Using about 700 genes that are common to all vertebrates, the researchers estimated the molecular evolutionary rate of the elephant shark and compared it to other vertebrates. The results showed that the nucleotide substitution rate of elephant shark is significantly slower than that of all other vertebrates examined, including the coelacanth which is the slowest evolving among bony vertebrates. Coelacanth is popularly known as a 'living fossil' because of its remarkable resemblance to fossil relatives indicating that its morphological features have virtually unchanged over hundreds of million years of evolution. Unfortunately, the skeletons of cartilaginous fishes fossilize poorly and therefore it is unclear if the elephant shark also qualifies to be called a 'living fossil'.

The lower rate of molecular evolution of the elephant shark is also evident in its genome organisation; it has experienced fewer intron gains or losses than all bony vertebrates analysed. The highest rates of intron changes, on the other hand, were found in teleost fishes such as the stickleback and zebrafish. The elephant shark genome has also experienced a relatively low rate of inter-chromosomal rearrangements, comparable to that of chicken, which has the most stable karyotypes within the tetrapods. When the genome structure of the elephant shark was compared with that of chicken and human, an extensive conservation of gene order was discovered. In contrast, the comparison with zebrafish revealed that the zebrafish has undergone a higher number of genomic rearrangements than previously thought. In addition, zebrafish and other fishes were found to have lost several hundred genes that are conserved in elephant shark and human.

(3) Genetic Basis of Bone Formation: Bone is the most widespread mineralised tissue in vertebrates and its formation represents a major leap in animal evolution. Although cartilaginous fishes produce dermal bones (e.g., teeth, fin spines) and calcified cartilages, unlike bony vertebrates, their cartilage is not replaced with ossified bone. The elephant shark genome sequence provided a unique opportunity to address the question of why the skeleton of cartilaginous fish is not converted to bone. Based on a whole genome screen, the researchers identified all but one gene family (secretory calcium-binding phosphoprotein or SCPP) that is critical for bone formation. Notably, a significant reduction in bone formation was observed when a member of this gene family was knocked out in zebrafish. Thus, it is inferred that the absence of this gene family in cartilaginous fishes explains why their skeleton remains cartilaginous.

(4) Primordial Adaptive Immune System: The immune system of cartilaginous fishes shares many features of the innate and adaptive immune systems of mammals. However, the genome analysis highlighted several unexpected findings to the primordial state of immune systems in cartilaginous fishes, particularly in adaptive immunity. The elephant shark, as well as an elasmobranch, the nurse shark (Ginglymostoma cirratum), appeared to be deficient in a special type of immune cells (T regulatory cells), which are essential for combating infections in humans. The researchers noted from their analyses that elephant shark and nurse shark lack the CD4 co-receptor, transcription factor RORC, several cytokines and cytokine receptors associated with helper and regulatory functions of immunity in mammals.

Despite these findings on the immune system, elephant shark and other sharks are long-lived with robust immunity. They seem to possess restricted subsets of T cells with unconventional antigen binding properties. The study also suggested that the adaptive immune system became more elaborate in bony vertebrates through the recruitment of CD4 co-receptor, RORC, conventional FOXP3 and a host of CD4-lineage-specific cytokines and cytokine receptors.

Annex B

About Professor Venkatesh and his Research Group

Professor Byrappa Venkatesh is a Research Director in IMCB, and an adjunct Professor of the Department of Paediatrics at the National University of Singapore (NUS). He is a co-Principal Investigator of A*STAR's strategic programme on rare genetic diseases. He is also a chairperson of 'Genome 10K', an international project which aims to sequence genomes of 10,000 vertebrates. For his work on the fugu genome, Prof Venkatesh was honoured with Singapore's National Science Award in 2004.

The Comparative Genomics Lab was set up in 1992 by Prof Venkatesh and Dr Sydney Brenner. The group sequenced the whole genome of the Japanese pufferfish fugu in 2002, the first vertebrate genome to be sequenced soon after the completion of the human genome. Since then, the group has sequenced whole genomes of the Japanese lamprey and elephant shark. In addition to these genomes, Prof Venkatesh's group is working on several 'model' vertebrates including the coelacanth, cichlid fishes, the spotted gar, and sunfish. Besides comparative genomics, Prof Venkatesh's group works on the causative variants associated with chronic metabolic disorders and rare genetic diseases.

For more information on Prof Venkatesh's lab, please visit http://www.imcb.a-star.edu.sg/php/venkatesh.php