Supplementary Information for

White shark : ancient elasmobranch adaptations associated with wound healing and the maintenance of genome stability.

Nicholas J. Marraa,b,1,2, Michael J. Stanhopeb,1,3,4, Nathaniel Juec, Minghui Wangd, Qi Sund, Paulina Pavinski Bitarb, Vincent P. Richardse, Aleksey Komissarovf, Mike Raykog, Sergey Kliverf, Bryce J. Stanhopeb, Chuck Winklerh, Stephen J. O’Brienf,i, Agostinho Antunes,j,k, Salvador Jorgensenl, Mahmood S. Shivjia,3,4.

authors to whom correspondence should be addressed: Michael J. Stanhope: [email protected] Mahmood S. Shivji: [email protected] Stephen J. O’Brien: [email protected]

This PDF file includes: Figs. S1 to S3: pgs. 2-4 Table S1: pgs. 5-14 RESULTS; Shark Vision: pg. 15 Additional Methods; Transcriptome and Genome Annotation: pgs. 15-17 Captions for databases S1 to S8: pgs. 17-18. References for SI reference citations: pgs. 18-26 1 www.pnas.org/cgi/doi/10.1073/pnas.1819778116

Supplementary figures

Fig. S1. K-mer plot of white shark genome sequence reads. Includes 23 bp lengths for all 2x150 bp reads (with the exclusion of the SWIFT kit sequences) included in the SOAPdenovo genome assembly, versus their frequency in the read pool. Several k-mer analyses were conducted to obtain an average estimate of genome size, of which this figure is representative. Genome size was calculated using the frequency of the k- mers present in the largest local maximum peak of the plot, using the following formula: G = Nk-mer/Ck-mer where G is genome size, N is the total number of error free k-mers expected from the sequence data, and C is the frequency of k-mers at the main peak. The plot also indicates a low level of heterozygosity ( with high heterozygosity have a secondary peak to the left of the local maximum), and a high repeat content, as shown by the large tail on the bottom right of the figure. There are also several areas of possible duplicated sequence, evidenced by the presence of several small secondary peaks to the right of the main genome size peak (magnified in the inset figure). The axes are plotted on a log-log scale to illustrate better the repeat tail.

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Fig S2. Benchmarking Universal Single-Copy Orthologs (BUSCO). Assessment of the presence and completeness of Metazoan and Vertebrate BUSCO in the independently, de novo constructed genomes, of C. milli, R. typus, and C. carcharias as well as a multi- tissue transcriptome of C. carcharias.

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Fig. S3. Break-down of the composition of the white shark genome assembly. The percent of the genome assembly belonging to introns and exons came from the MAKER output which provides an average intron and exon size along with the predicted number of . Segmental duplications were identified as sections of the genome that had >90% similarity to another portion of the genome over a continuous 1 kbp region as identified by BLASTN or that had 6 of 7 consecutive sliding windows of 1 kbp with a mean read depth of > mean + 3.5 SD, when the reads of the single end 150 bp library were mapped back to the genome. All repeat categories were determined by using the program RepeatMasker by using vertebrate + custom white shark repeats. The remaining percentage of the genome assembly not covered by these categories was defined as general 'Non-coding Regions'.

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Table S1. Annotated list of positively selected genome stability related genes presented in Table 1 of the manuscript.

Gene name Role in genome Genome stability stability relevant GO terms CHEK2* Serine/threonine- DNA repair, apoptosis, DNA damage checkpoint; protein tumor suppressor (1-8) double-strand break repair; Chk2 regulation of apoptotic process; regulation of signal transduction by class mediator RFC5* Replication factor DNA repair; translesion DNA damage response, C subunit 5 synthesis; nucleotide detection of DNA damage; excision repair (9) DNA repair; translesion synthesis; nucleotide- excision repair FBXO45*! F-box/SPRY Regulates/degrades cellular response to DNA domain- tumor suppressor TP73 damage stimulus containing protein (10) 1 DICER1* Endoribonuclease siRNA and miRNA apoptotic DNA Dicer biogenesis; DNA repair fragmentation; miRNA (11-15) loading onto RISC involved in silencing by miRNA; conversion of ds siRNA to ss siRNA involved in RNA interference INO80B* INO80 complex remodelling DNA repair; chromatin subunit B and DNA repair (16-18) remodelling; DNA recombination; cellular response to DNA damage stimulus DTL* Denticleless DNA damage response cellular response to DNA protein and translesion DNA damage stimulus; signal synthesis (19-25) transduction involved in G2 DNA damage checkpoint; POLD3* DNA DNA repair (26-30) nucleotide-excision repair, delta subunit 3 DNA incision, 5'-to lesion; maintenance; translesion synthesis FEM1B* Protein fem-1 Apoptosis and DNA regulation of extrinsic homolog B repair (31,32) apoptotic signaling pathway via death domain receptors; regulation of

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DNA damage checkpoint SIRT7* NAD-dependent DNA repair; chromatin chromatin organization; protein remodelling; apoptosis; NAD-dependent regulates p53 (33-37) deacetylase activity (H3- -7 K18 specific)

PLK2* Serine/threonine- Cell cycle control; DNA damage response, regulates tumor growth signal transduction by p53 PLK2 and apoptosis (38-42) class mediator resulting in cell cycle arrest; negative regulation of apoptotic process; CENPS* Centromere DNA repair (43-45) DNA repair; interstrand protein S cross-link repair; chromatin binding; cellular response to DNA damage stimulus; replication fork processing; CASS4* Cas scaffolding Cell adhesion and cell Cell adhesion protein family spreading; apoptosis member 4 (46-48) UFD1* Ubiquitin Protein negative regulation of recognition factor deubiquitination; core activity; in ER-associated component of p97- ubiquitin-dependent degradation UFD1-NPL4 complex ERAD pathway protein 1 involved in protein extraction from chromatin (49,50) AGT* Angiotensinogen Apotosis; cell regulation of apototic proliferation (51-54) process; regulation of cell proliferation; ERK1 and ERK2 cascade RPS6* 40S ribosomal Apoptosis (55,56) regulation of apoptotic protein S6 process; TOR signalling MYOG* Myogenin Regulation of cell positive regulation of cell proliferation and cell cycle arrest; regulation of cycle arrest (57,58) cell proliferation; chromatin DNA binding USP13* Ubiquitin Controls autophagy and regulation of autophagy; carboxyl-terminal p53 levels; cell cell proliferation; protein hydrolase 13 proliferation (59) deubiquitination PRIM1* DNA Okazaki fragment DNA replication, synthesis small subunit synthesis (60) of RNA primer; telomere maintenance via semi- conservative replication; G1/S transition of mitotic

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cell cycle ALKBH7* Alpha- Necrosis (61) cellular response to DNA ketoglutarate damage stimulus; dependent programmed cell death; dioxygenase alkB regulation of homolog7 mitochondrial membrane permeability involved in programmed necrotic cell death BUD23* 18S rRNA chromatin organization; (guanine-N(7))- (62) methyltransferase FIGL1# Fidgetin-like-1 Double strand break regulation of double-strand repair; regulation of break repair via meiotic recombination homologous (63,64) recombination; negative regulation of reciprocal meiotic recombination CTNNBL1# Beta-catenin-like coupled positive regulation of protein 1 repair; apoptosis (65-67) apoptotic process; mRNA splicing, via spliceosome CMTM7# CKLF-like Tumor suppressor (68) cytokine activity MARVEL transmembrane domain- containing protein 7 MDM4# Protein MDM4 p53 regulator (69-71) DNA damage response; signal transduction by p53 class mediator resulting in cell cycle arrest ARL6IP5# PRA1 family Apoptosis (72-79) intrinsic apoptotic protein 3 signaling pathway in response to oxidative stress; positive regulation of stress-activated MAPK cascade KIAA1324# UPF0577 protein Autophagy; tumor positive regulation of KIAA1324 suppressor (80-84) autophagosome assembly SALL4# Sal-like protein 4 DNA damage response; somatic stem cell stem cell maintenance population maintenance; (85-87) regulation of transcription, DNA-templated

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PDCD2@! Programmed cell Apoptosis; regulation of positive regulation of death protein 2 stem cell proliferation apoptotic process; positive (88-90) regulation of hematopoietic stem cell proliferation PDCD4! Programmed cell Apoptosis; tumor positive regulation of death protein 4 suppressor (91-94) vascular associated smooth muscle cell apoptotic process; positive regulation of endothelial cell apoptotic process; cell aging; negative regulation of apoptotic process NHP2! H/ACA Telomere maintenance telomere maintenance via ribonucleoprotein (95-97) telomerase complex subunit 2 RRS1! Ribosome p53 regulator (98-102) regulation of signal biogenesis transduction by p53 class regulatory protein mediator homolog

*= white shark # = whale shark @ = elephant shark ! = elasmobranchs

Annotation and references: Genome stability PS genes

CHK2 Activated when DNA undergoes a double-strand break (1); more specifically, phosphatidylinositol kinase family protein (PIKK) ATM, phosphorylates site Thr68 and activates CHK2 (1,2). CHK2 then in turn, phosphorylates targets such as CDC25 (cell division control protein 25), responsible for removing phosphate from active site residues and activating the cyclin-dependent (CDKs). Thus, CHK2’s inhibition of CDC25 phosphatases prevents entry of the cell into . The CHK2 protein also interacts with and stabilizes p53 leading to cell cycle arrest. CHK2 is known to regulate apoptosis through the phosphorylation of p53/TP53, MDM4 (protein MDM4) and PML (protein PML) (1-3). It also phosphorylates NEK6 (Serine/threonine-protein kinase Nek6) which is involved in G2/M cell cycle arrest (4). It promotes DNA repair through phosphorylation of BRCA2, enhancing the association of RAD51 (DNA repair protein RAD51 homolog 2) with chromatin (5). CHK2 also stimulates the transcription of genes involved in DNA repair (including BRCA2) through the phosphorylated activation of the FOXM1 (Forkhead box protein M1) (6). CHK2 phosphorylation of p53/TP53 alleviates inhibition by MDM2, leading to accumulation of active p53/TP53 and phosphorylation of MDM4 reduces p53/TP53 degradation (3). CHK2 is a tumor suppressor that also has a role in instability through its effect on mitotic

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spindle assembly, with the loss of CHK2-mediated phosphorylation of BRCA1 leading to spindle formation defects and chromosomal instability (7). Facilitates the association of CCAR2 (cell cycle and apoptosis regulator protein 2) and SIRT1 (NAD-dependent protein deacetylase sirtuin-1) and is required for CCAR2-mediated SIRT1 inhibition (8).

RFC5 (RF-C) is required for DNA replication and repair. It is essential for loading of proliferating cell nuclear antigen (PCNA) onto double-stranded DNA and for subsequent DNA elongation by DNA δ and ϵ (9).

FBXO45 A component of E3 ubiquitin ligase complex that recognizes TP73, promoting its ubiquitination and degradation (10).

DICER1 DICER1 plays a critical role in siRNA (small interfering RNA) and miRNA (microRNA) biogenesis by cleaving double-stranded RNA (dsRNA) and pre-microRNA (pre-miRNA) into these two microRNA types (11). It also facilitates the activation of the RNA-induced silencing complex (RISC), which is essential for RNA interference (12). miRNA depletion caused by genetic alterations in the components of miRNA biogenesis is often oncogenic (11). In addition, DICER1 terminates Pol II transcription at highly transcribed protein coding genes, as well as tRNA, and rRNA genes. These DICER1- terminated loci are at sites of replication stress and DNA damage, resulting from transcription-replication collisions. DICER1 resolves these collisions, preventing , which would otherwise result in loss of rDNA repeats, and as such, acts to maintain genome stability (13). DICER1 acts in response to DNA damage after recognition of DNA lesions and is one of the essential components for the secondary recruitment of DNA damage response factors (14). Dicer is essential for resolving replication-associated DNA damage in developing cerebellum and embryonic stem cells (15).

INO80B INO80B encodes a subunit of the chromatin remodeling INO80 complex which is involved in transcriptional regulation, DNA replication and DNA repair (16-18).

DTL Substrate-specific adapter of a DCX E3 ubiquitin-protein ligase complex essential for cell cycle control, DNA damage response and translesion DNA synthesis. The DCX(DTL) complex, facilitates the polyubiquitination and degradation of CDT1 (DNA replication factor Cdt1), CDKN1A/(CIP1; Cyclin-dependent kinase inhibitor 1), FBXO18/FBH1 (F-box DNA 1), KMT5A (N-lysine methyltransferase KMT5A) and SDE2 (Replication stress response regulator SDE2) (19-24). In response to DNA damage CDT1 degradation is required for proper cell cycle regulation of DNA replication (19,20). CDKN1A/p21(CIP1) degradation during is essential to control replication licensing (25). KMT5A degradation is important for proper regulation of TGF-beta signaling, cell cycle progression, DNA repair and cell migration (23). In undamaged

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proliferating cells, the DCX(DTL) complex promotes 'Lys-164' monoubiquitination of PCNA, and therefore is involved in PCNA-dependent translesion DNA synthesis (22-24).

POLD3 As a component of the trimeric and tetrameric DNA polymerase delta complexes, POLD3 plays a fundamental role in genome replication, lagging strand synthesis, and DNA repair. For example, concomitant with RFC1-replication factor C complex and PCNA (proliferating cell nuclear antigen), POLD3 is required to recruit POLD1, the catalytic subunit of the polymerase delta complex, to DNA damage sites (26). During DNA replication stress, POLD3 is required for the repair of broken replication forks through the break-induced replication (BIR) pathway (27). It is also involved in translesion synthesis, facilitating abasic site bypass by DNA polymerase delta (28,29). Pold3 plays important roles in DNA double strand break repair, telomere maintenance and genomic stability of mice embryonic stem cells and spermatocytes (30). Pold3 also mediates DNA replication and repair by regulating 53BP1 (p53-binding protein 1; promotes the non-homologous end-joining pathway of DNA double strand break repair), RIF1 (telomere-associated protein RIF1; involved in DNA damage response, organisation of chromatin architecture and regulation of replication timing), ATR (a central regulator of the replication checkpoint; recognizes and stabilizes stalled replication forks) and ATM (protein kinase recruited and activated by DNA double-strand breaks; phosphorylates several key that initiate activation of the DNA damage checkpoint, leading to cell cycle arrest, DNA repair or apoptosis; targets, include p53, CHK2, BRCA1, NBS1 and H2AX) (30).

FEM1B Involved in apoptosis by acting as a death receptor-associated protein; death receptors are a subgroup of the TNF-R (tumor necrosis factor receptor 1) superfamily that can induce apoptosis via a signaling module termed the death domain (31). FEM1B also functions as a regulator of replication stress-induced signaling that leads to the activation of CHEK1 (Checkpoint kinase 1, a protein kinase that coordinates DNA damage response and cell cycle checkpoint response; activation of CHEK1 results in the initiation of cell cycle checkpoints, cell cycle arrest, DNA repair and cell death) (32).

SIRT7 NAD-dependent protein deacetylase that acts on at 'Lys-18' (H3K18Ac). Hypoacetylation of H3K18 is a marker of malignancy in various and appears to maintain the transformed phenotype of cells. SIRT7 plays a role in oncogenic transformation by lowering expression of tumor suppressor genes by the -specific deacetylation of H3K18Ac at promoter regions (33). SIRT7 is recruited to sites of DNA damage, where it modulates H3K18Ac levels, which in turn controls recruitment of the damage response factor 53BP1 to DNA double‐strand breaks, thereby influencing the efficiency of non‐homologous end joining (34-36). SIRT7 promotes cellular survival under conditions of genomic stress, by attenuating DNA damage, SAPK (stress activated protein kinase) activation and p53 response (37).

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PLK2 A serine/threonine-protein kinase involved in genotoxic stress, synaptic plasticity, centriole duplication and G1/S phase transition (38). PLK2 is a wild type (wt) p53 target, involved in cell cycle control (39). PLK2 is transcriptionally induced by wt p53 in response to DNA damage (Burns et al. 2003) (40). In a wt p53 setting, PLK2 is tumor suppressive; however, with a mutant p53, PLK2 functions as an oncogene (39,41,42).

CENPS DNA-binding component of the Fanconi anemia (FA) core complex, required for activation of the pathway, leading to monoubiquitination of the FANCI-FANCD2 complex in response to DNA damage, cellular resistance to DNA cross-linking agents and acting to prevent chromosomal breakage (43-45).

CASS4 CASS4 regulates PTK2/FAK1 activity, and is involved in focal adhesion integrity, and cell spreading (46). PTK2/FAK1 plays an essential role in cell cycle progression, cell proliferation and apoptosis (47,48).

UFD1 UFD1 is one of the core adaptor (cofactor) proteins of the p97-UFD1-NPL4 complex. p97 is a segragase that is involved in a number of cellular functions and pathways, the specificity of which is determined by different p97 cofactor proteins. Chromatin- associated protein degradation (CAD) is regulated by p97–Ufd1–Npl4. Chromatin-bound proteins are polyubiquitinated and sumoylated by E3 ubiquitin and SUMO ligases. The p97–Ufd1–Npl4 complex is recruited to such polyubiquitinated substrates by the ubiquitin-binding domain in Ufd1, where conformational changes in p97 release these chromatin-bound substrates. Extracted substrates are degraded by proteasomes. The p97– Ufd1–Npl4 complex facilitates CAD and prevents PICHROS (protein-induced chromatin stress) (49,50).

AGT Angiotensingoen, is an essential component of the renin-angiotensin system and is a precursor molecule for angiotensin II. Angiotensin I is formed by the action of renin on angiotensinogen, and exists solely as a precursor to angiotensin II. Angiotensinogen has a wide diversity of functions. Its primary role in the area of genome stability falls within the realm of apoptosis. Angiotensin II, the physiologically active protein, induces apoptosis in a number of different cell types (51,52). Angiotensin II acts through at least two receptors, AT1 and AT2. AT2 mediates apoptosis (53). p53 has also been demonstrated to interact with the renin-angiotensin system to trigger and enhance myocyte apoptosis (54).

RPS6 RPS6 is a mediator of TRAIL-induced apoptosis. TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is an inducer of apoptosis in tumor cells (55). RPS6 is a target of the TOR signaling pathway. TOR signaling involves serine-threonine kinases that sense nutrient status and signal cells to grow and proliferate. TOR signaling plays a key 11

role in cancer and autophagy. One of the important functions of TOR signaling is in regulating protein translation and the phosphorylation of RPS6 is thought to play a key role in this process (56).

MYOG Myogenin is a myogenic transcription factor that promotes cell differentiation, initiated by the coordinated action of MYOD and MYOG. MYOD activates the expression of MYOG, which interacts with MEF2 transcription factors (myocyte enhancer factor-2 – important regulators of cell differentiation) to promote differentiation (57). Oncogenic RAS signaling via the MAPK pathway, can drive FN-RMS (Fusion-Negative Rhabdomyosarcoma) cell proliferation and inhibit the expression of MYOG, which prevents initiation of myogenic differentiation. This blockage is created by interaction between ERK2 and RNA Pol II at the MYOG locus, stalling transcription of MYOG (58).

USP13 Deubiquitinase that mediates deubiquitination of several proteins including BECN1, MITF, SKP2 and USP10. USP13 deubiquitinates USP10, an essential regulator of p53/TP53 stability; BECN1, USP10, and USP13 comprise the key components of a regulatory loop that controls the levels of p53 (59).

PRIM1 An essential polymerase that synthesizes small RNA primers for the (short DNA fragments formed on the lagging strand during replication) made during discontinuous DNA synthesis (series of short fragments) and can also interact with transcriptional regulators to control DNA replication. LIM-only 2 (LMO2) is a transcriptional regulator that functions in the erythroid-specific program, but can also function in controlling DNA replication via protein–protein interactions with essential DNA replication , such as PRIM1 (60). LMO2 is master regulator of cell fate and is a known oncogenic transcription factor.

ALKBH7: ALKBH7 induces programmed necrosis, in response to DNA damage caused by cytotoxic alkylating forces and prevents the accumulation of cells with DNA damage (61).

BUD23 A methyltransferase that specifically methylates the N7 position of a guanine in 18S rRNA. Is required for the maintenance of open chromatin at certain loci to facilitate protein loading and is required for the maintenance of dimethylation on histone H3 'Lys- 79' (H3K79me2) (62). An alternative name for this protein: Metastasis-related methyltransferase 1.

FIGL1 In addition to its role in generating genetic diversity, homologous recombination is also

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an essential DNA repair mechanism. FIGL1 is involved in DNA double-strand break repair through homologous recombination (63). It interacts with FLIP (fidgetin-like-1 interacting protein) to form a complex that interacts with RAD51 and DMC1, two recombinases responsible for catalyzing DNA strand exchange in homologous recombination (64). The FIGL1-FLIP complex is conserved from plants to mammals and acts to regulate the important step of strand invasion in homologous recombination.

CTNNBL1 Component of the PRP19-CDC5L complex involved in pre-mRNA splicing, transcription, and transcription coupled-repair (65,66). Evidence for a role in apoptosis (67).

CMTM7 A cytokine with tumor-suppressor function associated with its role in G1/S cell cycle arrest, via upregulating p27 and downregulating cyclin-dependent kinase 2 (CDK2) and 6 (CDK6) (68).

MDM4 Inhibits the cell cycle arrest and apoptosis mediated by TP73/p73 and p53/TP53, by binding the transcriptional activation domain. Also inhibits the degradation of MDM2 and can reverse MDM2-targeted degradation of TP53 (69-71).

ARL6IP5 ADP-ribosylation-like factor 6 interacting protein 5 (alternative names: PRA1 family 3; PRAF3; JWA; Arl6ip5), belongs to the prenylated rab-acceptor-family, has a key role in glutathione metabolism and exocytic protein trafficking. ARL6IP5 is also an important tumor suppressor gene, and apoptosis inducer for a variety of cancers including melanoma, gastric cancer, hepatocellular carcinoma, esophageal squamous cell carcinoma and ovarian cancer (72-77). Arl6ip5 also functions to regulate calcium in the ER and in controlling calmodulin signaling in osteoblast proliferation. Osteoblast deficiency of Arl6ip5 causes ER stress and enhances ER stress-mediated apoptosis (78). Arl6ip5 also interacts with the transcription factor CEBPA, which plays a key role in hematopoietic cell differentiation and causal role in hematological malignancies. CEBPA is also a key regulator of the apoptotic network activated in pancreatic β cells and Arl6ip5 is one of its targets (79).

KIAA1324 KIAA1324 regulates autophagy and under certain conditions of cell stress can promote cell survival (80). Autophagy is a process that can be activated by the DNA damage response (DDR), and play roles in both processing of genomic lesions and occasionally also in cell death (81). KIAA1324 acts as a gastric cancer tumor suppressor via inhibition of the oncogene GRP78 and inducing apoptosis (82); expression levels of KIAA1324 are regulated by micro RNA miR-18B, which is known to regulate other important cancer related proteins, such as MDM2 (83,84).

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SALL4 Transcription factor playing a fundamental role in self renewal and overall maintenance of both hematopoietic and embryonic stem cells (85). Human embryonic stem cells repair DNA lesions much more efficiently than differentiated cells, owing to the increased expression of a number of components of DNA repair machinery (86). One of these is SALL4 which is rapidly mobilized to sites of double strand breaks after DNA damage. SALL4 interacts with RAD50, stabilizing the MRE11–RAD50–NBS1 complex (binds to broken DNA ends providing enzymatic activities required for double-strand break repair), resulting in the activation of ATM (ATM serine/threonine kinase that phosphorylates key proteins involved in initiating DNA damage checkpoint) (87).

PDCD2 A DNA binding protein playing a regulatory role in cell death and cell proliferation (88). It also plays a key role in stem cell development (89,90).

PDCD4 PDCD4 is a tumor suppressor protein that interacts with translation initiator eIF4A to inhibit translation, resulting in the suppression of neoplastic transformation and tumor invasion (91-93). PDCD4 is negatively regulated by the important oncomiR, MIRN21 (94).

NHP2 A member of the H/ACA snoRNPs (small nucleolar ribonucleoproteins) gene family which are involved in rRNA processing and modification and are components of the telomerase complex (95), which is responsible for maintaining the homeostasis of telomere length (96). are regions of repetitive sequence at the end of , providing chromosome ends protection from deterioration; as such telomeres play a key role in genome stability (Hernandez-Sancheza et al. 2016) (97).

RRS1 Involved in assembly of the large ribosomal subunit and an essential component of 5S RNP (5S ribonucleoprotein particle; a ribosomal assembly intermediate) biogenesis (98). 5S RNP in turn, is essential for the activation of p53 by p14(ARF), a protein that is activated by oncogene (e.g. RAS or MYC) overexpression (99,100). p14(ARF) accumulates mainly in the nucleolus where it forms complexes with NPM (Nucleophosmin - involved in various processes including ribosome biogenesis, histone assembly, centrosome duplication, cell proliferation, and regulation of tumor suppressors p53/TP53 and p14ARF) or MDM2. p14ARF then can act as a tumor suppressor by inhibiting either ribosome biogenesis (in the case of NPM) or initiating p53-dependent cell cycle arrest and apoptosis (in the case of MDM2) (101). 5S RNP accumulates when ribosome biogenesis is blocked and the excess 5S RNP binds to MDM2, a principal p53- suppressor, inhibiting MDM2 function and leading to p53 activation (102). The abundance of 5S RNP, and therefore p53 levels, is determined by factors regulating 5S complex formation (100).

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RESULTS

Shark Vision

While visual acuity and black/white vision is driven by the presence of rhodopsin in rod cells of the eye, color vision is driven by the presence and absorption of light by opsin genes in the cone cells. In some pelagic predators such as the Pacific Bluefin tuna, these genes are expanded to ten different loci (103) (typically four different opsin genes are responsible for the molecules that absorb light in the visible spectrum). Conversely it has long been thought that sharks and other chondrichthyans have an absence of color vision despite possessing cone cells where the opsins are normally found (reviewed in (104)). Recently, however, several batoids (Subclass Elasmobranchii; Superorder Batoidea) have been shown to possess the ability to perceive several additional wavelengths of light (105) and the holocephalon, elephant shark, was shown to have several color absorbing opsin genes (106). Nonetheless, evidence in sharks still points to a lack of color vision (107). We interrogated the predicted protein products of genes of the white and whale sharks with a list of opsin and opsin like genes from teleosts and chondrichthyans to identify the presence of visual pigment genes. In each species we found the presence of Rhodopsin (Rh1) for black/white vision and similarity of two sequences to color absorbing opsins. In each case one of these was an ancient vertebrate opsin (wavelength sensitivity approximately 500 nm (108), and another was panopsin (responsible for light absorption and sensing photoperiod) but in none of the cases was the best match to one of the color absorbing opsins associated with broader spectrum. Thus, our results point to the likelihood of black/white or monochromatic vision in these two large pelagic sharks. Rh1 was also the sole opsin gene reported in the recent cloudy catshark genome analysis (109).

Additional Methods

Transcriptome

In order to generate a de novo transcriptome to facilitate gene prediction, we isolated RNA from 4 different cell/tissue types: blood, epidermis, subdermis, and muscle and added this to the previous heart transcriptome. RNA was isolated from these tissues using the Zymo Research Direct-zol kit. Total RNA was examined for RNA integrity numbers and all samples had values >7. To generate mRNA sequencing libraries, we used the Illumina mRNA TruSeq kit, which included a poly-A selection module. All libraries were then sequenced with Dovetail sequencing on an Illumina HiSeq-4000 to generate 150bp paired-end sequencing libraries. To assemble these reads into a de novo assembly, we utilized a multi-assembler/multi-kmer approach to mitigate any biases related to specific assemblers or selected kmer parameter values. To prepare reads for assembly, we trimmed adapter sequence and low quality regions from all reads using Trimmomatic v0.36 (110) and normalized all reads by kmer count using methods implemented in the Trinity de novo transcriptome assembly package (111). We then used those normalized reads to generate 6 assemblies with the Velvet-based Oases

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transcriptome assembly pipeline (112) for kmers 27, 33, 39, 45, 51, and 57, 6 assemblies with the TransAbyss transcriptome assembly pipeline (113) for kmers 26, 32, 38, 44, 50, and 56, and 1 assembly using the Trinity transcriptome assembly software (kmer size = 25). All resulting transcriptome assemblies were then collapsed into one final de novo assembly using the EviGene transcriptomics pipeline (114), which discards redundant predicted transcripts, collapsing identical transcripts, and selecting representative principal transcripts based on the length of coding region. Proteins were predicted from these “final” predicted transcripts using TransDecoder v3.0.1 tool (111) and examined for the likelihood of being full length coding sequence by comparing them to the UniProt protein database using BLASTX. The final transcriptome was then used to train gene prediction in Maker.

Transcriptome assembly efforts yielded a de novo transcriptome sequence with 104,592 predicted transcripts (N50 = 1,230 bp). Of those predicted transcripts, 59,978 (57.3%) were identified as having predicted protein coding region and 27,266 (26.1%) could be associated with a known protein sequence from the NCBI non-redundant protein database (BLASTX e-value < 1 x10-5).

Genome annotation

An initial white shark specific set of repeats and transposable elements were created de novo from read data and the initial SOAPdenovo assembly. This library was then used in conjunction with existing elephant shark repeats and basic repeats available on RepBase to conduct an initial masking of the genome with RepeatMasker. Subsequently, RepeatModeler was used to identify additional repetitive sequences in the genome according to the suggestions for Repeat Library Construction in the MAKER 2.0 wiki (115). This library from RepeatModeler and the de novo repeat library were then combined and condensed using TEclassifier. This final repeat library was used to mask the Dovetail assembly prior to genome annotation, and to obtain final estimates of repeat content using RepeatMasker.

The Dovetail genome assembly was annotated using the MAKER 2.0 pipeline using the final repeat library described above for masking, and using all chondrichthyan Swiss-Prot proteins, as well as evidence derived from our consensus RNA-seq Carcharodon carcharias transcriptome obtained from heart (116,117) blood, muscle, subdermis and epidermis. Training data for the gene predictor AUGUSTUS (118) was taken from the BUSCO analysis; the gene predictor SNAP (119) was trained by running the MAKER pipeline with a subset of the Dovetail assembly consisting of all scaffolds above 5 Mbp, and repeated for three iterations. Initially, we utilized all scaffolds greater than 1 Kbp in length (46,307 scaffolds covering 3.99 Gbp). Following recommendations from the MAKER documentation, and in order to provide a conservative assessment of gene annotations, we then filtered this initial set for only scaffolds that were greater than 10 Kbp in length. This comprised 9,222 scaffolds with a total length of 3.92 Gbp and contained 24,520 predicted genes.

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These 24,520 predicted genes were blasted (using both BLASTP for predicted proteins and BLASTX for predicted transcripts) against the Uniprot/Swiss-Prot db to obtain BLAST descriptions; any additional sequences that did not have BLAST hits were blasted against the nr database. All genes with BLAST hits that had an e-value < 1x10-6 were then assigned gene ontology terms using BLAST2GO V 3.0. For downstream comparisons Refseq protein datasets for elephant shark and whale shark were run through the same BLAST and GO assignment steps.

Additional data tables S1-S8 Dataset, Table S1. Genes under positive selection on each of the white shark, whale shark, elephant shark, elasmobranch, and chondricthyan branches. Each sheet of this workbook identifies the branch tested and lists the alignment ID, the Swissprot ID corresponding to that alignment, the protein name and the gene name. Also included is the q-value (FDR adjusted p-value) and the ω value for the proportion of sites in the category that allows for positive selection. Each alignment was manually checked for accuracy and only retained if the sequences judged to be under positive selection covered greater than 60% of the coding sequence length, as determined by their comparison to the Swissprot reference sequence for that gene.

Dataset, Table S2. (GO) enrichments of positively selected genes for the white shark, in comparisons back to its genome.

Dataset, Table S3. Gene Ontology (GO) enrichments of positively selected genes for the whale shark, in comparisons back to its genome.

Dataset, Table S4. Gene Ontology (GO) enrichments of positively selected genes for the elephant shark, in comparisons back to its genome.

Dataset, Table S5. Most specific gene content Gene Ontology (GO) term enrichments and under-representations for each of Biological Process, Molecular Function, and Cellular Component, as well as Reactome pathways, for white shark compared to five other vertebrates. This file includes all the most specific GO term enrichments and under- representations, as well as Reactome pathways, that arose from the inter-vertebrate Panther comparisons involving the white shark genome.

Dataset, Table S6. Most specific gene content Gene Ontology (GO) term enrichments and under-representations for each of Biological Process, Molecular Function, and Cellular Component, as well as Reactome pathways, for whale shark compared to five other vertebrates. This file includes all the most specific GO term enrichments and under- representations, as well as Reactome pathways, that arose from the inter-vertebrate Panther comparisons involving the whale shark genome.

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Dataset, Table S7. Most specific gene content Gene Ontology (GO) term enrichments and under-representations for each of Biological Process, Molecular Function, and Cellular Component, as well as Reactome pathways, for elephant shark compared to five other vertebrates. This file includes all the most specific GO term enrichments and under- representations, as well as Reactome pathways, that arose from the inter-vertebrate Panther comparisons involving the elephant shark genome.

Dataset, Table S8. Program settings and versions for software used in genome assembly, assessment, annotation and analysis.

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