Genome Duplication and Fish Models for Toxicology

Genome duplication and fish models for toxicology

John Postlethwait University of Oregon

DIVISION OF COMPARATIVE ~\. National Institute of Environmental Health Sciences ,'~ National Toxicology Program MEDICINE liilllr'J' Your Environment. Your Health. ~ U.S. Department of Health and Human Services Genome duplication and fish models for toxicology Loren Frank von Hippel Buck N AZ U N AZ U

Jesse Gologergen Sivuqaq Island

Pete Batzel UO Tom Ingo Braasch Titus Tiffany Immingen UO Sivuqaq Island··-·•

NIH ' DIVISION OF COMPARATIVE ~\. Nationa_l Institute of En~ir~nmental Hea~~S · - MEDICINE lilllr'/ Your Environment. Your Health. ciences The problem: People living a subsistence lifestyle in the Arctic are highly exposed to persistent organic pollutants.

Annie Alowa, Sivuqaq Islander ,; The problem: Sources: t'@~""'~ Formerly used defense sites SLI a 1 Global distillation VA,. . Point sources condense & fall move to ... -). _ Greenland . .. ·· ~i colder ·t.··.•~-- ... ~.• .. ,•.· ·••'.A. ...· . . . • . 11 . ·•. . . ' . . •' '• • Norway climates 1 \ . . . . . Canada. 'II,_ !'· . • ·•. -~ ... • •.• : ,. ~''-~.--...... - ---. \.- -:: l·· 4_· organochlorines ····: ...: . ··. "\ .:· . : ·- . ·~ :- - --- evaporate

aqumaps.org Biological magnification ' ' ~,v- ,; The problem: Point sources t@.,-Formerly used defense ~sites SLI : 1 Global distillation VA,. . ·. -:·J. . Greenland • ! ...... ·· ~ : .. ...a·· . •· .A . ·· • • 1 \ I •• ••:• .' • · ••' .:• • • • Norway , ...... ' •...... Canada ~...... •. ·• .. · .. .:.:, ~- \ ... . ' ... ;.-.~...... I•. 4_· 9-spine . _:-:·;l· .. . ~,.~ fyl stickleback ______..:, Teleost fish are convenient models for toxicology.

aqumaps.org Biological magnification

How to connect teleost genomes to human biology? How to connect teleost genomes to human biology?

• Genome duplication in vertebrates • Genome duplication complicates connectivity • Ohnologs gone missing and lineage-specific evolution • Application: St. Lawrence Island How to connect teleost genomes to human biology? • Genome duplication in zebrafish vertebrates

Ohnologs: duplicates from genome duplication

bony fish Susumo Ohno Two rounds of Evolution by Gene genome Duplication (1970) duplication

jawed vertebrates

1 Hox cluster e shark 4 Hox clusters cartilaginous fish How to connect teleost genomes to human biology? • Genome duplication in zebrafish vertebrates

rayfin fish

bony fish

Two rounds of coelacanth genome duplication xenopus lobefin jawed fish vertebrates chicken mouse

1 Hox human cluster e shark 4 Hox clusters cartilaginous fish How to connect teleost genomes to human biology? • Genome duplication in zebrafish teleost vertebrates fish medaka rayfin cavefish fish

goldeye

eel

bony fish gar

Two rounds of coelacanth genome duplication xenopus lobefin jawed fish vertebrates chicken mouse

1 Hox human cluster e shark 4 Hox clusters cartilaginous fish How to connect teleost genomes to human biology? • Genome duplication in zebrafish teleost vertebrates fish Teleost genome medaka 7-8 Hox clusters Amores… duplication Postlethwait rayfin 1998 Science 3R, TGD cavefish fish

goldeye

eel

1R, 2R 4 Hox clusters Amores…Postlethwait bony fish gar 2011 Genetics VGD Two rounds of coelacanth genome duplication xenopus lobefin jawed fish vertebrates chicken mouse

goldeye

eel 1R, 2R 4 Hox clusters bony fish gar VGD Spotted gar: Two rounds of coelacanth • Diverged before the TGD genome duplication • Biology similar to zebrafish xenopus jawed vertebrates chicken mouse

goldeye

eel 1R, 2R 4 Hox clusters bony fish gar VGD Spotted gar: Two rounds of coelacanth • Diverged before the TGD genome duplication • Biology similar to zebrafish xenopus jawed • Genomics similar to tetrapods vertebrates chicken • Same duplication as human • Amenable to laboratory culture mouse • Can grow from embryos

1 Hox human cluster e shark 4 Hox clusters What does duplication cartilaginous fish mean for toxicology? What does duplication mean for toxicology? • Genome duplication in zebrafish How to connect teleost vertebrates genomes to human biology? Teleost genome medaka duplication 3R, TGD cavefish

goldeye

eel dioxins 1R, 2R bony fish gar VGD AHR Two rounds of coelacanth genome duplication xenopus AHR ARNT jawed chicken dioxin response cyp1a1 vertebrates element mouse nucleus

1 Hox human cytoplasm cluster e shark cartilaginous fish What does duplication mean for toxicology?

dioxins How to connect teleost AHR genomes to human biology?

AHR ARNT arnt arntl1a dioxin response cyp1a1 element arntl1b nucleus TGD cytoplasm zebrafish arnt2 arntl2

VGD1 Arnt Arntl VGD2 Arnt Arntl Arnt Arntl Tandem duplication Arnt Arntl Arnt2 Arntl2 ARNT ARNTL human ARNT2 ARNTL2 How do we know? Catchen…Postlethwait 2009 Genome Res. 19:1497 How do we know?

gene tree ,-----Hsa.ARNTI. How to connect teleost ---Dre.amtl1a genomes to human biology? ---Dre.amtl1b r------Hsa.ARNTL2 100 '------Dre.amt12 ,______-Rfl .110703

,-----Hsa .ARNT2 arnt arntl1a 100 ,...... __ --l Dre.amt2 ,------Hsa.ARNT arnt2 arntl1b 81 ~-----Dre.amt orthologs TGD zebrafish arnt2 arntl2

arnt2b arntl2b VGD1 Arnt Arntl VGD2 Arnt Arntl Arnt Arntl Tandem duplication Arnt Arntl Arnt2 Arntl2 ARNT ARNTL Arnt3 Arntl3 human ARNT2 ARNTL2 Arnt4 Arntl4 Catchen…Postlethwait 2009 Genome Res. 19:1497 How do we know?

gene tree ,-----Hsa.ARNTI. How to connect teleost ---Dre.amtl1a genomes to human biology? ---Dre.amtl1b r------Hsa.ARNTL2 100 '------Dre.amt12 ,______-Rfl .110703

,-----Hsa .ARNT2 arnt arntl1a 100 ,...... __ --l Dre.amt2 ,------Hsa.ARNT arntl1b 81 ~-----Dre.amt orthologs TGD zebrafish arnt2 arntl2

VGD1 Arnt Arntl VGD2 Arnt Arntl Arnt Arntl Tandem duplication Arnt Arntl Arnt2 Arntl2 ARNT ARNTL human ARNT2 ARNTL2

Catchen…Postlethwait 2009 Genome Res. 19:1497 How do we know?

gene tree ,-----Hsa.ARNTI. How to connect teleost ---Dre.amtl1a genomes to human biology? ---Dre.amtl1b r------Hsa.ARNTL2 100 '------Dre.amt12 ,______-Rfl .110703

,-----Hsa .ARNT2 arnt arntl1a 100 ,...... __ --l Dre.amt2 ,------Hsa.ARNT orthologs arntl1b 81 ~-----Dre.amt TGD zebrafish arnt2 arntl2

VGD1 Arnt Arntl VGD2 Arnt Arntl Arnt Arntl Tandem duplication Arnt Arntl Arnt2 Arntl2 ARNT ARNTL human ARNT2 ARNTL2

Catchen…Postlethwait 2009 Genome Res. 19:1497 How do we know?

gene tree ,-----Hsa.ARNTI. How to connect teleost ---Dre.amtl1a genomes to human biology? ---Dre.amtl1b Tree shows co-orthologs 100 '------Dre.amt12 duplication after ,______-R fl .110703 human-fish ,-----Hsa .ARNT2 arnt arntl1a 100 ,...... __ --l divergence. Dre.amt2 ,------Hsa.ARNT arntl1b 81 ~-----Dre.amt TGD zebrafish arnt2 arntl2

VGD1 Arnt Arntl VGD2 Arnt Arntl Arnt Arntl Tandem duplication Arnt Arntl Arnt2 Arntl2 ARNT ARNTL human ARNT2 ARNTL2

Catchen…Postlethwait 2009 Genome Res. 19:1497 How do we know?

conserved syntenies How to connect teleost show duplicated genomes to human biology? chromosome ARNTL Tree shows arntl1b segments duplication after human-fish arnt arntl1a divergence. arntl1a arnt2 arntl1b TGD zebrafish arnt2 arntl2

arnt2b arntl2b VGD1 Arnt Arntl VGD2 Arnt Arntl Arnt Arntl Tandem duplication Arnt Arntl Arnt2 Arntl2 ARNT ARNTL Arnt3 Arntl3 human ARNT2 ARNTL2 How do we know? Arnt4 Arntl4 Catchen…Postlethwait 2009 Genome Res. 19:1497 How do we know?

♦ ARNT + +-++t++ + +♦ + ++++ + ♦ + + ++ + ♦++ + + + + -· -*• + + ♦ + ♦ How to connect teleost + * ·-+ + ♦ ++ ++ + + + ... + genomes to human biology? Human + + + + ++-I+ ♦ ++ + + + ++ + + ++ .....+ + + ..... + ++ chromosomes + -++- ++++ * ++ ♦ Duplicated + ++ ++ + + ARNTL++ + chromosome . +- _ __.._ - -- - ➔ -++- .. -+- ·+-·- ·- .... - - .,_.,... __ +-· ++ ♦ ♦ + ··-+++ ** + + + + segments in arnt arntl1a ARNTL2 -· + + human 0 + + + -HI-++ + ++ ARNT2 ♦ + + + + + + + + + + + + genome ++• arnt2 arntl1b + + + + + + + + - + + + + ...... +++++ + ·- .... TGD + + + ... + ♦ + ♦- zebrafish arnt2 arntl2 Genes on human chromosome 11 arnt2b arntl2b VGD1 Arnt Arntl VGD2 Arnt Arntl Arnt Arntl Tandem duplication Arnt Arntl Arnt2 Arntl2 ARNT ARNTL Arnt3 Arntl3 human ARNT2 ARNTL2 How do we know? Arnt4 Arntl4 Catchen…Postlethwait 2009 Genome Res. 19:1497 How do we know?

♦ ARNT + +-++t++ + +♦ + ++++ + ♦ + + ++ + ♦++ + + + + -· -*• + + ♦ + ♦ How to connect teleost + * ·-+ + ♦ ++ ++ + + + ... + genomes to human biology? Human + + + + ++-I+ ♦ ++ + + + ++ + + ++ .....+ + + ..... + ++ chromosomes + -++- ++++ * ++ ♦ Duplicated + ++ ++ + + ARNTL++ + chromosome How do . +- _ __.._ - -- - ➔ -++- .. -+- ·+-·- ·- .... - - .,_.,... __ +-· ++ ♦ ♦ + ··-+++ ** + + + + segments in arnt arntl1a fish ARNTL2 -· + + human genes 0 + + + -HI-++ + ++ ARNT2 ♦ + + + + + + + + + + + + genome relate to ++• arnt2 arntl1b + + + + + + + + - + + + + ...... +++++ + human ·- .... TGD genes? + + + ... + ♦ + ♦- zebrafish arnt2 arntl2 Genes on human chromosome 11 arnt2b arntl2b VGD1 Arnt Arntl VGD2 Arnt Arntl Arnt Arntl Tandem duplication Arnt Arntl Arnt2 Arntl2 • Genome ARNT ARNTL duplication Arnt3 Arntl3 human complicates ARNT2 ARNTL2 How do we know? connectivity Arnt4 Arntl4 Catchen…Postlethwait 2009 Genome Res. 19:1497 How to connect teleost genomes to human biology?

• Genome duplication in vertebrates • Genome duplication complicates connectivity • Ohnologs gone missing and lineage-specific evolution • Application: St. Lawrence Island • Genome duplication complicates connectivity Protein coding Functional domains Regulatory duplication

nonfunctionalization Gene loss: most frequent fate

Alan Force Force…Postlethwait 1999 Genetics • Genome duplication complicates connectivity Protein coding Functional domains Regulatory duplication

nonfunctionalization neofunctionalization subfunctionalization Gene loss: most New functions evolve Reciprocal loss of frequent fate from ancestral genes ancestral functions

Alan Force Force…Postlethwait 1999 Genetics

Genetics 1999 Postlethwait … Force

importance of these fates. these of importance

Alan Force Alan

We don’t yet know the relative relative the know yet don’t We

from ancestral genes genes ancestral from ancestral functions functions ancestral frequent fate fate frequent

New functions evolve evolve functions New Reciprocal loss of of loss Reciprocal Gene loss: most most loss: Gene

functionalization functionalization non functionalization functionalization neo functionalization functionalization sub

• . -L • -L • •

..... - • -L / .

• - human with orthologs

Most teleost genes are 1:1 1:1 are genes teleost Most

9.5 19,602

1,853 1,853

--L • • n o od a r t Te

9.7 19,699 1,910 1,910 Medaka

--L • •

duplication duplication 10.4 20,787

2,156 2,156 Stickleback

Functional domains domains Functional

8.4 26,459

2,228 2,228

t Zebrafish

s e n ge pairs pairs

Protein coding coding Protein

% retained Coding R S etained TGD TGD etained pecies

complicates connectivity complicates duplication Genome • • • • Genome duplication complicates connectivity

Species Retained TGD Coding % retained pairs genes Zebrafish 2,228 26,459 8.4 Problem: All can occur in a lineage-specific fashion. Stickleback 2,156 20,787 10.4 Medaka 1,910 19,699 9.7

Tetraodon 1,853 19,602 9.5

What does this mean for toxicology?

nonfunctionalization neofunctionalization subfunctionalization Gene loss: most New functions evolve Reciprocal loss of frequent fate from ancestral genes ancestral functions We don’t yet know the relative importance of these fates. Alan Force Force…Postlethwait 1999 Genetics What does this mean for toxicology?

dioxins nonfunctionalization neofunctionalization subfunctionalization AHR heterodimer with ligand- heterodimer with CLOCK, bound AHR, promotes promotes expression of expression of xenobiotic circadian rhythm genes AHR ARNT metabolism genes dioxin response cyp1a1 ARNT ARNTL element nucleus ARNT2 ARNTL2 cytoplasm heterodimer with hypoxia- heterodimer with inducible factor 1alpha, CLOCK & HIF1alpha promotes expression of oxygen-responsive genes.

neofunctionalization subfunctionalization To see if this is subfunctionalization Amphioxus or neofunctionalization, must look at a pre-duplication outgroup. What about AHR? Ascidians Appendicularians

http://marineinvaders.myspecies.info/taxonomy/term/70 evodevogenomics-unibarcelona.weebly.com/oikopleura www2.le.ac.uk/departments/geology/people/goodall-r/personal/images/lancelet. What does this mean for toxicology? What about AHR?

dioxins nonfunctionalization neofunctionalization subfunctionalization AHR one AHR gene AHR ARNT

dioxin response cyp1a1 three AHR-related genes element nucleus cytoplasm

Conserved syntenies show that Gga7 Gga2 chicken and zebrafish AHR- Dre16 related genes are orthologs Dre22 three AHR-related genes

Dre22 Dre16

Karchner 2005 Biochem J. 392:153 How did AHR-related genes evolve? How did AHR-related genes evolve?

dioxins nonfunctionalization neofunctionalization subfunctionalization AHR

AHR ARNT

dioxin response cyp1a1 ahr2 ahr1b element TGD zebrafish nucleus ahr1a ahr1b’ cytoplasm

VGD2 ahr2 ahr1b chicken AHR2 AHR1B ahr ahr2 ahr1 ahr1a ahr1b’ AHR ahr1b’

human ahr1a ahr1b

AHR ahr1b’ How did AHR-related genes evolve?

dioxins nonfunctionalization neofunctionalization subfunctionalization AHR The ‘primary mediator of toxicity’ for teleosts is not the ortholog of human AHR AHR ARNT

dioxin response cyp1a1 ahr2 ahr1b element TGD zebrafish nucleus ahr1a cytoplasm

VGD2 ahr2 ahr1b chicken AHR2 AHR1B ahr ahr2 ahr1 ahr1a AHR Zebrafish three AH Rs human • AHR2 primary mediator of toxicity ~ • AHRl A deficient in TCDD binding and - =---- • transactivation activity • AHRl B functional but no known toxicological AHR roles Robert Tanguay Oregon State University 2018 10 25 Zebrafish ortholog of human AHR is an ‘incipient pseudogene’. Karchner 2005 Biochem J. 392:153 How did AHR-related genes evolve?

dioxins nonfunctionalization neofunctionalization subfunctionalization AHR Study of ahr1b may reveal sub- functions of human AHR gene AHR ARNT subfunctionalization dioxin response cyp1a1 ahr2 ahr1b element TGD zebrafish nucleus ahr1a ahr1b’ cytoplasm

VGD2 ahr2 ahr1b chicken AHR2 AHR1B ahr ahr2 ahr1 ahr1a ahr1b’ AHR ahr1b’ Zebrafish three AH Rs human • AHR2 primary mediator of toxicity ahr1a ahr1b • AHRl A deficient in TCDD binding and • transactivation activity • AHRl B functional but no known toxicological AHR ahr1b’ roles Robert Tanguay Oregon State University 2018 10 25 How did AHR-related genes evolve?

dioxins nonfunctionalization neofunctionalization subfunctionalization AHR • Ohnologs gone missing and lineage-specific evolution Postlethwait 2007 J Exp Zool B Mol Dev Evol. 308:563 AHR ARNT

dioxin response cyp1a1 ahr2 ahr1b element TGD zebrafish nucleus ahr1a ahr1b’ cytoplasm

VGD2 ahr2 ahr1b chicken AHR2 AHR1B ahr ahr2 ahr1 ahr1a ahr1b’ AHR ahr1b’

human ahr1a ahr1b

AHR ahr1b’ How to connect teleost genomes to human biology?

Savunga St. Lawrence Island Northeast Cape Russia Alaska St. Lawrence Island • Application: St. Lawrence Island 2 3 air strip 4 5 Suqitughneq River 6

7 8 12 10 11 13 1985 - 2014, $120M spent on remediation 14 Main operations of FUD • Application: St. Lawrence Island 2 3 air strip 4 5 Suqitughneq River 6

7 8 12 10 11 13 1985 - 2014, $120M spent on remediation 14 Main operations of FUD Do upstream and downstream fish differ? Do upstream and 2 w downstream fish differ? 3 en +f 0 0) 0 0) ,- .s Alaska blackfish ~ IIl 4 0e:. ro § C: 0 5 ~ lO ::E 6

0 i i 2 3 4 5 0 7 8 7 Estuary Site number 8 12 10 11 => significantly more 13 PCBs in downstream fish

Are PCBs from global distillation or the FUD?

von Hippel 2018 Environmental Pollution 234:279 Are PCBs from global distillation or the FUD? 3 Greater 0000 00 00 00 0 => Upstream from global distillation upstream => Downstream PCBs from FUD + distillation. Greater 0 0 0 000 00000000 0 0000000000000 000000000 0 000000000 000 0 0 0000 0000000 000000000000 0 0000 0000 light heavy down stream 6

7 8 12 10 11 13

14 Main operations of FUD Danielle Dillon What are the biological effects? What are the biological effects? sex genotype PCR Gene expression differences?

4 females from highly contaminated sites 4 females from less contaminated sites

BIOO NEXTflex qRNA-seq

Strand-specific: guards against gDNA Unique molecular indexes: guards against PCR duplicates

Tom Titus UO Aligned 9-spine reads to 3-spine genome brca2 paired-end reads

3-spine genome 3’UTR unannotated polymorphisms ■ -- 9-spine PCBs low strand specific wrong way => genomic contamination

9-spine PCBs high

Pete Batzel Aligned 9-spine reads to 3-spine genome brca2 paired-end reads

3-spine genome

■ -- 9-spine PCBs low compared to

I I 9-spine PCBs high

DESeq2: Finds differentially expressed genes

Pete Batzel DESeq2: Finds differentially expressed genes

Differentially expressed genes Females: contamin/uncontam 2312 Males: contamin/uncontam 642

What do these genes do? DAVID 6.7: Database for Annotation, Visualization and Integrated Discovery What do these genes do? DAVID 6.7: brca2 (fancd1) = down Annotation Cluslllr1 Enrichment Sc:otr.15.17 k, Cowit P_Value BenJamlnl cdc23 = down UP _KEYWORDS Cell cycle RT 135 1.9E-20 S.2E-l8 UP _KEYWORDS - fbxo5 = down Cell division cell cycle genes 9 1 1.2E-17 l.7E-l5 UP _KEYWORDS Mitosis ..... 69 3.7E-16 3.0E-14 h2ax = down GOTERM_BP _DIRECT cell division RT - 83 1. l E-13 2.6E-10 haus6 = down - mad2l1 = down rab11fip4a = down llgl2 = down ccnb1 = down most tgf betas down What do these genes do? brca2 (fancd1) = down Annotation Cluslllr1 Enrichment Sc:otr.15.17 k, Cowit P_Value BenJamlnl cdc23 = down UP _KEYWORDS Cell cycle RT 135 1.9E-20 S.2E-l8 UP _KEYWORDS fbxo5 = down Cell division RT - 9 1 1.2E-17 l.7E-l5 UP _KEYWORDS Mitosis RT - 69 3.7E-16 3.0E-14 h2ax = down GOTERM_BP _DIRECT cell division RT - 83 1. l E-13 2.6E-10 haus6 = down GOTERM_BP _DIRECT mitotic nuclear division RT -• 63 1.SE-11 2.3E-8 mad2l1 = down Annotation Clusta, 2 Enrichment Scorw: 11.1, Ill,, Cowit P_Value B■nJ■mlnl UP _KEYWORDS - - --~------'UI rab11fip4a = down ii 1 ""'-'" Q RE-14 GOTERM_BP _DIRECT llgl2 = down ii DNA replication genes E-8 ccnb1 = down most tgf betas down mcm3,4,6,7 cdc45 hsc70 pcna rbbp4 tert terf1, terf2 What do these genes do? brca2 (fancd1) = down Annotation Cluslllr1 Enrichment Sc:otr.15.17 k, Cowit P_Value BenJamlnl cdc23 = down UP _KEYWORDS Cell cycle RT 135 1.9E-20 5.2E-l8 UP _KEYWORDS fbxo5 = down Cell division RT - 91 1.2E-17 l.7E-l5 UP _KEYWORDS Mitosis RT - 69 3.7E-16 3.0E-14 h2ax = down GOTERM_BP _DIRECT cell division RT - 83 1. lE-13 2.6E-10 haus6 = down GOTERM_BP _DIRECT mitotic nuclear division RT -• 63 1.5E-11 2.3E-8 mad2l1 = down Annotation Clusta, 2 Enrichment Scorw: 11.1, Ill,, Cowit P_Value B■nJ■mlnl rab11fip4a = down UP _KEYWORDS D A replication ii 38 1.5E-15 9.8E-l4 GOTERM_BP _DIRECT llgl2 = down Gl/5 transit ion of mitot ic cell cycle ii 36 3.lE-11 3.7E-8 GOTERM_BP _DIRECT D A replication ini iation i 19 1. 3E-10 l.2E-7 ccnb1 = down Annotation Cluslllr 3 Enrichment Score: 8.IM k, Cowit . P_ Value BenJ■ mlnl most tgf betas down UP _KEYWORDS AT 67 4 . 4E-9 l.8E-7 UP _KEYWORDS 58 1.8E-8 5.4E-7 mcm3,4,6,7 DNA repair genes cdc45 hsc70 pcna rbbp4 tert terf1, terf2 Fanconi anemia genes = down What do these genes do? brca2 (fancd1) = down Annotation Cluslllr1 Enrichment Sc:otr.15.17 k, Cowit P_Value BenJamlnl cdc23 = down UP _KEYWORDS Cell cycle RT 135 1.9E-20 5.2E-l8 UP _KEYWORDS fbxo5 = down Cell division RT - 9 1 1.2E-17 l.7E-l5 UP _KEYWORDS Mitosis RT - 69 3.7E-16 3.0E-14 h2ax = down GOTERM_BP _DIRECT cell division RT - 83 1. l E-13 2.6E-10 haus6 = down GOTERM_BP _DIRECT mitotic nuclear division RT -• 63 1.5E-11 2.3E-8 mad2l1 = down Annotation Clusta, 2 Enrichment Scorw: 11.1, Ill,, Cowit P_Value B■nJ■mlnl rab11fip4a = down UP _KEYWORDS D A replication ii 38 1.5E-15 9.8E-l4 GOTERM_BP _DIRECT llgl2 = down Gl/5 transition of mitotic cell cycle ii 36 3. l E-11 3.7E-8 GOTERM_BP _DIRECT D A replication ini iation i 19 1. 3E-10 l.2E-7 ccnb1 = down Annotation Cluslllr 3 Enrichment Score: 8.IM k, Cowit . P_ Value BenJ■ mlnl most tgf betas down UP _KEYWORDS D Adamaqe • 67 4 . 4E-9 l.8E-7 UP _KEYWORDS D A repair 58 1.8E-8 5.4E-7 mcm3,4,6,7 • GOTERM_BP _DIRECT D A repair • 46 1.9E-5 5.7E-3 cdc45 Annotation Cluster• Enrichment Scan: 8.39 Ila. Cowit P_ Yalu ■ BenJ ■ mlnl hsc70 UP _KEYWORDS Centromere RT ii 36 1.0E-8 3.6E-7 pcna UP _KEYWORDS Chromosome chromosome behavior 8 3.7E-7 rbbp4 GOTERM_BP _DIRECT sister chromatid cohesion 8 6.9E-6 UP _KEYWORDS tert Kinetochore genes 7 2.8E-6 GOTERM_CC_DIRECT kinetochore RT ii 20 1.7E-4 9.4E-3 terf1, terf2 Fanconi anemia genes = down ercc4 msh6 pola1, pola2 What about duplicated genes? prim1 What about duplicated genes?

nonfunctionalization neofunctionalization subfunctionalization 246 TGD gene pairs had at least one copy that was differentially expressed. 21 pairs had both copies differentially expressed. 4 of those 21 pairs had one copy up, other copy down in contaminated fish.

Gene baseMean log2FC pvalue padj 225 TGD pairs had only one copy DE. camklga 27.345 -2.275 0.003 0.037 Study of how they differ with toxicants camklgb 60.386 1.587 0.010 may give insight into mechanisms. fabpl0a 5935.662 2.457 0.000 fabpl0b 32.048 -1.766 0.001

fam65a 91.364 1.367 0.002 fam65b 64.561 -1.156 0.009 neofunctionalization subfunctionalization TGD pairs provide that important advantage kcnj2a 97.195 -2.508 0.001 for analysis of toxicity. .r'_ ~-- --"'."-·- ,. ' .,-.~ . . -Ji.. kcn '2b 18.028 " "" .' ,;...... " ' ..,._,. ' - !►....;~. ·_ ' .._.. -_ -· Water at North East Cape is toxic to stickleback genome. Water at North East Cape is toxic to stickleback genome. DNA repair pathways: low expression in contaminated sites. Cell cycle genes: low expression in contaminated sites. DNA replication: low in contaminated sites. Estrogenic effect on males.

Even after site remediation: • contaminants persist, • accumulate in fish, Health of indigenous • alter endocrine pathways, people is put at risk. • alter expression of genome stability genes. How to connect teleost genomes to human biology?

• Genome duplication in vertebrates • Genome duplication complicates connectivity • Ohnologs gone missing and lineage-specific evolution • Application: St. Lawrence Island Genome duplication and fish models for toxicology Loren Frank von Hippel Buck N AZ U N AZ U

Jesse Gologergen Sivuqaq Island

Pete Batzel UO Tom Ingo Braasch Titus Tiffany Immingen UO Sivuqaq Island··-·•

NIH ' DIVISION OF COMPARATIVE ~\. Nationa_l Institute of En~ir~nmental Hea~~S · - MEDICINE lilllr'/ Your Environment. Your Health. ciences

What are the biological effects?

sex genotype PCR Tapi River

Suqi River Ninespine stickleback

800

FUD 600

[vitellogenin] in 400 males (µg/g) 200 => estrogenic effect on Suqi males 0 Sq2 Sq4 Sq5 Sq6 Sq7 Sq11 Sq13 Tapi What is the genetic response? : . Mlfouplolo Ind Placental mommolo: 3 homolog1 What does this mean for toxicology? ,-., AHR2. Chicken ENSMGAG00000002128, Turkey ENSAPLG00000003921 , Duck ENSFALG00000003870, Flycalcher What about AHR? ENSOANG000000135-48, Pllltypul Eu1eleol1eomorpha: 42 homolog1 nonfunctionalization neofunctionalization subfunctionalization ENSPNAG00000005817, Recl,belllod piranha ENSAMXG00000043051 , Cave fllh ENSIPUG00000002619, Channel Clttllh ENSPNAG00000015192. Recl,bellled piranha dioxins OOleoglonttonnH: 4 homolog1 EutlleolleOmOrphl: 44 homologa 1hr2, Red-bellied pln1nh1 AHR ahr2, Cove lllh ■ hr2. Channel catfish lhr2. Zebrllflah Teleoat fl111H : 8 homologo ENSPNAG00000003288, Recl,belllod piranha ahr2, Spotted gar ENSLACG00000018685, Coel1<1nlh Cheroclphy-: 2 homologl ~ ~--- lhr1a. Zebr1fl1h AHR ARNT lhr11, Northern plkl Oateoglo11tt0nnff: 2 homolog1 lhr1 I. Spotted gar Placental mammala: 14 homotog1 r Homlnlnes: 3 homolog1 AC019117.2, Humon dioxin response cyp1a1 AHR, Hurrn,n ENSPPYG0000001TT58, orangutan element ENSNLEG00000016550, Gibbon f Old World monkeyl: 10 homolog1 New Wor1d monkeys: 4 homologI 0 ENSTSYG00000010830, Tarsler nucleus Wet noae lemura: 3 homologa ENST8EG00000007210, Tree Shrew Rodents: 9 homolog1 cytoplasm Leurul1therl1n m1mma11: HS h0mok>g1 Placental ■ : 5 homologa Marauplala: 3 homok>ga ENSOANG00000009e11 . Ptatypu1 ENSACAG00000023372, Anole lizard e AHR, Chicken • • , ENSMGAG00000010174, Tur1cey • S ENSAPLG00000008789. Duck . Perching birds : 2 homologa LENSPSIG00000010051 , Chlneoe eottohell lurtle ahr, Xenopua .... ahr1 ■ , Coelecanth Lamprey: 3 homologa ver1e1>r,1eo: 5 homolog1 Perching blrdo: 2 homologo ENSMGAG00000002118, Tur1cey AHR1B, Chicken ENSPSIG00000018138, ChlneH aott1hell lurtle r ENSMODG00000027484, Opo11um • ENSOCUG00000028055, Rabbit Ohr! b, Coelocanth

EuleleOateornOg1 Placental ■ : 5 homologa Marauplala: 3 homok>ga ENSOANG00000009e11 , PlatypUI ENSACAG00000023372, Anole lizard e AHR, Chicken • • , ENSMGAGOOOOOOI0174, Tur1cey • S ENSAPLG00000008789. Duck . Perching birds : 2 homologa LENSPSIG00000010051 , Chlneoe eottohell turtle ahr, Xenopua .... ahr1 ■ , Coelecanth Lamprey: 3 homologa ver1e1>r,1eo: 5 homolog1 Perching blrdo: 2 hornologo ENSMGAG00000002118, Tur1cey AHR1B, Chicken ENSPSIG00000018136, C:~!oeH aottahetll tun.Ml r ENSMODG000000274&ii, Opo11um • ENSOCUG0000..'!128055, Rabbit Ohr1 b, Coelocanth EuteleOateornorpha: 33 homolog1 lhr1 b, Red-bellied pln1nha lhrl b, Cave fllh ahr1b, Channel cattloh ahr1 b, Zebralllh • Aalan bonytongue: 2 homolog1 zebrafish and chicken have ahr1b Euteleoateomorph1: 45 homologo ENSPNAG0000000528 I, Recl,bellled piranha ENSAMXG00000035088, Cava floh ahr1 b, Pan1mormyropo klng1ieyN Karchner 2005 Biochem J. 392:153 ENSLBEG000000111998, Ballan wraaae