Comparative / Evolutionary Genomics

Transcription

1 Canestro et al 2003 Genome Biology Comparative / Evolutionary Genomics What processes have shaped metazoan genomes? What genes are responsible for anatomical & physiological differences among metazoan taxa? How can comparative genomics inform studies of gene regulation and function? Evolution of biochemical processes/pathways? Evolution and diversification of multi-gene familes?

2 Comparative / Evolutionary Genomics Human <----> Biology (marine) Comparative / Evolutionary Genomics Introduction Cross-species sequence comparisons Genome structure and evolution Genome duplication in chordate evolution - The 2R hypothesis - A fish-specific duplication? - Origin and fate of duplicated genes

3 Cross-species sequence comparisons What can we learn? Chromosome structure: conserved synteny; conserved contiguity Gene identification & structure: - exon-intron boundaries (e.g. mouse) Conservation of gene structure: variation across a typical gene Based on 3,165 human-mouse pairs of mrnas Waterston et al 2002 Nature 420:520 (Figure 25)

4 Cross-species sequence comparisons What can we learn? Chromosome structure: conserved synteny; conserved contiguity Gene identification & structure: - exon-intron boundaries (e.g. mouse) Gene function Regulatory elements promoter; intronic; distal Cross-species sequence comparisons: some considerations Question being asked

5 Cross-species sequence comparisons: some considerations Question being asked Genes: true orthologs or paralogs? (draw) Cross-species sequence comparisons: some considerations Question being asked Genes: true orthologs or paralogs? (draw) Chromosomal segments: do you expect conserved contiguity? (e.g. human-mouse)..conserved synteny? (e.g. human-fugu)

6 Conserved synteny and contiguity between human and mouse (diverged 75 MYA) Waterston et al 2002 Nature 420:520 Conserved synteny between human and Fugu (diverged 450 MYA) scaffold # Fugu genes Aparicio et al 2002 Science 297:1283

7 Cross-species sequence comparisons: some considerations Question being asked Genes: true orthologs or paralogs? Chromosomal segments do you expect conserved contiguity? (e.g. human-mouse) conserved synteny? (e.g. human-fugu) Choice of species? - evolutionary distance (conserved sequences by selection or chance) Cross-species sequence comparisons: Strategies & Methods Reduce false positives (conservation by chance) - increase evol. dist. (phylogenetic footprinting) - add more (closely related) species (phylogenetic shadowing) Alignments - local (e.g. PipMaker) - global (e.g. VISTA) (examples)

8 Comparative Sequence Analysis: MultiPipMaker (Local alignments) Percent Identity Plot (PIP) of the Human ST7 (Suppression of tumorigenicity 7) gene (CFTR region of Hs7q31) Frazer et al 2003 Genome Res. 13:1-12 Comparative Sequence Analysis: MultiPipMaker Percent Identity Plot (PIP) of the Human ST7 (Suppression of tumorigenicity 7) gene (CFTR region of Hs7q31) Blue: coding exons Yellow: introns Red: conserved non-coding sequences (>100 bp and >70% ID) Conserved non-coding sequence (CNS) Frazer et al 2003 Genome Res. 13:1-12

9 Comparative Sequence Analysis: VISTA (Global alignments) b 8 9 Frazer et al 2003 Genome Res. 13:1-12 Phylogenetic footprinting (long-range comparative sequence analysis) Fickett & Wasserman (2000) Cur Op Biotechnol 11:19 Based on Aparicio et al (1995) 92: 1684

10 Comparative Sequence Analysis: Phylogenetic shadowing Phylogenetic shadowing enables multiple comparisons among DNA sequences from closely related species. In this way, the least variable regions of the genome, which should include exons and regulatory elements, can be identified. Gibbs & Nelson (2003) Science 299:1331 Boffelli et al (2003) Science 299: 1391 Comparative Sequence Analysis: Phylogenetic shadowing Figure 1. Likelihood ratios under a fast- versus slow-mutation regime for genomic intervals containing apo-b exon 19 (A), CETP exon 8 (B), LXR- exon 3 (C), and plasminogen exon 6 (D). The x axis represents the position in the multiple alignment consensus sequence, and the y axis the log likelihood ratio at that position. Boffelli et al (2003) Science 299: 1391

11 rvista Frazer et al 2003 Genome Res. 13:1-12 Genome structure and evolution What processes shape metazoan genomes?

12 Genome structure and evolution What processes shape metazoan genomes? Gene duplication Gene loss (deletion / inactivation) Genome duplication Intra-chromosomal rearrangements (inversions, duplications) Inter-chromosomal rearrangements Mobile genetic elements - Class I - retrotransposons LINES SINES processed pseudogenes - Class II - DNA transposons Domain shuffling / accretion Gene conversion Genome duplication: A common theme in eukaryotic genome evolution Taxon Xenopus laevis Salmonids Catastomids Yeast Arabidopsis Oryza (rice) Vertebrates? Bony Fishes? Estimated date of tetraploidization 30 MYA MYA 150 MYA 65 MYA MYA 450 MYA MYA

13 Genome duplication Why? Mechanisms: - allotetraploidy - autotetraploidy Consequences: - re-diploidization - divergent resolution (reciprocal silencing) Susumu Ohno City of Hope National Medical Center and Beckman Research Institute

14 Susumu Ohno Evolution by Gene Duplication With 28 Figures 1970 Springer-Verlag New York - Heidelberg - Berlin Genome size Taxon pg DNA/haploid cell Ciona 0.21 Amphioxus 0.60 Eptatretus (hagfish) 2.8 Lampetra 1.4 Oncorhynchus (salmon) 3.15 Fundulus 1.5 Spheroides (puffer) 0.50 Lepidosiren (lungfish) Homo 3.5 The state of apparent chaos which prevails with regard to genome sizes in fish is merely a reflection of nature s great experiment with genome duplication (Ohno, p. 125) Data from Ohno and/or from Hinegardner

15 Urochordates Hemichordates Chordates Cephalochordates Craniates Vertebrates Hagfish Lamprey Gnathostomes Cartilag. fish Bony fish Lungfish Tetrapods 400 Echinoderms Genome duplication Genome duplication 550 (MYA) Invertebrate (protostome) ancestor Ohno 1970 Carroll 1995 Nature 376:479

16 Urochordates Hemichordates Chordates Cephalochordates Craniates Vertebrates Hagfish Lamprey Gnathostomes Cartilag. fish Bony fish Lungfish Tetrapods Echinoderms Gen(om)e duplication Gen(om)e duplication 550 (MYA) Invertebrate (protostome) ancestor Holland et al 1994 Extensive gene duplication in early vertebrate evolution When did it occur? What mechanisms were involved? - Gene or genome duplications? - Auto- or allo-tetraploidy? - Octaploidy?

17 Evidence bearing on the question of tetraploidy in vertebrate evolution Gene number (overall: vertebrates vs invertebrates) Modified from Furlong & Holland 2001 Gene numbers in sequenced metazoan genomes Species # Genes C. elegans 19,000 Drosophila 13,500 Strongylocentrotus (urchin) (22,000) Ciona 16,000 Amphioxus? Lamprey? Fugu (puffer) 31,000 Mus (mouse) 27,000 Homo 32,000

18 Evidence bearing on the question of tetraploidy in vertebrate evolution Gene number (overall: vertebrates vs invertebrates) Genes per gene family in sequenced genomes ( tetralogy 1-to-4?) Modified from Furlong & Holland 2001 Diversification of Gene Families in Vertebrates Hox Hox Wnt Dlx clusters genes genes genes Drosophila Amphioxus Bony fish 4 ~ Mammals Source: Carroll (1995); Holland et al. (1994)

19 Lander et al 2001 Figure 39 Simplified cladogram of the 'many-to-many' relationships of classical nuclear receptors. Triangles indicate expansion within one lineage; bars represent single members. Numbers in parentheses indicate the number of paralogues in each group The Metazoan bhlh-pas Gene Superfamily Human Drosophila C. elegans Fundulus MP Majority rule (Hahn 2001) ARNT ARNT2 DmARNT CeARNT BMAL1 / MOP3 BMAL2 / MOP9 DmCYCLE DmMET DmMet-Like MOP22 CeC15C8 AHR FhAHR1 FhAHR2 AHRR FhAhRR DmAHR CeAHR CLOCK1 CLOCK2 / NPAS2 DmCLOCK DmTAIMAN NRC1 NRC2 NRC3 HIF1 HIF2 HIF3 SIM1 SIM2 DmSIM NPAS1 NPAS3 / MOP6 DmTRH CeT01D3 DmSIMA CeHIF DmPer Per1 Per3 Per2 AtZEITLUPE ARNT BMAL MET/MOP22 AHR CLOCK NRC HIF SIM TRH PER

20 Venter et al 2001 Figure 12. Gene duplication in complete protein clusters. The predicted protein sets of human, worm, and fly were subjected to Lek clustering (27). The num bers of clusters with varying ratios (whole number) of human versus worm and human versus fly proteins per cluster were plotted. Lander et al 2001 Figure 49. Number of human paralogues of genes having single orthologues in worm and fly. Ortholog groups : 2.4 genes per group (human) versus 1.1 (fly or worm)

21 Lander et al 2001 One of the most controversial hypotheses about vertebrate evolution is the proposal that two WGD events occurred early in the vertebrate lineage, around the time of jawed fishes some 500 Myr ago...we analysed the draft genome sequence for evidence that might bear on this question. The analysis provides many interesting observations, but no convincing evidence of ancient WGD. We also examined human proteins in the IPI for which the orthologues among fly or worm proteins occur in the ratios 2:1:1, 3:1:1, 4:1:1 and so on (Fig. 49). The number of such families falls smoothly, with no peak at four and some instances of five or more homologues. Although this does not rule out two rounds of WGD followed by extensive gene loss and some unrelated gene duplication, it provides no support for the theory. Furlong & Holland: strong evidence for extensive gene duplication in the vertebrate lineage, but they do not support a strict 1:4 rule. Furlong & Holland 2001 (Using Amphioxus as a starting point)

22 Evidence bearing on the question of tetraploidy in vertebrate evolution Gene number (overall: vertebrates vs invertebrates) Genes per gene family in sequenced genomes ( tetralogy 1-to-4?) Gene families: topologies (when did duplication occur?) - (AB)(CD) versus asymmetric arrangements Modified from Furlong & Holland 2001 (AB)(CD) vs. (A,(B,(C,D))) Lander et al if two successive rounds of genome duplication occurred, phylogenetic analysis of the proteins having 4:1:1 ratios between human, fly and worm would be expected to show more trees with the topology (A,B)(C,D) for the human sequences than (A,(B,(C,D))). However, of 57 sets studied carefully, only 24% of the trees constructed from the 4:1:1 set have the former topology; this is not significantly different from what would be expected under the hypothesis of random sequential duplication of individual loci. (Hughes; Martin make similar arguments) (what are the assumptions of the (A,B)(C,D) prediction??)

23 Evidence bearing on the question of tetraploidy in vertebrate evolution Gene number (overall: vertebrates vs invertebrates) Genes per gene family in sequenced genomes ( tetralogy 1-to-4?) Gene families: topologies (when did duplication occur?) - (AB)(CD) versus asymmetric arrangements* Paralogy regions (paralogons) within a genome (linked, unrelated sets of paralogs) - existence - coincidence of duplication dates? - congruent tree topologies for linked gene families? Modified from Furlong & Holland 2001 MYOD IGF-2 LDH-A LDH-C RAS(1) AAAH1 AAAH2 PTH-2 LDH-B RAS(2) AAAH3 PTH-1 MYOD3 MYOD2 IGF-1 Hsa11 Hsa12 Patton et al 1998 Mol Biol Evol.

24 MYOD IGF-2 LDH-A LDH-C RAS(1) AAAH1 AAAH2 PTH-2 invert vert LDH-B RAS(2) AAAH3 PTH-1 MYOD3 MYOD2 IGF-1 Hsa11 Hsa12 Patton et al 1998 Mol Biol Evol. IGF-1 AAAH1 Ancestral invertebrate Patton et al 1998 Mol Biol Evol.

25 IGF-1 AAAH1 AAAH2 AAAH3 Ancestral invertebrate after tandem duplications of AAAH Patton et al 1998 Mol Biol Evol. IGF-1 IGF-2 AAAH1 AAAH2 AAAH3 AAAH4 AAAH5 AAAH6 Ancestral chordate after genome/chromosome duplication Patton et al 1998 Mol Biol Evol.

26 IGF-1 vert IGF-2 AAAH1 AAAH2 invert AAAH6 Divergent gene loss Tandem gene duplications and divergent gene loss complicate the phylogenetic signal from paralogy regions. Patton et al 1998 Mol Biol Evol. systematic analysis of human genome for paralogous chromosomal regions (paralogons) many more paralogons that expected by chance (6120 genes in 1,642 paralogons) timing: burst of gene duplication activity MYA Our results support the contention that many of the gene families in vertebrates were formed or expanded by large-scale DNA duplications in an early chordate...the results are compatible with at least one round of polyploidy.

27 Dated 1,739 gene duplication events from phylogenetic analysis of 749 vertebrate gene families Two waves: one recent (tandem/segmental); one in early vertebrate evolution supports the idea of genome duplication(s) large- and small-scale gene duplications both make a significant contribution during the early stage of vertebrate evolution. Urochordates Hemichordates Chordates Cephalochordates Craniates Vertebrates Hagfish Lamprey Gnathostomes Cartilag. fish Bony fish Lungfish Tetrapods Echinoderms Autoautooctoploid 550 (MYA) Invertebrate (protostome) ancestor Furlong & Holland 2001

28 auto-tetraploidy auto-tetraploidy allo-tetraploidy auto-tetraploidy Furl ong & Holland 2001 from Furlong & Holland 2001

29 Zebrafish hox Clusters and Vertebrate Genome Evolution Amores, et al. Science 1998 November 27; 282: Genome duplication in ray-finned fishes?

30 Zebrafish hox Clusters and Vertebrate Genome Evolution Amores, et al. Science 1998 November 27; 282: Zebrafish hox Clusters and Vertebrate Genome Evolution Amores, et al. Science 1998 November 27; 282:

31 Smith et al 2002 Genome Res. 12:776

32 22 20 of 49 gene families outgroup topology phylogeny and synteny data suggest that the common ancestor of zebrafish and pufferfish experienced a large-scale gene or complete genome duplication event and that the pufferfish has lost many duplicates that the zebrafish has retained. Taylor, et al. (2003) Genome Res : Gene duplication: fate of duplicated genes

33 Gene duplication: fate of duplicated genes Non-functionalization Neo-functionalization Gene duplication: fate of duplicated genes Non-functionalization Neo-functionalization Sub-functionalization

34 Duplication, degeneration, complementation (DDC) model of gene evolution 1 H L B Original gene with multiple functions (expression pattern) H L B H L B Fully redundant duplicates H B L B Complementary duplicates Regulatory subfunctionalization Diagram by Hahn 2003, based on the hypothesis of Force, et al. (1999) Genetics 151: 1531; Lynch & Force (2000) Genetics 154:459. Force et al Genetics 151: 1531

35 Force et al Genetics 151: 1531 Force et al Genetics 151: 1531

36 DDC model of gene evolution A B A B kinase Original protein with multiple functions (substrates) A B kinase A B kinase Fully redundant duplicates B kinase A kinase Complementary duplicates Structural subfunctionalization Diagram by Hahn 2003, based on the hypothesis of Force, et al. (1999) Genetics 151: 1531; Lynch & Force (2000) Genetics 154:459. Implications of the DDC model Use of duplicated genes (e.g. zebrafish) to dissect multiple functions of single human gene Fewer duplicates retained after second round of tetraploidization

37 Summary Cross-species sequence comparisons - various strategies; gene-dependent - methods evolving Genome duplication in chordate evolution? - strong evidence for extensive duplication of genes and chromosomal segments - 2R possible but mechanism unclear - combination of gene & genome duplic, gene loss complicates assessment Genome duplication in fish evolution - strong evidence Origin and fate of duplicated genes - Most lost - DDC model may explain higher-than-expected rate of retention of duplicated genes Terms Synteny Conserved synteny Conserved contiguity (conserved segment) Paralogon Homolog Ortholog Paralog Co-ortholog (Semi-ortholog)

38 References Sequence comparisons: Frazer et al (2003) Cross-species sequence comparisons: a review of methods and available resources. Genome Res. 13:1-12. Fickett & Wasserman (2000) Discovery and modeling of transcriptional regulatory regions. Curr.Opin. Biotechnol. 11: Genome duplication in vertebrates: Wolfe (2001) Yesterday s polyploids and the mystery of diploidization. Nature Rev. Genet. 2: 333. Furlong and Holland (2002) Were vertebrates octoploid? Phil. Trans. R. Soc. London B 357:531 Genome duplication in fishes: Taylor, et al. (2001) Comparative genomics provides evidence for an ancient genome duplication event in fish. Phil. Trans. R. Soc. Lond. B 356: Taylor, et al. (2003) Genome duplication: a trait shared by 22,000 species of ray-finned fish. Genome Res. 13:382. Amores, et al (1998) Zebrafish hox clusters and vertebrate genome evolution. Science 282: Fate of gene duplicates: Force, et al (1999) Preservation of duplicate genes by complementary, degenerative mutations. Genetics 151: Lynch, M. and Conery, J.S. (2000) The evolutionary fate and consequences of duplicate genes. Science 290: Lynch, M. and Force, A. (2000) The probability of duplicate gene preservation by subfunctionalization. Genetics 154: