1 / 53

Genome Evolution in Yeast

27 th January 2009 | European Course on. Genome Evolution in Yeast. Gilles Fischer. INTRODUCTION: Comparative genomics Yeasts as model organisms GENOME EVOLUTION: DNA duplications Chromosome dynamics Nucleotide composition. A brief introduction to the field of Comparative Genomics.

crescent
Download Presentation

Genome Evolution in Yeast

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. 27th January 2009 | European Course on Genome Evolution in Yeast Gilles Fischer

  2. INTRODUCTION: Comparative genomics Yeasts as model organisms GENOME EVOLUTION: DNA duplications Chromosome dynamics Nucleotide composition

  3. A brief introduction to the field of Comparative Genomics Comparing genomes is a very old idea… DNA carries the genetic information: Avery (1943) and Hershey-Chase (1952) Vendrely and Vendrely (1950): "Il ne fait aucun doute que l'étude systématique de la teneur absolue du noyau en acide désoxyribonucléique, à travers de nombreuses espèces animales puisse fournir des suggestions intéressantes en ce qui concerne le problème de l'évolution" Jacques Monod: "Tout ce qui est vrai pour le colibacille est vrai pour l'éléphant"

  4. A brief introduction to the field of Comparative Genomics identical divergent different time or quantity of evolutionary changes Looking for differences Looking for similarities

  5. A brief introduction to the field of Comparative Genomics identical divergent different time or quantity of evolutionary changes Looking for differences Looking for similarities NEED FOR ADEQUATELY RELATED ORGANSIMS

  6. Experimental Biology Genetic screens Mechanistic hypotheses Molecular mechanisms functional genomics A brief introduction to the field of Comparative Genomics Bio-informatics Looking for differences Rules governing genome evolution Genome sequences Looking for similarities

  7. A brief introduction to the field of Comparative Genomics Bio-informatics Looking for differences Rules governing genome evolution Genome sequences Looking for similarities SMALL GENOMES AND EXPERIMENTALLY TRACTABLE Experimental Biology Genetic screens Mechanistic hypotheses Molecular mechanisms functional genomics

  8. A brief introduction to the field of Yeast Genomics Organisms with small genomes, phylogenetically related and experimentally tractable = YEASTS • Eukaryotic micro-organisms classified in the kingdom Fungi • About 1,500 species currently described (only 1% of all yeast) • Yeasts are unicellular, typically measuring 3–4 µm in diameter (up to over 40 µm) • Saccharomyces cerevisiae used in baking and fermenting alcoholic beverages for thousands of years • Other species of yeast, such as Candida albicans, are opportunistic human pathogens • Yeasts have recently been used to generate electricity in microbial fuel cells and produce ethanol for the biofuel industry. • Yeasts are found in both divisions Ascomycota and Basidiomycota • The budding yeasts ("true yeasts") are classified in the Saccharomycotina subphylum

  9. A brief introduction to the field of Yeast Genomics Organisms with small genomes, phylogenetically related and experimentally tractable = YEASTS The Tree of Eukaryotes (Keeling et al., 2005)

  10. Saccharomycotina Saccharomyces paradoxus Saccharomyces mikatae Saccharomyces cerevisiae Saccharomyces kudriavzevii Saccharomyces bayanus Saccharomyces pastorianus Saccharomyces exiguus Saccharomyces servazzii Saccharomyces castellii Candida glabrata Vanderwaltozyma polyspora Zygosaccharomyces rouxii Lachancea thermotolerans Lachancea waltii Lachancea kluyveri Kluyveromyces lactis Kluyveromyces marxianus Eremothecium gossypii Saccharomycodes ludwigii Brettanomyces bruxellensis Pichia angusta Candida lusitaniae Debaryomyces hansenii Pichia stipitis Pichia sorbitophila Candida guilliermondii Candida tropicalis Candida parapsilosis Lodderomyces elongisporus Candida albicans Candida dubliniensis Arxula adeninivorans Yarrowia lipolytica Schizosaccharomyces pombe A brief introduction to the field of Yeast Genomics The first eukaryotic genome sequence: The genome of S. cerevisiae André Goffeau 8 years, 120 labs, 641 people Life with 6000 genes Science (1996)

  11. Saccharomycotina Saccharomyces paradoxus Saccharomyces mikatae Saccharomyces cerevisiae Saccharomyces kudriavzevii Saccharomyces bayanus Saccharomyces pastorianus Saccharomyces exiguus Saccharomyces servazzii Saccharomyces castellii Candida glabrata Vanderwaltozyma polyspora Zygosaccharomyces rouxii Lachancea thermotolerans Lachancea waltii Lachancea kluyveri Kluyveromyces lactis Kluyveromyces marxianus Eremothecium gossypii Saccharomycodes ludwigii Brettanomyces bruxellensis Pichia angusta Candida lusitaniae Debaryomyces hansenii Pichia stipitis Pichia sorbitophila Candida guilliermondii Candida tropicalis Candida parapsilosis Lodderomyces elongisporus Candida albicans Candida dubliniensis Arxula adeninivorans Yarrowia lipolytica Schizosaccharomyces pombe A brief introduction to the field of Yeast Genomics Whole Genome Duplication Gain of Megasatellites Gain of HO gene Gain of mating type cassettes and small centromeres frequent tandem duplications Extensive loss of transposable elements and spliceosomal introns

  12. Genome annotation # genes # introns # chr size (Mb) # tRNA 16 12,1 5769 287 274 13 12,3 5204 131 207 7 9,8 4998 167 272 8 11,3 5308 322 258 8 10,4 5104 286 231 6 10,7 5084 175 162 7 12,1 6273 475 200 6 20,5 6434 1070 510 A brief introduction to the field of Yeast Genomics Saccharomyces cerevisiae Candida glabrata Zygosaccharomyces rouxii Lachancea kluyveri (WashU seq center M. Jonhston) Lachancea thermotolerans Kluyveromyces lactis Debaryomyces hansenii Yarrowia lipolytica

  13. amino acid identity % 100 * Homo sapiens 65 - - - Takifugu rubripes Tetraodon negroviridis 60 51 Ciona intestinalis 48 Evolutionary scale A brief introduction to the field of Yeast Genomics Saccharomyces cerevisiae 100 * 90 70 50 100 MYr 100 MYr Candida glabrata Zygosaccharomyces rouxii 450 MYr 100 - 300 MYr Lachancea kluyveri Mus musculus Lachancea thermotolerans 550 MYr 300 - 1000 MYr Kluyveromyces lactis Debaryomyces hansenii Yarrowia lipolytica *Dujon et al., et * Jaillon et al., Nature, 2004 Berbee and Taylor, 2006; James et al., 2006

  14. A brief introduction to the field of Yeast Genomics Genome redundancy 1.40 Saccharomyces cerevisiae 1.35 WGD 1.30 mean family size Candida glabrata 1.25 1.20 1.15 Zygosaccharomyces rouxii 1.10 ZYRO DEHA YALI LATH KLLA CAGL LAKL SACE Lachancea kluyveri (WashU seq center M. Jonhston) • important level of redundancy (in all eukaryotic phyla) • Gene order changes (differential loss of duplicates, translocation breakpoints) • several mechanisms of duplication Lachancea thermotolerans Kluyveromyces lactis Debaryomyces hansenii Yarrowia lipolytica Wolfe and Shields, 1997

  15. Small, compact and specialized: • small intergenic sequences • few transposable elements • few introns • limited RNA interference • Large evolutionary scale • High level of genome redundancy • Numerous evolutionary novelties in all clades • High number of sequenced genomes Yeast Genomes ===> good model organisms to study genome evolution

  16. Degeneration Complementation Pseudogenization Neofunctionalization Conservation Genome evolution: DNA duplications Most eukaryotic genomes contain high proportion of duplicated genes S. c.A. t.C. e. D. m.H. s. s. duplication Duplicated Genes 43% 65% 49% 40% 50% Loss of function (most frequent fate) Gain of a new function Specialization of the 2 copies Gene dosage increase Genetic robustness ===> Strong evolutionary potential

  17. Genome evolution: DNA duplications Adaptative value of DNA duplications: Adaptation to sulfate-limited conditions in chemostats for 200 generations: CGH SDs containing between 1 to 22 genes No homology at the junctions (microhomologies) Gresham et al., PLoS Genet 2008

  18. ??? RPL20B XV RPL20B XIII ==> WT growth rate rpl20A∆ délétion ==>slow growth Genome evolution: DNA duplications A duplication assay: XV and so on… RPL20B XIII 3days - YPD - 30° RPL20A ==> WT growth rate

  19. Karyotype Comparative Genomic Hybridization RPL20B IV - XII Hybridization XV 143 kb VII,XV RPL20B Molecular combing V, XIII II XIV X XI direct tandem V - VIII PCR and sequence IX III VI I A duplication assay: Genome evolution: DNA duplications Molecular characterization of segmental duplications: Despite the selection of a single gene duplication event, only large segmental duplications were recovered

  20. T T T T T T Raghuraman et al. Science, 2001 time (min) 0 5 • Lately replicated regions • tRNAs • LTRs • microsatellites • a connection with replication? 10 15 20 25 30 35 40 Molecular mechanisms: Genome evolution: DNA duplications strain rate of SDs (/cell/division) type of SDs breakpoint sequences (%) Intra-chromosomal Inter-chromosomal microhomologies (2 to 11 bp) microsatellites (poly A/T or répét trinucleotides) LTRs (300bp) WT 10-7 (1) 42 6 48 52 pol32∆ 0 (<0.07) - - - - REPLICATION clb5∆ 7x 10-5 (730) 66 3 62 38 CPT 3 x 10-5 (320) 22 0 54 56 rad52∆ 3 x 10-7 (3) 70 1 0 100 DSB REPAIR rad52∆ rad1∆ dnl4∆ 8 x 10-8 (0.8) 15 0 0 100 Koszul et al. EMBO J., 2004

  21. 7x 10-5 (730) 66 3 62 38 Nick McElhinny, Cell 2008 Pol32 pol32∆ 0 (<0.07) - - - - Bloom and Cross, 2007 Clb5 • Pol32 is required for initiating BIR reaction (Lydeard et al, 2007) • SDs are generated through replication-based mechanisms Replication-based mechanisms strain rate of SDs (/cell/division) type of SDs breakpoint sequences (%) microhomologies microsatellites LTRs Intra-chromosomal Inter-chromosomal WT 10-7 (1) 42 6 48 52 clb5∆ • defect in the firing of late replication origins (Schwob et al , 1993) • S-phase lasts twice longer (Epstein et al, 1992) • Rad9-dependent activation of the replication checkpoint indicative of DNA damages (Gibson et al, 2004) • RPL20B lies in Clb5-dependent region (CDR; McCune et al, 2008) • replication perturbations strongly induce SD formation

  22. 3 x 10-5 (320) 22 0 54 56 Replication-based mechanisms strain rate of SDs (/cell/division) type of SDs breakpoint sequences (%) microhomologies microsatellites LTRs Intra-chromosomal Inter-chromosomal WT 10-7 (1) 42 6 48 52 CPT CPT Top1 Top1 =>broken forks promote SD formation Broken forks as precursor lesions leading to SDs

  23. The DSB repair pathways Dnl4 NHEJ Resection HR Rad52 Rad1 pas d’homologies, religature simple Rad51 Pol32 MMEJ SSA BIR Microhomologies (5-12pb) >30pb d’homologies SDSA DSBR

  24. rad52∆ 3 x 10-7 (3) 70 1 0 100 => ====> HR-dependent HR-independent => HR-mediated SDs result from BIR Rad51-independent => Non HR-mediated SDs result from ? Two different replication-based mechanisms strain rate of SDs (/cell/division) type of SDs breakpoint sequences (%) microhomologies microsatellites LTRs Intra-chromosomal Inter-chromosomal WT 10-7 (1) 42 6 48 52

  25. The DSB repair pathways X Dnl4 Resection X X Rad52 Rad1

  26. 8 x 10-8 (0.8) 15 0 0 100 • SD are still being formed in the absence of all known DSB repair pathways existence of a new DSB repair pathway? • Sequences found at breakpoints: microhomologies between 2 and 11 bp poly (A/T)13-23 trinucleotide repeats (GTT)3-20 • Extremely high density of microhomologies and microsatelites in the genome • often intragenic • Formation of chimeric genes at breakpoints (in 13 out of 26 junctions) MMIR: microhomology microsatellite-induced replication strain rate of SDs (/cell/division) type of SDs breakpoint sequences (%) microhomologies microsatellites LTRs Intra-chromosomal Inter-chromosomal WT 10-7 (1) 42 6 48 52 rad52∆ 3 x 10-7 (3) 70 1 0 100 rad52∆ rad1∆ dnl4∆ • HR requires Rad52 • MMEJ requires Rad1 • NHEJ requires Dnl4

  27. The DSB repair pathways X Dnl4 Resection X X Rad52 Rad1

  28. The DSB repair pathways X Dnl4 Resection X X Rad52 Rad1 A new pathway? MMIR Microhomology/microsatellites Induced Replication - independent from all known DSB repair pathways (HR, NHEJ, MMEJ) - dependent from Pol32 - Replication template switching between microhomologies and microsatellites

  29. Conclusions Genome evolution: DNA duplications • SDs are spontaneously generated at high frequency:10-7 SD/cell/divisionfor the RPL20B locus • SDs arise from two alternative replication-based mechanisms:BIRandMMIR • MMIRrepresents anew mechanismdifferent from known DSB repair pathways (HR, NHEJ): • between microhomologie (between 2 to 11 nt) and microsatellites (poly A/T, trinucleotide repeats) • independent from Rad52 • requires Pol32 • MMIR induces the formation ofchimerical genesat the rearrangement junctions

  30. In human, FoSTeS/MMBIR: Genome evolution: DNA duplications Hastings et al, Nature Review Genetics, 2009 Complex structural variations: - Lissencephaly (Nagamani et al., J. Med Genet 2009) - Miller-Dieker syndrome - Charcot-Marie-Tooth disease (Lupski and Chance, 2005) - Pelizaeus Merzbacherdisease (Lee et al., Cell 2007) - XLMR syndrome (Bauters et al., Genome Res 2008) - SDs and CNVs (Kim et al., Genome Res 2008)

  31. Genome evolution: Chromosome Dynamics • Duplications: high evolutionary potential (creation of new genes, adaptation, specialization,…) • Translocations, inversions, deletions: very low evolutionary potential? (Loss of genes, deregulation of gene expression, modification of sub-nuclear architecture,…) Species 1 • translocations • Inversions • duplications • deletions • rates of rearrangements Species 2 # x #

  32. Sensu stricto S. serevisiae S. bayanus Candida glabrata Zygosaccharomyces rouxii S. cerevisiae S. cariocanus Lachancea kluyveri S. paradoxus S. mikatae Lachancea thermotolerans S. kudriavzevii S. bayanus Kluyveromyces lactis Debaryomyces hansenii Yarrowia lipolytica Genome evolution: Chromosome Dynamics Saccharomyces sensu stricto complex: - monophyletic group - very closely related species - hybrids viable but sterile - 16 chromosomes

  33. S. cerevisiae S. cerevisiae S. paradoxus S. kudriavzevii S. mikatae S. cariocanus S. bayanus S. cariocanus S. paradoxus S. mikatae S. kudriavzevii S. bayanus Genome evolution: Chromosome Dynamics • only few translocations: • low reorganization • recombination between repeated sequences • no chromosomal speciation • variable rate of rearrangements? (4) (0) (2) (0) (4) S. cerevisiae S. paradoxus S. kudriavzevii S. mikatae S. cariocanus S. bayanus Fischer et al. , Nature 2000

  34. Sensu stricto S. serevisiae S. bayanus Candida glabrata Zygosaccharomyces rouxii Lachancea kluyveri Lachancea thermotolerans Kluyveromyces lactis Debaryomyces hansenii Yarrowia lipolytica Genome evolution: Chromosome Dynamics S. cerevisiae C. glabrata K. lactis D. hansenii Y. lipolytica S. bayanus 1 5 7 9 11 13 3 5 15 8 2 4 6 2 4 5 6 A D G I J 1 4 6 8 10 12 chr VIII 88% 77% 5% 98% 11%

  35. Genome evolution: Chromosome Dynamics S. cerevisiae C. glabrata K. lactis D. hansenii Y. lipolytica S. bayanus 1 5 7 9 11 13 3 5 15 8 2 4 6 2 4 5 6 A D G I J 1 4 6 8 10 12 chr VIII 88% 77% 5% 98% 11%

  36. Genome evolution: Chromosome Dynamics Fischer S. cerevisiae C. glabrata K. lactis D. hansenii Y. lipolytica S. bayanus 1 5 7 9 11 13 3 5 15 8 2 4 6 2 4 5 6 A D G I J 1 4 6 8 10 12 chr VIII F. Brunet 88% 77% 98% Fischer et al. , PLoS Genet 2006

  37. Genome evolution: Chromosome Dynamics S. cerevisiae C. glabrata K. lactis D. hansenii Y. lipolytica S. bayanus 1 5 7 9 11 13 3 5 15 8 2 4 6 2 4 5 6 A D G I J 1 4 6 8 10 12 chr VIII 88% 77% 5% 98% 11%

  38. Genome evolution: Chromosome Dynamics S. cerevisiae C. glabrata K. lactis D. hansenii Y. lipolytica S. bayanus 1 5 7 9 11 13 3 5 15 8 2 4 6 2 4 5 6 A D G I J 1 4 6 8 10 12 chr VIII 88% 77% 5% 98% 11%

  39. C. glabrata "UNSTABLE" GENOMES S.cerevisiae Mean amino acid identity: 58% • moderate reshuffling • 91 translocations, 22 inversions • large chromosomal segments • (up to 670 kb) L. thermotolerans "STABLE" GENOMES L. kluyveri at genome scale: Genome evolution: Chromosome Dynamics Saccharomyces cerevisiae Mean amino acid identity: 65% • comprehensive reshuffling • 509 translocations, 104 inversions • no homologous chromosomes Candida glabrata Zygosaccharomyces rouxii Lachancea kluyveri Lachancea thermotolerans

  40. ? Genome evolution: Chromosome Dynamics Quantitative estimation of the relative genome stability: GOC (gene order conservation) species 1 =5 # neighboring orthologues If yes: +1 If no: 0 GOC = Total # orthologues species 2 =5 - GOL : Gene Order Loss = 1 - GOC GOL ( ( - Rate of rearrangements = mean rate Dist phylogénétique Rocha, Trends Genet, 2003,

  41. Species instability scale 0.7 D. hansenii 0.6 S. cerevisiae 0.5 C. glabrata Z. rouxii 0.4 K. lactis L. kluyveri L. thermot 0.3 Genome evolution: Chromosome Dynamics Rearrangement branch rate WGD 1.5 Saccharomyces cerevisiae 2.7 1.3 0.4 Candida glabrata 0.6 Zygosaccharomyces rouxii 1.7 0.3 Lachancea kluyveri (WashU seq center M. Jonhston) 0.4 Lachancea thermotolerans 0.0 0.9 Kluyveromyces lactis 1.7 Debaryomyces hansenii 1.7 Yarrowia lipolytica Fischer et al. , PLoS Genet 2006

  42. Genome evolution: Chromosome Dynamics moderate massive low Sensu stricto differential gene loss S. serevisiae S. bayanus Unstable genome Candida glabrata Zygosaccharomyces rouxii Lachancea kluyveri (WashU seq center M. Jonhston) Stable genomes Lachancea thermotolerans Kluyveromyces lactis TGA expansion Debaryomyces hansenii No synteny Y. lipolytica

  43. Conclusions Genome evolution: Chromosome Dynamics • High level of chromosome plasticity • Hundreds of translocations and inversions • Gene order is not very constrained • Highly variable rates of chromosome rearrangements between lineages but also within a given lineage • Is there a selective advantage associated to these rearrangements? Are they accumulated by genetic drift? • usually considered as deleterious • few examples of the adaptative role of rearrangements (proliferation of cancer cells (O’Neil and Look, 2007), growth advantage of translocated yeast cells (Colson et al, 2004), adaptative gene loss (Domergue, 2005). • Creation of genetic novelties requires chromosome plasticity?

  44. Base substitution mutations: C T transitions : cytosinedeamination Kreutzer and Essigmann, PNAS, 1998 G T transversions : 8-oxo-guanine Shibutani et al., Nature, 1991 Global AT-enrichment Biased Gene Conversion (BGC): Duret and Galtier, Annu Rev Genomics Human Genet, 2009 not in yeast? AT GC mutations Global GC-enrichment Marsolier-Kergoat and Yeramian, Genetics, 2009 Genome evolution: Nucleotide composition GC% Saccharomyces cerevisiae 38.3 38.8 Candida glabrata 39.1 Zygosaccharomyces rouxii 41.5 Lachancea kluyveri Lachancea thermotolerans 47.3 38.8 Kluyveromyces lactis > Eremothecium gossypii 52.0 < 36.3 Debaryomyces hansenii Yarrowia lipolytica 49.0 The Génolevures Consortium,Genome Res.,2009

  45. Lachancea thermotolerans A B C D E F G H 47.3 Lachancea kluyveri GC% 80 1 Mb C-left 60 A B C D E F G 52.9 40 41.5 20 Mb 1 2 3 4 5 6 7 8 9 10 11 A B C E F G H D GC% 80 60 40 39.1 20 Mb 1 2 3 4 5 6 7 8 9 10 Zygosaccharomyces rouxii

  46. RNA 1st 2nd 3rd 1st 2nd 3rd 1st 2nd 3rd AAAAAA GC% in C-left: 53.3 46.4 41.0 37.0 68.3 42.7 • strong bias in codon usage GC% out of C-left: Protein A G P R I N K F 84 84 84 72 GC% in synonymous codons 11 16 16 16 0.7 0.8 0.9 0.9 1.3 1.2 1.1 1.2 relative use in C-left • bias in protein composition Payen et al., Genome Res., 2009 Genome evolution: Nucleotide composition DNA GC% in C-left: 46.1 37.4 54.2 42.0 46.8 36.5 • global GC increase GC% out of C-left:

  47. 19 families (4631 residues) in C-left S. cerevisiae C. glabrata Z. rouxii L. kluyveri L. waltii L. thermotolerans K. lactis E. gossypii Phylogeny: Genome evolution: Nucleotide composition • Alignments of universally conserved proteins : • 17 families (6688 residues) outside C-left 100 100 100 0.05 100 96 100 100 100 100 98 • C-left has the same phylogentic origin than the rest of the genome Payen et al., Genome Res., 2009

  48. Synteny: Genome evolution: Nucleotide composition LAWA_S33 LAWA_S27 LAWA_S56 LAWA_S55 LAKL_C 670 kb LATH_F LATH_G LATH_C LATH_E LATH_A C-left share a common ancestral origin with the genomes of L. waltii (LAWA) and L. thermotolerans (LATH)

  49. - Time course analysis of copy number variation during S-phase: G1 DNACy3 DNACy5 S G2 Replication: Genome evolution: Nucleotide composition - Design of custom microarrays (Agilent 2 x 105k): 200bp fragments

  50. Replication: Genome evolution: Nucleotide composition ChrA ChrB

More Related