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

Genome Evolution in Yeast

Gilles Fischer

27th January 2009 | European Course on

INTRODUCTION:

Comparative genomics

Yeasts as model organisms

GENOME EVOLUTION:

DNA duplications

Chromosome dynamics

Nucleotide composition

A brief introduction to the field of Comparative Genomics

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"

Comparing genomes is a very old idea…

DNA carries the genetic information: Avery (1943) and Hershey-Chase (1952)

"Tout ce qui est vrai pour le colibacille est vrai pour l'éléphant"

Jacques Monod:

identical divergent different

timeor

quantity of evolutionary changes


Looking for differences Looking for similarities

identical divergent different

timeor

quantity of evolutionary changes


Looking for differences Looking for similarities

NEED FOR ADEQUATELY RELATED ORGANSIMS

Looking for differences

Looking for similarities


Genome sequences

Bio-informatics

Rules governing genome evolution

Mechanistic hypotheses

Genetic screens

functional genomics

Experimental Biology

Molecularmechanisms

Looking for differences

Looking for similarities


Genome sequences

Bio-informatics

Rules governing genome evolution

Mechanistic hypotheses

Genetic screens

functional genomics

Experimental Biology

Molecularmechanisms

SMALL GENOMESAND

EXPERIMENTALLY TRACTABLE

•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

Organisms with small genomes, phylogenetically related and

experimentally tractable = YEASTS

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)


The genome of S. cerevisiae

André Goffeau

8 years, 120 labs,641 people

Life with 6000 genesScience (1996)

The first eukaryotic genome sequence:

Saccharomyces paradoxusSaccharomyces mikataeSaccharomyces cerevisiaeSaccharomyces kudriavzevii Saccharomyces bayanus

Saccharomyces pastorianusSaccharomyces exiguus Saccharomyces servazzii Saccharomyces castelliiCandida glabrata Vanderwaltozyma polysporaZygosaccharomyces rouxiiLachancea thermotoleransLachancea waltiiLachancea kluyveri

Kluyveromyces lactisKluyveromyces marxianusEremothecium gossypiiSaccharomycodes ludwigiiBrettanomyces bruxellensisPichia angustaCandida lusitaniae Debaryomyces hanseniiPichia stipitisPichia sorbitophilaCandida guilliermondiiCandida tropicalisCandida parapsilosisLodderomyces elongisporusCandida albicansCandida dubliniensisArxula adeninivoransYarrowia lipolytica

Schizosaccharomyces pombe

Saccharomycotina

Saccharomyces paradoxusSaccharomyces mikataeSaccharomyces cerevisiaeSaccharomyces kudriavzevii Saccharomyces bayanus

Saccharomyces pastorianusSaccharomyces exiguus Saccharomyces servazzii Saccharomyces castelliiCandida glabrata Vanderwaltozyma polysporaZygosaccharomyces rouxiiLachancea thermotoleransLachancea waltiiLachancea kluyveri

Kluyveromyces lactisKluyveromyces marxianusEremothecium gossypiiSaccharomycodes ludwigiiBrettanomyces bruxellensisPichia angustaCandida lusitaniae Debaryomyces hanseniiPichia stipitisPichia sorbitophilaCandida guilliermondiiCandida tropicalisCandida parapsilosisLodderomyces elongisporusCandida albicansCandida dubliniensisArxula adeninivoransYarrowia lipolytica

Schizosaccharomyces pombe

SaccharomycotinaA brief introduction to the field of Yeast Genomics

Whole Genome Duplication

Extensive loss of transposable elements and spliceosomal introns

Gain of mating type cassettesand small centromeres

frequent tandem duplications

Gain of Megasatellites

Gain of HO gene

5769

5204

4998

5308

5104

5084

6273

6434

# genes

274

207

272

258

231

162

200

510

# tRNA

287

131

167

322

286

175

475

1070

# introns

12,1

12,3

9,8

11,3

10,4

10,7

12,1

20,5

size (Mb)# chr

16

13

7

8

8

6

7

6

Genome annotation

Yarrowia lipolytica

Saccharomyces cerevisiae

Candida glabrata

Lachancea kluyveri(WashU seq center M. Jonhston)

Debaryomyces hansenii

Kluyveromyces lactis

Lachancea thermotolerans

Zygosaccharomyces rouxii


Yarrowia lipolytica


Candida glabrata

Lachancea kluyveri





300

- 10

00 M

Yr

100

- 30

0 M

Yr

100

MY

r

Berbee and Taylor, 2006; James et al., 2006

100 *

65

-

-

-

60

51

48

amino acididentity %

Evolutionary scale

Mus musculus

Takifugu rubripesTetraodon negroviridis

Homo sapiens

100 *

90

70

50

450 MY

r

100 MY

r

550 MY

r

Ciona intestinalis

*Dujon et al., et * Jaillon et al., Nature, 2004


Yarrowia lipolytica


Candida glabrata






1.10

1.15

1.20

1.25

1.30

1.35

1.40

me

an

fa

mily

siz

e

Genome redundancy

YALI

SACE

LAK

L

DEH

A

KLL

A

LATH

ZYR

O

CA

GL

WGD

Wolfe and Shields, 1997

- important level of redundancy (in all

eukaryotic phyla)

- Gene order changes (differential loss of

duplicates, translocation breakpoints)

- several mechanisms of duplication


- 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

Most eukaryotic genomes contain high proportion of

duplicated genes

Duplicated Genes 43% 65% 49% 40% 50%

S. c. A. t. C. e. D. m. H. s. s. duplication

Gene dosage increaseGenetic robustness

Gain of a new function

Specialization of the 2 copies

Loss of function(most frequent fate)

Pseudogenization Neofunctionalization ConservationDegenerationComplementation

===> Strong evolutionary potential

Genome evolution: DNA duplications

CGH

SDs containing between 1 to 22 genesNo homology at the junctions (microhomologies)

Gresham et al., PLoS Genet 2008

Adaptation to sulfate-limited conditions in chemostats for 200 generations:


Adaptative value of DNA duplications:

3days - YPD - 30°

and so on…RPL20B

XV

XIII

RPL20A

==> WT growth rate

RPL20B

XV

XIII

==>slow growth

rpl20A∆délétion

???

RPL20B

==> WT growth rate

A duplication assay:


IVI

III

IX

V - VIII

XIX

XIVII

V, XIII

VII, XV

IV - XII

XV

Karyotype Hybridization

RPL20B

Molecular combing

direct tandem

PCR and sequence

A A C C T A G A G C T T ( G T T ) 14 G T G G A T T G T T T

Despite the selection of a single gene duplication event, only large segmental duplications were recovered

Molecular characterization of segmental duplications:

Comparative Genomic Hybridization

143 kb

RPL20B

Genome evolution: DNA duplications A duplication assay:

Inter-chromosomalIntra-chromosomal

strain rate of SDs (/cell/division)

type of SDs breakpoint sequences (%)

LTRs(300bp)

microhomologies(2 to 11 bp)

microsatellites(poly A/T or

répét trinucleotides)

WT 10-7 42 6 48 52(1)

pol32∆ 0 - - - -(<0.07)

RE

PL

ICA

TIO

N

clb5∆ 7x 10-5 66 3 62 38(730)

CPT 3 x 10-5 22 0 54 56(320)

rad52∆ 3 x 10-7 70 1 0 100(3)

DS

B R

EP

AIR

rad52∆rad1∆dnl4∆

8 x 10-8 15 0 0 100(0.8)

Genome evolution: DNA duplications Molecular mechanisms:

Koszul et al. EMBO J., 2004

T T T T TT0

5

10

15

20

25

30

35

40

time (min)

Lately replicated regionstRNAsLTRsmicrosatellites

a connection with replication?

Raghuraman et al. Science, 2001

Clb5

Replication-based mechanisms

Inter-chromosomalIntra-chromosomal



LTRs microhomologiesmicrosatellites

WT 10-7 42 6 48 52(1)

clb5∆ 7x 10-5 66 3 62 38(730)

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

Bloom and Cross, 2007

Pol32Nick McElhinny, Cell 2008

pol32∆ 0 - - - -(<0.07)

Pol32 is required for initiating BIR reaction (Lydeard et al, 2007)

SDs are generated through replication-based mechanisms

Broken forks as precursor lesions leading to SDs



WT 10-7 42 6 48 52(1)

CPT 3 x 10-5 22 0 54 56(320)

Top1CPT

Top1

=>broken forks promote SD formation

Inter-chromosomalIntra-chromosomal LTRs microhomologiesmicrosatellites

Replication-based mechanisms

pas d’homologies, religature simple

NHEJ

Dnl4

Resection

Rad52 Rad1

MMEJ

SSA BIR

SDSA DSBR

Rad51

The DSB repair pathways

Pol32

Microhomologies (5-12pb)

>30pb d’homologies

HR

Two different replication-based mechanisms



WT 10-7 42 6 48 52(1)

HR-dependent

rad52∆ 3 x 10-7 70 1 0 100(3)=

>

==

==

>

HR-independent


=> HR-mediated SDs result from BIR Rad51-independent

=> Non HR-mediated SDs result from ?

Dnl4

Resection

Rad52 Rad1


X

X X



WT 10-7 42 6 48 52(1)

rad52∆ 3 x 10-7 70 1 0 100(3)

MMIR: microhomology microsatellite-induced replication


rad52∆rad1∆dnl4∆

8 x 10-8 15 0 0 100(0.8)

SD are still being formed in the absence of all known DSB repair pathways

existence of a new DSB repair pathway?

HR requires Rad52MMEJ requires Rad1NHEJ requires Dnl4

Sequences found at breakpoints: microhomologies between 2 and 11 bp

poly (A/T)13-23

trinucleotide repeats (GTT)3-20

Formation of chimeric genes at breakpoints (in 13 out of 26 junctions)

Extremely high density of microhomologies and microsatelites in the genome

often intragenic

Dnl4

Resection

Rad52 Rad1


X

X X

Dnl4

Resection

Rad52 Rad1


X

X X

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

SDs are spontaneously generated at high frequency: 10-7 SD/cell/division for the RPL20B locus

SDs arise from two alternative replication-based mechanisms: BIR and MMIR

MMIR represents a new mechanism different 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 of chimerical genes at the rearrangement junctions

ConclusionsGenome evolution: DNA duplications

Hastings et al, Nature Review Genetics, 2009

In human, FoSTeS/MMBIR:

Complex structural variations: - Lissencephaly (Nagamani et al., J. Med Genet 2009)

- Miller-Dieker syndrome

- Charcot-Marie-Tooth disease (Lupski and Chance, 2005)

- Pelizaeus Merzbacher disease (Lee et al., Cell 2007)

- XLMR syndrome (Bauters et al., Genome Res 2008)

- SDs and CNVs (Kim et al., Genome Res 2008)


Genome evolution: Chromosome Dynamics

translocations

Inversions

duplications

deletions

rates of rearrangements

Species 1

Species 2

#

# x

-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,…)

S. paradoxus

S. kudriavzevii

S. cariocanus

S. mikatae

S. bayanus

S. cerevisiae

Saccharomyces sensu stricto complex:

- monophyletic group

- very closely related species

- hybrids viable but sterile

- 16 chromosomes


Yarrowia lipolytica

S. serevisiaeS. bayanus

Candida glabrata

Lachancea kluyveri





Sensu stricto

S. paradoxus

S. kudriavzevii

S. cariocanus

S. mikatae

S. bayanus

S. cerevisiae

Fischer et al. , Nature 2000

S. cerevisiaeS. paradoxus S. cariocanusS. mikataeS. kudriavzevii S. bayanusS. cerevisiaeS. paradoxus S. cariocanusS. mikataeS. kudriavzevii S. bayanus


(4)

(4)

(2)

only few translocations:• low reorganization• recombination between repeated sequences• no chromosomal speciation• variable rate of rearrangements?

(0)

(0)

C. glabrata K. lactis D. hanseniiS. cerevisiae

chr VIII

1 45678910111213 1

4562

3A D G IJ 245 6

88% 77% 11% 5%

Y. lipolyticaS. bayanus8 15

98%

Yarrowia lipolytica


Candida glabrata

Lachancea kluyveri





Sensu stricto



chr VIII

1 45678910111213 1

4562

3A D G IJ 245 6

88% 77% 11% 5%


98%



chr VIII

1 45678910111213 1

4562

3A D G IJ 245 6

88% 77%


98%

Fischer

Fischer et al. , PLoS Genet 2006

F. Brunet



chr VIII

1 45678910111213 1

4562

3A D G IJ 245 6

88% 77% 11% 5%


98%



chr VIII

1 45678910111213 1

4562

3A D G IJ 245 6

88% 77% 11% 5%


98%



Candida glabrata

Lachancea kluyveri



at genome scale:

S.cerevisiae

C.

glab

rata

- comprehensive reshuffling

- 509 translocations, 104 inversions

- no homologous chromosomes

"UNSTABLE" GENOMES

"STABLE" GENOMES


L. kluyveri

L.

ther

mo

tole

ran

s

-moderate reshuffling

-91 translocations, 22 inversions

- large chromosomal segments (up to 670 kb)

Mean amino acid identity: 58%

Mean amino acid identity: 65%

Quantitative estimation of the relative genome stability: GOC (gene order conservation)

species 1

species 2

?

=5

=5

If yes: +1

If no: 0

Rocha, Trends Genet, 2003,

GOC =

# neighboring orthologues

Total # orthologues

- GOL : Gene Order Loss = 1 - GOC

- Rate of rearrangements = GOL

Dist phylogénétique(

(

mean rate


Yarrowia lipolytica


Candida glabrata






1.5

1.32.7

WGD

1.7

1.7

1.7

0.6

0.9

0.4

0.3

0.0

0.4

Rearrangement branch rate

S. cerevisiae

C. glabrata

Z. rouxiiK. lactisL. kluyveri

L. thermot

D. hansenii

Species instability scale

0.3

0.4

0.5

0.6

0.7


Fischer et al. , PLoS Genet 2006

Y. lipolytica


Candida glabrata






Sensu stricto

lowmassive

Unstable genome

Stable genomes

differential gene loss

No synteny

moderate

TGA expansion


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?

ConclusionsGenome evolution: Chromosome Dynamics

Base substitution mutations:

C T transitions : cytosine deamination

QuickTime™ et undécompresseur

sont requis pour visionner cette image.

Kreutzer and Essigmann, PNAS, 1998

G T transversions : 8-oxo-guanine

Shibutani et al., Nature, 1991

Global AT-enrichment

Biased Gene Conversion (BGC):

Global GC-enrichment

AT GC mutations Duret and Galtier, Annu RevGenomics Human Genet, 2009

GC%

38.3

38.8

39.1

41.5

47.3

38.8

36.3

49.0Yarrowia lipolytica


Candida glabrata

Lachancea kluyveri





Eremothecium gossypii 52.0

The Génolevures Consortium, Genome Res., 2009

>

<

Marsolier-Kergoat and Yeramian, Genetics, 2009

not in yeast?

Genome evolution: Nucleotide composition

20

40

60

80

1 2 3 4 5 6 7 8 9 10

A B C D E F G

GC%

Mb

39.1

A B C D E F G H

47.3



QuickTime™ et undécompresseur

sont requis pour visionner cette image.

A B C D E F G H

1 2 3 4 5 6 7 8 9 10 Mb11

20

40

60

80

GC% Lachancea kluyveri

41.5

52.9

C-left

1 Mb

DNA

46.137.4

54.242.0

46.836.5

GC% in C-left:GC% out of C-left:

• global GC increase

RNA 1st 1st 1st2nd 2nd 2nd3rd 3rd 3rd AAAAAA

53.346.4

41.037.0

68.342.7

• strong bias in codon usageGC% in C-left:

GC% out of C-left:

Protein A G P R I N K F

84 84 84 72 11 16 16 16 GC% in synonymous codons

1.3 1.2 1.1 1.2 0.7 0.8 0.9 0.9relative use in C-left

• bias in protein compositionPayen et al., Genome Res., 2009

Genome evolution: Nucleotide composition

100

98

E. gossypii

K. lactis

L. thermotolerans

L. waltii

L. kluyveri

Z. rouxii

C. glabrata

S. cerevisiae

100

100

100

100

96

100

100

100

0.05

Payen et al., Genome Res., 2009

• C-left has the same phylogentic origin than the rest of the genome

Alignments of universally conserved proteins :

• 17 families (6688 residues) outside C-left

• 19 families (4631 residues) in C-left

Genome evolution: Nucleotide compositionPhylogeny:

LATH_GLATH_E

LATH_CLATH_A

LATH_F

LAKL_C

LAWA_S27 LAWA_S56 LAWA_S55LAWA_S33

670 kb

C-left share a common ancestral origin with the genomes of L. waltii (LAWA) and L. thermotolerans (LATH)

Genome evolution: Nucleotide compositionSynteny:

- Design of custom microarrays (Agilent 2 x 105k):

200bp fragments

G1

S

G2

DNACy3

DNACy5

- Time course analysis of copy number variation during S-phase:

Genome evolution: Nucleotide compositionReplication:

ChrA

ChrB


ChrC

ChrD


• Global GC increase (codon usage bias and protein composition bias)

• harbors a normal gene density

• Phylogenetic origin consistent with the rest of the genome

• presents a very high level of synteny conservation with sister species genomes

• encompasses the MAT locus but has lost the silent cassettes HMR and HML

• is devoid of Transposable Elements (203 insertions in the rest of the genome)

• harbors the same compositional bias in all 11 L. kluyveri strains tested

• The replication program is modified (more origins and delayed firing)

=> a cause or a consequence of the unusual GC composition?

• Meiotic recombination and BGC?

Genome evolution: Nucleotide compositionConclusions

L. kluyveri offers a unique opportunity to understand the mechansims of evolution of genome nucleotide composition

Merci

- Plateforme Puces ADN, Génopole Pasteur Odile Sismeiro, Jean-Yves Coppé

- Génopole Pasteur-Ile de France Christiane Bouchier, Lionel Frangeul

- Centre National de Séquençage, Evry

Jean-Luc Souciet Univ. Louis Pasteur, Strasbourg

Jean Weissenbach, Patrick Winker

- Génolevures consortium:

- Unité de Génétique Moléculaire des Levures, Institut Pasteur

Celia Payen

Romain Koszul

- Unité de Génomique des Microorganismes, équipe Biologie des

Génomes

Nicolas Agier

Guénola Drillon

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

Documents

field of yeast genomics

brief introduction

comparative genomics

yeast yeasts

tractable slide

related organsims slide

species of yeast

budding yeasts true