EPIGENOMICS

André Goffeau

Institut Pasteur/EMBO/CNPq course

Florianopolis, July 11, 2008.

Epigenomics is any regulation (on/off) of

gene expression that is not due to DNA mutations and is heritable

Epigenetic jargon

• Paramutation• Bookmarking• Imprinting• Gene silencing• X chromosome inactivation• Position effect• Reprogramming• Transvection• Maternal effects• Carcinogenesis• Teratogen effects• Histone and chromatin modifications• Parthenogenesis • Cloning • Prions• Embryogenesis

Jean-Baptiste Lamark 1744-1829

Charles Darwin 1809-1882

According to Lamarck's theory, acquired characteristics can be passed to subsequent generations.

According to Darwin's (and Wallace's) theory of natural selection, a population of giraffes will have individuals with variations in neck length. If having a longer neck is advantageous in feeding, longer necked giraffes will be more successful and reproduce more.

Two views about the type of mechanism that promotes evolution.

RNA interference, Histone acetylation and DNA methylation

DNA METHYLATION

Cytosine methylation occurs at CpG and is mutagenic

It prevents activation of promoters

Methylation of CpG islands

and the informatician?

they try to predict which

cytosines

are methylated

in DNA

EMBRIO EPIGENETICS

Reprogramming in Germ Cells and Embryos

CHROMATIN CODE

Chromatin chemistryAcetylation or methylation

Histone modifications

Methylation Genomics

Aberrant methylation in human and mouse leukemia

DISEASES and DRUGS

 

                                                                                                                                                                    

                

Epigenetic diseases

  Epigenetic drugs

Gene silencing and pharmacology

siRNA

RNA silencing

and YEAST??S.cerevisiae has no DNA methylationS.cerevisiae has no siRNAS.cerevisiae has chromatin modification S.pombe siRNA controls heterochromatinN.crasa DNA methylation depends on a histone methyl transferaseS.cerevisiae has other epigenetic systems such: Mating type silencing, FLO11 a pseudohyphal telomeric gene, Prions

RNA interference, Histone acetylation and DNA methylation

For elucidation of mechanism, use S.pombe, N.crassa or Y.lipolytica ?? but not at S.cerevisiae

Epigenetics References Pennisi E. Behind the scenes of gene expression. 2001 Science, 293:1604-1607.

Egger G, Liang G, Aparicio A & Jones PE. Epigenetics in human disease and prospects for epigenetic therapy. 2004. Nature,429:457-463.

Jenuwein T and Allis CD. Translating the Histone Code 2001 Science, 293:1074-1080.

Matzke M, Matzke AJM, Kooter JM RNA: Guiding gene silencing. 2001 Science, 293:1080-1083.

Reik W, Dean W, WalterJ. Epigenetic reprogramming in mammaliandevelopment. 2001 Science, 293:1089-1093

Hatada I et al. A genomic scanning method for higher organisms using restriction sites as landmarks. 1991. P.N.A.S.,88,9523-9527

Kimura et al. Methylation profiles of genes utilizing newly developed CpG island methylation microarray on colorectal cancer patients 2005 Nucleic Acids Research, 20, E pub

Agrawal et al. RNA interference: biology, mechanism and applications. 2003 Microb. and Molec Biology Reviews, 67, 657-685

QuickTime™ et undécompresseur Sorenson Video 3

sont requis pour visionner cette image.

http://www.nature.com/focus/rnai/animations/animation/animation.htm

PARENTAL DIFFERENTIAL METHYL TAGGING

Hinny and Dolly

EARLY EXAMPLES

• agouti mice (folic acid)

• cancer human (p16)

• diseases human (BWS)

• eye apendage fly (Hsp90)

Methyl detector

Yellow: hyper-methylated; Blue: under-methylated

Restriction Landmark Genomic Scanning

Reprogramming and Imprinting

Deoxynucleoside analogue inhibition

GENOME EVOLUTION

Genomesequencing

Deletomics(systematic)

Overexpressionics(systematic)

Transcriptomics(DNA chips)

Proteomics(2D gels/2 hybrids)

PhysiologistsPathologists

Structuralists Biologists BiochemistsREDUCTIONICS

(SPECIFIC)

NEW TOOLS(GLOBAL)

GENOMICS(GLOBAL)

Genomemapping

Genomecomparisons

Genomology

Databases

Schematic alternating signature for Whole Genome Duplication

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 2211 22 33 44 55 66 77 88 99 1010 1111 1212 1313 1414 1515 1616 1717 1818 1919 2020 2121 2222

4 5 6 7 10 11 12 13 14 15 16 20 21 224 5 6 7 10 11 12 13 14 15 16 20 21 22

1 2 3 4 8 9 10 11 16 17 18 191 2 3 4 8 9 10 11 16 17 18 19

Duplicated copy 1in S. cerevisiae

Duplicated copy 2 in S. cerevisiae

Reference blockin K. waltii

The dark grey genes are contiguous in the non-duplicated reference species (K. waltii, K. lactis or A. gossypii). Yellow genes are conserved in both S. cerevisiae copies. Red genes are conserved only in S. cerevisiae copy 1 .Blue genes are conserved only in S. cerevisiae copy 2.The lost genes are in light grey.

EMERGENCE OF SPECIES-SPECIFIC TRANSPORTERS

DURING EVOLUTION OF THE HEMIASCOMYTE PHYLUM

Benoît De Hertogh*[1], Frédéric Hancy†[2], André Goffeau‡ and Philippe V. Baret*

Université catholique de Louvain

www.gena.ucl.ac.be

Evolution of the yeast genome

Wolfe KH, Shields DC. Molecular evidence for an ancient duplication of the entire yeast genome. Nature. 1997;387:708-13.

Kellis M, Birren BW, Lander ES. Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae. Nature. 2004;428:617-24.

Dujon B, Sherman D, Fischer G, Durrens P, Casaregola S, Lafontaine I, De Montigny J, Marck C, Neuveglise C, Talla E, Goffard N, Frangeul L, Aigle M, Anthouard V, Babour A, Barbe V, Barnay S, Blanchin S, Beckerich JM, Beyne E, Bleykasten C, Boisrame A, Boyer J, Cattolico L, Confanioleri F, De Daruvar A, Despons L, Fabre E, Fairhead C, Ferry-Dumazet H, Groppi A, Hantraye F, Hennequin C, Jauniaux N, Joyet P, Kachouri R, Kerrest A, Koszul R, Lemaire M, Lesur I, Ma L, Muller H, Nicaud JM, Nikolski M, Oztas S, Ozier-Kalogeropoulos O, Pellenz S, Potier S, Richard GF, Straub ML, Suleau A, Swennen D, Tekaia F, Wesolowski-Louvel M, Westhof E, Wirth B, Zeniou-Meyer M, Zivanovic I, Bolotin-Fukuhara M, Thierry A, Bouchier C, Caudron B, Scarpelli C, Gaillardin C, Weissenbach J, Wincker P, Souciet JL. Genome evolution in yeasts. Nature. 2004;430:35-44.

Epigenomics

Egger G, Liang G, Aparicio A, Jones PA. Epigenetics in human disease and prospects for epigenetic therapy. Nature. 2004;429:457-63.

Costello JF. Comparative epigenomics of leukemia. Nat Genet. 2005;37:211-2.

Membrane Classification (MC)

Membrane Proteins

A

C

10

F

10.A Lipid Metabolism

10.B Anchoring

10.C Polysaccharide Metabolism

10.D Trafficking

10.E Signaling

10.F Oxidoreductases

10.G Subtelomeric Conserved

10.H Chaperones

B

D

E

G

H

Genomesequencing

Deletomics(systematic)

Overexpressionics(systematic)

Transcriptomics(DNA chips)

Proteomics(2D gels/2 hybrids)

PhysiologistsPathologists

Structuralists Biologists BiochemistsREDUCTIONICS

(SPECIFIC)

NEW TOOLS(GLOBAL)

GENOMICS(GLOBAL)

Genomemapping

Genomecomparison

Genomology

Databases

Conclusions

• Analysis of the 28.000 protein sequences obtained from 14 hemiascomycetes illustrates the usefulness of the functional/ phylogenetic TC system proposed by MILTON SAIER • A similar system for non - transport membrane proteins is proposed (179 members)• S. cerevisiae contains contains 11 channels, 211 permeases,

•16 P-ATPases and 22 ABC-ATPases•They contain also 28 putative transporters families and 112 singletons of unknown function• Speciation of hemiascomycetes is accompanied by the emergence of membrane proteins not represented in S. cerevisiae• Similar analysis of TMS 1 and 2 proteins is required•Our database has been used for identification of novel putative yeast transporters• Our database will serve as reference for the automatic annotation of membrane proteins from recently sequenced yeast genomes

TC - Class 9

Incompletely Characterized Transport Systems

Functionally Characterized Transporters Lacking Identified Sequences

9.C

Recognized Transporters of Unknown Biochemical Mechanism

9.A

Putative Uncharacterized Transport Proteins9.B

The Membrane Proteins of Unknown Function

9.D

9

9.E Questionable ORFs with TMS>2

10.A Lipid Metabolism 13 28

10.B Anchoring 9 10

10.C Polysaccharide Metabolism 8 32

10.D Trafficking 11 39

Total : 55 146

10.E Signaling 4 7

10.F Oxidoreductases 5 11

10.G Subtelomeric Conserved 1 12

10.H Chaperones 4 7

Subfamilies ORFs

S. Cerevisiae Membrane Classification

Table 1 Global statistics of membrane proteins in Hemiascomycete species

Species Y. lipolytica D. hansenii K. lactis C. glabrata S. cerevisiae

Total

Code YALI DEHA KLLA CAGL SACE

Strain CLIB122 CBS767 CLIB210 CBS138 S288c

Database Génolevures Génolevures Génolevures

Génolevures

SGD

Release 22 may 2004 22 may 2004 22 may 2004

22 may 2004

22 may 2004

Natural substrate fats salted fish milk blood grapes

ORFs 6666 6896 5331 5272 5800 29965

Classified transporters 597 538 439 398 508 2480

% 9.0 7.8 8.2 7.5 8.8 8.3

Possible transporters (9.B.X.Y.Z) still unannotated

296 295 226 236 243 1296

Table 2 Functional distribution of the “established and putative” transporters in the Hemiascomycete phylum

YALI DEHA KLLA CAGL SACE Total

1.A Alpha-Type channels 46 32 26 26 38 168

1.B Beta Barrel porins. 1 1 1 2 2 7

2.A Porters (uniporters, symporters, antiporters)

316 281 206 162 218 1183

3.A P-P-bond-hydrolysis-driven transporters 94 89 91 87 107 468

3.B Decarboxylation-driven transporters 2 1 1 2 0 6

3.D Oxidoreduction-driven transporters 21 27 18 15 25 106

3.E Light absorption-driven transporters 0 0 0 3 3 6

8.A Auxiliary transport proteins 5 7 5 3 10 30

9.A Recognized transporters of unknown mechanism

82 75 76 81 85 399

9.B Putative uncharacterized transport proteins 30 25 15 17 20 107

Total 597 538 439 398 508 2480

Table 4 Mean and standard deviation of the subfamily size according to the different modes of evolution within the Hemiascomycete phylum

Mode of evolution Number of subfamilies

Mean number of ORF per subfamily

Minimum number of ORF

Maximum number of ORF

UBIQUITOUS 107 19.9 ± 25.8 5 146

SPECIES-SPECIFIC UNIQUE

15 1.5 ± 1.6 1 7

SPECIES-SPECIFIC ABSENT

20 7.9 ± 7.0 4 28

PHYLUM-GAINED 13 3.2 ± 1.0 2 6

PHYLUM- LOST 36 2.6 ± 2.6 1 16

HOMOPLASIC 13 3.1 ± 1.0 2 5

Table 7 Hemiascomycete Mitochondrial Carrier subfamilies that are absent in S. cerevisiae

 

Y. lipolytica D. hansenii K. lactis C. glabrata  

Subfamily  

2.A.29.6 YALI0A20944g DEHA0G14454g KLLA0E02750g  

2.A.29.Y14 DEHA0E08349g KLLA0A09383g CAGL0F08305g  

2.A.29.Y15 CAGL0B03883g  

2.A.29.Y16 YALI0A16863g  

2.A.29.Y17 YALI0A20988g DEHA0G19437g KLLA0E09680g  

2.A.29.Y18 YALI0B05852g  

2.A.29.Y19 YALI0E33341g YALI0F00418g

 

2.A.29.Y20 YALI0F20262g DEHA0E11022g  

2.A.29.Y21 YALI0F15609g DEHA0B16401g DEHA0E09691g

 

2.A.29.Y22 YALI0E06897g  

2.A.29.Y23 YALI0D06798g  

ORF number

10 6 3 2

Figure 2B Identification principles of the different evolution patterns distinguished in Figure 2A

Symbols used in

Figure 2 A

Mode of evolution Species A Species B Species C Species D

Ubiquitous

? 1 ORF

? 1 ORF

? 1 ORF

? 1 ORF

Unique

no ORF

? 1 ORF

no ORF

no ORF

Absent

? 1 ORF

no ORF

? 1 ORF

? 1 ORF

Gained

? 1 ORF

? 1 ORF

no ORF

no ORF

Lost

no ORF

no ORF

? 1 ORF

? 1 ORF

Homoplasic

? 1 ORF

no ORF

? 1 ORF

no ORF

Main characteristics Species Y. lipolytica D. hansenii K. lactis C. glabrata S. cerevisiae Total

Code YALI DEHA KLLA CAGL SACE

Natural substrate fats salted fish milk blood grapes

ORFs 6666 6896 5331 5272 5800 29965

Classified transporters

597 538 439 398 508 2480

% 9.0 7.8 8.2 7.5 8.8 8.3

Possible transporters (9.B.X.Y.Z) still unannotated

296 295 226 236 243 1296

Our objective : a consistent annotation

• Key elements– Consistent databases– The TCDB system of classification– A well-known evolutive context

• Output– A subfamily by subfamily discussion– Dynamic species vs. Quiet species

• Extension– Other species– Different levels of annotation

Databases Knowledge Models

DescriptionProcesses

Annotation Evolution

The TCDB Classification

• Based on five digits• Consistent across species• Extensible• An example

– 2 Electrochemical Potential-driven transporte• 2.A Porters (uniporters, symporters,

antiporters)– 2.A.1 The Major Facilitator (MFS) Superfamily

» 2.A.1.Y2 Undefined Subfamily

In practice – the most variable families

Subfamily YALI DEHA KLLA CAGL SACE Mean Variance

2.A.1.1 Sugar Porter (SP) 27 48 20 17 34 29.2 153.7

2.A.1.14 Anion Cation Symporter (ACS) 39 27 13 6 10 19.0 187.5

2.A.1.2 Drug Proton Antiporter 1 (DHA-1) 33 24 8 10 12 17.4 114.8

9.A.5.1 Peroxisomal Protein Importer (PPI) 27 7 10 11 10 13.0 63.5

2.A.67.1 Oligopeptide Transporter (OPT) 17 4 3 0 2 5.2 45.7

2.A.1.16 Ferrioxamine H+ symporter (SIT) 14 5 4 1 6 6.0 23.5

2.A.1.13 Fructose uniporter (FRU) 5 8 12 3 0 5.6 21.3

9.B.17.1 The Putative Fatty Acid Transporter (FAT-1)

14 3 3 5 5 6.0 21.0

3.D.1.2 NADH Dehydrogenase I (NDH 1) 8 8 0 0 0 3.2 19.2

2.A.3.10 AminoAcid-Polyamine-Organocation Yeast Transporter( APC-YAT )

14 24 16 14 18 17.2 17.2

1.A.20.5 Yeast Metal Channel ( Cyt B-FRE ) 11 7 5 1 7 6.2 13.2

Our objective : a consistent annotation

• Three elements– The Genolevure database– The TCDB system of classification– A well-known evolutive context

• Our material– Five species of Hemiascomycetes– 2480 identified transporter proteins

• Our objective– To understand how subfamilies of transporters emerge along the

evolutionary process

The chosen phylum

Figure 2. The Yeast MIT Family (Metal Ion Channels). TC # 1.A.35.

0.1

DEHA-0E11616g

YALI-0B05148g

KLLA-0F26895g

CAGL-0M13233g

SACE-MNR2

YALI-0F06248g

DEHA-0B05445g

KLLA-0F02519g

CAGL-0E05368gSACE-MRS2

DEHA-0E05731g

YALI-0D19514g

CAGL-0M07249gKLLA-0F28017g

SACE-LPE10

DEHA-0F17776g

YALI-0D00319g

YALI-0E00462g

KLLA-0E07249g

SACE-ALR2

SACE-ALR1

CAGL-0E01617g

1.A.35.5mitochondriaMg, (Zn, Mn, Cu?)

1.A.35.2plasma membrane Mg, Zn, Mn, Cu

Figure 4. The Yeast CDF Family (Cation Diffusion Facilitator). TC # 2.A.4.

0.1

DEHA-0G14113g

KLLA-0F20746g0

CAGL-0F05401g

SACE-MSC2

CAGL-0E06006g

SACE-MMT2

KLLA-0C16181g

CAGL-0H08822gSACE-MMT1

DEHA-0A03553g

YALI-0C12254g

YALI-0C18359gSACE-COT1

DEHA-0G03828g

YALI-0F00176g KLLA-0F08723g

CAGL-0K07392gSACE-ZRC1

2.A.4.2vacuoles, mitochondriaZn, Co

2.A.4.4endoplasmic reticulum, nucleusZn

2.A.4.Y1mitochondriaFe

Figure 5. The Yeast ZIP Family (ZINC Iron Porters). TC # 2.A.5.

0.1

DEHA-0B16335g

DEHA-0E25388g

SACE-ZRT1

YALI-0F21659gDEHA-0B07337g

CAGL-0E01353gKLLA-0D16434g

CAGL-0M04301g

SACE-ZRT2

YALI-0D00759g

YALI-0E00748g

YALI-0F15411g

SACE-YKE4

KLLA-0F17886g

YALI-0D19008g

DEHA-0E06105g

KLLA-0A07601g

SACE-ATX2

CAGL-0K05577g

2.A.5.Y1GolgiMn

2.A.5.2endoplasmic reticulumZn

2.A.5.1plasma membraneZn

2.A.5.Y2no datano data

Figure 6. The Yeast Nramp Family (Metal Ion Transporters). TC # 2.A.55.1

0.1

DEHA-0D06996g

KLLA-0D09581g

CAGL-0J00407g

SACE-SMF2

YALI-0D26818g

DEHA-0G09251g

KLLA-0F17391g

CAGL-0A03476g

SACE-SMF3

YALI-0C04411g

DEHA-0F25234g KLLA-0A03740g

CAGL-0E01969g

SACE-SMF1

2.A.55.1.2vesicles, mitochondriaMn

2.A.55.1.3vacuolesFe

2.A.55.1.1plasma membrane, vacuolesMn

0.1

DEHA-0D05269gDEHA-0E13211g

KLLA-0A03025g

CAGL-0I06743g

SACE-FTR1

YALI-0A04917g

DEHA-0C06226g

DEHA-0C07117g

KLLA-0F28039gCAGL-0M05511gSACE-FTH1YALI-0D06688gYALI-0D07304g

YALI-0D06754g

YALI-0D07282g

YALI-0A20273g

CAGL-0J08481g

KLLA-0C01694g

SACE-YDR506

KLLA-0F26400gDEHA-0E13332gKLLA-0D05489gCAGL-0K12738gSACE-FET5DEHA-0G05720gCAGL-0F06413gSACE-FET3

Figure 8. The Yeast OFeT Family (Oxydase-dependant Iron Transporters). TC # 9.A.10.

9.A.10.1plasma membrane, vacuolesFe

9.A.10.Y1plasma membrane, vacuolesFe

Figure 9. The Yeast CTR (Copper Transporters). TC # 9.A.12.

0.1

SACE-CTR3

KLLA-0A09207gDEHA-0G15268g

SACE-CTR2

CAGL-0I02508g

DEHA-0B00407g

DEHA-0F16390g

YALI-0C20295g KLLA-0B11407g

CAGL-0D04708g

SACE-CTR1

9.A.12.Y1,2,3no datano data

9.A.12.1vacuolesCu

9.A.12.Y4plasma membraneCu

9.A.12.2plasma membraneCu

CVYALI DEHA KLLA CAGL SACE name

39 27 13 6 10 9,87 2.A.1.14 The Anion: Cation Symporter (ACS) Family17 4 3 0 2 8,79 2.A.67.1 Subfamily of the Oligopeptide Transporter (OPT) Family0 0 0 0 7 7,00 8.A.9.Y1 Subfamily of the rBAT Transport Accessory Protein (rBAT) Family

33 24 8 10 12 6,60 2.A.1.2 The Drug:H+ Antiporter-1 (12 Spanner) (DHA1) Family8 8 0 0 0 6,00 3.D.1.2 Subfamily of the Proton-translocating NADH Dehydrogenase (NDH) Family

27 48 20 17 34 5,26 2.A.1.1 The Sugar Porter (SP) Family27 7 10 11 10 4,88 9.A.5.1 Subfamily of the Peroxisomal Protein Importer (PPI) Family14 5 4 1 6 3,92 2.A.1.16 The Siderophore-Iron Transporter (SIT) Family5 8 12 3 0 3,80 2.A.1.13 The Monocarboxylate Porter (MCP) Family

14 3 3 5 5 3,50 9.B.17.1 The Putative Fatty Acid Transporter (FAT-1) Type 1 Subfamily3 0 0 0 0 3,00 3.A.1.201 The Multidrug Resistance Exporter (MDR) Family (ABCB)7 3 1 1 1 2,62 2.A.17.2 Subfamily of the Proton-dependent Oligopeptide Transporter (POT) Family0 0 0 3 3 2,25 3.E.1.4 The Fungal Subfamily of the Ion-translocating Microbial Rhodopsin Subfamily6 3 2 0 1 2,21 2.A.1.12 The Sialate:H+ Symporter (SHS) Family

11 7 5 1 7 2,13 1.A.20.5 Subfamily of the Human Phagocyte NADPH Oxidase Cyt b558 H+ Channel

Nbr of Orfs in each subfamily

The variation coefficients of transporters in subfamilies

TC/YETI Family or Subfamily Identificator