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EPIGENOMICS André Goffeau Institut Pasteur/EMBO/CNPq course Florianopolis, July 11, 2008.

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Presentation on theme: "EPIGENOMICS André Goffeau Institut Pasteur/EMBO/CNPq course Florianopolis, July 11, 2008."— Presentation transcript:

1 EPIGENOMICS André Goffeau Institut Pasteur/EMBO/CNPq course Florianopolis, July 11, 2008.

2 Epigenomics is any regulation (on/off) of gene expression that is not due to DNA mutations and is heritable

3 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

4 Jean-Baptiste Lamark 1744-1829 Charles Darwin 1809-1882

5 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.

6 RNA interference, Histone acetylation and DNA methylation



9 Cytosine methylation occurs at CpG and is mutagenic It prevents activation of promoters


11 Methylation of CpG islands

12 and the informatician? they try to predict which cytosines are methylated in DNA


14 Reprogramming in Germ Cells and Embryos


16 Chromatin chemistry Acetylation or methylation

17 Histone modifications


19 Methylation Genomics



22 Aberrant methylation in human and mouse leukemia


24 P rv a c y P ol ic y Epigenetic diseases

25 Epigenetic drugs

26 Gene silencing and pharmacology

27 siRNA

28 RNA silencing

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

30 RNA interference, Histone acetylation and DNA methylation For elucidation of mechanism, use S.pombe, N.crassa or Y.lipolytica ?? but not at S.cerevisiae

31 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 mammalian development. 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


33 /animation/animation.htm





38 Hinny and Dolly

39 EARLY EXAMPLES agouti mice (folic acid) cancer human (p16) diseases human (BWS) eye apendage fly (Hsp90)


41 Methyl detector Yellow: hyper-methylated; Blue: under-methylated

42 Restriction Landmark Genomic Scanning

43 Reprogramming and Imprinting

44 Deoxynucleoside analogue inhibition




















64 Genome sequencing Deletomics (systematic) Overexpressionics (systematic) Transcriptomics (DNA chips) Proteomics (2D gels/2 hybrids) Physiologists Pathologists StructuralistsBiologists Biochemists REDUCTIONICS (SPECIFIC) NEW TOOLS (GLOBAL) GENOMICS (GLOBAL) Genome mapping Genome comparisons Genomology Databases

65 Schematic alternating signature for Whole Genome Duplication Duplicated copy 1 in S. cerevisiae Duplicated copy 2 in S. cerevisiae Reference block in 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.

66 EMERGENCE OF SPECIES-SPECIFIC TRANSPORTERS DURING EVOLUTION OF THE HEMIASCOMYTE PHYLUM Benoît De Hertogh* [1], Frédéric Hancy† [2], André Goffeau‡ and Philippe V. Baret* [1] [2] Université catholique de Louvain

67 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.

68 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

69 Genome sequencing Deletomics (systematic) Overexpressionics (systematic) Transcriptomics (DNA chips) Proteomics (2D gels/2 hybrids) Physiologists Pathologists StructuralistsBiologists Biochemists REDUCTIONICS (SPECIFIC) NEW TOOLS (GLOBAL) GENOMICS (GLOBAL) Genome mapping Genome comparison Genomology Databases

70 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

71 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 Proteins 9.B The Membrane Proteins of Unknown Function 9.D 9 9.E Questionable ORFs with TMS>2

72 10.A Lipid Metabolism1328 10.B Anchoring 910 10.C Polysaccharide Metabolism 832 10.D Trafficking1139 Total: 55146 10.E Signaling 4 7 10.F Oxidoreductases 511 10.G Subtelomeric Conserved 112 10.H Chaperones 4 7 SubfamiliesORFs S. Cerevisiae Membrane Classification

73 Table 1 Global statistics of membrane proteins in Hemiascomycete species SpeciesY. lipolyticaD. hanseniiK. lactisC. glabrataS. cerevisiae Total CodeYALIDEHAKLLACAGLSACE StrainCLIB122CBS767CLIB210CBS138S288c DatabaseGénolevures SGD Release22 may 2004 Natural substratefatssalted fishmilkbloodgrapes ORFs6666689653315272580029965 Classified transporters5975384393985082480 % Possible transporters (9.B.X.Y.Z) still unannotated 2962952262362431296

74 Table 2 Functional distribution of the “established and putative” transporters in the Hemiascomycete phylum YALIDEHAKLLACAGLSACETotal 1.A Alpha-Type channels463226 38168 1.B Beta Barrel porins.111227 2.A Porters (uniporters, symporters, antiporters) 3162812061622181183 3.A P-P-bond-hydrolysis-driven transporters94899187107468 3.B Decarboxylation-driven transporters211206 3.D Oxidoreduction-driven transporters2127181525106 3.E Light absorption-driven transporters000336 8.A Auxiliary transport proteins57531030 9.A Recognized transporters of unknown mechanism 8275768185399 9.B Putative uncharacterized transport proteins3025151720107 Total5975384393985082480

75 Table 4 Mean and standard deviation of the subfamily size according to the different modes of evolution within the Hemiascomycete phylum Mode of evolutionNumber of subfamilies Mean number of ORF per subfamily Minimum number of ORF Maximum number of ORF UBIQUITOUS10719.9 ± 25.85146 SPECIES-SPECIFIC UNIQUE 151.5 ± 1.617 SPECIES-SPECIFIC ABSENT 207.9 ± 7.0428 PHYLUM-GAINED133.2 ± 1.026 PHYLUM- LOST362.6 ± 2.6116 HOMOPLASIC133.1 ± 1.02 5

76 Table 7 Hemiascomycete Mitochondrial Carrier subfamilies that are absent in S. cerevisiae Y. lipolyticaD. hanseniiK. lactisC. glabrata Subfamily 2.A.29.6YALI0A20944gDEHA0G14454gKLLA0E02750g 2.A.29.Y14DEHA0E08349gKLLA0A09383gCAGL0F08305g 2.A.29.Y15CAGL0B03883g 2.A.29.Y16YALI0A16863g 2.A.29.Y17YALI0A20988gDEHA0G19437gKLLA0E09680g 2.A.29.Y18YALI0B05852g 2.A.29.Y19YALI0E33341g YALI0F00418g 2.A.29.Y20YALI0F20262gDEHA0E11022g 2.A.29.Y21YALI0F15609gDEHA0B16401g DEHA0E09691g 2.A.29.Y22YALI0E06897g 2.A.29.Y23YALI0D06798g ORF number 10632

77 Figure 2B Identification principles of the different evolution patterns distinguished in Figure 2A ? 1 ORF Homoplasic ? 1 ORF no ORF ? 1 ORF no ORF

78 Main characteristics Species Y. lipolyticaD. hanseniiK. lactisC. glabrataS. cerevisiaeTotal Code YALIDEHAKLLACAGLSACE Natural substratefatssalted fishmilkbloodgrapes ORFs6666689653315272580029965 Classified transporters 5975384393985082480 % Possible transporters (9.B.X.Y.Z) still unannotated 2962952262362431296

79 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

80 Databases Knowledge Models Description Processes AnnotationEvolution

81 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

82 In practice – the most variable families Subfamily YALIDEHAKLLACAGLSACEMeanVariance 2.A.1.1Sugar Porter (SP)274820173429.2153.7 2.A.1.14Anion Cation Symporter (ACS)39271361019.0187.5 2.A.1.2Drug Proton Antiporter 1 (DHA-1)33248101217.4114.8 9.A.5.1Peroxisomal Protein Importer (PPI)27710111013.063.5 2.A.67.1Oligopeptide Transporter (OPT)1743025.245.7 2.A.1.16Ferrioxamine H+ symporter (SIT)1454166.023.5 2.A.1.13Fructose uniporter (FRU)5812305.621.3 9.B.17.1The Putative Fatty Acid Transporter (FAT-1)1433556.021.0 3.D.1.2NADH Dehydrogenase I (NDH 1)880003.219.2 2.A.3.10AminoAcid-Polyamine-Organocation Yeast Transporter( APC-YAT ) 142416141817.2 1.A.20.5Yeast Metal Channel ( Cyt B-FRE )1175176.213.2

83 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

84 The chosen phylum

85 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- 0E05368g SACE- MRS2 DEHA- 0E05731g YALI- 0D19514g CAGL- 0M07249g KLLA- 0F28017g SACE- LPE10 DEHA- 0F17776g YALI- 0D00319g YALI- 0E00462g KLLA- 0E07249g SACE- ALR2 SACE- ALR1 CAGL- 0E01617g 1.A.35.5 mitochondria Mg, (Zn, Mn, Cu?) 1.A.35.2 plasma membrane Mg, Zn, Mn, Cu

86 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- 0H08822g SACE- MMT1 DEHA- 0A03553g YALI- 0C12254g YALI- 0C18359g SACE- COT1 DEHA- 0G03828g YALI- 0F00176g KLLA- 0F08723g CAGL- 0K07392g SACE- ZRC1 2.A.4.2 vacuoles, mitochondria Zn, Co 2.A.4.4 endoplasmic reticulum, nucleus Zn 2.A.4.Y1 mitochondria Fe

87 Figure 5. The Yeast ZIP Family (ZINC Iron Porters). TC # 2.A.5. 0.1 DEHA- 0B16335g DEHA- 0E25388g SACE- ZRT1 YALI- 0F21659g DEHA- 0B07337g CAGL- 0E01353g KLLA- 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.Y1 Golgi Mn 2.A.5.2 endoplasmic reticulum Zn 2.A.5.1 plasma membrane Zn 2.A.5.Y2 no data

88 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.55.1.2 vesicles, mitochondria Mn 2.A.55.1.3 vacuoles Fe 2.A.55.1.1 plasma membrane, vacuoles Mn

89 0.1 DEHA- 0D05269g DEHA- 0E13211g KLLA- 0A03025g CAGL- 0I06743g SACE- FTR1 YALI- 0A04917g DEHA- 0C06226g DEHA- 0C07117g KLLA- 0F28039g CAGL- 0M05511g SACE- FTH1 YALI- 0D06688g YALI- 0D07304g YALI- 0D06754g YALI- 0D07282g YALI- 0A20273g CAGL- 0J08481g KLLA- 0C01694g SACE- YDR506 KLLA- 0F26400g DEHA- 0E13332g KLLA- 0D05489g CAGL- 0K12738g SACE- FET5 DEHA- 0G05720g CAGL- 0F06413g SACE- FET3 Figure 8. The Yeast OFeT Family (Oxydase-dependant Iron Transporters). TC # 9.A.10. 9.A.10.1 plasma membrane, vacuoles Fe 9.A.10.Y1 plasma membrane, vacuoles Fe

90 Figure 9. The Yeast CTR (Copper Transporters). TC # 9.A.12. 0.1 SACE- CTR3 KLLA- 0A09207g DEHA- 0G15268g SACE- CTR2 CAGL- 0I02508g DEHA- 0B00407g DEHA- 0F16390g YALI- 0C20295g KLLA- 0B11407g CAGL- 0D04708g SACE- CTR1 9.A.12.Y1,2,3 no data 9.A.12.1 vacuoles Cu 9.A.12.Y4 plasma membrane Cu 9.A.12.2 plasma membrane Cu


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