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
Two views about the type of mechanism that promotes evolution.
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.
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 chemistry
Acetylation 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 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
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 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
QuickTime™ et un
décompresseur 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
Genomology
Genome
mapping
Genome
sequencing
GENOMICS
(GLOBAL)
Genome
comparisons
Databases
NEW TOOLS
(GLOBAL)
REDUCTIONICS
(SPECIFIC)
Deletomics Overexpressionics Transcriptomics
(systematic)
(systematic)
(DNA chips)
Physiologists
Pathologists
Structuralists
Biologists
Proteomics
(2D gels/2 hybrids)
Biochemists
Schematic alternating signature for
Whole Genome Duplication
4
5
6
7
5
6
7
1
2
3
4
1
2
3
4
10 11 12
13
14
15
16
13
14
15
16 17 18
19
16 17 18
19
8
9
10 11 12
8
9
10 11
20
21 22
20
21 22
Duplicated copy 1
in S. cerevisiae
Reference block
in K. waltii
Duplicated copy 2
in S. cerevisiae
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, FerryDumazet 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, OzierKalogeropoulos 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, BolotinFukuhara 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)
10
Membrane Proteins
A
10.A
Lipid Metabolism
B
10.B
Anchoring
C
10.C
Polysaccharide Metabolism
D
10.D Trafficking
E
10.E
Signaling
F
10.F
Oxidoreductases
G
10.G Subtelomeric Conserved
H
10.H Chaperones
Genomology
Genome
mapping
Genome
sequencing
GENOMICS
(GLOBAL)
Genome
comparison
Databases
NEW TOOLS
(GLOBAL)
REDUCTIONICS
(SPECIFIC)
Deletomics Overexpressionics Transcriptomics
(systematic)
(systematic)
(DNA chips)
Physiologists
Pathologists
Structuralists
Biologists
Proteomics
(2D gels/2 hybrids)
Biochemists
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
9
Incompletely Characterized Transport Systems
9.A
Recognized Transporters of Unknown
Biochemical Mechanism
9.B
Putative Uncharacterized Transport Proteins
9.C
Functionally Characterized Transporters
Lacking Identified Sequences
9.D
The Membrane Proteins of Unknown Function
9.E
Questionable ORFs with TMS>2
S. Cerevisiae Membrane Classification
Subfamilies
13
ORFs
28
10.A
Lipid Metabolism
10.B
Anchoring
9
10
10.C
Polysaccharide Metabolism
8
32
11
39
10.D Trafficking
10.E
Signaling
4
7
10.F
Oxidoreductases
5
11
10.G Subtelomeric Conserved
1
12
10.H Chaperones
4
7
Total :
55
146
Table 1 Global statistics of membrane proteins in Hemiascomycete species
Species
Y. lipolytica
D. hansenii
K. lactis
C. glabrata
S.
cerevisiae
Code
YALI
DEHA
KLLA
CAGL
SACE
Strain
CLIB122
CBS767
CLIB210
CBS138
S288c
Database
Génolevures
Génolevures
Génolevure
s
Génolevure
s
SGD
Release
22 may 2004
22 may 2004
22 may
2004
22 may
2004
22 may
2004
fats
salted fish
milk
blood
grapes
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
Natural substrate
ORFs
Total
Table 2 Functional distribution of the “established and putative” transporters in the Hemiascomycete phy
YALI
DEHA
KLLA
CAGL
46
32
26
26
38
168
1
1
1
2
2
7
316
281
206
162
218
1183
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
597
538
439
398
508
2480
1.A Alpha-Type channels
1.B Beta Barrel porins.
2.A Porters (uniporters, symporters,
antiporters)
3.A P-P-bond-hydrolysis-driven transporters
Total
SACE
Total
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
YALI0A20944g
DEHA0G14454g
KLLA0E02750g
DEHA0E08349g
KLLA0A09383g
Subfamily
2.A.29.6
2.A.29.Y14
2.A.29.Y15
CAGL0B03883g
2.A.29.Y16
YALI0A16863g
2.A.29.Y17
YALI0A20988g
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
CAGL0F08305g
10
DEHA0G19437g
6
KLLA0E09680g
3
2
Figure 2B Identification principles of the different evolution patterns distinguished in Figure 2A
Symbols used in
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
Figure 2 A
Main characteristics
Species
Y. lipolytica
D. hansenii
K. lactis
C. glabrata
S. cerevisiae
Code
YALI
DEHA
KLLA
CAGL
SACE
Natural substrate
fats
salted fish
milk
blood
grapes
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
ORFs
Total
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
Description Processes
Annotation
unannotated
(9.B.X.Y.Z) still
Possible transporters
296
295
226
236
243
%
9.0
7.8
8.2
7.5
8.8
8.3
transporters
Classified
597
538
439
398
508
2480
29965
ORFs
6666
6896
5272
5800
Natural substrate
fats
salted fish
milk
blood
grapes
Code
YALI
DEHA
KLLA
CAGL
SACE
Y. lipolytica
D. hansenii
K. lactis
C. glabrata
S. cerevisiae
Species
5331
1296
Total
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
YALI
DEHA
KLLA
CAGL
SACE
Mean
Variance
Subfamily
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
SACE-ALR2
KLLA-0E07249g
YALI-0E00462g
YALI-0D00319g
1.A.35.2
plasma membrane
Mg, Zn, Mn, Cu
SACE-ALR1
CAGL-0E01617g
DEHA-0E11616g
YALI-0B05148g
DEHA-0F17776g
KLLA-0F26895g
CAGL-0M13233g
SACE-MNR2
SACE-LPE10
KLLA-0F28017g
CAGL-0M07249g
YALI-0D19514g
0.1
DEHA-0E05731g
SACE-MRS2
YALI-0F06248g
DEHA-0B05445g
KLLA-0F02519g
CAGL-0E05368g
Figure 2. The Yeast MIT Family (Metal Ion Channels). TC # 1.A.35.
1.A.35.5
mitochondria
Mg, (Zn, Mn, Cu?)
YALI-0F00176g
DEHA-0G03828g
KLLA-0F08723g
CAGL-0K07392g
SACE-ZRC1
YALI-0C18359g
SACE-COT1
2.A.4.2
vacuoles, mitochondria
Zn, Co
DEHA-0G14113g
KLLA-0F20746g0
CAGL-0F05401g
SACE-MSC2
2.A.4.4
endoplasmic reticulum, nucleus
Zn
YALI-0C12254g
CAGL-0E06006g
SACE-MMT2
DEHA-0A03553g
0.1
SACE-MMT1
KLLA-0C16181g
CAGL-0H08822g
Figure 4. The Yeast CDF Family (Cation Diffusion Facilitator). TC # 2.A.4.
2.A.4.Y1
mitochondria
Fe
2.A.5.Y1
Golgi
Mn
YALI-0D19008g
KLLA-0F17886g
DEHA-0E06105g
2.A.5.Y2
no data
no data
KLLA-0A07601g
SACE-YKE4
SACE-ATX2
CAGL-0K05577g
2.A.5.2
endoplasmic reticulum
Zn
YALI-0F15411g
DEHA-0B16335g
YALI-0E00748g
YALI-0D00759g
0.1
DEHA-0E25388g
SACE-ZRT2
CAGL-0M04301g
2.A.5.1
plasma membrane
Zn
KLLA-0D16434g
SACE-ZRT1
YALI-0F21659g
DEHA-0B07337g
CAGL-0E01353g
Figure 5. The Yeast ZIP Family (ZINC Iron Porters). TC # 2.A.5.
DEHA-0F25234g
KLLA-0A03740g
2.A.55.1.1
plasma membrane, vacuoles
CAGL-0E01969g Mn
YALI-0C04411g
SACE-SMF1
2.A.55.1.3
vacuoles
Fe
SACE-SMF3
DEHA-0D06996g
CAGL-0A03476g
KLLA-0D09581g
KLLA-0F17391g
CAGL-0J00407g
DEHA-0G09251g
SACE-SMF2
0.1
YALI-0D26818g
re 6. The Yeast Nramp Family (Metal Ion Transporters). TC # 2.A.55.1
2.A.55.1.2
vesicles, mitochondria
Mn
KLLA-0C01694g
CAGL-0J08481g
YALI-0A20273g
SACE-YDR506
YALI-0D07282g
YALI-0D06754g
KLLA-0F26400g
DEHA-0E13332g
KLLA-0D05489g
CAGL-0K12738g
SACE-FET5
DEHA-0G05720g
CAGL-0F06413g
SACE-FET3
9.A.10.Y1
plasma membrane, vacuoles
Fe
YALI-0D07304g
YALI-0D06688g
SACE-FTH1
CAGL-0M05511g
KLLA-0F28039g
DEHA-0C07117g
DEHA-0C06226g
YALI-0A04917g
0.1
DEHA-0D05269g
DEHA-0E13211g
KLLA-0A03025g
CAGL-0I06743g
SACE-FTR1
9.A.10.1
plasma membrane, vacuoles
Fe
Figure 8. The Yeast OFeT Family (Oxydase-dependant Iron Transporters). TC # 9.A.10.
9.A.12.Y1,2,3
no data
no data
YALI-0C20295g
KLLA-0B11407g
CAGL-0D04708g
DEHA-0F16390g
9.A.12.2
plasma membrane
Cu
SACE-CTR1
DEHA-0B00407g
9.A.12.Y4
plasma membrane
Cu
SACE-CTR3
CAGL-0I02508g
SACE-CTR2
KLLA-0A09207g
DEHA-0G15268g
0.1
Figure 9. The Yeast CTR (Copper Transporters). TC # 9.A.12.
9.A.12.1
vacuoles
Cu
The variation coefficients of transporters in subfamilies
Nbr of Orfs in each subfamily CV
YALI DEHA KLLA CAGL SACE
39
17
0
33
8
27
27
14
5
14
3
7
0
6
11
27
4
0
24
8
48
7
5
8
3
0
3
0
3
7
13
3
0
8
0
20
10
4
12
3
0
1
0
2
5
6
0
0
10
0
17
11
1
3
5
0
1
3
0
1
10
2
7
12
0
34
10
6
0
5
0
1
3
1
7
TC/YETI Family or Subfamily Identificator
name
9,87
8,79
7,00
6,60
6,00
5,26
4,88
3,92
3,80
3,50
3,00
2,62
2,25
2,21
2,13
2.A.1.14 The Anion: Cation Symporter (ACS) Family
2.A.67.1 Subfamily of the Oligopeptide Transporter (OPT) Family
8.A.9.Y1 Subfamily of the rBAT Transport Accessory Protein (rBAT) Family
2.A.1.2 The Drug:H+ Antiporter-1 (12 Spanner) (DHA1) Family
3.D.1.2 Subfamily of the Proton-translocating NADH Dehydrogenase (NDH) Family
2.A.1.1 The Sugar Porter (SP) Family
9.A.5.1 Subfamily of the Peroxisomal Protein Importer (PPI) Family
2.A.1.16 The Siderophore-Iron Transporter (SIT) Family
2.A.1.13 The Monocarboxylate Porter (MCP) Family
9.B.17.1 The Putative Fatty Acid Transporter (FAT-1) Type 1 Subfamily
3.A.1.201 The Multidrug Resistance Exporter (MDR) Family (ABCB)
2.A.17.2 Subfamily of the Proton-dependent Oligopeptide Transporter (POT) Family
3.E.1.4 The Fungal Subfamily of the Ion-translocating Microbial Rhodopsin Subfamily
2.A.1.12 The Sialate:H+ Symporter (SHS) Family
1.A.20.5 Subfamily of the Human Phagocyte NADPH Oxidase Cyt b558 H+ Channel