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Evolution of regulatory interactions in bacteria

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Evolution of regulatory
interactions in bacteria
Mikhail Gelfand
Institute for Information Transmission Problems, RAS
4th Bertinoro Computational Biology (BCB) Meeting
“Evolution of and Comparative Approaches to Gene Regulation”
24-30 June 2006
Это – ряд наблюдений. В углу – тепло.
Взгляд оставляет на вещи след.
Вода представляет собой стекло.
Человек страшней, чем его скелет.
Иосиф Бродский
A list of some observations. In a corner, it’s warm.
A glance leaves an imprint on anything it’s dwelt on.
Water is glass’s most public form.
Man is more frightening than its skeleton.
Joseph Brodsky
Plan
• Evolution of individual sites
• Coevolution of transcription factors and
their binding signals
• Distribution of transcription factor families
in various genomes
• Evolution of simple and complex
regulatory systems
Birth and death of sites
is a very dynamic process
NadR-binding sites upstream of pnuB seem absent in
Klebsiella pneumoniae and Serratia marcescens
… but there are candidate sites further upstream …
… and they are clearly diferent (not simply misaligned).
Loss of regulators and cryptic sites
Loss of the RbsR in Y. pestis
(ABC-transporter also is lost)
RbsR binding site
Start codon of rbsD
Unexpected conservation of non-consensus
positions in orthologous sites
regulatory site of LexA upstream of lexA
consensus nucleotides are in caps
Escherichia coli
Salmonella typhi
Yersinia pestis
Haemophilus influenzae
Pasteurella multocida
Vibrio cholerae
TgCTGTATATActcACAGcA
aACTGTATATActcACAGcA
agCTGTATATActcACAGcA
atCTGTATAcAatacCAGTt
TtCTGTATATAataACAGTt
cACTGgATATActcACAGTc
wrong consensus?
TF PurR, gene purL
Escherichia coli
Salmonella typhi
Yersinia pestis
Haemophilus influenzae
Pasteurella multocida
Vibrio cholerae
A C G C A A A C Gg T T t C G T
A C G C A A A C Gg T T t C G T
A C G C A A A C Gg T T t C G T
A t G C A A A C G T T T G Ct T
A C G C A A A C G T T Tt C G T
A C G C A A A C Gg T T G C t T
TF PurR, gene purM
Escherichia coli
Salmonella typhi
Yersinia pestis
Haemophilus influenzae
Pasteurella multocida
Vibrio cholerae
t C G C A A A C G T T T G Ct T
t C G C A A A C G T T T G Ct T
t C G C A A A C G T T T G Cc T
t C G C A A A C G T T T G Ct T
t C G C A A A C G T T T G Ct T
A C G C A A A C G T T Tt C c T
Non-consensus positions are more conserved
than synonymous codon positions
Relative conservation of non-consensus
nucleotides may be higher than conservation
of consensus nucleotides
Regulators and their signals
• Subtle changes at close evolutionary
distances
• Cases of signal conservation at
surprisingly large distances
• Changes in spacing / geometry of dimers
• Correlation between contacting
nucleotides and amino acid residues
The LacI family:
subtle changes in signals at close distances
G
A
CG
Gn GC
n
NrdR (regulator of ribonucleotide reducases
and some other replication-related genes):
conservation at large distances
BirA (biotin regulator in eubacteria and
archaea): conserved signal, changed spacing
Profile 1: Gram-positive bacteria, Archaea
Profile 2: Gram-negative bacteria
DNA signals and protein-DNA interactions
Entropy at aligned sites and the number of contacts
(heavy atoms in a base pair at a distance <cutoff from a protein atom)
CRP
PurR
IHF
TrpR
Specificity-determining positions
in the LacI family
• Training set: 459 sequences,
average length: 338 amino acids,
85 specificity groups
– 44 SDPs
10 residues contact NPF (analog of
the effector)
7 residues in the effector contact zone
(5Ǻ<dmin<10Ǻ)
6 residues in the intersubunit
contacts
5 residues in the intersubunit
contact zone (5Ǻ<dmin<10Ǻ)
7 residues contact the operator
sequence
6 residues in the operator contact
zone (5Ǻ<dmin<10Ǻ)
LacI from E.coli
CRP/FNR family of regulators
TGTCGGCnnGCCGACA
CooA
D es u lfo vib rio
TTGTGAnnnnnnTCACAA
FNR
G am m a
TTGATnnnnATCAA
HcpR
D es u lfo vib rio
TTGTgAnnnnnnTcACAA
Correlation between contacting
nucleotides and amino acid residues
•
•
•
•
DD
DV
EC
YP
VC
DD
DV
EC
YP
VC
CooA in Desulfovibrio spp.
CRP in Gamma-proteobacteria
HcpR in Desulfovibrio spp.
FNR in Gamma-proteobacteria
COOA
COOA
CRP
CRP
CRP
HCPR
HCPR
FNR
FNR
FNR
ALTTEQLSLHMGATRQTVSTLLNNLVR
ELTMEQLAGLVGTTRQTASTLLNDMIR
KITRQEIGQIVGCSRETVGRILKMLED
KXTRQEIGQIVGCSRETVGRILKMLED
KITRQEIGQIVGCSRETVGRILKMLEE
DVSKSLLAGVLGTARETLSRALAKLVE
DVTKGLLAGLLGTARETLSRCLSRMVE
TMTRGDIGNYLGLTVETISRLLGRFQK
TMTRGDIGNYLGLTVETISRLLGRFQK
TMTRGDIGNYLGLTVETISRLLGRFQK
Contacting residues:
REnnnR
TG: 1st arginine
GA: glutamate and 2nd
arginine
TGTCGGCnnGCCGACA
TTGTGAnnnnnnTCACAA
TTGTgAnnnnnnTcACAA
TTGATnnnnATCAA
The
correlation
holds for
other
factors in
the family
Distribution of TF families in bacterial genomes
Pseudomonas aeruginosa
TetR
LysR
LuxR
Streptomyces coelicolor
LacI
GntR
AraC
ExtraTrain
database
Agrobacterium tumefaciens
Escherichia coli
Bacillus subtilis
Strategies of successful TF families
• One ortholog per genome:
– LexA, NrdR, HrcA, ArgR
– present even in archaea: BirA (also enzyme), ModE
• Several (2-3) orthologs per genome
– CRP/FNR, FUR
• Local explosions
– LacI in alpha- and gamma-proteobacteria
– 2CS systems in delta-proteobacteria
– sigma-factors in Streptomyces
• Because TF in a family tend to have related functions
and these might depend on the lifestyle?
LacI family regulons in closely related strains
(top: TFs, bottom: regulated genes)
Seven Escherichia and
Shigella spp.
Four Bacillus cereus and
B. anthracis strains
Five Salmonella spp.
1
1
2
3
2
2
4
3
5
4
3
6
7
1
2
3
4
5
6
7
1
5
1
2
3
4
5
4
1
2
3
4
What are the driving forces for the
present-day state?
• Expansion and contraction of regulons
• Duplications of regulators with or without
regulated loci
• Loss of regulators with or without
regulated loci
• Re-assortment of regulators and structural
genes
• … especially in complex systems
• Horizontal transfer
Regulon expansion:
how FruR has become CRA
Mannose
Glucose
manXYZ
ptsHI-crr
edd
epd
eda
adhE
aceEF
Mannitol
mtlA
gapA
fbp
Fructose
pykF
mtlD
fruBA
fruK
pfkA
pgk
gpmA
icdA
ppsA
pckA
aceA
tpiA
aceB
Gamma-proteobacteria
Common ancestor of Enterobacteriales
Mannose
Glucose
manXYZ
ptsHI-crr
edd
epd
eda
adhE
aceEF
Mannitol
mtlA
gapA
fbp
Fructose
pykF
mtlD
fruBA
fruK
pfkA
pgk
gpmA
icdA
ppsA
pckA
aceA
tpiA
aceB
Gamma-proteobacteria
Enterobacteriales
Common ancestor of Escherichia and Salmonella
Mannose
Glucose
manXYZ
ptsHI-crr
edd
epd
eda
adhE
aceEF
Mannitol
mtlA
gapA
fbp
Fructose
pykF
mtlD
fruBA
fruK
pfkA
pgk
gpmA
icdA
ppsA
pckA
aceA
tpiA
aceB
Gamma-proteobacteria
Enterobacteriales
E. coli and Salmonella spp.
Trehalose/maltose catabolism
in alpha-proteobacteria
Duplicated LacI-family regulators: lineagespecific post-duplication loss
The binding signals are very similar (the blue branch is
somewhat different: to avoid cross-recognition?)
Utilization of an unknown galactoside in
gamma-proteobacteria
Yersinia and Klebsiella: two regulons, GalR (not shown,
includes genes galK and galT) and Laci-X
Erwinia: one regulon, GalR
Loss of regulator and merger of
regulons: It seems that laci-X was
present in the common ancestor
(Klebsiella is an outgroup)
Utilization of maltose/maltodextrin
in Firmicutes
Two different ABC transporters (shades of red)
PTS (pink)
Glucoside hydrolases (shades of green)
Two regulators (black and grey)
Modularity of the functional subsystem
Two different ABC systems
Three hydrolases in one operon (E. faecalis) or separately
Changes of regulation
Two different ABC systems
Displacement: invasion of a regulator from a
different subfamily (horizontal transfer from a
related species?) – blue sites
Orthologous TFs
with completely
different regulons
Utilization of xylose in alpha-proteobacteria
xylBA
Three different ABC transporters
Three regulators: two from the LacI family and one from the ROK family
Changes in operon structure
Changes in regulation
Displacement: Operon regulation
changed from XylR-1 to XylR-2
(different subfamily)
Duplication and displacement:
Duplicated XylR-1a assumed the role
of the ROK-family regulator
Catabolism of gluconate in proteobacteria
extreme variability of regulation of “marginal” regulon members
β
Pseudomonas spp.
γ
Regulation of amino acid biosynthesis in
Firmicutes
• Interplay between regulatory RNA
elements and transcription factors
• Expansion of T-box systems (normally
RNA structures regulating aminoacyltRNA-synthetases)
Aromatic amino acid regulons
Five
regulatory
systems for
the
methionine
biosynthesis
A.
SAMdependent
RNA
riboswitch
B. Met-tRNAdependent
T-box (RNA)
C,D,E.
repressors of
transcription
Methionine regulatory systems:
loss of S-box regulons
• S-boxes (SAM-1 riboswitch)
– Bacillales
– Clostridiales
– the Zoo:
•
•
•
•
•
•
ZOO
Petrotoga
actinobacteria (Streptomyces, Thermobifida)
Chlorobium, Chloroflexus, Cytophaga
Fusobacterium
Deinococcus
proteobacteria (Xanthomonas, Geobacter)
• Met-T-boxes (Met-tRNA-dependent attenuator)
+ SAM-2 riboswitch for metK
– Lactobacillales
• MET-boxes (candidate transcription signal)
– Streptococcales
Lact.
Strep. Bac. Clostr.
Mapping the events to the phylogenetic tree
loss of S-boxes
(SAM-I riboswitches)
expansion of Met-T-boxes,
emergence of SAM-2 riboswitches
Trp-T-boxes TRAP
Tyr-T-boxes PCE
Bacillus
subtilis
and
related
species
emergence of MtaR
Tyr-T-boxes ARO
Bacillus
cereus
and
related
species
Lactobacillus
spp.
Streptococcus
spp.
Clostridium
spp.
Combined regulatory network for iron homeostasis genes in in a-proteobacteria.
[- Fe]
[+Fe]
[ - Fe]
[+Fe]
RirA
RirA
Irr
Irr
FeS
heme
degraded
Siderophore
uptake
2+
3+
Fe / Fe
uptake
Iron uptakesystems
Fur
[- Fe]
Iron storage
ferritins
FeS
synthesis
Heme
synthesis
Iron-requiring
enzymes
[ironcofactor]
Fur
IscR
Fe
FeS
Transcription
factors
FeS status
of cell
[+Fe]
The connecting line denote regulatory interactions, which the thickness reflecting the frequency of the interaction in the
analyzed genomes. The suggested negative or positive mode of operation is shown by dead-end and arrow-end of the line.
Fe and Mn regulons
Rhizobiaceae
Organism
Abb.
Irr
MUR /
FUR
MntR
RirA
IscR
Sinorhizobium meliloti
SM
+
+
-
+
-
+ +
+
-
+
-
Rhizobium leguminosarum
RL
Rhizobium etli
RHE
+
+
-
+
-
Agrobacterium tumefaciens
AGR
+
+
-
+
-
ML
+
-
+
+
-
MBNC
+
+ +
-
+
-
+
-
+
-
+
-
Mesorhizobium loti
Mesorhizobium
sp. BNC1
Brucella melitensis
Rhizobiales
Rhodobacteraceae
BQ
+
+
Bradyrhizobium japonicum
BJ
Rhodopseudomonas palustris
RPA
+ +
+ +
+
+
-
-
-
Nitrobacter hamburgensis
Nham
+
+
-
-
-
Nitrobacter winogradskyi
Nwi
+
+
-
-
-
Rhodobacter capsulatus
RC
-
Rhodobacter sphaeroides
Rsph
+
+
+
+
-
+
+
+
+
Silicibacter
STM
+
+
-
+
+
Silicibacter pomeroyi
S PO
+
+
-
+
+
Jannaschia
Jann
+
+
-
#?
+
+
+
+
quintana
and spp.
sp. TM1040
sp.CC51
HTCC2654
Rhodobacterales bacterium
Roseobacter
sp. MED193
Roseovarius nubinhibens
- proteobacteria
Rhodobacterales
Roseovarius
ISM
sp.217
Loktanella vestfoldensis
Sulfitobacter sp.
SKA53
EE-36
RB2654
+
+
-
MED193
+
+
-
ISM
+
+
-
+
#?
ROS217
+
+
-
+
+
SKA53
+
+
-
#?
+
EE36
+
+
-
#?
#?
+
OB2597
+
+
OA2633
-
+
-
-
+
CC
-
+
-
-
+
PB2503
-
+
-
-
+
Erythrobacter litoralis
ELI
-
-
Novosphingobium aromaticivorans
Saro
-
+
+
-
-
+
+
Sphinopyxis
g
alaskensis
HTCC2597
Oceanicola batsensis
HTCC2633
Oceanicaulis alexandrii
Caulobacterales
Caulobacter crescentu
s
Parvularculales
Parvularcula bermudensis
Rhodospirillales
SAR11 cluster
Rickettsiales
HTCC2503
Sala
-
+
-
-
+
ZM
-
+
-
-
+
Gluconobacter oxydans
GOX
-
+
-
+
Rhodospirillum rubrum
Rrub
-
+
+
-
-
+ +
Magnetospirillum magneticum
Amb
-
+ +
-
-
+
PU1002
+
+
-
-
+
-
-
-
-
+
Pelagibacter ubique
Rickettsia
HTCC1002
and Ehrlichia
species
B.
C.
+
Zymomonas mobilis
RB2256
A.
Distribution of
Irr,
Fur/Mur,
MntR,
RirA, and
IscR regulons
in α-proteobacteria
+
-
Hyphomonadaceae
Sphingomonadales
+
-
Bartonella
Bradyrhizobiaceae
BME
Group
D.
#?' in RirA column denotes
the absence of the rirA gene
in an unfinished genomic sequence
and the presence of candidate
RirA-binding sites upstream of
the iron uptake genes.
Distribution of the conserved members of the Fe- and Mn-responsive regulons
and the predicted RirA, Fur/Mur, Irr, and DtxR binding sites in a-proteobacteria
Genes Functions:
Iron uptake
Iron storage
FeS synthesis
Iron usage
Heme biosynthesis
Regulatory genes
Manganese uptake
Phylogenetic tree of the Fur family of transcription factors in a-proteobacteria - I
Fur
sp|
Escherichia coli: P0A9A9
ECOLI
Pseudomonas aeruginosa
PSEAE
NEIMA
Neisseria meningitidis
: sp|Q03456
: sp|P0A0S7
Fur in g- and b- proteobacteria
HELPY Helicobacter pylori : sp|O25671
Bacillus subtilis : P54574
sp|
BACSU
SM mur
Sinorhizobium meliloti
Mesorhizobium sp. BNC1 (I)
MBNC03003179
BQ fur2
Bartonella quintana
BMEI0375
Brucella melitensis
EE36 12413 Sulfitobacter sp. EE-36
MBNC03003593Mesorhizobium sp. BNC1 (II)
HTCC2654
Rhodobacterales bacterium
RB2654 19538
Agrobacterium
tumefaciens
AGR C 620
RHE_CH00378 Rhizobium etli
Rhizobium leguminosarum
RL mur
Nham 0990 Nitrobacter hamburgensis X14
Nwi 0013
Nitrobacter winogradskyi
Rhodopseudomonas palustris
RPA0450
Bradyrhizobium japonicum
BJ fur
Roseovarius sp.217
ROS217 18337
Jannaschia sp. CC51
Jann 1799
Silicibacter pomeroyi
SPO2477
STM1w01000993Silicibacter sp. TM1040
MED193 22541 Roseobacter sp. MED193
OB2597 02997 Oceanicola batsensis HTCC2597
Loktanella vestfoldensisSKA53
SKA53 03101
Rhodobacter sphaeroides
Rsph03000505
Roseovarius nubinhibens ISM
ISM 15430
PU1002 04436Pelagibacter ubiqueHTCC1002
GOX0771 Gluconobacter oxydans
Zmomonas
y
mobilis
ZM01411
Novosphingobium aromaticivorans
Saro02001148
Sphinopyxis alaskensis RB2256
Sala 1452
ELI1325
Erythrobacter litoralis
Oceanicaulis alexandrii HTCC2633
OA2633 10204
PB2503 04877 Parvularcula bermudensis HTCC2503
CC0057
Caulobacter crescentus
Rhodospirillum rubrum
Rrub02001143
(I)
Magnetospirillum magneticum
Amb1009
Magnetospirillum magneticum (II)
Amb4460
Fur in e- proteobacteria
Fur in Firmicutes
Mur
in a-proteobacteria
Regulator of manganese
uptake genes (sit, mntH)
Fur
in a-proteobacteria
Regulator of iron uptake
and metabolism genes
Irr
a-proteobacteria
Erythrobacter litoralis
Caulobacter crescentus
Zymomonas mobilis
Novosphingobium aromaticivorans
Oceanicaulis alexandrii
Sphinopyxis alaskensis
Gluconobacter oxydans
Rhodospirillum rubrum
Parvularcula bermudensis -
Magnetospirillum magneticum
Identified Mur-binding sites
The A, B, and C groups
of a - proteobacteria
-
Sequence logos for
the identified
Fur-binding sites
in the D group of
a-proteobacteria
Bacillus subtilis
Mur
Escherichia coli
Sequence logos for
the known
Fur-binding sites
in Escherichia coli
and Bacillus subtilis
Phylogenetic tree of the Fur family of transcription factors in a-proteobacteria - II
Fur
Escherichia coli : P0A9A9
sp|
ECOLI
Pseudomonas aeruginosa : sp|Q03456
PSEAE
NEIMA
Fur in g- and b- proteobacteria
Neisseria meningitidis : sp|P0A0S7
HELPY Helicobacter pylori : sp|O25671
sp|
BACSU Bacillus subtilis : P54574
Fur in e- proteobacteria
Fur in Firmicutes
a-proteobacteria
Mur / Fur
Agrobacterium tumefaciens
AGR C 249
Sinorhizobium meliloti
SM irr
Rhizobium etli
RHE CH00106
Rhizobium leguminosarum (I)
RL irr1
RL irr2 Rhizobium leguminosarum (II)
Mesorhizobium loti
MLr5570
MBNC03003186 Mesorhizobium sp. BNC1
BQ fur1 Bartonella quintana
Brucella melitensis (I)
BMEI1955
Brucella melitensis (II)
BMEI1563
BJ blr1216 Bradyrhizobium japonicum (II)
RB2654 182 Rhodobacterales bacterium HTCC2654
Loktanella vestfoldensis SKA53
SKA53 01126
Roseovarius sp.217
ROS217 15500
Roseovarius nubinhibens ISM
ISM 00785
OB2597 14726 Oceanicola batsensis HTCC2597
Jann 1652 Jannaschia sp. CC51
Rsph03001693Rhodobacter sphaeroides
Sulfitobacter sp. EE-36
EE36 03493
STM1w01001534 Silicibacter sp. TM1040
Roseobacter sp. MED193
MED193 17849
SPOA0445
Silicibacter pomeroyi
Rhodobacter capsulatus
RC irr
RPA2339
Rhodopseudomonas palustris (I)
RPA0424*
Rhodopseudomonas palustris (II)
Bradyrhizobium japonicum (I)
BJ irr*
Nwi 0035* Nitrobacter winogradskyi
Nham 1013* Nitrobacter hamburgensis X14
PU1002 04361
Pelagibacter ubique HTCC1002
Irr in a-proteobacteria
regulator of iron
homeostasis
Sequence logos for the identified Irr binding sites in a-proteobacteria.
The A group (8 species) - Irr
The B group (4 species) - Irr
The C group (12 species) - Irr
Phylogenetic tree of the Rrf2 family of transcription factors in a-proteobacteria
Nitrite/NO-sensing regulator NsrR
(Nitrosomonas europeae, Escherichia coli)
ROS217_15206
Rsph03001477
RC NsrR
GOX0860
Amb1318
Nwi_0743
Iron repressor RirA
(Rhizobium leguminosarum)
SPOA0186
Ricket.
Sala_1049
Saro02000305
NE NsrR
OB2597_05195
ROS217_02155
ROS217_14291
SMc00785
RHE CH00735
AGR_C_344
Cysteine metabolism
repressor CymR
(Bacillus subtilis)
AGR_L_1131
SPO3722
RHE_CH02777
RL_3336
SPO1393
MBNC02000669
MLl1642
SMc02238
AGR_C_872
RHE_CH00547
OA2633_11510
RL RirA
BMEII0707
MLr1147
MBNC02002196
BQ04990
RC 0780
RB2654_19993
Rsph023178
SPO0432
MED193_09800
STM_634
Positional clustering of rrf2-like genes with:
iron uptake and storage genes;
Fe-S cluster synthesis operons;
genes involved in nitrosative stress protection;
sulfate uptake/assimilation genes;
CC0132
thioredoxin reductase;
SMc01160
BJ blr7974
carboxymuconolactone
RL_5159
AGR_L_2343
decarboxylase-family genes;
AGR_C_402
hmc cytochrome operon
NsrR
RirA
RL_619
ZMO0116
ROS217_16231
GOX0099
BS CymR
IscR-II
Rrub02000219
ZMO0422
Sala_1236
IscR
ELI0458
Saro3534
DV Rrf2
OA2633_03246
CC1866
EC IscR
Jann_2366
STM_3629
EE36_14302
SPO2025
Rsph023725
RC_0477
Rrub_1115
Amb0200
GOX1196
RPA0663
Ricket.
Cytochrome complex
regulator Rrf2
(Desulfovibrio vulgaris)
Iron-Sulfur cluster
synthesis repressor IscR
(Escherichia coli)
PB2503_ 09884
proteins with the conserved C-X(6-9)-C(4-6)-C motif within effector-responsive domain
proteins without a cysteine triad motif
Sequence logos for the identified RirA-binding sites in a-proteobacteria
The A group - RirA (8 species)
The C group - RirA (12 species)
An attempt to reconstruct the history
Open problems
• Model the evolution of regulatory systems (a catalog of elementary events,
estimates of probabilities)
–
–
–
–
–
Birth of a binding site; what are the mechanisms?
Loss of a binding site
Duplication of a regulated gene and/or a regulator
Horizontal transfer of a regulated gene and/or a regulator
Loss of structural a gene and/or a regulator
• Develop an evolutionary model that would converge to the present state
(that is, have the same properties)
–
–
–
–
Distribution of TF families sizes
Distribution of regulon sizes
Other graph-theoretical properties (node degrees etc.)
General properties? E.g. stable cores and flexible margins of functional systems
(in terms of gene presence and regulation)
• “Microevolution” (strains):
– “metagenomic” regulatory systems?
• Co-evolution of TFs and DNA sites:
– “Neutral” model for the evolution of binding sites (with invariant functional
pressure from the bound protein)
– How do the signals evolve? What is the driving force – changes in TFs?
– TF-family, position-specific protein-DNA recognition code?
All that needs to take into account the incompleteness and noise in the data
Acknowledgements
•
•
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Andrei A. Mironov
Dmitry Rodionov (now at Burnham Institute)
Olga Laikova
Alexei Vitreschak
Anna Gerasimova
Ekateina Kotelnikova (now at Ariadne Genomics)
Ekaterina Panina (now at UCLA)
• Leonid Mirny (MIT)
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Howard Hughes Medical Institute
Russian Fund of Basic Research
Russian Academy of Sciences, program “Molecular and Cellular Biology”
INTAS
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