Integration of data to uncover evolutionary trends
and infer protein function: The tale of Rcs1
M. Madan Babu
MRC Laboratory of Molecular Biology
Cambridge
Overview of research
Evolution of biological systems
Evolution of networks within and across genomes
Evolution of transcription factors
Evolutionary of transcriptional networks
Nuc. Acids. Res (2003)
Nature Genetics (2004)
J Mol Biol (2006a)
Structure and function of biological systems
Structure and dynamics of transcriptional networks
J Mol Biol (2006b)
J Mol Biol (2006c)
Uncovering a distributed architecture in networks
Methods to study network dynamics
Nature (2004)
Data integration, function prediction and classification
Discovery of transcription factors in Plasmodium
Discovery of novel DNA binding proteins
C
Evolution of a global regulatory hubs
H
H
C
Nuc. Acids. Res (2005)
Cell Cycle (2006)
Rcs1 – regulator of cell size 1
S. cerevisiae - wild type
Size of mutant cells are
twice that of the parental
strain
S. cerevisiae - Rcs1 mutant
The critical size for
budding in the mutant is
similarly increased
Rcs1 binds specific DNA
sequences
The following parameters that were used to define cell-size for the Rcs1 mutant
were at least 2 Standard deviation (2 s) from the mean values of the wild-type
Mother
cell-size
874
760
Contour length
of mother cell
108
100
Long axis length
of mother cell
36
33
Short axis length
of mother cell
30
27
Roundness of
mother cell
1.29
1.20
Micrographs and data from SCMD
Rcs1 is a global regulatory hub – Network analysis I
P53
Tigger
Dal82
Ime1
Tea
Abf1
Tig
AT-Hook
Ace1
Rcs1
Gcr1
LisH
HMG1
Mads
Myb
Apses
Hsf
Fkh
bHLH
Gata
Homeo
bZip
C2H2-Zn
C6-Fungal
No. of members
Rcs1p and Aft2p are global regulatory hubs
with an as yet uncharacterized DNA binding
domain
Distribution of DNA binding domains in yeast transcription factors
Transcriptional regulatory
network in yeast
Sub-network of Rcs1 and Aft2
Aft2p
123
41
Rcs1p
314
Number of target genes regulated
How did the paralogous hubs that regulate distinct sets of genes evolve?
Relationship to WRKY DNA binding domain – Sequence analysis I
...
+
.
Non-redundant
database
Candida albicans (ascomycete)
Yarrowia lipolytica (ascomycete)
Ustilago maydis (basidiomycete)
Cryptococcus sp (basidiomycetes)
E. cuniculi (microsporidia)
Giardia lamblia (diplomonad)
Dictyostelium discoideum
Entamoeba histolytica
Lineage specific expansion in several fungi and is seen in lower eukaryotes
WRKY domain
(Arabidopsis)
+
FAR-1 type transposase
(Medicago truncatula)
Profiles + HMM
of this region
Non-redundant
database
Globular region maps to WRKY DNA-binding domain
Confirmation of relationship to WRKY DBD – Sequence analysis II
Rcs1
(S. cerevisiae)
+
WRKY DNA-binding
Domain from
Arabidopsis
WRKY4
Gcm1
(Drosophila)
Non-redundant
database
WRKY DNA-binding domain maps to the same globular region
S1
S2
S3
S4
JPRED/PHD
Multiple sequence
alignment of all globular
domains
Sequence of secondary structure is similar
to the WRKY DNA-binding domain
and GCM1 protein seen in mouse
Homologs of the conserved globular domain constitutes a
novel family of the WRKY DNA-binding domain
Characterization of the globular domain – structural analysis I
Predicted SS of Rcs1 DBD
S1
S2
S3
Predicted SS of Rcs1 DBD
S4
S1
SS of WRKY4
S1
S2
S3
S2
S3
S4
SS of GCM1
S4
S1
S2
S3
S4
Template structure
A. thaliana transcription factor
(WRKY4:1wj2:NMR structure)
Mus musculus Glial Cell Missing - 1
(GCM-1:1odh:X-ray structure)
Both WRKY and GCM1 have similar network of stabilizing interactions
Characterization of the globular domain – structural analysis II
S1
S2
S3
S4
4 residues involved in metal co-ordination and
10 residues involved in key stabilizing hydrophobic
interactions that determine the path of the backbone
in the four strands of the GCM1-WRKY domain
show a strong pattern of conservation.
Core fold of the Rcs1 DBD
will be similar to the WRKY-GCM1
domain and may bind DNA in a
similar way
Classification of WRKY-GCM1 superfamily – Cladistic analysis I
S1
S2
S3
S4
C
H
Zn2+
+
H
C
S1
S2
S3
S4
Template structure
Classical WRKY
(C)
C
Insert containing
version (I)
HxC containing
version (HxC)
C
H
C
Zn2+
Zn2+
C
C
C
S2
S3
S4
WRKY motif in S1
Short loop
between S2 & S3
C
WRKY4
S1
C
H
W
S2
S3
H
C
I
S1
S2
H
S3
S1
S4
S2
Rcs1
HxC instead of HxH
N-terminal helix
Short insert
between S2 & S3
HxC
H
C
S3
S4
Conserved W in S2
Sequence features
F
H
Zn2+
C
W
S4
N-terminal helix
Conserved W in S4
Large insert
between S2 & S3
Far1
H
Zn2+
C
S1
GCM domain
(G)
H
Zn2+
H
FLYWCH domain
(F)
Mdg
S1
S2
S3
S4
Insertion of Zn
ribbon between
S2 and S3
G
Gcm1
Domain context for the different families – network analysis I
HxC containing
version (HxC)
C
H
Zn2+
C
H
C
Zn2+
H
Zn2+
H
C
W
S2
S3
OUT
protease
MULE
Tpase
Zn
knuckle
SMBD
Stand
alone
I
Stand
alone
Tandem
e.g. WRKY4
I
Tandem
C
I
Zn
cluster
C
e.g. Rcs1
S2
S3
S1
S4
S2
S3
S4
S1
S2
S3
e.g. At2g23500
HxC
F
G
HxC
F
G
e.g. 101.t00020
H
H
C
S4
e.g. Far1
C
S1
BED
finger
S1
Zn2+
Mobile
element
S4
Stand
alone
S3
MULE
Tpase
S2
H
C
W
C
S1
H
H
Zn2+
C
C
GCM domain
(G)
e.g. Mod (mdg)
Stand
alone
C
FLYWCH domain
(F)
POZ
C
Insert containing
version (I)
Stand
alone
Classical WRKY
(C)
e.g. Gcm1
S4
C
I
TF only
TF only
TF + TP
Phyletic distribution – Comparative genome analysis I
HxC
F
Transcription factor
G
Transposase
Human
Fly
Higher
Eukaryotes
GCM1 and FLYWCH versions
evolved from an insert containing
version that is a transposase
Worm
Fungi
Fungi
Entamoeba
Lower
eukaryotes
Slim mould
Plants
Plants
HxC and Insert containing versions
are seen as both transcription factors
and as transposases
Classical version of the WRKY
evolved from an insert containing
version that is a transposase
-explain that there has been multiple
transitions from transposase to TFs in the
fungal genomes
-explain how this could have happened by
showing the snapshot of the breakup of
selfish elements into two distinct products
-explain that the transposase can itself
regulate the gene expression of itself
Outline of the presentation
Rcs1 and aft2 have a distinct version of the WRKY type DNA binding domain
Sensitive sequence search reveals that
Oryza sativa (monocot)
Arabidopsis thaliana (dicot)
Medicago truncatula (dicot)
Nicotiana tabacum (dicot)
Structural equivalences of WRKY-GCM1 domain proteins with Bed and Zn finger
WRKY
(1wj2)
GCM-type WRKY
(1odh)
Zn
C
Zn2+
S2
C
C
H
Zn2+
H
C
S1
H
C
H
S1
S4
S2
C
C
H
C
H
Zn2+
Zn2+
C
S3
Classical Zn-finger
(1m36)
C
C
C
Bed-finger
(2ct5)
H
H
S3
S4
S1
S2
S3
S4
S1
S2
H1
Why Rcs1? While systematically analyzing the genes which gave rise to abnormal
cell size, We and the other noted that mutants of Rcs1 give abnormal cell shape.
It was known to be an important transcription factor involved in cell size regulation
– explain showing graphs and images
Independently, during the analysis of the TNET in yeast
We looked at the hubs and the DNA binding domains
That were present in them. Interestingly, there were two
Hubs that did not have any known DNA binding domain
Identified in them, but the region which mediates DNA
was known – explain showing the family relationship
Of the hubs
-only two members, and both are hubs
-how and when did they evolve?
Standard search procedures using Pfam and other databases did not
provide any clue about the domain. So we set out to characterize the
DNA binding region from Rcs1p and its paralog Aft2p using sensitive
sequence search and other computational methods.
-show output from Pfam hits
WRKY DNA binding domain – Structure analysis I
Structural aspects of the DNA binding domain
Explain the residues involved in metal chelating
-DNA contacting surface
-Inserts in the loops
-Stabilizing contacts involved
WRKY DNA binding domain – Structure analysis II
Structure comparisons identify several other
Known transcription factors including the GCM protein in eukaryotes
-Explain the insert of a zinc ribbon in the loop
In fact sequence comparison without the insert can pick these WRKY proteins
Classification of WRKY domains – Cladistic analysis I
Multiple starting points identified all homologs in the different species
This allowed us to classify the sequences into different families
Each with a specific feature suggesting common evolutionary relationship
Based on shared and derived features of the domains
- List the 5 families and point to features involved using a structure template
Phylogenetic distribution and domain architecture
for the different families - I
Phyletic profiles of the different domains points to the possibility that these
transcription factors could have evolved from transposases
With at least two distinct recruitment into transcription factors.
-In plants in one case
-In the base of the fungal genomes in the other case
Phylogenetic distribution and domain architecture
for the different families - II
Comparative genomics using the fungal genomes
provides the clue for the evolution of these TFs
-explain that there has been multiple
transitions from transposase to TFs in the
fungal genomes
-explain how this could have happened by
showing the snapshot of the breakup of
selfish elements into two distinct products
-explain that the transposase can itself
regulate the gene expression of itself
Comparative genomics using the fungal genomes
provides the clue for the evolution of these TFs
-extensive recruitment of the transposase
in the different fungal lineages
-multiple jumps within the fungal lineage
-very recent duplication event in the order
Saccharomycetales suggest hubs could
Evolve rapidly
-Candida rbf1 and other TFs independently
duplicated and evolved as global
regulators
Analysis of the gene expression data in plants
Since it happened in fungal genomes, we ask how
does this behave in the plants.
-show the gene expression patterns for the
different subfamilies.
We see two trends one where divergence has
primarily occurred in the expression changes
rather than in the protein sequence, and the other
in which proteins with the same expression pattern
have different binding site residues.
-spatio-temporal changes in gene expression
-It is experimentally well known that the FLYWCH
and the GCM proteins are developmentally
important regulatory proteins.
So in three lineages there has been recruitment of
the transposase into becoming a developmentally
important global regulator.
Analysis of the gene expression data in plants
There are interesting traces of gene expression
pattern when we see for the different WRKY
containing proteins. TPases are expressed in the
root and in the pollen enhancing the possibility of
rapidly expanding themselves during evolution.
Acknowledgements
Aravind group
L Aravind
S Balaji
Lakshminarayan Iyer
MtrDRAFT_AC146590g49v2_Mtru_92891293
*
I
C
hGCMa_Hsap_1769820
*
C
*
NtEIG-D48_Ntab_10798760
mod(mdg4)_Dmel_24648712
F
1- 5
CG13845_Dmel_24649011
Homo sapiens
I
Drosophila
melanogaster
I
I
*
*
I
YALI0C00781g_Ylip_50547661
I
C26E6.2_Cele_32565510
F
I
HxC
I
Ci-ZF-1_Cint_93003122
F
1- 5
KIAA1552_Hsap_10047169
LOC_Os11g31760_Osat_77551147 C20orf164_Hsap_13929452
C
CHGG_00311_Cglo_88184608
I
LOC411361_Amel_66547010
F
HxC
CHGG_08318_CGLO_88179597
I
I
T24C4.2_Cele_17555262
*
UM03656.1_Umay_71019145
I
Caenorhabditis elegans
AN6124.2_ANID_67539908
FAR1_Atha_18414374
I
AT4g19990_Atha_7268794
C
F
G
WRKY41_Osat_46394336
At2g23500_Atha_3242713
*
I
F54C4.3_Cele_3790719
*
TTR1_Atha_30694675
I
gcm_Dmel_17137116
MtrDRAFT_AC126008g21v1_Mtru_92876827
C
YALI0A02266g_Ylip_50543034
T24C4.7_Cele_17555272
G
*
I
I
HxC
Fungi
Plants
HxC
C
C
Animals
WRKY58_Atha_22330782 At2g34830_Atha_27754312
I
mutA_Ylip_49523824
I
Afu2g08220_Afum_71000950
AFT2_Scer_6325054
Encephalitozoon
cuniculi
ECU05_0180_Ecun_19173554
Ciliates
HxC
Apicomplexa
I
Giardia
lamblia
I
101.t00020_Ehis_67474280
GLP_9_36401_35940_Glam_71071693)
Entamoeba histolytica
C
C
C
Classical
WRKY
GCM-type
G
WRKY
C
I
F
Insert-containing HxC
WRKY
FLYWCH-type
WRKY
HxC-type
WRKY
MULE
transposase
C
Dictyostelium
dd_03024_Ddis_28829829 discoideum
GLP_79_64671_67418_Glam_71077115)
Plant specific
Zn-cluster
Zinc
knuckle
BED
finger
SWIM
domain
Plant-specific
mobile domain
PHD
finger
C2H2
finger
LRR
STAND
ATPase
Isochoris
matase
Plant specific
N-all-beta
TIR
domain
AT-hook
OTU
POZ
Expression profiles of WRKY-GCM1 domain proteins in Arabidopsis
Gene expression profiles for the
light exposure conditions in
Arabidopsis thaliana
+
WRKY proteins
show light
specific expression
15 Far1-type
proteins
15 Far1-type
proteins
5 WRKY domain
Proteins with TIR/LRR
5 WRKY domain
Proteins with TIR/LRR
60 WRKY domain
containing proteins
b
+
40 HxC type WRKY
domain proteins
40 HxC type WRKY
domain proteins
WRKY proteins
show tissue
specific expression
Gene expression profiles for the
developmental stages in
Arabidopsis thaliana
60 WRKY domain
containing proteins
a
ot tem
S
Ro
f
a
Le
ex
er
al
Ap low
or
F
Fl ans
g
or
Se
s
ed
ess ous
rkn tinu
a
D Con ght
li
lse
Pu ht
lig
Relationship between Rcs1p and Aft2p homologs
Multiple independent evolution of TFs from Transposons
UM03656.1 Umay 71019145
CAGL0H03487G CGLA 49526254
CAGL0G09042G CGLA 49526062
CaO19.2272 Calb 68482460
DEHA0F25124g Dhan 50425555
KLLA0D03256g Klac 50306475
AFL087C AGOS 44984319
ORFP Sklu Contig1830.2 kluyveri
Kwal 24045 waltii
ORFP Scas Contig720.21 castelli
ORFP Skud Contig2057.12 kudriavzeii
ORFP 7853 mikatae
*
ORFP 8601 paradoxus
RCS1 SCER 51830313
ORFP Scas Contig690.14 castelli
Rcs1
Aft2p
cluster
ORFP Skud Contig1659.3 kudriavzeii
Animals
Rbf1
cluster
ORFP 21513 mikatae
Plants
*
ORFP 22109 paradoxus
Entamoeba
AFT2 SCER 6325054
Fungi
AAL026Wp Agos 44980144
UM03656.1 Umay 71019145
CHGG 06963 CGLO 88178242
CHGG 06785 CGLO 88182698
CHGG 09478 CGLO 88177996
CHGG 00175 CGLO 88184472
CHGG 10902 CGLO 88175616
FG05699.1 Gzea 46122643
NCU06551.1 Ncra 85106835
NCU05145.1 Ncra 85081010
YALI0F07128g Ylip 50555399
MG05295.4 Mgri 39939890
FG04147.1 Gzea 46116610
NCU07855.1 Ncra 85109845
MG06795.4 Mgri 39977821
NCU08168.1 Ncra 85093270
CHGG 09951 CGLO 88176079
CHGG 08318 CGLO 88179597
NCU04492.1 Ncra 32406464
FG09606.1 Gzea 46136181
NCU06975.1 Ncra 85108658
CHGG 05063 CGLO 88180976
HOP78 FOXY 30421204
CHGG 00311 CGLO 88184608
CIMG 00825 CIMM 90305840
AN6124.2 Anid 67539908
ISOCHOR AFUM 71001046
CNC00740 CNEO 57225606
CNBH2400 Cneo 50256416
AN0859.2 ANID 67517161
YALI0A16269g Ylip 50545173
CaO19 12424 Calb 68467239
DEHA0E17127g Dhan 50422877
RBF1P CALB 2498834
DEHA0A05258g Dhan 50405817
CaO19.2272 Calb 68482460
DEHA0F25124g Dhan 50425555
CAGL0H03487G CGLA 49526254
AFL087C AGOS 44984319
KLLA0D03256g Klac 50306475
CAGL0G09042G CGLA 49526062
RCS1 SCER 51830313
AFT2 SCER 6325054
YALI0A05313g Ylip 50543230
YALI0A02266g Ylip 50543034
Mutyl Ylip 50545163
YALI0C17193g.c Ylip 50548927
Mutyl.c Ylip 50545161
YALI0C00781g.d Ylip 50547661
YALI0C00781g.a Ylip 50547661
YALI0C00781g.b Ylip 50547661
YALI0C00781g.c Ylip 50547661
YALI0C17193g.a Ylip 50548927
Mutyl.a Ylip 50545161
YALI0D22506g Ylip 50551361
Mutyl.b Ylip 50545161
YALI0C17193g.b Ylip 50548927
MG07557.4 Mgri 39972511
MG09992.4 Mgri 39965911
101.T00020 EHIS 67474280
4.T00052 EHIS 67483840
FAR1 ATHA 18414374
AT2G27110 ATHA 18401324
AT2G43280 ATHA 30689328
AT4G38180 ATHA 15233732
AT3G59470 ATHA 18411179
AT5G28530 ATHA 22327146
AT1G52520 ATHA 15219020
AT1G80010 ATHA 15220043
C20ORF164 HSAP 13929452
LOC428161 GGAL 50759053
T24C4.2 CELE 17555262
SJCHGC04823 SJAP 56758936
6330408A02RIK MMUS 50053999
LOC374920 HSAP 27694337
Transcriptional network involving Aft2p and Rcs1p
Aft2p
Aft2p
Rcs1p
Rcs1p
123 41
314
Number of target genes regulated
Conclusion
Integration of different types of experimental data allowed us to
Identify the DNA binding domain in Rcs1
Sequence
Structure
Expression
Interaction