Evolution of transcription factors from selfish elements: The tale of Rcs1, a global regulator of cell size in yeast

MRC Laboratory of Molecular BiologyCambridge

MRC Laboratory of Molecular BiologyCambridge

M. Madan BabuM. Madan Babu

Overview of researchEvolution of biological systems

Evolution of transcriptional networks Evolution of networks within and across genomes

Nature Genetics (2004) J Mol Biol (2006a)

Evolution of transcription factors

Nuc. Acids. Res (2003)

Discovery of novel DNA binding proteins

Data integration, function prediction and classification

Nuc. Acids. Res (2005) Cell Cycle (2006)

C

C

H

H

Discovery of transcription factors in Plasmodium

Evolution of global regulatory hubs

Structure and dynamics of transcriptional networks

Structure and function of biological systems Uncovering a distributed architecture in networks

Methods to study network dynamics

J Mol Biol (2006b) J Mol Biol (2006c) Nature (2004)

A fundamental developmental process we are interested

in understanding is theregulation of cell size

Rcs1: DNA binding domain not known

Reasons why we became interested in Rcs1

What is the DNA binding domain in Rcs1?

Transcriptional regulatory network in yeast

123 41 314

Aft2p Rcs1p

Number of target genes regulated

Sub-network of Rcs1 and Aft2

How did Rcs1 and its paralog Aft2, which are two global regulators, evolve?

Rcs1: regulator of cell size

Micrographs and data from SCMD

Roundness of mother cell

1.291.20

We find that the following parameters that were used to define cell-sizewere at least 2 Standard deviation (2 ) from the mean values of the wild-type

Mother cell-size

874760

Contour length of mother cell

108100

Long axis length of mother cell

3633

Short axis length of mother cell

3027

S. cerevisiae - wild type S. cerevisiae - Rcs1 mutantSize of mutant cells are

twice that of the parental strain

The critical size for budding in the mutant is

similarly increased

Rcs1 binds specific DNA sequences

Outline: Data integration to infer function & evolution

Sequence analysis to identify members and distant homologs

Structural analysis to infer function and distant homologs

Cladistic analysis to group proteins into families and infer relationship

Domain context analysis to infer function of individual members

Comparative genomics and phylogenetic analysis to infer evolution of the family

Expression data and network analysis to infer spatio-temporal behaviour

Relationship to WRKY DNA binding domain – Sequence analysis I

Non-redundant database

+

...

.

Lineage specific expansion in several fungi and is seen in lower eukaryotes

Candida albicans (ascomycete)Yarrowia lipolytica (ascomycete)Ustilago maydis (basidiomycete)Cryptococcus sp (basidiomycetes)E. cuniculi (microsporidia)

Giardia lamblia (diplomonad)Dictyostelium discoideumEntamoeba histolytica

Rcs1

Profiles + HMMof this region

Non-redundant database

+

WRKY domain(Arabidopsis)

FAR-1 type transposase(Medicago truncatula)

Globular region maps to WRKY DNA-binding domain

Non-redundant Database & PDB

+

WRKY DNA-bindingDomain fromArabidopsis

WRKY4

Rcs1(S. cerevisiae)

Gcm1 (Mouse)PEB-1 (C. elegans)

WRKY maps to the same globular region, Gcm1 & FLYWCH

Confirmation of relationship to WRKY DBD – Sequence analysis II

Homologs of the conserved globular domain constitutes a novel family of the WRKY DNA-binding domain

Multiple sequence alignment of all globular

domains

JPRED/PHD

Sequence of secondary structure is similar to the WRKY DNA-binding domainand GCM1 protein seen in mouse

S1 S2 S3 S4

Characterization of the globular domain – structural analysis I

A. thaliana transcription factor(WRKY4:1wj2:NMR structure)

S1 S2 S3

S1 S2 S3

Predicted SS of Rcs1 DBD

SS of WRKY4

S4

S4 S1 S2 S3

S1 S2 S3

Predicted SS of Rcs1 DBD

SS of GCM1

S4

S4

Mus musculus Glial Cell Missing - 1(GCM-1:1odh:X-ray structure)

Both WRKY and GCM1 have similar network of stabilizing interactions

Template structure

Characterization of the globular domain – structural analysis II

S1 S2 S3

4 residues involved in metal co-ordination and10 residues involved in key stabilizing hydrophobic interactions that determine the path of the backbone

in the four strands of the GCM1-WRKY domainshow a strong pattern of conservation.

S4

Core fold of the Rcs1 DBDwill be similar to the WRKY-GCM1

domain and may bind DNA in a similar way

Outline: Data integration to infer function & evolution

Sequence analysis to identify members and distant homologs

Structural analysis to infer function and distant homologs

Cladistic analysis to group proteins into families and infer relationship

Domain context analysis to infer function of individual members

Comparative genomics and phylogenetic analysis to infer evolution of the family

Expression data and network analysis to infer spatio-temporal behaviour

Classification of WRKY-GCM1 superfamily – Cladistic analysis I

S1 S2 S3 S4

S1 S2 S3S4

C

C

H

H

Zn2+

Template structure

+

S1 S2 S3S4

C

C

H

C

Zn2+

HxC containing version (HxC)

HxC instead of HxHN-terminal helixShort insert between S2 & S3

HxC

S1 S2 S3S4

CH

H

Zn2+

N-terminal helixConserved W in S4Large insert between S2 & S3

Insert containingversion (I)

W

C

I Rcs1Far1

S1 S2 S3S4

C

C

H

H

Zn2+

FLYWCH domain(F)

Conserved W in S2Sequence features

W

F Mdg

S1 S2 S3S4

CH

H

Zn2+

Insertion of Zn ribbon between S2 and S3

GCM domain(G)

C

G Gcm1

... > 4500 proteins

from over 450

genomes

S1 S2 S3S4

C

C

H

H

Zn2+

Classical WRKY (C)

WRKY motif in S1Short loop between S2 & S3

C WRKY4

Domain context for the different families – Domain network analysis I

S1 S2 S3S4

C

C

H

H

Zn2+

Classical WRKY (C)

C

e.g. WRKY4

C C

Tan

dem

Stan

dal

one

Zn

clus

ter

S1 S2 S3S4

CH

H

Zn2+

Insert containingversion (I)

W

C

e.g. Rcs1

e.g. Far1

I I

I

Tan

dem

Stan

dal

one

MU

LE

Tpa

se

OT

Upr

otea

seSM

BD

Znkn

uckl

e

S1 S2 S3S4

C

C

H

C

Zn2+

HxC containing version (HxC)

HxC

MU

LE

Tpa

se

Mob

ile

elem

ent

Stan

dal

one

HxC

e.g. 101.t00020

e.g. At2g23500

S1 S2 S3S4

C

C

H

H

Zn2+

FLYWCH domain(F)

W

e.g. Mod (mdg)

F

BE

Dfi

nger

Stan

dal

one

PO

Z

F

S1 S2 S3S4

CH

H

Zn2+

GCM domain(G)

C

G

G

Stan

dal

one

e.g. Gcm1

WRKY is seen both in transcription factors and transposases

Phyletic distribution – Comparative genome analysis I

GC HxC I F

TF o

nly

TF o

nly

TF +

TP

Human

Fly

Worm

Fungi

Plants

Entamoeba

Slime mould

Plants

Lowereukaryotes

Fungi

HigherEukaryotes

GCM1 and FLYWCH versionsevolved from an insert containingversion that is a transposase

Classical version of the WRKYevolved from an insert containingversion that is a transposase

HxC and Insert containing versionsare seen as both transcription factorsand as transposases only in fungi e.g. Rcs1

Domain context and phyletic analysis suggests that transcription factors could have evolved from transposases

Transcription factor

Transposase

Comparative genomics using >30 different fungal genomes provides convincing evidence

Evolutionary relationship of the insert containing WRKY domains

TFs have evolved from TPsin multiple instances within fungi

Rcs1Aft2

Recent duplication event within Saccharomycetales has resulted

in two hubs Independent duplicationin candida

MULE TransposaseInsert-WRKY

Insert-WRKY

Subsequently recruited as transcription factors by the host

Functional transition in evolutioncaptured by genomic studies

MULE TransposaseMULE Selfishelements in Yarrowia

are seen as standalone ORFs & can regulate their own expression

Insert-WRKY

Transposases have been recruited to become developmentally important

global regulatory proteins in all the three eukaryotic kingdoms of life

WRKY domain is seen in developmentally important proteins

Classical type WRKY has expanded in plants and are expressed in a tissue specific

manner across all developmental stages

RootStem Leaf

ApexFlower

Floral

organs Seeds

Plants

Insert containing WRKY domainshave been recruited to be regulators of

cell size and morphology in yeast

Fungi

GCM1 and FLYWCH type WRKYdomains have been recruited inthe differentiation of stem-cells

Animals

Conclusion

Integration of different types of publicly experimental data allowed us to identify that Rcs1 and several other developmentally important proteins in

different lineages contains a WRKY-type DNA binding domain

Sequence Structure Expression InteractionCladistics &phylogenetics

Data integration allowed us to elucidate that developmentally important transcription factors in the different lineages have evolved from

transposases

Acknowledgements

S Balaji

Lakshminarayan Iyer

National Center for Biotechnology InformationNational Institutes of Health

L Aravind

Structural equivalences of WRKY-GCM1 domain proteins with Bed and Zn finger

S1 S2 S3 S4

C

C

HZn2+

H

ZnC

C

C

C

S1 S2 S3S4

C

C

H

H

Zn2+

WRKY (1wj2)

GCM-type WRKY(1odh)

S1 S2 S3

CC

H

HZn2+

S4S1 S2 H1

CC

H

HZn2+

Bed-finger(2ct5)

Classical Zn-finger(1m36)

Rcs1 regulates genes involved in metal ion transport, specifically ironSiderophore transportCu ion homeostasis

Vacuolar protein catabolism

Intracellular transportVesicle mediated transportGolgi vesicle transportMembrane fusionSecretory pathway

Aft2 regulates genes involved in metal ion transport, again specifically ironIron homeostasisCu ion homeostasis

Vacuolar protein catabolism

Co-factor synthesisVitamin B6 biosynthesisPyridoxine metabolismThiamin biosynthesis

Common targets include:

Genes involved in metal ion transport, again specifically ironIron homeostasisCu ion homeostasis

Vacuolar protein catabolism

Aft2 (171 genes) Rcs1 (381 genes)

Common targets (41 genes)

Ciliates

Apicomplexa

WRKY domain

GCM-type WRKY

MudR transposase

Plant specificZn-cluster

SWIMdomain

POZ

Giardia lamblia

GLP_79_64671_67418_Glam_71077115)

GLP_9_36401_35940_Glam_71071693)

Fungi

mutA_Ylip_49523824

AFT2_Scer_6325054

Encephalitozoon cuniculi

Dictyostelium discoideum

Entamoebahistolytica

101.t00020_Ehis_67474280

dd_03024_Ddis_28829829

ECU05_0180_Ecun_19173554

Caenorhabditis elegans

C26E6.2_Cele_32565510

T24C4.2_Cele_17555262

mod(mdg4)_Dmel_24648712

LOC411361_Amel_66547010

CG13845_Dmel_24649011

Homo sapiens

Drosophilamelanogaster

Animals

1- 5

LOC374920_Hsap_27694337

C20orf164_Hsap_13929452

KIAA1552_Hsap_10047169

hGCMa_Hsap_1769820

gcm_Dmel_17137116

FLYWCH-type WRKY

Zincknuckle

BEDfinger

NtEIG-D48_Ntab_10798760

Plants

TTR1_Atha_30694675

WRKY41_Osat_46394336

WRKY58_Atha_22330782

At2g34830_Atha_27754312

FAR1_Atha_18414374AT4g19990_Atha_7268794

LOC_Os11g31760_Osat_77551147

At2g23500_Atha_3242713

**

Plant specificN-all-beta

TIRdomain

LRR

DUF1723

STANDATPAse

Domain architectures of WRKY-GCM1 domain proteins

60 W

RK

Y do

mai

n co

ntai

ning

pro

tein

s15

Far

1-ty

pe

prot

eins

40 H

xC ty

pe W

RK

Ydo

mai

n pr

otei

ns5

WR

KY

dom

ain

Pro

tein

s w

ith

TIR

/LR

R

+

60 W

RK

Y do

mai

n co

ntai

ning

pro

tein

s15

Far

1-ty

pe

prot

eins

40 H

xC ty

pe W

RK

Ydo

mai

n pr

otei

ns5

WR

KY

dom

ain

Pro

tein

s w

ith

TIR

/LR

R

+

Gene expression profiles for the developmental stages in

Arabidopsis thaliana

Gene expression profiles for the light exposure conditions in

Arabidopsis thaliana

RootStem Leaf

Apex

Flower

Floral

organs Seeds

Darkness

Continuous

light

Pulse

light

a b

Expression profiles of WRKY-GCM1 domain proteins in Arabidopsis

WRKY proteinsshow tissue

specific expression

WRKY proteinsshow light

specific expression

123 41 314

Aft2p Rcs1p

Number of target genes regulated

Aft2p

Rcs1p

Transcriptional network involving Aft2p and Rcs1p

UM

03656.1 Um

ay 71019145

CA

GL

0H03487G

CG

LA

49526254

CA

GL

0G09042G

CG

LA

49526062

CaO

19.2272 Calb 68482460

DE

HA

0F25124g D

han 50425555

KL

LA

0D03256g K

lac 50306475

AF

L087C

AG

OS 44984319

OR

FP

Sklu Contig1830.2 kluyveri

Kw

al 24045 waltii

OR

FP

Skud Contig2057.12 kudriavzeii

OR

FP

Scas Contig720.21 castelli

RC

S1 S

CE

R 51830313

OR

FP

7853 mikatae

OR

FP

8601 paradoxus

OR

FP

21513 mikatae

OR

FP

Scas Contig690.14 castelli

OR

FP

22109 paradoxus

AF

T2 S

CE

R 6325054

OR

FP

Skud Contig1659.3 kudriavzeii

Relationship between Rcs1p and Aft2p homologs

* *

AAL026Wp Agos 44980144UM03656.1 Umay 71019145CHGG 06963 CGLO 88178242CHGG 06785 CGLO 88182698CHGG 09478 CGLO 88177996CHGG 00175 CGLO 88184472CHGG 10902 CGLO 88175616FG05699.1 Gzea 46122643NCU06551.1 Ncra 85106835NCU05145.1 Ncra 85081010YALI0F07128g Ylip 50555399MG05295.4 Mgri 39939890FG04147.1 Gzea 46116610NCU07855.1 Ncra 85109845MG06795.4 Mgri 39977821NCU08168.1 Ncra 85093270CHGG 09951 CGLO 88176079CHGG 08318 CGLO 88179597NCU04492.1 Ncra 32406464FG09606.1 Gzea 46136181NCU06975.1 Ncra 85108658CHGG 05063 CGLO 88180976HOP78 FOXY 30421204CHGG 00311 CGLO 88184608CIMG 00825 CIMM 90305840AN6124.2 Anid 67539908ISOCHOR AFUM 71001046CNC00740 CNEO 57225606CNBH2400 Cneo 50256416AN0859.2 ANID 67517161YALI0A16269g Ylip 50545173CaO19 12424 Calb 68467239DEHA0E17127g Dhan 50422877RBF1P CALB 2498834

DEHA0A05258g Dhan 50405817CaO19.2272 Calb 68482460DEHA0F25124g Dhan 50425555CAGL0H03487G CGLA 49526254AFL087C AGOS 44984319KLLA0D03256g Klac 50306475CAGL0G09042G CGLA 49526062RCS1 SCER 51830313AFT2 SCER 6325054YALI0A05313g Ylip 50543230YALI0A02266g Ylip 50543034Mutyl Ylip 50545163YALI0C17193g.c Ylip 50548927Mutyl.c Ylip 50545161YALI0C00781g.d Ylip 50547661YALI0C00781g.a Ylip 50547661YALI0C00781g.b Ylip 50547661YALI0C00781g.c Ylip 50547661YALI0C17193g.a Ylip 50548927Mutyl.a Ylip 50545161YALI0D22506g Ylip 50551361Mutyl.b Ylip 50545161YALI0C17193g.b Ylip 50548927MG07557.4 Mgri 39972511MG09992.4 Mgri 39965911101.T00020 EHIS 674742804.T00052 EHIS 67483840FAR1 ATHA 18414374AT2G27110 ATHA 18401324AT2G43280 ATHA 30689328AT4G38180 ATHA 15233732AT3G59470 ATHA 18411179AT5G28530 ATHA 22327146AT1G52520 ATHA 15219020AT1G80010 ATHA 15220043C20ORF164 HSAP 13929452LOC428161 GGAL 50759053T24C4.2 CELE 17555262SJCHGC04823 SJAP 567589366330408A02RIK MMUS 50053999LOC374920 HSAP 27694337

Multiple independent evolution of TFs from Transposons

Animals

Plants

Entamoeba

Fungi

Rcs1Aft2p

cluster

Rbf1cluster

CIN5

YAP5

GCN4

YAP6YAP7 YAP1

CAD1

MET4

CST6

SKO1

ARR1

YAP3MET28

HAC1

ACA1

(227)

Fig 3

SWI4 SWI4

MBP1 MBP1

XBP1 XBP1

PHD1 PHD1

SOK2 SOK2

XBP1

SWI4

SOK2(471)

PHD1

MBP1

STE12

YOX1

TOS8

YHP1

PHO2

CUP9

HML2

HMRa1HML1

HMRa2

(357)

Basic Leucine Zipper family Homeodomain family Apses familya b c

MET4MET4

MET28MET28

GCN4GCN4

ARR1ARR1

YAP3YAP3

YAP1YAP1

CAD1CAD1

CIN5CIN5

YAP6YAP6

YAP5YAP5

YAP7YAP7

HAC1HAC1

SKO1SKO1

ACA1ACA1

CST6CST6

HMLALPHA2 HMLALPHA2

HMRA2 HMRA2

CUP9 CUP9

TOS8 TOS8

HMRA1 HMRA1

PHO2 PHO2

YOX1 YOX1

YHP1 YHP1

MET4MET4MET28MET28GCN4GCN4ARR1ARR1YAP3YAP3YAP1YAP1CAD1CAD1CIN5CIN5YAP6YAP6YAP5YAP5YAP7YAP7HAC1HAC1SKO1SKO1ACA1ACA1CST6CST6

HML2 HML2

HMRA2 HMRA2

CUP9 CUP9

TOS8 TOS8

HMRA1 HMRA1

PHO2 PHO2

YOX1 YOX1

YHP1 YHP1

SWI4 SWI4

MBP1 MBP1

XBP1 XBP1

PHD1 PHD1

SOK2 SOK2

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AscomycotaFungi

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40 -

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10 -

20 -

30 -

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Fig 1

0 100

0 100

Fraction of the 14 fungal genomes in which a non-hub transcription factor is evolutionarily conserved (i.e. an ortholog exists)

Fraction of the 14 fungal genomes in which a regulatory hub is evolutionarily conserved (i.e. an ortholog exists)

* Fungal specific DNA-binding domain

DNA-binding domain family which evolved from a transposon

Each box represents a TF member with a specific DBD family, arranged according to evolutionary conservationA red box represents a regulatory hub (A TF regulating > 150 genes), and a blue box represents a non-hub regulatorThe intensity of color represents the fraction of the 14 fungal genomes in which the protein has an ortholog

Possible evolutionary trajectories of transcriptional regulators

Common ancestor was nota regulatory hub. One of the extant

proteins is a regulatory hub

Common ancestor wasa regulatory hub. One of the extant

proteins is a regulatory hub

Common ancestor wasa regulatory hub. Both extant proteins

are regulatory hubs

Common ancestor was nota regulatory hub. Extant proteins

are not regulatory hubs

Extant proteins share less target genes than expected by chance

XXXY ZZZW

Extant proteins share less target genes than expected by chance

XXXY ZZZW

Extant proteins share less target genes than expected by chance

XXXY ZZZW

Extant proteins share less target genes than expected by chance

XXXY ZZZW

Extant proteins share more target genes than expected by chance

Sok2 Phd1

Extant proteins share more target genes than expected by chance

XXXY ZZZW

Extant proteins share more target genes than expected by chance

XXXY ZZZW

Extant proteins share more target genes than expected by chance

XXXY ZZZW

Gene duplication Gene duplication Gene duplication Gene duplication

a b c d