1. Evolution of transcription factors from selfish elements: The tale of Rcs1, a global regulator of cell size in yeast MRC Laboratory of Molecular Biology Cambridge MRC Laboratory of Molecular Biology Cambridge M. Madan Babu

2. Overview of research Evolution 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)

3. A fundamental developmental process we are interested in understanding is the regulation 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 Aft2pRcs1p Number of target genes regulated Sub-network of Rcs1 and Aft2 How did Rcs1 and its paralog Aft2, which are two global regulators, evolve?

4. Rcs1: regulator of cell size Micrographs and data from SCMD Roundness of mother cell 1.29 1.20 We find that the following parameters that were used to define cell-size were at least 2 Standard deviation (2 ) 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 S. cerevisiae - wild type S. cerevisiae - Rcs1 mutant Size 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

5. 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

6. 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 discoideum Entamoeba histolytica Rcs1 Profiles + HMM of this region Non-redundant database + WRKY domain (Arabidopsis) FAR-1 type transposase (Medicago truncatula) Globular region maps to WRKY DNA-binding domain

7. Non-redundant Database & PDB + WRKY DNA-binding Domain from Arabidopsis 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 domain and GCM1 protein seen in mouse S1S2S3S4

8. Characterization of the globular domain – structural analysis I A. thaliana transcription factor (WRKY4:1wj2:NMR structure) S1S2S3 S1S2S3 Predicted SS of Rcs1 DBD SS of WRKY4 S4 S1S2S3 S1S2S3 Predicted SS of Rcs1 DBD SS of GCM1 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

9. Characterization of the globular domain – structural analysis II S1S2S3 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. S4 Core fold of the Rcs1 DBD will be similar to the WRKY-GCM1 domain and may bind DNA in a similar way

10. 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

11. Classification of WRKY-GCM1 superfamily – Cladistic analysis I S1S2S3S4 S1 S2 S3 S4 C C H H Zn 2+ Template structure + S1 S2 S3 S4 C C H C Zn 2+ HxC containing version (HxC) HxC instead of HxH N-terminal helix Short insert between S2 & S3 HxC S1 S2 S3 S4 C H H Zn 2+ N-terminal helix Conserved W in S4 Large insert between S2 & S3 Insert containing version (I) W C I Rcs1 Far1 S1 S2 S3 S4 C C H H Zn 2+ FLYWCH domain (F) Conserved W in S2 Sequence features W F Mdg S1 S2 S3 S4 C H H Zn 2+ Insertion of Zn ribbon between S2 and S3 GCM domain (G) C G Gcm1... > 4500 proteins from over 450 genomes S1 S2 S3 S4 C C H H Zn 2+ Classical WRKY (C) WRKY motif in S1 Short loop between S2 & S3 C WRKY4

12. Domain context for the different families – Domain network analysis I S1 S2 S3 S4 C C H H Zn 2+ Classical WRKY (C) C e.g. WRKY4 CC Tandem Stand alone Zn cluster S1 S2 S3 S4 C H H Zn 2+ Insert containing version (I) W C e.g. Rcs1 e.g. Far1 I I I Tandem Stand alone MULE Tpase OTU protease SMBD Zn knuckle S1 S2 S3 S4 C C H C Zn 2+ HxC containing version (HxC) HxC MULE Tpase Mobile element Stand alone HxC e.g. 101.t00020 e.g. At2g23500 S1 S2 S3 S4 C C H H Zn 2+ FLYWCH domain (F) W e.g. Mod (mdg) F BED finger Stand alone POZ F S1 S2 S3 S4 C H H Zn 2+ GCM domain (G) C G G Stand alone e.g. Gcm1 WRKY is seen both in transcription factors and transposases

13. Phyletic distribution – Comparative genome analysis I GC HxC IF TF only TF + TP Human Fly Worm Fungi Plants Entamoeba Slime mould Plants Lower eukaryotes Fungi Higher Eukaryotes GCM1 and FLYWCH versions evolved from an insert containing version that is a transposase Classical version of the WRKY evolved from an insert containing version that is a transposase HxC and Insert containing versions are seen as both transcription factors and 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

14. Comparative genomics using >30 different fungal genomes provides convincing evidence Evolutionary relationship of the insert containing WRKY domains TFs have evolved from TPs in multiple instances within fungi Rcs1 Aft2 Recent duplication event within Saccharomycetales has resulted in two hubs Independent duplication in candida MULE Transposase Insert- WRKY Insert- WRKY Subsequently recruited as transcription factors by the host Functional transition in evolution captured by genomic studies MULE Transposase MULE Selfish elements in Yarrowia are seen as standalone ORFs & can regulate their own expression Insert- WRKY

15. 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 Root Stem Leaf Apex Flower Floral organs Seeds Plants Insert containing WRKY domains have been recruited to be regulators of cell size and morphology in yeast Fungi GCM1 and FLYWCH type WRKY domains have been recruited in the differentiation of stem-cells Animals

16. 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 SequenceStructureExpressionInteractionCladistics & phylogenetics Data integration allowed us to elucidate that developmentally important transcription factors in the different lineages have evolved from transposases

17. Acknowledgements S Balaji Lakshminarayan Iyer National Center for Biotechnology Information National Institutes of Health L Aravind

18. Structural equivalences of WRKY-GCM1 domain proteins with Bed and Zn finger S1 S2 S3 S4 C C H Zn 2+ H Zn C C C C S1 S2 S3 S4 C C H H Zn 2+ WRKY (1wj2) GCM-type WRKY (1odh) S1 S2 S3 C C H H Zn 2+ S4 S1 S2 H1 C C H H Zn 2+ Bed-finger (2ct5) Classical Zn-finger (1m36)

19. Rcs1 regulates genes involved in metal ion transport, specifically iron Siderophore transport Cu ion homeostasis Vacuolar protein catabolism Intracellular transport Vesicle mediated transport Golgi vesicle transport Membrane fusion Secretory pathway Aft2 regulates genes involved in metal ion transport, again specifically iron Iron homeostasis Cu ion homeostasis Vacuolar protein catabolism Co-factor synthesis Vitamin B6 biosynthesis Pyridoxine metabolism Thiamin biosynthesis Common targets include: Genes involved in metal ion transport, again specifically iron Iron homeostasis Cu ion homeostasis Vacuolar protein catabolism Aft2 (171 genes)Rcs1 (381 genes) Common targets (41 genes)

20. Ciliates Apicomplexa WRKY domain GCM-type WRKY MudR transposase Plant specific Zn-cluster SWIM domain 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 Entamoeba histolytica 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 Drosophila melanogaster Animals 1- 5 LOC374920_Hsap_27694337 C20orf164_Hsap_13929452 KIAA1552_Hsap_10047169 hGCMa_Hsap_1769820 gcm_Dmel_17137116 FLYWCH-type WRKY Zinc knuckle BED finger NtEIG-D48_Ntab_10798760 Plants TTR1_Atha_30694675 WRKY41_Osat_46394336 WRKY58_Atha_22330782 At2g34830_Atha_27754312 FAR1_Atha_18414374 AT4g19990_Atha_7268794 LOC_Os11g31760_Osat_77551147 At2g23500_Atha_3242713 * * Plant specific N-all-beta TIR domain LRR DUF1723 STAND ATPAse Domain architectures of WRKY-GCM1 domain proteins

21. Expression profiles of WRKY-GCM1 domain proteins in Arabidopsis WRKY proteins show tissue specific expression WRKY proteins show light specific expression

22. 123 41 314 Aft2pRcs1p Number of target genes regulated Aft2p Rcs1p Transcriptional network involving Aft2p and Rcs1p UM03656.1 Umay 71019145CAGL0H03487G CGLA 49526254CAGL0G09042G CGLA 49526062CaO19.2272 Calb 68482460DEHA0F25124g Dhan 50425555KLLA0D03256g Klac 50306475AFL087C AGOS 44984319ORFP Sklu Contig1830.2 kluyveriKwal 24045 waltiiORFP Skud Contig2057.12 kudriavzeiiORFP Scas Contig720.21 castelliRCS1 SCER 51830313ORFP 7853 mikataeORFP 8601 paradoxusORFP 21513 mikataeORFP Scas Contig690.14 castelliORFP 22109 paradoxusAFT2 SCER 6325054ORFP Skud Contig1659.3 kudriavzeii Relationship between Rcs1p and Aft2p homologs * *

23. CIN5 YAP5 GCN4 YAP6 YAP7 YAP1 CAD1 MET4 CST6 SKO1 ARR1 YAP3 MET28 HAC1 ACA1 (227) Fig 3 SWI4 MBP1 XBP1 PHD1 SOK2 XBP1 SWI4 SOK2 (471) PHD1 MBP1 STE12 YOX1 TOS8 YHP1 PHO2 CUP9 HML  2 HMRa1 HML  1 HMRa2 (357) Basic Leucine Zipper familyHomeodomain familyApses family ab c MET4 MET28 GCN4 ARR1 YAP3 YAP1 CAD1 CIN5 YAP6 YAP5 YAP7 HAC1 SKO1 ACA1 CST6 HMLALPHA2 HMRA2 CUP9 TOS8 HMRA1 PHO2 YOX1 YHP1

24. MET4 MET28 GCN4 ARR1 YAP3 YAP1 CAD1 CIN5 YAP6 YAP5 YAP7 HAC1 SKO1 ACA1 CST6 HML  2 HMRA2 CUP9 TOS8 HMRA1 PHO2 YOX1 YHP1 SWI4 MBP1 XBP1 PHD1 SOK2

25. 40 - 0 - 10 - 20 - 30 - C6-Fungal C2H2-Zn bZip Homeo Gata bHLH Fkh Hsf Apses Myb Mads HMG1 LisH+CTLH Gcr1p+Msn1p Rcs1Ace1 AT-Hook Tig Abf1 Tea Ime1 Dal82 Tigger P53-Cytochrome Number of members in the family (non-hub : hub) * * Fig 1 0100 0 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 conservation A red box represents a regulatory hub (A TF regulating > 150 genes), and a blue box represents a non-hub regulator The intensity of color represents the fraction of the 14 fungal genomes in which the protein has an ortholog

26. Possible evolutionary trajectories of transcriptional regulators Common ancestor was not a regulatory hub. One of the extant proteins is a regulatory hub Common ancestor was a regulatory hub. One of the extant proteins is a regulatory hub Common ancestor was a regulatory hub. Both extant proteins are regulatory hubs Common ancestor was not a regulatory hub. Extant proteins are not regulatory hubs Extant proteins share less target genes than expected by chance XXXYZZZW Extant proteins share less target genes than expected by chance XXXYZZZW Extant proteins share less target genes than expected by chance XXXYZZZW Extant proteins share less target genes than expected by chance XXXYZZZW Extant proteins share more target genes than expected by chance Sok2Phd1 Extant proteins share more target genes than expected by chance XXXYZZZW Extant proteins share more target genes than expected by chance XXXYZZZW Extant proteins share more target genes than expected by chance XXXYZZZW Gene duplication a bc d