1. Identifying conserved promoter motifs and transcription factor binding sites in plant promoters Endre Sebestyén, ARI-HAS, Martonvásár, Hungary 26th, November, 2009 RCPGD Annual Meeting

2. Transcription factor binding sites TFs bind short, often degenerate DNA sequences Promoters are variable length 5’ sequences ▫With TFBSs TFBSs are usually conserved in a nonconserved surrounding sequence Some well known TFBSs ▫TATA box ▫GC box ▫CpG island Lots of other, less genereal TFBSs Similarly expressed genes, or homologues should contain similar TFBSs

3. Transcription

4. TFBS search and promoter analysis Wet-lab methods ▫DNAse footprinting ▫Electrophoretic mobility shift assay ▫ChIP-Chip, ChIP-Seq In silico methods ▫Experimentally verified sites  Consensus sequences  Consensus matrices ▫De novo motif discovery  Oligo frequency  Phylogenetic footprinting  Other methods

5. Experimentally verified sites TRANSFAC JASPAR PLACE PlantCARE

6. De novo motif discovery Orthologous gene groups ▫Evolutionary conserved functional sites Co-regulated genes ▫Same tissue, body part ▫Same developmental stage ▫Etc

7. „Real” promoter structure No general motifs ▫No TATA-box, GC-box, etc Lots of false positive TFBS ▫With wet-lab and in silico methods Sometimes no apparent common TFBSs between coregulated genes

8. Database of Orthologous Promoters Orthologous promoter sequence collections ▫Based on a BLAST search with first exons of reference species  Plants (Viridiplantae)  Reference species: Arabidopsis thaliana  Chordates  Reference species: Homo sapiens ▫500/1000/3000 bp 5’ upstream regions  Conserved sequence regions  Annotations  Xrefs to other databases  Annotated transcription start sites

9. DoOP http://doop.abc.hu

10. DoOP cluster number

11. DoOP subsets Cluster > Subset ▫Subset: collection of evolutionary monophyletic sequences in a cluster ▫Plant subsets  Brassicaceae  Arabidopsis thaliana  Brassicaceae species  Eudicotyledons  Grape, Solanum species, papaya, tobacco  Magnoliophyta  Maize, rice  Viridiplantae

12. DoOP subsets

15. Gene types – Gene Ontology Standardized annotation for genes ▫Biological process  What does it do?  Transcription, translation, stress response, etc ▫Cellular component  Where is it located?  Membrane, ribosome, cytosol, etc ▫Molecular function  How does it work?  Dehydrogenase, ATP binding, etc

16. Gene types – Gene Ontology 500 bp promoters ▫Search for significantly enriched terms in annotation  Brassicaceae  Eudicotyledons  Magnoliophyta  Viridiplantae  BP: transcription, translation, protein folding, stress response  CC: plasma membrane, ribosome parts  MF: ATP/GTP binding, DNA binding, ribosome parts

17. Motif generation Phylogenetic footprinting Functional TFBSs should be conserved Local sequence alignment Define conserved regions

18. Motif generation Magnoliophyta eudicotyledons Brassicaceae

19. Motif statistics Motif number50010003000 Brassicaceae323411410720893788 eudicotyledons138632019234353 Magnoliophyta200922111938 Viridiplantae589565372

20. Motif statistics % conserved50010003000 Brassicaceae221916 eudicotyledons532 Magnoliophyta652 Viridiplantae421 Avg length50010003000 Brassicaceae999 eudicotyledons777 Magnoliophyta898 Viridiplantae999

21. TFBS databases DatabaseTFBSs TRANSFAC977 JASPAR18 PLACE416 PlantCARE646 ABS650 AGRIS72 Lots of redundant data Low quality, not updated More than a 100 different version for TATA box

22. Synthetic biology ▫iGEM competition ▫BioBricks ▫MIT Registry of Standard Biological Parts  UV responsive promoter  Promoter expressed in roots  Etc Synthetic promoters ▫Define basic promoter elements ▫Build and use custom made promoters ▫Gene expression more or less when and where you want it

23. SNP conservation Gene expression levels change because ▫Regulatory elements change ▫Usually NOT protein coding regions Conserved promoter regions might be functional regulatory elements ▫Search for SNPs in this regions ▫These SNPs might be interesting for breeders as theye are likely to be functional ones

24. A real example Vilmos Soós, Endre Sebestyén, Angéla Juhász, János Pintér, Marnie E. Light, Johannes Van Staden, Ervin Balázs (2009) Stress-related genes define essential steps in the response of maize seedlings to smoke-water. Functional and Integrative Genomics, Volume 9, Number 2, Pages 231-242; doi:10.1007/s10142- 008-0105-8 Microarray experiments ▫Maize kernels (Mv 540) ▫24 and 48 h – control vs smoke treated samples ▫Up and downregulated genes  Promoter sequences up to 1500 bp were extracted if available

25. Analysis of promoters TRANSFAC database version 12.1 ▫Collection of TFBSs ▫More than a 100 plant TFBSs  DRE-element: GCCGAC Scan for the TFBSs in the maize promoters ▫Up and downregulated Also count the frequencies of all 5-8mer sequences ▫In all available maize promoters, not only the up or downregulated Calculate the over or underrepresentation of a TFBS by the following ▫Observed frequency in up or downregulated promoters divided by the expected frequency in all promoters ▫If ratio > 1 : overrepresented ▫If ratio < 1 : underrepresented

26. Analysis of promoters Results ▫Binding sites related to  Organogenesis  Meristem development  Housekeeping functions  Biotic stress  Cold and dehydration stress  ABA related motifs

27. Thank you for your attention!