identifying conserved promoter motifs and transcription factor binding sites in plant promoters...
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Identifying conserved promoter motifs and transcription factor binding sites in plant promotersEndre Sebestyén, ARI-HAS, Martonvásár, Hungary
26th, November, 2009RCPGD Annual Meeting
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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
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Transcription
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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
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Experimentally verified sites
• TRANSFAC• JASPAR• PLACE• PlantCARE
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De novo motif discovery• Orthologous gene groups
▫ Evolutionary conserved functional sites
• Co-regulated genes▫ Same tissue, body part▫ Same developmental
stage▫ Etc
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„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
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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
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DoOP http://doop.abc.hu
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DoOP cluster number
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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
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DoOP subsets
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v1.5 v1.6 v1.80
5000
10000
15000
20000
25000
30000
35000
40000
45000 Other
Solanum tuberosum
Arabidopsis lyrata
Sorghum bicolor
Physcomitrella patens
Capsella rubella
Glycine max
Zea mays
Oryza sativa
Solanum lycopersicum
Nicotiana tabacum
Brassica napus
Lotus japonicus
Medicago truncatula
Vitis vinifera
Ricinus communis
Populus trichocarpa
Carica papaya
Boechera stricta
Brassica oleracea
Brassica rapa
Arabidopsis thaliana
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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
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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
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Motif generation
• Phylogenetic footprinting• Functional TFBSs should be conserved• Local sequence alignment• Define conserved regions
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Motif generation
Magnoliophyta
eudicotyledons
Brassicaceae
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Motif statistics
Motif number 500 1000 3000
Brassicaceae 323411 410720 893788
eudicotyledons 13863 20192 34353
Magnoliophyta 2009 2211 1938
Viridiplantae 589 565 372
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Motif statistics% conserved 500 1000 3000
Brassicaceae 22 19 16
eudicotyledons 5 3 2
Magnoliophyta 6 5 2
Viridiplantae 4 2 1
Avg length 500 1000 3000
Brassicaceae 9 9 9
eudicotyledons 7 7 7
Magnoliophyta 8 9 8
Viridiplantae 9 9 9
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TFBS databasesDatabase TFBSs
TRANSFAC 977
JASPAR 18
PLACE 416
PlantCARE 646
ABS 650
AGRIS 72
• Lots of redundant data• Low quality, not updated
• More than a 100 different version for TATA box
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Synthetic biology• 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
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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
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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
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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
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Analysis of promoters
•Results▫Binding sites related to
Organogenesis Meristem development Housekeeping functions
Biotic stress Cold and dehydration stress
ABA related motifs
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Thank you for your attention!