Identifying conserved promoter motifs and transcription factor binding sites in orthologous plant promoter collections Endre Sebestyén, ARI-HAS, Martonvásár 8th International Symposium on “New development in green gene technology” 1-4 September 2009, Szeged, Hungary

Transcription factors and binding sites TFs bind short, often degenerate DNA sequences, the TFBSs Variable length upstream sequence as promoter, with TFBSs The TFBSs are usually conserved, in a nonconserved surrounding sequence Some well known TFBSs TATA-box GC-box CpG islands Lots of other, less general TFBSs Similarly expressed genes, or homologs should contain similar TFBSs in their promoters

TFs bind short, often degenerate DNA sequences, the TFBSs

Variable length upstream sequence as promoter, with TFBSs

The TFBSs are usually conserved, in a nonconserved surrounding sequence

Some well known TFBSs

TATA-box

GC-box

CpG islands

Lots of other, less general TFBSs

Similarly expressed genes, or homologs should contain similar TFBSs in their promoters

Transcription factors and binding sites

Promoter analysis/TFBS search Wet-lab methods DNAse footprinting Electrophoretic mobility shift assay ChIP-Chip, ChIP-Seq Bioinformatic methods Search for experimentally verified sites Consensus sequences Matrices De novo motif discovery Oligo frequency Phylogenetic footprinting Other methods A 30 0 0 0 20 C 0 25 1 0 2 G 0 1 31 0 3 T 2 6 0 32 7

Wet-lab methods

DNAse footprinting

Electrophoretic mobility shift assay

ChIP-Chip, ChIP-Seq

Bioinformatic methods

Search for experimentally verified sites

Consensus sequences

Matrices

De novo motif discovery

Oligo frequency

Phylogenetic footprinting

Other methods

Experimentally verified sites TRANSFAC, JASPAR, PLACE, PlantCARE

TRANSFAC, JASPAR, PLACE, PlantCARE

De-novo motif discovery Orthologous gene groups Evolutionary conserved functional sites Co-regulated genes In same tissue, body part Developmental stage Etc

Orthologous gene groups

Evolutionary conserved functional sites

Co-regulated genes

In same tissue, body part

Developmental stage

Etc

“Real” promoter structure No general motifs No TATA box, GC-box, etc Lots of false positive TFBSs With wet lab and most of the in silico methods Sometimes no apparent common TFBSs between co-regulated genes

No general motifs

No TATA box, GC-box, etc

Lots of false positive TFBSs

With wet lab and most of the in silico methods

Sometimes no apparent common TFBSs between co-regulated genes

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: human 500/1000/3000 bp 5’ upstream regions Conserved sequence regions Annotations Xrefs to other databases Annotated transcription start sites

Orthologous promoter sequence collections

Based on a BLAST search with first exons of reference species

Plants (Viridiplantae)

Reference species: Arabidopsis thaliana

Chordates

Reference species: human

500/1000/3000 bp 5’ upstream regions

Conserved sequence regions

Annotations

Xrefs to other databases

Annotated transcription start sites

Database of Orthologous Promoters http://doop.abc.hu

DoOP - Clusters

DoOP - Subsets Cluster > Subset Subset: collections of evolutionary monophyletic sequences in a cluster Plant subsets Brassicaceae Arabidopsis thaliana Brassica species Eudicotyledons Grape, Solanum species, papaya, tobacco Magnoliophyta Maize, rice Viridiplantae

Cluster > Subset

Subset: collections of evolutionary monophyletic sequences in a cluster

Plant subsets

Brassicaceae

Arabidopsis thaliana

Brassica species

Eudicotyledons

Grape, Solanum species, papaya, tobacco

Magnoliophyta

Maize, rice

Viridiplantae

DoOP - Subsets

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

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

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

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

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

Phylogenetic footprinting

Functional TFBSs should be conserved

Local sequence alignment

Define conserved regions

Motif generation Brassicaceae eudicotyledons Magnoliophyta

Motif statistics Motif number 500 1000 3000 Brassicaceae 323411 410720 893788 eudicotyledons 13863 20192 34353 Magnoliophyta 2009 2211 1938 Viridiplantae 589 565 372

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

TFBS databases Lots of redundant data Low quality, not updated More than a 100 different version for TATA box Database 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

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 where and when you want it

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 where and when you want it

SNP conservation Gene expression level changes 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 they are likely to be functional ones

Gene expression level changes 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 they are likely to be functional ones

Thank you for your attention! Acknowledgements: Endre Barta Tibor Nagy