Approaches
24
description
Transcript data from detailed lab studies Transcript data from past experiments reveals co-expression. Large transcript data-sets Clustering of array data over development, biotic/abiotic stresses in order to discover co-regulation through co-expression. - PowerPoint PPT Presentation
Transcript of Approaches
Approaches
Different resources used to identify regulatory promoter motifs:
Transcript data from detailed lab studiesTranscript data from past experiments reveals co-expression.
Comparative genomics: Inter-species promoter comparisonComparison of promoter sequences of a gene between different species can identify elements highly conserved since divergence.
Large transcript data-setsClustering of array data over development, biotic/abiotic stresses in order to discover co-regulation through co-expression.
Network discovery driven by large-scale transcriptional profiling
Vast publicly-available Arabidopsis transcript data sets
Identify tightly co-expressed mitochondrial genes and predict regulatory motifs
Test activity of motifs
Cluster genes with a motif of interest based on motif organisation and expression
Test motif activity and importance of organisation
O2 H2O
AOX
NAD(P)H
NAD(P)+
int
ext
NAD(P)H
NAD(P)+
NADH
NAD+
ADP + Pi ATP
O2 H2O
succinate
fumarate
H+ H+ H+
H+
Respiratory chain
UQ
IMS
MATRIX
I
II
III IV V
c
Number of genes in Arabidopsis:• cytochrome pathway – approx. 150• AOX – 5• alternative NAD(P)H DHs - 7
The alternative respiratory pathway was induced
ModuleFinder - Biclustering
Salicylic acid (100M)Salicylic acid (10M)Rotenone (3hr)Rotenone (12hr)Chloramphenicol (3 hr)Chloramphenicol (12 hr)CysteineH2O2
Oligomycin (1.25M)Oligomycin (0.125M)Anoxia (N2)
0% 50%
100%
SR2
SR1
SR3
SR4
SR5
SR6
SR6
SR8
Identification of regulatory motifs
Model of module promoters:
Models of regulation - testable
Leaf development transcript profiles
0 1 2 3 4 5 60 1 2 3 4 5 60 1 2 3 4 5 6
QRT-PCR to measure transcripts over 2˚ rosette leaf development
Identification of Import Component Regulatory Elements
Leaf Development
0 1 2 3 4 5 6
0 1 2 3 4 5 6
Normalised Transcript Abundance
Leaf age (Weeks)
Tom20-2 / Tom20-3
Tom9-1 / Tom20-4
Identification of Import Component Regulatory Elements
0 1 2 3 4 5 6
0 1 2 3 4 5 6
Tom20-2 / Tom20-3
Tom9-1 / Tom20-4
Test predictions
Measure reporter gene
Testing functionality
Phylogenetic analysis AtAox1c and GmAox2b motif
AtAox1a
Distance from TSS
motifs identified in the common co-expression environmentmotifs identified in the AOX1a co-expression environmentmotifs identified in the upstream regions of AOX1a and NDB2
motif identified oncemotif identified twicemotif identified three timesmotif identified four timesmotif identified five timesmotif identified six times
Colour key: Motifs predicted by co-expression analysis
Line width key:
1000 GTTATGGTTTTGTATAAAAACCCATTTGATTGTAATTTTTATTTAAGTTTTGGTTTAGGTTTGGTTTGATTAAAAACCGTAAAACCGAACGTTTTTTTTG
900 TTTTTGATTTAAATTAAAAATAATTGTATATATATATATATAATGTTCATTTGATAACATGATATCTATCAAACTATCGAAAAACAAAACCCTAACTGTA
800 ACCTAAACTAAAATTCTATATAAATTACATGCCGTCATTTAGGATTTGAGTTTACAAATTAGATTTTGATTTTATTTATGCATCACACTTATAATTTTTT
700 TTGGTAAAAACATGAAAAAACCGGAACCAAACCGGAACCGATCCGAACCAAAATACATATGGTTTTTAAATGGTTTTAATTTTTTAAAACCAAAAACTGT
600 AAAACTGTTAAAACCGAACCGTAACCAAACCGAATTTTATATGGTTTTTATATGGTTTTACTTTTCTTAAAATCGAAAAACCGTAAAACCTAAAACCGAA
500 ACGAAACCAAACCGAAAAACTGAACACCCAGCCCTTAAATATAATGAAAATCGAATAAATTTGTTTGAAAGAATCGAACAAAATTGACAATAAAATCTAA
400 TTAGGACTATTTTCGTCTAATTTTGACTTAGTTGAAACAGAATATTAGCAAAAATACTAAAACACCACAACGCGTAATAATACCCACACACGATATCATT
300 AAATTTGACCAATAAGAATCTAGCTCTTGGCGACCACGCAAGTATCTTCCATCTTGCTCTCCAAGAAAAATCTACACCGGCTTTAAATTTACATAAACAC
200 CCTCAGTCAAAGAAAAGTCGTAAACATAGTCTCTCTCATGACCACAAGGGTAACACAGTCATCCTAAATATAAACCACACAAGAAAACTGTTATACTTTA
100 TACACGTGTCATAGTCTCATTACATCTACGTGAAGAGTTTCGATCATCAACCGTTCGTTTTCTTACTATATAAACCTTGCTCGAGACCTGCGTGTGAAGC TSS
A1 B1 A2 B2
D
E
F G H
KJ L
GGAGACTTGAAGCTGATGCGACGCTTCGTGATGTCGTCATGGTGGTCCGTGCTGATGAAGCGCATCACCGTGATGTTAACCACTATGCATCCGTAAGTAG
CTTGAGGAAGAAGCAATCCACTCTTACACTGAGTTTCTTAAAGAACTCGATAATGGTAACATCGAAAATGTGCCTGCACCGGCTATTGCCATTGATTACT
1750
1850
motifs identified by comparison to soybean Aox
motifs identified by bi-clustering
I1
C
I2
AtAox1a
Distance from TSS
motifs identified in the common co-expression environmentmotifs identified in the AOX1a co-expression environmentmotifs identified in the upstream regions of AOX1a and NDB2
motif identified oncemotif identified twicemotif identified three timesmotif identified four timesmotif identified five timesmotif identified six times
Colour key: Motifs predicted by co-expression analysis
Line width key:
1000 GTTATGGTTTTGTATAAAAACCCATTTGATTGTAATTTTTATTTAAGTTTTGGTTTAGGTTTGGTTTGATTAAAAACCGTAAAACCGAACGTTTTTTTTG
900 TTTTTGATTTAAATTAAAAATAATTGTATATATATATATATAATGTTCATTTGATAACATGATATCTATCAAACTATCGAAAAACAAAACCCTAACTGTA
800 ACCTAAACTAAAATTCTATATAAATTACATGCCGTCATTTAGGATTTGAGTTTACAAATTAGATTTTGATTTTATTTATGCATCACACTTATAATTTTTT
700 TTGGTAAAAACATGAAAAAACCGGAACCAAACCGGAACCGATCCGAACCAAAATACATATGGTTTTTAAATGGTTTTAATTTTTTAAAACCAAAAACTGT
600 AAAACTGTTAAAACCGAACCGTAACCAAACCGAATTTTATATGGTTTTTATATGGTTTTACTTTTCTTAAAATCGAAAAACCGTAAAACCTAAAACCGAA
500 ACGAAACCAAACCGAAAAACTGAACACCCAGCCCTTAAATATAATGAAAATCGAATAAATTTGTTTGAAAGAATCGAACAAAATTGACAATAAAATCTAA
400 TTAGGACTATTTTCGTCTAATTTTGACTTAGTTGAAACAGAATATTAGCAAAAATACTAAAACACCACAACGCGTAATAATACCCACACACGATATCATT
300 AAATTTGACCAATAAGAATCTAGCTCTTGGCGACCACGCAAGTATCTTCCATCTTGCTCTCCAAGAAAAATCTACACCGGCTTTAAATTTACATAAACAC
200 CCTCAGTCAAAGAAAAGTCGTAAACATAGTCTCTCTCATGACCACAAGGGTAACACAGTCATCCTAAATATAAACCACACAAGAAAACTGTTATACTTTA
100 TACACGTGTCATAGTCTCATTACATCTACGTGAAGAGTTTCGATCATCAACCGTTCGTTTTCTTACTATATAAACCTTGCTCGAGACCTGCGTGTGAAGC TSS
A1 B1 A2 B2
D
E
F G H
KJ L
GGAGACTTGAAGCTGATGCGACGCTTCGTGATGTCGTCATGGTGGTCCGTGCTGATGAAGCGCATCACCGTGATGTTAACCACTATGCATCCGTAAGTAG
CTTGAGGAAGAAGCAATCCACTCTTACACTGAGTTTCTTAAAGAACTCGATAATGGTAACATCGAAAATGTGCCTGCACCGGCTATTGCCATTGATTACT
1750
1850
motifs identified by comparison to soybean Aox
motifs identified by bi-clustering
I1
C
I2
Aox1a - motif stress specific
Functional analysis of motifs in leaves
Aox 2kbAox 2kb 1683-1677
Aox 1kb
Aox1a
NDB2
Identification of common active elements
Model of gene expression for Aox1a and NDB2 upon treatment with rotenone
Identification of Import Component Regulatory Elements
0 1 2 3 4 5 6
0 1 2 3 4 5 6
Tom20-2 / Tom20-3
Tom9-1 / Tom20-4
Identification of Import Component Regulatory Elements
0 1 2 3 4 5 6
0 1 2 3 4 5 6
Tom20-2 / Tom20-3
Tom9-1 / Tom20-4
Site II Elements
Promoter deletion/biolistics Electrophoretic mobilityshift assays
Enhancer function+ +
+
+
+
Reporter gene activity
20-4 60-merO
ligo
Olig
o+
nu
c e
xtra
ct
Olig
o +
co
mp
+ n
uc
ext
rct
Olig
o
Olig
o+
nu
c e
xtra
ct
Olig
o +
co
mp
+ n
uc
ext
ract
20-4 60-mer∆TGGGCT
Motif identification
