Promoter sequence analysis of genes, differentiallyexpressed in sheep, following a nematode parasite
challenge

Shivashankar Nagaraj
CSIRO Livestock Industries

Poster 69

Outline¾ Background¾ Previous experimental work
¾ Goals¾ Results¾ Future directions
CSIRO. Promoter sequence analysis of differentially expressed genes

Regulation of Transcription¾ Regulatory proteins, binding to the up-stream region of a gene act aseither promoters or suppressors of transcription

Transcription
FactorTranscription
FactorGene

ACAGTGA

GeneACAGTGA

ProteinProtein¾ Regulation of transcription is time, cell and tissue specific
CSIRO. Promoter sequence analysis of differentially expressed genes

Promoter architecture¾ Organization of promoter motifs represents a "footprint" of thetranscriptional regulatory mechanisms¾ Complex transcriptional control modules

Levine et al, 2003

Active promoters have a unique 3-dimensional structure
Changing the order or spacing of transcription
factor binding sites (TFBS) can change theoverall structure of the promoter and thus
affect transcription
Werner et al, 2003
CSIRO. Promoter sequence analysis of differentially expressed genes

Previous workCSIRO. Promoter sequence analysis of differentially expressed genes

Data Sets of differentially expressed (DE) genes in resistant and susceptible
animals following nematode challenge
XDH cluster
TLR cluster

Cathepsin Cluster

DUOX Cluster

Co-expression

Transcriptional
co-regulation

CSIRO. Promoter sequence analysis of differentially expressed genes

Common regulatory
elements

Goals
¾ To conduct a comprehensive analysis of promoter sequences forDE genes¾ To identify potential TF binding sites involved in regulation* To find common motifs shared by the majority of TFs* Are there any global regulators (TFs) that turns on DE genes in sheep?
¾ To benchmark currently available methods and establish a broad
protocol for regulatory sequence analysis¾ To extensively use human promoter sequence data (well studied)using comparative genomics approaches¾ To apply this protocol to other related projects at CSIRO as a starting
point to understand transcription regulation
CSIRO. Promoter sequence analysis of differentially expressed genes

Promoter sequence analysis - schema
Desired genes
Retrieve 2KB upstream region from TSS

Identify statisticallyover-represented
oligonucleotides in eachgeneset

Search for TFBS
1. Known TFBS data (database)
2. Identify common framework
3. Position weight matrices (PWM)
4. Literature evidence

Rank the potential
regulatory elements

Experimental Validation
CSIRO. Promoter sequence analysis of differentially expressed genes

Comparative Promoter
Analysis
Compare oligonucleotides,
TFBS with closely related
mammals (eg: human)

Over-represented consensus binding sites discoveredin 22 Toll Like Receptor (TLR) pathway genes

Motif 1: TCAGAAA
P-value : 3.5e-05

Motif 2: AGAGAAA
P-value : 3.5e-06

Motif 3: GGGAGGA
P-value : 2.1e-05

CSIRO. Promoter sequence analysis of differentially expressed genes

Promoter sequence analysis - schema
Desired genes
Retrieve 2KB upstream region from TSS

Identify statisticallyover-represented
oligonucleotides in eachgeneset

Search for TFBS
1. Known TFBS data (database)
2. Position weight matrices (PWM)
3. Identify common framework
4. Literature evidenceRank the potential
regulatory elements

Experimental Validation
CSIRO. Promoter sequence analysis of differentially expressed genes

Comparative PromoterAnalysis
Compare oligonucleotides,
TFBS with closely relatedmammals (eg: human)

Common patterns (frameworks) of TFBS identified in promotersfrom a set of three differentially expressed genes in TLR pathway

TFBS common to all sequences in a set of three promoters from TLR pathway

Selected hits from matrix search for transcription
factor binding sites

Literature evidence
in humanQuery GenePosition(strand)Sequence

Transcription
FactorName

Resistant: TLR2150 (-)

aCACTTga

Nkx2-5 (homeoboxgene)

Resistant:
DUOX1

948 (-)

acaAACAAac

FOXD3(fork head)

Susceptible:
IKBKB

686 (-)

atGGAAAttcc
c

NF-kappaB

Susceptible:
TGFB2

121 (+)

gatAACGGtc

v-Myb

CSIRO. Promoter sequence analysis of differentially expressed genes

Promoter sequence analysis - schema
Desired genes
Retrieve 2KB upstream region from TSS

Identify statisticallyover-represented
oligonucleotides in eachgeneset

Search for TFBS
1. Known TFBS data (database)
2. Position weight matrices (PWM)
3. Identify common framework
4. Literature evidence

Comparative PromoterAnalysis
Compare oligonucleotides,
TFBS with closely relatedmammals (eg: human)

Rank the potential
regulatory elements

Experimental Validation
CSIRO. Promoter sequence analysis of differentially expressed genes

Promoter region– conserved in mammalian species
List of TFs

Common TF matches located in aligned regions
TFBS1

TFBS2

TFBS3
CSIRO. Promoter sequence analysis of differentially expressed genes

Conclusions• We believe that the promoter motifs identified
in this study have regulatory potential• Binding of transcription factors to these motifs
might explain the differential expression
observed following nematode challenge• Functional variation in these motifs is therefore
likely to contribute to an individual’s ability toresist infection

Future directions
Computational¾ To establish a roadmap for regulatory sequence analysis based ongene expression data

Experimental validation for initial results¾ In-vitro experiments such as Electrophoretic Mobility Shift Assay(EMSA)¾ In-vivo experiments (ChIP-Seq experiments)¾ The second set of RT-PCR data spanning 102 genes

CSIRO. Promoter sequence analysis of differentially expressed genes

Acknowledgments

Antonio Reverter
CSIRO Livestock Industries

 OCE Post-doctoral fellowship SheepGenomics

CSIRO. Promoter sequence analysis of differentially expressed genes

Aaron Ingham
CSIRO Livestock Industries

Transcription Factors (TF) - Factsheet
Found in all living organisms¾ Approximately 10% of genes in the mammalian genome code for TFs¾ Have affinity for short, degenerate DNA sequences (5-15 bp)¾ Contain one or more DNA binding domains (DBDs)¾ Mutated TF genes have been shown to cause numerous diseases(Eg: Haemophilia B Leyden)¾ Potential Targets for several therapeutic drugs (Eg: breast cancer)¾

http://www.biochem.arizona.edu/CSIRO. Promoter sequence analysis of differentially expressed genes

Butt et al 1995, Latchman et al 2000

Challenges¾ Ovine genome incomplete, therefore we have chooses bovine asstarting point¾ Several physical binding sites comes up in predictions as aphysical TFBS is found every 10 to 15 bps throughout any mammaliangenome¾ A single isolated TF binding site carries no function¾ TFs work through complexes which are represented on sequencelevel through sets of TF binding sites in certain distance relationshipand orientation ->promoter frameworks¾ Therefore, we have to decipher gene anatomy landmarks , rather thanjust promoter motifs¾ So wee are talking promoter model/ framework and not individual TFBSCSIRO. Promoter sequence analysis of differentially expressed genes

Why are we doing what we are doing ?
¾ Promoter features provide clues to gene function that are not obvious
from the protein sequence alone¾ The organizational features of promoters derived from promoter
sequences contain information about the spatial and temporal ‘functional
context’ of expression.
¾ Genes having similar expression patterns contain common motifs intheir promoter regions. Thus, a common set of TFs is likely to control
these genes¾ Understanding the gene regulation in livestock species is still in primitive
stages , so Starting point .. **CSIRO. Promoter sequence analysis of differentially expressed genes

Vilo et al., 2000, Klok et al. 2002)

Different tissues and families of genes differentially use distinct typesof promoters.

Tamoxifen – drug for breast cancer

CSIRO. Promoter sequence analysis of differentially expressed genes

GeneCluster

Number ofgenes per
cluster

Sequence

observed
occurrences

expected
occurrencesP-value

TLR
Cluster25

TCAGAAA25

9.81

3.5e-05

XDH
cluster

10

AGAGAAA16

3.88

3.3e-06

XDH
cluster10

ccgcgc18

5.29

1.1e-05

Cathepsincluster9

cccccg17

4.42

4.2e-06