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Gireedhar V; Kumar AP; Loo SY; Pervaiz S; Clement MV; and Sakharkar MK. Computational identification and experimental validation of PPRE motifs in NHE1 and MnSOD genes of Human. Presenting author: Gireedhar Venkatachalam. International Conference on Bioinformatics (InCoB). - PowerPoint PPT Presentation
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Computational identification and experimental validation of PPRE motifs in NHE1 and MnSOD
genes of Human
Presenting author: Gireedhar Venkatachalam
Gireedhar V; Kumar AP; Loo SY; Pervaiz S; Clement MV; and Sakharkar MK
International Conference on Bioinformatics (InCoB)
Brief overview1) Introduction to PPAR (Peroxisome Proliferator Activated Receptor) and PPRE (Peroxisome Proliferator Response elements) 2) Aims and PPRE prediction3) PPARg, NHE1 and MnSOD in Breast cancer4) Computational Prediction of PPREs in NHE1 and MnSOD5) Experimental Validation of PPREs in NHE1 and MnSOD6) Conclusion
Brief overview1) Introduction to PPAR (Peroximsome Proliferator Activated Receptor) and PPRE (Peroximsome Proliferator Response elements) 2) Aims and PPRE prediction3) PPARg, NHE1 and MnSOD in breast cancer4) Computational Prediction of PPREs in NHE1 and MnSOD5) Experimental Validation of PPREs in NHE1 and MnSOD6) Conclusion
PPAR AND PPREs PPARs belong to the nuclear receptor super family and are ligand
activated transcription factors, regulating a wide variety of genes Three isoforms (α, β and g) for PPAR PPARs are involved in lipid metabolism and induces
differentiation and inhibit proliferation in a variety of cancer cells
C A A A A C T A G G T C A N A G G T C AFlanking Hexamer 1 Spacer Hexamer 2
PPAR
PPRE TATA TARGET GENE
RXR
DBD DBD
Direct Repeat 1
Co activators or Co repressors
Peroxisome Proliferator Respose element
Ligand
Brief overview1 ) Introduction to PPAR (Peroximsome Proliferator Activated Receptor) and PPRE (Peroximsome Proliferator Response elements) 2) Aims and PPRE prediction3) PPARg, NHE1 and MnSOD-Breast cancer4) Computational Prediction of PPREs in NHE1 and MnSOD5) Experimental Validation of PPREs in NHE1 and MnSOD6) Conclusion
Aim of the study
• Recently it is shown that PPARs also bind to DR2 repeats (AGGTCA NN AGGTCA) (Fontaine et al.,2003; AP Kumar et al.,2004)
• Flanking sequence plays a significant role in PPARs specificity and binding (Palmer et al., 1995)
• Nuclear receptor- competitive binders (Harikrishna et al., 1998)
PPRE Prediction• Collection of PPRE database
Contains 414 reported PPRE motifs from literature.
The sequences reported only with experimental validation were added to this database.
PPRE element in database - reported consensus, isoform specificity, in vivo and in vitro binding efficiencies and Pubmed IDs
PPRE
Pre
dicti
on-Te
xt m
inin
g
PPRESearch Webserver
Brief overview1) Introduction to PPAR (Peroximsome Proliferator Activated Receptor) and PPRE (Peroximsome Proliferator Response elements) 2) PPRE prediction3) PPARg, NHE1 and MnSOD-Breast cancer4) Computational Prediction of PPREs in NHE1 and MnSOD5) Experimental Validation of PPREs in NHE1 and MnSOD6) PPARg as novel therapeutic approach in breast cancer therapy
Breast cancer
Tumor breast tissue expresses
PPARg higher than normal breast
epithelium
Breast cancer - PPARg, NHE1 and MnSOD
PPARg
MnSOD (Manganese Superoxide dismustase)
NHE1 ( Sodium Hydrogen Exchange 1)
Function : NHE1 deficient cells- either fail to grow or show retarded growth ( Liu et al., 2008)
Function : Downregulaion of MnSOD expression decreases cancer cells invasive property (Kattan et al., 2008)
? ?
??
Brief overview1) Introduction to PPAR (Peroximsome Proliferator Activated Receptor) and PPRE (Peroximsome Proliferator Response elements) 2) PPRE prediction3) Breast cancer-PPARg, NHE1 and MnSOD4) Computational Prediction of PPREs in NHE1 and MnSOD5) Experimental Validation of PPREs in NHE1 and MnSOD6) Conclusion
Sequence ID:NCBI-GI: 27777632NCBI-GeneID: 6548Ensembl: ENSG00000090020
PPRE1
PPRE2
PPREs in NHE1PPRE1
PPRE2
-2742 TGCAGAGGACATCCTGAGCTGGCTGGAGTAACTTGGGACACAGGTCAAT
-1673 ACTTGAGGTCAGGCGTTCGAGACCATCCTGACCAACATAGTGAAACCCCGT
Sequence ID:NCBI-GI: 67782305NCBI-GeneID: 6648Ensembl: ENSG00000112096
PPRE1
PPRE2
PPRE3
PPREs in MnSOD
PPRE1
PPRE3PPRE2
Brief overview1) Introduction to PPAR (Peroximsome Proliferator Activated Receptor) and PPRE (Peroximsome Proliferator Response elements) 2) PPRE prediction3) Breast cancer-PPARg, NHE1 and MnSOD4) Computational Prediction of PPREs in NHE1 and MnSOD5) Experimental Validation of PPREs in NHE1 and MnSOD6) Conclusion
NHE1
PPAR gamma
NHE1 MnSOD
AGGTCA G AGGTCA
Nucleus
Maintains pH
MnSOD ROS Balance
15d-PGJ2
Breast cancer cells (MCF7,MDA-MB-
231,MDA-MB-468)
Cytoplasm
Experimental validation setup
mRNA, protein level, Promoter activity and in vitrobinding assay
?
0
20
40
60
80
100
120
MCF-7 MDA-MB-231
NH
E1 m
RN
A 3µM 15d-PGJ2
5µM 15d-PGJ2
% fr
om u
ntre
ated
NHE1
15d-PGJ2/µM
0 1 3 5
NHE1
β-actin
15d-PGJ2/µM 0 1 3 5
β-actin
MD
A-M
B-2
31M
CF-
7 MnSOD
β-actin
MnSOD
β-actinMD
A-M
B-4
68
0 3 5 10
15d-PGJ2/µM
0 3 5 10
15d-PGJ2/µMM
DA
-MB
-231
MDA-MB-231
MDA-MB-468
50525456586062646668
% fr
om u
ntre
ated
MnS
OD
mRN
A
5µM 15d-PGJ2
10µM 15d-PGJ2
NHE1 MnSOD
NHE1 and MnSOD repression upon PPARg activation
CATTATA
CATTATA
TGAGGTCAGGAGTTCGAG
PPRE 1
-1374/+16
-850/+16
CAAGGTCACACGGTAACT
PPRE 2
Human NHE1 promoter constructs
-1374 has PPRE1, and PPRE2-850 has only PPRE2
0µM
3µM
5µM
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Hum
an N
HE1
pro
mot
er a
ctiv
ity
abso
rban
ce a
t 405
nm/μ
g to
tal p
rote
in
-1374 -850
PPRE1 - PPARg binding site in NHE1
LUCIFERASETATA
LUCIFERASETATA
-3400 to +24 pGL3
-1605 to +24 pGL3
PPRE 1TGAGGTCAGGCGTTCGAG
PPRE 2ATAGGTCCCAAGGTCGGC
PPRE 3
LUCIFERASETATA -555 to +24 pGL3
CTTGGGACACAGGTCAAT
-3405 -1605 -5550
500
1000
1500
2000
2500
3000
3500
RLU
/reni
lla/u
g to
tal p
rote
inHu
man
MnS
OD
prom
oter
act
ivity
Human MnSOD promoter constructs
-3405 has PPRE1, PPRE2, and PPRE3-1605 has PPRE2 and PPRE3-555 has no PPRE1, PPRE2, or PPRE3
0µM3µM5µM
PPARg binding site in MnSOD
PPRE 2 PPRE 30
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
DNA binding activity of PPARγ at PPRE2 and PPRE3 of MnSOD promoter (n=2)
Abs
orba
nce
at 4
50nm
PPA
Rg
bind
ing
assa
y
0µM3µM5µM
PPRE3-PPARg binding site in MnSOD
Brief overview1) Introduction to PPAR (Peroximsome Proliferator Activated Receptor) and PPRE (Peroximsome Proliferator Response elements) 2) PPRE prediction3) Breast cancer-PPARg, NHE1 and MnSOD4) Computational Prediction of PPREs in NHE1 and MnSOD5) Experimental Validation of PPREs in NHE1 and MnSOD6) Conclusion
Conclusion• We have constructed a better in silico
approach to finding genes containing PPRE in their promoter region
• Our approach helps us to identify both DR1 and DR2 sites
• Importance of flanking sequence were incorporated.
• It is our hope with this PPRESearch database, researchers in the field of PPARs would better identify new target genes which could then be translated into the clinic for intervention.
Thank you
Questions?
NHE1PPAR gamma
15d- PGJ2
MnSODInvasive
property
Breast cancer death
represses
represses
PPARg as novel therapeutic approach in breast cancer therapy
cell proliferation
sensitizes
sensitizes
PPARs and PPRE• PPAR α, β, and g isoforms share a highly conserved DNA binding
domain that recognizes specific DNA sequences known as Peroxisome Proliferator Response Elements (PPREs)
• PPAR/RXR complex then binds to PPRE composed of a Direct Repeat (DR) preferably spaced by one nucleotide (DR1) with a
consensus sequence of AGGTCA-A-AGGTCA Direct Repeat (DR) preferably spaced by two nucleotide (DR2) with a
consensus sequence of AGGTCA-GG-AGGTCA.
Transcription factor analysis
• Transcription factor might be defined as any molecule participating, alone or as part of a complex, in the binding to a gene’s enhancer response element or promoter, with the ultimate outcome being the up- or down-regulation of expression of that gene.
• Transcription factors participate in stress pathways in cancer by causing the up- or down-regulation of specific genes.
Transcription factor analysisSignal
Pathways
Transcription factors
Gene expression up and downregulation
Target proteins
Cellular processes affected
Targeting cancer