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