Interpreting DNA Methylation in terms of TFBS
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Transcript Interpreting DNA Methylation in terms of TFBS
Meng Li
School of Informatics
Interdisciplinary Program of Biochemistry
Indiana University
Advisors: Dr. Sun Kim, Dr. Kenneth Nephew
4/25/2008
1
Overview
Biology background and experimental model
Objective
High-throughput data and data analysis
Combinatorial study and data mining
Conclusions
2
Ovarian cancer and drug resistance
Ovarian cancer
Most deadly gynecological malignancy
Cisplatin
Widely used chemotherapeutic drug
DNA intercalating agent
Cisplatin resistance
70% to 80% of patients develop resistance after 2year treatment
3
DNA methylation
CpG dinucleotides: 5’- AATACGCCACGA
4
DNA methylation
CpG island
CG
CG
MCG
MCG
MCGMCG MCG MCG
CG
CG
CG
CG CG CG
Normal
Cancer
X
C: cytosine
mC:
De novo methylation: acquired methylation
methylcytosine
5
Objective
Does de novo DNA methylation plays a role in the
development of chemotherapeutic drug resistance
in ovarian cancer cells?
How does de novo DNA methylation affect drug
resistance development?
6
In vitro drug resistance system
Cisplatin
Cisplatin
Drug-sensitive
parental cells
A2780 R-
IC50 (uM)
Drug-resistant
Cells
A2780 R+
Rounds of Cisplatin treatment
7
High-throughput data and data analysis
Global promoter methylation data
Global gene expression data
Louis Staudt, The nation’s investment in cancer research (NCI)
8
Global promoter methylation profiling
A2780 R-
A2780 R+
Differential Methylation
Hybridization (DMH)
44,000 probes representing
10,000 genes
Two-color microarray analysis
Data processing
Estimate methylation level
CpG Island Microarray (44K)
9
Global promoter methylation profiling
Loess normalization: correcting technical bias
Raw Data
Normalized Data
Fold-change analysis: extracting gene methylation level
M =
Red
log 2
Green
10
Global gene expression profiling
Affymetrix U133 plus 2.0
microarray
54,675 probes representing
20,606 genes
mRNA
Single-color microarray
cRNA
analysis
Data processing
Estimate gene expression
levels
U133 plus 2.0 array
11
Global gene expression profiling
Clustering
Fold change
A2780R+ expression
A2780R- expression
Welch’s t-test p-values
A2780 R+ A2780 RCutoffs:
- p-value < 0.01
- fold change >= 1.5
12
Combinatorial study and data mining
Does de novo DNA methylation plays a role in the
development of chemotherapeutic drug resistance
in ovarian cancer cells?
How does de novo DNA methylation affect drug
resistance development?
13
The number of hypermethylated genes
positively correlated with the increase of IC50
14
DNA methyl-transferases (DNMT) are
up_regulated in resistant cells
Welch's t-test
Fold change
Gene title
DNMT1
0.0011
1.63
DNA (cytosine-5-)-methyltransferase 1
DNMT2
0.3673
1.17
DNA (cytosine-5-)-methyltransferase 2
DNMT3A
0.1009
1.20
DNA (cytosine-5-)-methyltransferase 3 alpha
DNMT3B
0.0004
1.80
DNA (cytosine-5-)-methyltransferase 3 beta
DNMT1: maintain genomic DNA methylation
DNMT3B: de novo methylation
15
Resistant cells re-establish cisplatin sensitivity
after methylation inhibitor treatment
16
Key questions
Does de novo DNA methylation plays a role in the
development of chemotherapeutic drug resistance?
Yes
How does de novo DNA methylation affect drug
resistance development?
Does de novo methylation selectively blocking transcription factor
binding?
x
17
Surveying Transcription Factor Binding Sites
(TFBS) on differentially methylated regions
Scan TFBS on hypermethylation (S+), hypomethylation (S-),
or hypermethylated CGI (SCpG) regions with match program
against TRANSFAC database
.
.
.
.
.
*
S+
.*
.
.
.
.
*
S-
*
*
.
.
.
.
.
*
SCpG
18
Methylation selectively occurs at
certain TFBS
32.5
Occurrence percentage
30
Hypermethylated
27.5
25
Hypomethylated
22.5
20
17.5
15
12.5
10
7.5
5
2.5
0
19
Statistical scoring
• Fisher exact test
• Multiple test correction – False discovery rate (FDR)
TFBS
S+ vs. S-
S+ vs. Sr
S+ vs. SCpG
NCX
HMGIY
CEBP
BRCA
0.000698
0.00113
0.008758
0.01259
5.49E-10
6.14E-16
1.53E-09
1.67E-06
0.0007561
2.88E-05
0.0001531
0.001406
20
Key questions
Does de novo DNA methylation plays a role in the
development of chemotherapeutic drug resistance?
Yes
How does de novo DNA methylation affect drug
resistance development?
Does de novo methylation selectively blocking transcription factor
binding? Yes
Does de novo methylation selectively regulates certain pathways?
21
Methylation regulated pathways
• Hypomethylation up-regulated pathways
UpRegulated Genes fc > 1.5 (1037)
HypoMethyl fc <-1.5 and UpReg genes
p<0.01 fc >1.5 (55)
Impact
Input genes
in pathway
Corrected
p-value
Impact
Input genes in
pathway
Corrected
p-value
fisher.test
p-value
Glioma
6.09
6
0.016
11.909
3
8.69E-5
0.013
Melanoma
4.516
5
0.060
10.268
3
3.91E-4
0.009
Pancreatic cancer
5.237
8
0.033
9.09
3
0.0011
0.023
Prostate cancer
5.064
9
0.038
8.991
3
0.0012
0.029
Colorectal cancer
3.108
6
0.184
8.758
3
0.0015
0.012
Chronic myeloid leukemia
3.169
6
0.175
8.221
3
0.0025
0.012
Non-small cell lung cancer
1.376
2
0.600
5.59
2
0.0246
0.044
All are human cancer related pathways
22
Hypomethylated and up-regulated
pathways
Genes involved for each pathway:
PIK3R3, PDGFRA, E2F1, TGFBR2
E2F
Smad2/3
Smad4
23
Methylation regulated pathways
• Hypermethylation down-regulated pathways
DownReg Genes fc < -1.5 (1286)
HyperMethyl fc >1.5 and DownReg
genes p<0.01 fc <-1.5 (55)
Input
genes in
pathway
Corrected
p-value
Impact
Cell adhesion molecules
384.485
(CAMs)
14
1.95E-164
897.568
4
0
0.005052
Tight junction
8.868
13
0.0014
17.229
3
9.93E-7
0.02503
PPAR signaling pathway
3.68
6
0.1172
7.377
2
0.0052
0.03946
Impact
Input genes Corrected
in pathway p-value
fisher.test
p-value
Cell adhesion molecules (CAMs): Environmental information processing
Tight junction: Experimental processes -> Cell communication
PPAR signaling pathway: Experimental processes -> Endocrine system
24
Hypermethylated and down-regulated
pathways
Genes involved for each pathway:
CAMs (ITGAV, CLDN11, NEO1, CDH2)
Tight junction (CLDN11, PPP2R4, INADL)
PPAR signaling (CPT1A, SLC27A6)
25
Key questions
Does de novo DNA methylation plays a role in the
development of chemotherapeutic drug resistance?
Yes
How does de novo DNA methylation affect drug
resistance development?
Does de novo methylation selectively blocking transcription factor
binding? Yes
Does de novo methylation selectively regulates certain pathways?
Yes
26
Conclusion
Promoter CpG island methylation
Participates in the development of drug resistance of
ovarian cancer cells
Regulates gene expression alteration through drug
resistance development by selectively occurring at
certain TFBS
Regulates cellular functions by methylating key players
in certain pathways
27
Acknowledgements
Advisors
Sun Kim
Kenneth Nephew
Committee
Curt Balch
Haixu Tang
OSU ICBP center
Dustin Potter
Pearlly Yan
Tim H-M. Huang
IUPUI
Lang Li
Jeanette McClintick
Colleagues
Fang Fang
Shu Zhang
Henry Paik
John Montgomery
Mikyoung Jeong
Fuxiao Xin
Nicolas Berry
Xinghua Long
Nicole Nickerson
Xi Rao
Cori Hartiman-Frey
Funding Agencies
NCI U54 CA11300
NCI R01 CA85289
28
F. Coste, J. M. Malinge, L. Serre, W. Shepard, M. Roth, M. Leng and C. Zelwer, "Crystal structure
of a double-stranded DNA containing a cisplatin interstrand cross-link at 1.63 A resolution:
hydration at the platinated site", Nucleic Acids Res, 1999, 27, 1837.
29