Transcript No Slide Title - Interdisciplinary Graduate Program in Genetics

The Role and Mechanism of PPAR in the Transcriptional Regulation of its Target Genes
1
Cai ,
2
Keen ,Thomas
3,4,5
Casavant ,
2
Sigmund
Jinlu
Henry L.
L.
and Curt D.
1Interdisciplinary Program in Genetics, Departments of 2Internal Medicine, 3Electrical and Computer Engineering, 4Biomedical Engineering,
5
and the Center for Bioinformatics and Computational Biology, University of Iowa, Iowa City.
Background
Peroxisome proliferator-activated receptors
(PPARs) are transcription factors belonging to
the nuclear receptor superfamily that
heterodimerize with the retinoid X receptor
(RXR). The activation of its target genes
depends on the binding of the ligand, such as
TZDs (thiazolidinediones).
PPARγ is expressed predominantly in adipose
tissue and promotes adipocyte differentiation
and glucose homeostasis. PPARγ is also
expressed in endothelial and vascular smooth
muscle cells and has been shown to have an
important role in the regulation of vascular
function and blood pressure. Patients with
dominant negative mutations (P467L or
V290M) in the ligand binding domain of
PPARγ have been reported to have severe
insulin resistance leading to full-blown type II
diabetes mellitus and early onset hypertension.
Mice model and Microarray Design
Combine both expression and exon arrays by quantile normalization
S PPAR mice
Transgenic mice with dominant negative PPARγ (P465L equivalent to P467L in
human) targeted to vascular smooth muscle cell (VSMC) have been generated and these
transgenic mice have been shown to exhibit severe vascular dysfunction.
Platform:
Murine Genome U74 Version 2 (Affymetrix)
3T3-L1 adipocytes harvested at day 10 postdifferentiation
3 biological replicates for PPARγ and control
IgG (Immunoglobulin G)
Mouse Tiling 2.0R Array Set (Affymetrix)
After normalization:
(Array1-5 from expression array)
(Array6-17 from exon array)
For each hybridization, RNA pooled from 8 different mouse aortas was used.
S PPAR mice exon array experiment
Five wild-type control and seven transgenic mice hybridizations were performed.
For each hybridization, RNA exacted from a single mouse aorta was used.
Power Calculation
Motivation for conducting exon array after expression – increase power.
More probe-sets have high power with the increasing of sample size, at alpha=0.05.
Microarray data with comprehensive
annotations and p-value from T test.
Example of processing
a.) Delete duplication and concatenate all the annotations
Gene symbol
mRNA accession
Nap1l1 /// Nap1l1
D12618
a.) For each probe-set, concatenate all the
annotations after deletion of duplications
Gene symbol
mRNA accession
Nap1l1
D12618
Gene annotations
b.) Merge probe-sets with overlapping annotation:
Probe-sets with unique annotations
Result of processing
Gene annotations
p-value
D12618 /// Nap1l1
0.041
NM_015781 /// Nap1l1
0.813
Expression array: 45101  26599
Exon array: 101176  33797
PPRE (PPAR Response Element) enrichment
Gene annotations
Up or Down genes not enriched by PPRE compared to no-change genes in S PPAR.
Genes from Adipocyte microarray
Nap1l1 /// D12618
b.) Merge probe-sets with overlapping
annotation: p-value dependent
power cutoff 2wt v.s. 3mut 3wt v.s. 4mut 4wt v.s. 5mut 5wt v.s. 7mut 6wt v.s. 8mut 7wt v.s. 10mut
0.9
28
27
50
44
72
84
0.8
38
44
77
90
105
125
0.7
48
74
108
128
149
191
0.6
55
114
157
183
211
281
0.5
68
154
210
237
308
391
0.4
87
211
296
339
456
537
0.3
119
308
460
536
673
837
0.2
185
533
820
972
1201
1385
0.1
477
1416
2078
2341
3019
3159
PPRE identified from ChIP-chip experiment using adipocyte.
Delete probe-sets without any annotations of
gene symbol or mRNA accession information
p-value dependent
ChIP-chip data
Before normalization:
Two wild-type control and three transgenic mice hybridizations were performed.
Schema of processing
3T3-L1 adipocyte cells treated 24 hours with
either 0.1% DMSO (12 replicates), 20uM
pioglitazone (7 replicates), 1uM rosiglitazone
(10 replicates) or 20uM troglitazone (10
replicates)
Apply quantile normalization to merged dataset.
S PPAR mice expression array experiment
Data analysis 1 – probe-set processing
Microarray data
Merge probe-sets from both arrays according to annotations and result in 18309
probe-sets.
Affymetrix GeneChip Mouse Genome 430 2.0 array.
However, the molecular mechanism by which
PPARγ exerts its effect in the genome-wide
transcriptional regulatory network of its target
genes remains to be elucidated.
Pub resource
Data analysis 2 – combined schema
p-value
Distance
284 up-regulated
50k
100k
321 down-regulated
50k
100k
282 not changed
50k
100k
PPRE
Percentage
90
132
31.69%
46.48%
62
101
19.31%
31.46%
57
96
20.21%
34.04%
Future Plan
Genes from S PPAR microarray
Distance
217 up-regulated
50k
100k
141 down-regulated
50k
100k
225 not changed
50k
100k
PPRE
Percentage
39
76
17.97%
35.02%
38
52
26.95%
36.87%
45
80
20.00%
35.56%
Application of Bayesian framework, in which expression
array data serve as prior.
D12618 /// Nap1l1 /// NM_015781 0.041
Integration of protein-protein and protein-DNA network information to dissect pathways.