gene expression profiling of potential pparj target genes in ... - CiteSeerX

Articles in PresS. Physiol Genomics (March 30, 2004). 10.1152/physiolgenomics.00027.2004

PG-00027-2004 Final Accepted Version

GENE EXPRESSION PROFILING OF POTENTIAL PPARJ TARGET GENES IN MOUSE AORTA Henry L. Keen1,2, Michael J. Ryan1, Andreas Beyer1, Satya Mathur1, Todd E. Scheetz2, Barry D. Gackle2, Frank M. Faraci1, Thomas L. Casavant2 and Curt D. Sigmund1

1

Departments of Internal Medicine and Physiology & Biophysics, and the 2Center for Bioinformatics and Computational Biology University of Iowa College of Medicine Iowa City, IA 52242

Running Title: PPAR J regulated genes in mouse aorta

*Address correspondence and reprint requests to Curt D. Sigmund, Ph.D. Departments of Internal Medicine and Physiology & Biophysics 3181B Medical Education and Biomedical Research Facility (MEBRF) Roy J. and Lucille A. Carver College of Medicine University of Iowa Iowa City, Iowa 52242 Tel: 319-335-7604 FAX: 319-353-5350 E-mail: [email protected]

Copyright © 2004 by the American Physiological Society.


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ABSTRACT Diminished activity of PPARJ may play a role in the pathogenesis of hypertension and vascular dysfunction. To better understand what genes are regulated by PPARȖ, an experimental dataset was generated by microarray analysis, in duplicate, of pooled aortic mRNA isolated from mice treated for 21 days with a PPARȖ agonist (rosiglitazone) or vehicle. Of the 12488 probe sets present on the array (Affymetrix MG-U74Av2), 181 were differentially expressed between groups according to a statistical metric generated using Affymetrix software. A significant correlation was observed between the microarray results and real time RT-PCR analysis of 39 of these genes. Cluster analysis revealed 3 expression patterns, 29 transcripts of moderate abundance that were decreased (-93%) to very low levels, 106 transcripts that were downregulated (-42%), and 46 transcripts that were upregulated (+70%). Functional groups that were decreased included inflammatory response (-93%, n=6), immune response (-86%, n=7), and cytokines (-82%, n=7). There was an overall upregulation in the oxidoreductase activity group (+47%, n=9). Individually, 6 transcripts in this group were increased (+72%) and 3 were decreased (-34%). 14 of the genes map to regions in the rat genome that have been linked to increased blood pressure and of 142 upstream regions analyzed, sequences resembling the DNA binding site for PPARJ were identified in 101 of the differentially expressed genes.

Key words: gene expression analysis, bioinformatics, vasculature, mouse, transcription factor


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INTRODUCTION PPARJ is a member of the nuclear hormone receptor subfamily of transcription factors and acts by forming heterodimers with retinoid X receptors.

Ligands that activate PPARJ include a

naturally occurring prostaglandin J metabolite (15d-PGJ(2)) and a group of synthetic compounds, thiazolidinediones (TZD). Several lines of evidence demonstrate an important role for PPARJ in lipid and glucose metabolism. PPARJ is highly expressed in adipose tissue and induces adipocyte differentiation (20). Chronic administration of TZDs in humans with type II diabetes decreases release of free-fatty acids (FFA) from adipose tissue (17) and significantly improves insulin sensitivity (18). In addition to these well-characterized metabolic actions of PPARJ, recent experimental evidence suggests involvement in a number of other pathways including cell-cycle regulation, inflammation, and vascular function (3).

It has recently been demonstrated that PPARJ is expressed in all cell types (vascular smooth muscle cells (13), endothelial cells (5), and macrophages (22)) in the vasculature and current evidence suggests that PPARJ may normally mediate an important vascular protective action. Pharmacological activation of PPARJ with TZDs reduces vascular lesion formation in animal models of atherosclerosis (4), and inhibits vascular smooth muscle cell proliferation and neointima formation after balloon injury (14). Chronic reduction in blood pressure and improvement in endothelial function has been observed during TZD administration in humans and animal models (21, 27, 29). Moreover, naturally occurring loss-of-function mutations in human PPARJ are associated with hypertension (2).


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We and others hypothesize that PPARJ plays an important role in vascular function and hypertension. Although many targets for PPARJ have been identified in skeletal muscle and fat, the classical PPARJ target organs, little is known about the genes or gene networks that are directly regulated by this factor in the vasculature. Because PPARJ is a transcription factor, its physiological actions depend on binding to a specific DNA sequence, recruiting co-factors, and then activating/repressing transcription of target genes. Given the availability of genomic sequence, some investigators have employed a strategy of searching for transcription factor binding sites as a means to identify potential new downstream targets of various transcription factors (10, 25). In the present study, we have combined this and other types of computational approaches with large-scale gene expression analysis using microarrays to identify potential downstream targets of PPARJ and to generate hypotheses regarding the biological function and transcriptional regulation of PPARJ regulated genes in the vascular wall.


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METHODS The microarray data described in the present study complies with the MIAME (Minimum Information About Microarray Experiment) standard, has been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database, and can be accessed at http://www.ncbi.nlm.nih.gov/geo (Accession Number: GSE1011).

Animal Care: All procedures and care of the mice were conducted in accordance with National Institutes of Health guidelines using protocols approved by the Animal Care and Use Committee of the University of Iowa. Mice were fed standard mouse chow and received water ad libitum.

Experimental Protocols: To activate PPARJ, the TZD rosiglitazone was administered for 21 days by oral gavage to adult male mice (C57BL/6J strain) at a dose of 25 mg/kg/day. Control mice were given vehicle (sterile water). This dose is similar to that which has been reported in the literature for animal studies, but is higher than the standard dose reported for the treatment of non-insulin dependent diabetes mellitus. At the end of the experiment, mice were killed by CO2 asphyxiation and the aorta was immediately immersed in RNAlater solution (Qiagen). Total RNA was isolated from aortic samples using a commercially available Rneasy Miniprep kit (Qiagen). The quality of the RNA was confirmed by visual inspection of the 18S and 28S ribosomal RNA bands on an ethidium bromide stained 1.5% agarose gel. In order to have sufficient RNA for both the microarray hybridizations (in duplicate) and the real-time RT-PCR analysis, RNA from 4-5 mice in each group were pooled.


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Microarray analysis: Because the amount of starting RNA for each array experiment was low (less than 1 ug), two rounds of linear amplification were performed to produce sufficient material for hybridization to the array chip. The hybridization, washing steps, and scanning of the array were performed in the University of Iowa Microarray Facility using standard protocols. The relative amount of transcript was determined by comparing the perfect match and mismatch signals from all probe pairs. This signal value is a weighted mean value, relatively insensitive to outlier values, calculated using the one-step Tukey’s biweight estimate. Determination of whether the difference in transcript intensity between two arrays was statistically significant was accomplished by using Wilcoxon’s signed rank test. Only transcripts that were different in all between-group (i.e., vehicle vs. rosiglitazone) comparisons and that were not changed in all within-group comparisons (i.e., replicates) were considered to be differentially expressed. The Affymetrix microarray analysis suite was used to perform all of the above statistical tests.

Sequence analysis: Genomic DNA sequences (5kb) immediately upstream of the transcriptional start site from known and predicted transcripts from mouse (22,444) and human (24,847) were downloaded from Ensembl (http://www.ensembl.org) using the EnsMart tool (12). Additional gene information including chromosomal location, and human/mouse orthology was obtained from the same site. Processing of these data files was accomplished using custom Perl scripts. PPARJ has been shown to have important actions on blood pressure and vascular function. It is possible that some of the PPARJ target genes identified herein play a causal role in genetic hypertension. Therefore, to identify these potential candidate genes, we determined if any of the differentially expressed genes are located within known rat blood pressure quantitative trait loci (QTL) regions. This was accomplished by interspecies comparisons using syntenic maps from


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the Jackson Laboratories (http://www.jax.org) and from the Rat Genome Database (http://rgd.mcw.edu). To search for sequences closely resembling the PPAR response element (PPRE), we created a probabilistic-based model (Hidden Markov Model) based on the sequences of 19 known PPREs (Supplemental Table 1). The software used to build the model is part of a freely available package (HMMER 2, Eddy, S.R., http://hmmer.wustl.edu/).

Non-coding

sequences that are highly conserved between orthologous genomic sequences from different species are likely to contain important regulatory elements. To prioritize our list of potential PPREs, evolutionarily conserved sequences were identified by pairwise alignment of upstream sequences using a locally installed version of MegaBlast (http://ncbi.nlm.nih.gov/blast). Sequence fragments with 80% identity over at least 100 bp were considered to be highly conserved. Percent identity plots graphically depicting sequence conservation were downloaded from the Penn State University Center for Comparative Genomics and Bioinformatics (http://bio.cse.psu.edu/).

Real-time RT-PCR analysis: To validate the gene expression changes observed in the microarray experiment, expression of 39 genes was determined using a real-time quantitative PCR system (iCycler iQ Multi-Color Real Time PCR Detection System, Bio-Rad). These 39 genes were selected based on several criteria: 1) a representative mix of genes that included both up- and downregulated transcripts, 2) genes with known relevance to vascular physiology, and 3) genes with predicted PPREs in the regions upstream of the genes. Real-time reactions employed sequence-specific probes designed using commercial software (Applied Biosystems, Foster City, CA). Input samples were the same pooled RNA used in the microarray analysis.


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Other analysis: Functional categories were generated by assigning each transcript a function based on the Gene Ontology (http://geneontology.org) database.

We further examined the

patterns of gene expression changes with cluster analysis using hierarchical clustering followed by more detailed grouping using the k-means algorithm. The software used is available for download free of charge to academic users (Cluster and TreeView, Eisen M., http://rana.lbl.gov/EisenSoftware.htm).

Statistical Analysis: Correlation analysis was performed by calculating the Pearson correlation coefficient using commercially available software (SigmaStat). P values less than 0.05 represent statistical significance.


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RESULTS

To examine the PPARJ pathway in mouse aorta, we followed the steps outlined in Figure 1. Gene expression profiling with microarrays was used as a large-scale screening step to determine in a non-biased manner what pathways are activated/inhibited by rosiglitazone and to generate a list of potential PPARJ target genes. Subsequently, quantitative real-time RT-PCR was used to determine the expression changes in 39 individual genes, and computational methods were used to identify potentially important regulatory sequences.

As a measure of the quality of the microarray experiment, the signal intensities of transcripts from each chip were plotted as a histogram (Supplemental Figure 1).

They correctly

approximated a normal distribution with an expected modest skewing toward lower abundance transcripts. Under baseline conditions (vehicle-treated mice), 3,034 transcripts on the array (12,488 total) were expressed in both replicates. The expression levels of most transcripts were similar between replicates demonstrating the reproducibility of the microarray assay (Supplemental Figure 2).

We also noticed that when we mapped the accession number

associated with each probe set to the Unigene dataset, that many genes were represented more than once on the microarray. For example, among genes that were expressed in all samples, 105 unigenes were represented exactly 2 times on each array and there was a statistically significant correlation (R2 = 0.68, p < 0.05) between the fold change values (rosiglitazone versus vehicle) calculated from these two probe sets (data not shown).


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Using the criteria outlined in the Methods section, we identified 181 transcripts that were differentially expressed during chronic treatment with rosiglitazone (Supplemental Tables 2 and 3). To validate the microarray results, we performed real-time RT-PCR (using the same input RNA) on 39 of the differentially expressed genes (Table 1). There was a statistically significant correlation between these two different assays with an agreement of about 90 % on the up/downregulated calls (Figure 2). In an additional study, we examined gene expression levels of these same 39 genes in pooled aortic RNA prepared from independent groups of mice treated with the same protocol as the mice used for the initial microarray analysis. In these samples, there was an agreement of about 70-80 % on the up/down-regulated calls compared to the initial microarray results (data not shown).

To categorize these genes, each transcript was assigned a function based on the Gene Ontology database and sorted by expression cluster. Cluster analysis (k-means) revealed 3 basic patterns of expression, 29 transcripts of moderate abundance that were decreased (cluster 0, -93%) to very low levels, 106 transcripts of high abundance that were downregulated (cluster 1, -42%), and 46 transcripts of high abundance that were upregulated (cluster 2, +70%). The functional groups with the most significant changes in expression are highlighted in Figure 3. Transcripts from inflammatory response (-93%, n=6), immune response (-86%, n= 7), and cytokine activity (-82%, n=7) were primarily in cluster 0. Those from actin-binding activity (-52%, n=9), calciumbinding activity (-65%, n=10) and cytoskeleton (-46%, n = 6) grouped mainly to cluster 1. For the oxidoreductase activity group, 6 transcripts were in cluster 2 (+72%) and 3 were present in cluster 1 (-34%). A list of the individual genes in these functional groups whose expression is changed during rosiglitazone can be found in Table 2.


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To gain insight into possible mechanisms by which PPARȖ regulates these target genes, we compiled a list of upstream sequences (5 kb) from 142 of the differentially expressed genes. Using a sensitive model to search for PPAR response elements (PPREs), we detected a total of 117 PPREs in 101 of the sequences examined (Supplemental Table 4). When we examined a similar set of upstream sequences selected randomly from genes not differentially expressed in the microarray experiment, we detected 98 sequences resembling a PPRE in 86 genes. These PPRE-like sequences had more mismatches (3 or more) from consensus as compared to the potential PPREs identified from the set of differentially expressed genes, which were a much closer match to the consensus sequence with 50% more (42 versus 28) having two or fewer mismatches. Comparative sequence analysis was then used to prioritize the search for important regulatory sequences. From the upstream sequences examined, 31 sequence fragments were found to be highly conserved between human and mouse (Table 3). Conserved fragments upstream of basic helix-loop-helix domain containing protein, class B2 (BHLHB2) and frizzled homolog 4 (FZD4) contained a sequence closely resembling a PPRE (Figure 4). Because PPARJ might also regulate gene transcription by indirect means (activating/inhibiting secondary transcription factors), we looked for the presence of transcription factors among the differentially expressed genes. Of the 6 transcription factors identified, one was upregulated and 5 were downregulated (Table 4).

Using genomic location information and orthology between mouse and rat, we mapped 14 of the differentially expressed genes to genomic regions in the rat that have been associated with increased blood pressure (Table 5). We also examined if groups of differentially expressed genes are closely linked to a blood pressure QTL (1 MB). Several members of a small inducible


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cytokine family (known as CCL or chemokine (C-C motif) ligand) are located in a 400 kb stretch on mouse chromosome 11. This region is syntenic to a region on rat chromosome 10 containing a blood pressure QTL. As expected, the rat ortholog of mouse CCL3 has been mapped to this region. CCL3 (accession: J04491) was decreased by rosiglitazone. Another member of the family, CCL9 (accession: U49513) was also decreased by rosiglitazone; however, the rat ortholog of mouse CCL9 has not been conclusively determined at this time.

In the present study, RNA was prepared from the whole aorta making it difficult to discern the contribution of each of the vascular cell types to the overall gene expression profile.

In

particular, it would be attractive if existing publicly available resources could be integrated with our data to ascertain the cell expression pattern of the differentially expressed genes. The Cancer Genome Anatomy Project at the NIH has compiled Serial Analysis of Gene Expression (SAGE) results from several different tissue/cell types under both control and experimental/diseased conditions. The only available library derived from cells present in the vessel wall was from human microvascular endothelial cells (ID=29). We combined the information from this library with our complete list of differentially expressed genes (Supplemental Tables 2 and 3) to generate a subset of genes that are likely to be highly expressed in endothelial cells (Table 6).


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DISCUSSION

The major finding of the present study is that chronic treatment with a PPARJ agonist is associated with significant changes in a number of biologically important pathways in mouse aorta. A significant correlation was observed between the microarray results and real time RTPCR analysis of 39 of these genes suggesting that oligonucleotide chip-based assays of gene expression are a reliable way to interrogate gene expression of a large number of genes in an unbiased manner.

The actions of the TZDs, including rosiglitazone, are believed to be mediated primarily by activation of PPARJ, although results from some studies suggest that a contribution from PPARD activation or from a non-PPAR related mechanism might exist as well (3). We recognize that the dose of rosiglitazone used herein is similar to that which has been reported in the literature for animal studies, but is higher than the standard dose used in the treatment of noninsulin dependent diabetes mellitus. Therefore, we can not definitely rule out the possibility that some of the changes in gene expression may be due to this. This dose was effectively in lowering blood pressure and improving vascular function in a hypertensive mouse model (24). Indeed, in some experimental hypertensive models, PPARJ agonists significantly lower blood pressure and it has been proposed that this action is responsible for at least part of the vascular protective actions of PPARJ (7). Although, we did not measure blood pressure in the mice used in the microarray experiment; we did not observe any change in blood pressure in previous studies of normal control mice administered rosiglitazone at the same dose used herein (24).


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PPARJ in Blood Vessel Wall PPARJ is expressed in vascular smooth muscle (13), vascular endothelium (5), and macrophages (22), and the mechanisms responsible for the known actions of PPARJ in the vasculature appear to involve functional changes in each of these cell types.

In cultured vascular smooth muscle

cells, PPARJ has actions similar to that observed in animal models.

That is, it inhibits cell

migration, and attenuates the induction of matrix metalloproteinases (MMPs), a key step in cell invasion (16).

The finding of a significant decrease in MMP-9 mRNA in aorta from

rosiglitazone-treated mice in the present study is consistent with those results. Recent evidence suggests that downregulation of the type 1 receptor for angiotensin II in the VSMC may play a role in the blood pressure lowering and vascular protective actions of PPARJ (26). However, in the present study we did not detect any changes in expression of components of the reninangiotensin system, all of which were on the array chip.

Alterations in endothelial cell function may contribute to the vascular actions of PPARJ. PPARJ may protect the endothelium against high levels of reactive oxygen species such as superoxide by increasing expression of scavenger enzymes including Cu-superoxide dismutase (11) and catalase (8) and by decreasing expression of the p22 and p47 phox subunits of the superoxide-generating enzyme NADPH oxidase (11). In the present study, we identified in mouse aorta 9 differentially expressed genes involved in oxidative-reduction reactions. The functional contribution of these pathways to the chronic regulation of vascular tone remains to be elucidated. PPARJ has several other endothelial specific actions including decreased secretion of endothelin (5), a potent vasoconstrictor, and reduced expression of adhesion molecules on the


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endothelial cell surface (7), a key step in the atherogenic process. In our rosiglitazone-treated mice, there were no significant changes in endothelin or in any of the nitric oxide synthase isoforms.

There was, in contrast, a marked decrease in expression of several adhesion

molecules, a finding consistent with previous reports (7).

One of the first steps in vascular lesion formation is the attachment of circulating monocytes to the endothelium of the vascular wall followed by the release of pro-inflammatory mediators from activated macrophages. Recent studies with animal models of atherosclerosis have demonstrated that TZDs reduce the number and size of lesions formed in the vascular wall, in part, by attenuating the inflammatory responses of infiltrating macrophages (4). PPARJ agonists have recently been shown to have anti-inflammatory actions in other disease conditions including inflammatory bowel disease (1) and allergic encephalomyelitis (6) suggesting that this action is not vascular specific.

Moreover, these responses to PPARJ agonists may not require the

presence of atherosclerosis or a severe inflammatory condition. For example, in the present study, under basal conditions in which expression of inflammatory mediators would be expected to be relatively low, rosiglitazone treatment was associated with almost complete repression of several genes involved in the inflammatory response.

Genomic Approaches to Complex Pathways Sequence-based approaches can be used to gain insight into possible mechanisms by which PPARJ regulates the target genes identified in this study. To do this, we compiled a list of upstream sequences (5 kb) from 142 of the differentially expressed genes. Using a sensitive model to search for PPAR response elements (PPREs), we were unable to detect PPREs in about

PG-00027-2004 Final Accepted Version 30% of the sequences examined.

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The fact that not all of the regions examined contained a

binding sequence for PPARJ might suggest either that the response element is located outside of the sequence analyzed or that these genes are indirectly regulated by PPARȖ. The presence of 6 transcription factors among the differentially expressed genes provides support for the idea that PPARJ regulates target gene transcription via indirect (activating/inhibiting secondary transcription factors) and direct (binding to PPREs) mechanisms.

Comparative sequence analysis has been used to prioritize the genomic region in which to search for important regulatory sequences. Those locations in the genome most highly conserved between different species are likely to be enriched in biologically important sequences (28). Using this approach, previously unknown regulatory elements for the stem cell leukemia gene and for several genes important in the inflammatory response were discovered (9, 15). From the upstream sequences examined in this study, 31 sequence fragments were found to be highly conserved between human and mouse, and 2 of these contained a sequence closely resembling a PPRE. Of course, this does not rule out the functional importance of the other 99 PPRE sequences.

The two conserved PPREs are located upstream of basic helix-loop-helix domain containing protein, class B2 (BHLHB2) and frizzled homolog 4 (FZD4). There are no published reports of a linkage between FZD4 and PPARJ, although several recent studies in human patients have provided evidence that lack of FZD4, a member of the Wnt-signaling pathway, impairs normal retinal vascularization (23).

BHLHB2, a transcription factor, appears to be an important

downstream mediator of hypoxia-induced changes in gene expression, and inhibits expression of

PG-00027-2004 Final Accepted Version the PPARJ gene in adipocytes (30).

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significant decreases in expression of hypoxia inducible factor 1 (-56 %) and in BHLHB2 (-84 %). There was a tendency for decreased expression of PPARJ (-29 %) that was not statistically significant. These findings suggest the possibility that a negative feedback relationship between PPARJ and hypoxia-induced genes may exist in the vasculature, although future studies are required to test this hypothesis.

PPARJ, Blood Vessel Wall, and Hypertension In several disease conditions including atherosclerosis and hypertension, relatively large genomic regions associated with a particular disease trait (QTL regions) have been defined. However, because these chromosomal regions are large, elucidation of the exact genetic variations responsible for these associations has been difficult.

In a recent report, microarray

data and computational methods were combined to prioritize the list of candidate genes for human cytochrome c oxidase deficiency and the causative gene was discovered (19). Given the established importance of the PPARJ pathway in vascular function and blood pressure, the 14 differentially expressed genes from the present study that map to rat blood pressure QTL regions should be considered as potential candidate genes for further study.

Thus, chronic administration of a PPARJ agonist elicits changes in numerous genetic pathways in mouse aorta.

By combining large-scale gene expression analysis with oligonucleotide

microarrays and bioinformatic approaches, we were able to identify, in a non-biased manner, 181 differentially expressed genes, 14 of which map to rat blood pressure QTL regions. Further studies are required to determine the functional consequence of over/under expression of these


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genes, to evaluate the importance of genomic DNA sequences upstream of these genes predicted to contain important regulatory domains, and to dissect the contribution of the various vascular cell types to the integrated response.


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ACKNOWLEDGMENTS We thank Deborah R. Davis for assistance with the mice used in this study. This work was supported by National Institutes of Health grants HL48058, HL61446, HL55006 (to CDS) and The Center for Bioinformatics and Computational Biology at the University of Iowa. We gratefully acknowledge the generous research support of the Roy J. Carver Trust.


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FIGURE LEGENDS Figure 1. Basic schema of the experimental and informatics methods used in the present study to examine the PPARJ pathway in mouse aorta.

Figure 2. Correlation between expression differentials (rosiglitazone signal relative to vehicle control) as measured by microarray analysis (x-axis) or by quantitative real-time RT-PCR (yaxis). Red and blue circles represent up- and downregulated genes, respectively.

Figure 3. Functional groups with the most significant changes in expression during rosiglitazone administration sorted by expression cluster.

Cluster 0 contains 29 transcripts of moderate

abundance that were decreased (-93%) to very low levels, Cluster 1 contains 106 transcripts of high abundance that were downregulated (-42%), and Cluster 2 contains 46 transcripts of high abundance that were upregulated (+70%).

Figure 4. Percent identity plots showing potential PPAR response elements (PPREs) upstream of two genes differentially expressed during rosiglitazone administration. Both of these potential PPREs are located in regions of highly conserved non-coding sequence (indicated by the red color. Panel A: Frizzled homolog 4 (FZD4). Panel B: Basic helix-loop-helix containing protein, class B2 (BHLHB2).

Table 1: Correlation Between Microarray and Real Time RT-PCR Probe Set 93100_at 96573_at 161359_s_at 95356_at 101859_at 99642_i_at 98543_at 98088_at 160358_at 94939_at 101160_at 95286_at 96020_at 103353_f_at 96912_s_at 94492_at 104371_at 92836_at 94214_at 93459_s_at 101676_at 99598_g_at 93277_at 98629_f_at 98773_s_at 102658_at 99491_at 93753_at 94278_at 99957_at 102668_at 160481_at 104538_at 101030_at 103887_at 94259_at 160547_s_at 160532_at 103001_at

Accession X13297 M21495 AV212934 D00466 AB010100 X61232 AJ223208 X13333 AI847784 X97227 X53798 D14077 M22531 D50834 X15591 AB025406 AF078752 AA919594 X14961 AW122897 U13705 AI841629 X53584 Y09085 AI323667 X59769 U53696 AI852632 D37837 X72795 X57638 AF009605 AB001607 X99963 M83219 AB024935 AI839138 M22479 U43836

Description actin, alpha 2, smooth muscle, aorta actin, gamma, cytoplasmic apolipoprotein A-I binding protein apolipoprotein e aquaporin 7 carboxypeptidase E cathepsin S CD14 antigen CD34 antigen CD53 antigen chemokine (C-X-C motif) ligand 2 clusterin complement component 1, q subcomponent, ȕ polypeptide cytochrome P450, subfamily IV B, polypeptide 1 cytotoxic T lymphocyte-associated protein 2 alpha destrin diacylglycerol O-acyltransferase 1 elastin fatty acid binding protein 3, muscle and heart frizzled homolog 4 (Drosophila) glutathione peroxidase 3 guanine nucleotide binding protein, alpha inhibiting 2 heat shock protein, 60 kDa hypoxia inducible factor 1 alpha subunit immunoresponsive gene 1 interleukin 1 receptor, type II interleukin 10 receptor, beta LPS-induced TN factor lymphocyte cytosolic protein 1 matrix metalloproteinase 9 peroxisome proliferator activated receptor alpha phosphoenolpyruvate carboxykinase (pepck) gene co prostaglandin I2 (prostacyclin) synthase rhob gene S100 calcium binding protein A9 (calgranulin B) telomerase binding protein, p23 thioredoxin interacting protein tropomyosin 1, alpha vascular endothelial growth factor B

Array 0.45 0.55 1.82 0.71 2.80 0.42 0.47 0.13 0.37 0.26 0.01 0.48 0.58 1.87 0.42 0.44 1.80 0.60 2.90 1.46 0.67 0.56 1.36 0.45 0.05 0.23 0.55 0.56 0.42 0.12 1.89 1.56 0.44 0.54 0.02 0.53 2.07 0.50 1.48

RT-PCR 0.51 0.89 1.84 1.20 5.59 0.90 0.77 0.06 0.85 0.34 0.00 0.54 0.90 3.14 0.68 0.63 1.39 0.70 4.85 1.32 1.11 0.91 1.98 0.84 0.00 1.21 0.00 0.56 0.29 0.27 2.74 2.23 0.65 1.07 0.01 1.07 2.59 0.78 1.95

Table 2: Functional Groups Identified by Cluster Analysis Probe Set

Accession

Description

Change

actin binding activity (GO:0003779) 93499_at 93730_at 95142_s_at 160532_at 94492_at 160150_f_at 94278_at 97384_at 97990_at

U16740 AF085809 U10407 M22479 AB025406 AW125626 D37837 AA791012 D85923

capping protein alpha 1 synapsin I capping protein beta 1 tropomyosin 1, alpha destrin calponin 3, acidic lymphocyte cytosolic protein 1 glia maturation factor, gamma myosin heavy chain 11, smooth muscle

0.80 0.71 0.57 0.50 0.45 0.43 0.42 0.36 0.34

calcium ion binding activity (GO:0005509) 99536_at 98600_at 103721_at 92770_at 94278_at 101393_at 93281_at 93866_s_at 161703_f_at 103887_at 99536_at 98600_at

AB016080 U41341 AA592182 X66449 D37837 AJ001633 AF049125 D00613 AV003419 M83219 AB016080 U41341

kinase interacting protein 2 S100 calcium binding protein A11 (calizzarin) nephronectin S100 calcium binding protein A6 (calcyclin) lymphocyte cytosolic protein 1 annexin A3 reticulocalbin 2 matrix gamma-carboxyglutamate (gla) protein annexin A1 S100 calcium binding protein A9 (calgranulin B) kinase interacting protein 2 S100 calcium binding protein A11 (calizzarin)

1.76 0.66 0.49 0.48 0.42 0.40 0.40 0.40 0.38 0.02 1.76 0.66

cytokine activity (GO:0005125) 98600_at 104388_at 95348_at 102424_at 97519_at 101160_at 103486_at

U41341 U49513 J04596 J04491 X13986 X53798 M15131

S100 calcium binding protein A11 (calizzarin) chemokine (C-C motif) ligand 9 chemokine (C-X-C motif) ligand 1 chemokine (C-C motif) ligand 3 secreted phosphoprotein 1 chemokine (C-X-C motif) ligand 2 interleukin 1 beta

0.66 0.38 0.25 0.06 0.02 0.01 0.01

cytoskeleton (GO:0005200) 93499_at 101578_f_at 95142_s_at 96573_at 160532_at 93100_at

U16740 M12481 U10407 M21495 M22479 X13297

capping protein alpha 1 actin, beta, cytoplasmic capping protein beta 1 actin, gamma, cytoplasmic tropomyosin 1, alpha actin, alpha 2, smooth muscle, aorta

0.80 0.58 0.57 0.55 0.50 0.45

immune response (GO:0006955) 101054_at

X00496

Ia-associated invariant chain

0.61

104388_at 95348_at 98088_at 102424_at 101160_at 103486_at

U49513 J04596 X13333 J04491 X53798 M15131

chemokine (C-C motif) ligand 9 chemokine (C-X-C motif) ligand 1 CD14 antigen chemokine (C-C motif) ligand 3 chemokine (C-X-C motif) ligand 2 interleukin 1 beta

0.38 0.25 0.13 0.06 0.01 0.01

inflammatory response (GO:0006954) 95348_at 98988_at 98088_at 102424_at 101160_at 103486_at

J04596 AA614971 X13333 J04491 X53798 M15131

chemokine (C-X-C motif) ligand 1 molecule possessing ankyrin-repeats induced by lipopolysaccharide CD14 antigen chemokine (C-C motif) ligand 3 chemokine (C-X-C motif) ligand 2 interleukin 1 beta

0.25 0.20 0.13 0.06 0.01 0.01

oxidoreductase activity (GO:0016491) 93844_at 103353_f_at 95693_at 101525_at 95053_s_at 96899_at 101676_at 100068_at 97013_f_at

AW061302 D50834 U51167 AI848871 AA674669 AW123802 U13705 M74570 AW046124

RIKEN cDNA 1100001F06 gene cytochrome P450, subfamily IV B, polypeptide 1 isocitrate dehydrogenase 2 (NADP+), mitochondrial RIKEN cDNA 0610011B04 gene RIKEN cDNA 0710008N11 gene NADH dehydrogenase (ubiquinone) Fe-S protein 3 glutathione peroxidase 3 aldehyde dehydrogenase family 1, subfamily A1 cytochrome b-245, alpha polypeptide

1.94 1.88 1.54 1.47 1.46 1.38 0.67 0.61 0.51

Table 3: Conserved Sequences Between Human and Mouse in 5’ Flanking Region Of Differentially Expressed Genes Accession X13297 M12481 U19118 D00466 Y07836 Y07836 D14077 AW047343 AW047343 V00727 V00727 V00727 AW122897 AW122897 U57524 U57524 U69543 M69260 AW123802 AW125185 Z38110 X52046 X58251 X58251 X58251 X99963 AI853864 M83219 U88567 AI840996 M22479

Description actin, alpha 2, smooth muscle, aorta actin, beta, cytoplasmic activating transcription factor 3 apolipoprotein e basic helix-loop-helix domain containing, class B2 basic helix-loop-helix domain containing, class B2 clusterin D site albumin promoter binding protein D site albumin promoter binding protein fbj osteosarcoma oncogene fbj osteosarcoma oncogene fbj osteosarcoma oncogene frizzled homolog 4 (Drosophila) frizzled homolog 4 (Drosophila) i kappa b alpha gene exons 2-6 partial cds i kappa b alpha gene exons 2-6 partial cds lipase hormone sensitive lipocortin 1 NADH dehydrogenase (ubiquinone) Fe-S protein 3 open reading frame 18 peripheral myelin protein, 22 kDa procollagen type iii alpha 1 procollagen, type I, alpha 2 procollagen, type I, alpha 2 procollagen, type I, alpha 2 rhob gene RIKEN cDNA 2010100O12 gene S100 calcium binding protein A9 (calgranulin B) secreted frizzled-related sequence protein 2 selenoprotein R tropomyosin 1, alpha

Location 333-1 135-7 315-155 3901-3791 1833-1676 999-834 121-3 1955-1791 1700-1526 528-412 2086-1923 1894-1657 3572-3296 899-693 499-135 129-13 4850-4744 147-20 1595-844 1155-983 2686-2541 275-111 247-0 2598-2486 465-316 563-362 4503-4384 202-63 187-5 964-833 285-62

Start Sequence GCTGGCATCTTC TGCTGCACTGTG CAACCTAGCGGA CCAGCTGCAGGT CAGCCAGGTCAC GGCCACGTGAAG CCCCCACCTCTA CGCAGATGATGC GCAGATGCACTC CTGCACCCTCAG GCCTAAATTCCC TCAGCCCCCCGA TAATAATGAATC TTGTGCGCCTTC AAGCGAATCCCT GAGGACGAGCCA TTTATTTGTGCC GAGTAGTTTTGC CTTACCTGAGAT CAGTCCTGTGCA CCTGCAAGGCCT CTCCAGATGTGC CCCGGGCCCCTA CCACCAAGTGCT GTGTCCTAAAGT GCCTCTCCCAGC TCCCTCCTCCTA AACCAGTTTCCC GGGGTGGGGGCG CCCAGTCCCTTC TCCCAGGCGTGC

PPRE N N N N 1739 N N N N N N N N 878 N N N N N N N N N N N N N N N N N

Table 4: Differentially Regulated Transcription Factors Probe Set 102668_at 104155_f_at 98628_f_at 160841_at 98988_at 104701_at

Accession X57638 U19118 AF003695 AW047343 AA614971 Y07836

Description peroxisome proliferator activated receptor alpha activating transcription factor 3 hypoxia inducible factor 1, alpha subunit D site albumin promoter binding protein molecule possessing ankyrin-repeats induced by LPS basic helix-loop-helix domain containing, class B2

Change 1.90 0.46 0.44 0.32 0.20 0.16

Table 5: Differentially Expressed Genes in Blood Pressure QTLs Probe Set 161689_f_at 102658_at 98451_at 93277_at 100566_at 95477_at 103353_f_at 100437_g_at 95787_s_at 160108_at 102395_at 94469_at 92471_i_at 102424_at 101054_at

Accession AV223216 X59769 AI843164 X53584 L12447 AW125185 D50834 M27008 X87685 AI852641 Z38110 AW120950 AF099973 J04491 X00496

Description interleukin 1 receptor, type II interleukin 1 receptor, type II DnaJ (Hsp40) homolog, subfamily B, member 10 heat shock protein, 60 kDa insulin-like growth factor binding protein 5 open reading frame 18 cytochrome P450, subfamily IV B, polypeptide 1 orosomucoid 1 sterol carrier protein 2-pseudogene nuclear protein 1 peripheral myelin protein, 22 kDa expressed sequence AI182287 schlafen 2 chemokine (C-C motif) ligand 3 Ia-associated invariant chain

Mm Chr 1 1 1 1 1 4 4 4 4 7 11 11 11 11 18

Rn Chr 9 9 9 9 9 5 5 5 5 1 10 10 10 10 18

Change 0.13 0.24 0.58 1.36 0.42 0.63 1.88 0.85 1.55 0.65 0.63 0.63 0.28 0.06 0.61

Table 6: Differentially Expressed Genes in Endothelial SAGE Library Probe Set 160547_s_at 95690_at 104164_at 160558_at 93780_at 160904_at 97422_at 95736_at 95693_at 96348_at 103001_at 95053_s_at 97880_at 96899_at 104598_at 98600_at 93593_f_at 94469_at 96657_at 95542_at 97013_f_at 96146_at 100088_at 99642_i_at 94061_at 100566_at 93281_at 160358_at 97384_at 102779_at 161666_f_at 97519_at

Accession AI839138 AW047363 AI181257 U22445 AW060827 AI841484 AW048882 AI847546 U51167 AW121217 U43836 AA674669 AW061024 AW123802 X61940 U41341 U87948 AW120950 L10244 AI835858 AW046124 D83745 M27073 X61232 M13018 L12447 AF049125 AI847784 AA791012 X54149 AV138783 X13986

Description thioredoxin interacting protein RIKEN cDNA 1110030L07 gene RIKEN cDNA 1300019N10 gene thymoma viral proto-oncogene 2 RIKEN cDNA 0610006O17 gene hypothetical protein, clone 1-82 Mus musculus, clone IMAGE:3982770, mRNA, partial cds mitochondrial ribosomal protein L4 isocitrate dehydrogenase 2 (NADP+), mitochondrial RIKEN cDNA 0610039C21 gene vascular endothelial growth factor B RIKEN cDNA 0710008N11 gene RIKEN cDNA 4930529O08 gene NADH dehydrogenase (ubiquinone) Fe-S protein 3 dual specificity phosphatase 1 S100 calcium binding protein A11 (calizzarin) epithelial membrane protein 3 expressed sequence AI182287 spermidine/spermine N1-acetyl transferase ESTs, Moderately similar to S11390 tropomyosin 5 - mouse cytochrome b-245, alpha polypeptide B-cell translocation gene 3 protein phosphatase 1, catalytic subunit, beta isoform carboxypeptidase E cysteine rich intestinal protein insulin-like growth factor binding protein 5 reticulocalbin 2 CD34 antigen glia maturation factor, gamma growth arrest and DNA-damage-inducible 45 beta growth arrest and DNA-damage-inducible 45 beta secreted phosphoprotein 1

Mm Unigene Mm.77432 Mm.29651 Mm.27139 Mm.177194 Mm.2125 Mm.5166 Mm.29999 Mm.155033 Mm.2966 Mm.29998 Mm.15607 Mm.29141 Mm.28365 Mm.30113 Mm.2404 Mm.175848 Mm.20829 Mm.28848 Mm.2734 Mm.27685 Mm.448 Mm.2823 Mm.4572 Mm.31395 Mm.22049 Mm.578 Mm.1782 Mm.29798 Mm.29766 Mm.1360 Mm.1360 Mm.321

Hs Unigene Hs.179526 Hs.4877 Hs.15386 Hs.326445 Hs.9676 Hs.351871 Hs.197289 Hs.279652 Hs.429 Hs.118463 Hs.78781 Hs.64 Hs.296348 Hs.429506 Hs.171695 Hs.417004 Hs.9999 Hs.54642 Hs.28491 Hs.250641 Hs.68877 Hs.77311 Hs.21537 Hs.75360 Hs.423190 Hs.380833 Hs.79088 Hs.374990 Hs.5210 Hs.110571 Hs.110571 Hs.313

Change 2.07 2.03 1.92 1.81 1.80 1.79 1.74 1.68 1.54 1.52 1.49 1.46 1.40 1.38 0.66 0.66 0.64 0.63 0.62 0.57 0.51 0.46 0.43 0.42 0.42 0.42 0.40 0.37 0.36 0.26 0.05 0.02

Supplemental Table 1: Sequences of 19 known PPREs Used to Create Probabilistic-based Model to Search for RRPEs. Description Acyl-CoA oxidase Acyl-CoA oxidase Acyl-CoA synthase Adipocyte lipid binding protein (ALBP/aP2) Adipocyte lipid binding protein (ALBP/aP2) Apolipoprotein CIII Bifunctional enzyme c-Cbl-associating protein Cytochrome P450 A1 Cytochrome P450 A6 Fatty Acid Binding Protein (L-FABP) Fatty acid transport protein HMG-CoA synthase Lipoprotein lipase Malic enzyme Muscle-type carnitine palmitoyltransferase Phosphoenolpyruvate carboxykinase (PEPCK) Phosphoenolpyruvate carboxykinase (PEPCK) Uncoupling protein I

Species Rat Rat Rat Mouse Mouse Human Rat Mouse Rat Rabbit Mouse Rat Rat Rat Rat Human Rat Rat Mouse

Sequence AGGTACAAGGTCA AGGACAAAGGTCA AGGGCATCAGTCA GGATCAGAGTTCA GGGTGAAATGTGC TGGGCAAAGGTCA AGGTCCTAGTTCA AGGCTAAAGGTCA AGGGTAAAGTTCA AGGGCAAAGTTGA GGGGCAAAGGGCA GGGCCAAAGGTCT AGGCCATAGGTCA GGGGGAAAGGGCA GGGTCAAAGTTGA AGGGAAAAGGTCA CGGCCAAAGGTCA GGGTGAAATGTGC TGGTCAAGGGTGA

Pubmed 1537328, 2025260 1537328, 2025260 7642600 7926726 7926726 8274443 1502166 10734046 7887901 1326542 9933587 7913466 1487072 8895578 7929410 9535828 7799943 7799943 8668156

Supplemental Table 2: Genes Up-Regulated by Rosiglitazone Description solute carrier family 25, member 5 fatty acid binding protein 3, muscle and heart adp-ribosylation-like 4 aquaporin 7 cysteine sulfinic acid decarboxylase hypothetical protein MGC29978 sorbin and SH3 domain containing 1 lipase hormone sensitive RIKEN cDNA 2310016A09 gene thioredoxin interacting protein amyotrophic lateral sclerosis 2 (juvenile) homolog (human) RIKEN cDNA 1110030L07 gene RIKEN cDNA 6530401D17 gene RIKEN cDNA 1100001F06 gene plasma membrane associated protein, S3-12 RIKEN cDNA 1300019N10 gene peroxisome proliferator activated receptor alpha cytochrome P450, subfamily IV B, polypeptide 1 apolipoprotein A-I binding protein thymoma viral proto-oncogene 2 RIKEN cDNA 0610006O17 gene diacylglycerol O-acyltransferase 1 hypothetical protein, clone 1-82 lysosomal apyrase-like 1 kinase interacting protein 2 Mus musculus, clone IMAGE:3982770, mRNA, partial cds keratinocyte lipid binding protein mitochondrial ribosomal protein L4 ubiquitin C, related sequence 2 cytochrome c oxidase, subunit XVII assembly protein homolog (yeast) phosphoenolpyruvate carboxykinase (pepck) gene co sterol carrier protein 2-pseudogene isocitrate dehydrogenase 2 (NADP+), mitochondrial RIKEN cDNA 0610039C21 gene carboxylesterase 3 vascular endothelial growth factor B RIKEN cDNA 0610011B04 gene similar to peroxisomal biogenesis factor 6 RIKEN cDNA 0710008N11 gene

Change 7.83 2.91 2.90 2.81 2.30 2.19 2.18 2.17 2.07 2.07 2.04 2.03 2.00 1.94 1.93 1.92 1.90 1.88 1.83 1.81 1.80 1.80 1.79 1.77 1.76 1.74 1.73 1.68 1.67 1.62 1.56 1.55 1.54 1.52 1.50 1.49 1.47 1.46 1.46

Rosi Avg 810 14167 11635 4425 14310 29909 18990 25797 6062 10646 107972 12622 23873 208661 6108 6981 19104 9551 30900 15540 12831 8206 4191 27806 9135 5822 6646 9991 3817 45463 16998 6451 18313 141957 75951 12808 17190 3592 66211

Vehicle Avg 103 4876 4019 1577 6234 13687 8716 11863 2934 5133 52888 6215 11919 107314 3161 3641 10080 5086 16897 8584 7110 4558 2345 15685 5181 3355 3832 5965 2289 28039 10864 4161 11875 93225 50657 8606 11709 2459 45260

Unigene Mm.658 Mm.22220 Mm.8728 Mm.41853 Mm.224885 Mm.29030 Mm.28108 Mm.77432 Mm.30114 Mm.29651 Mm.27579 Mm.21625 Mm.12966 Mm.27139 Mm.1373 Mm.1840 Mm.205996 Mm.177194 Mm.2125 Mm.22633 Mm.5166 Mm.20806 Mm.42192 Mm.29999 Mm.155033 Mm.196580 Mm.27396

Mm.2966 Mm.29998 Mm.120807 Mm.15607 Mm.8688 Mm.41268 Mm.29141

frizzled homolog 4 (Drosophila) apolipoprotein A-I binding protein RIKEN cDNA 4930529O08 gene NADH dehydrogenase (ubiquinone) Fe-S protein 3 RIKEN cDNA 2010100O12 gene heat shock protein, 60 kDa RIKEN cDNA 2010003O02 gene

1.46 1.45 1.40 1.38 1.36 1.36 1.30

25192 12818 8957 32000 57242 40133 29445

17206 8846 6387 23260 41980 29486 22584

Mm.68712 Mm.205996 Mm.28365 Mm.30113 Mm.27477 Mm.1777 Mm.1103

Supplemental Table 3: Genes Down-Regulated by Rosiglitazone Description serum amyloid a 3 interleukin 1 beta chemokine (C-X-C motif) ligand 2 24p3 gene secreted phosphoprotein 1 S100 calcium binding protein A9 (calgranulin B) arginase 1, liver immunoresponsive gene 1 growth arrest and DNA-damage-inducible 45 beta chemokine (C-C motif) ligand 3 arginase type II germline interleukin 1 receptor antagonist (il-1rn C-type lectin, superfamily member 8 matrix metalloproteinase 9 secretory leukoprotease inhibitor gene complete c CD14 antigen interleukin 1 receptor, type II basic helix-loop-helix domain containing, class B2 molecule possessing ankyrin-repeats induced by lipopolysaccharide serine (or cysteine) proteinase inhibitor, clade A, member 3N P lysozyme structural cysteine rich protein 1 osteoglycin interleukin 1 receptor, type II chemokine (C-X-C motif) ligand 1 growth arrest and DNA-damage-inducible 45 beta CD53 antigen procollagen, type III, alpha 1 schlafen 2 pleckstrin homology, Sec7 and coiled/coil domains, binding protein major urinary protein group 1 gene 5 prime end c D site albumin promoter binding protein proteoglycan, secretory granule myosin heavy chain 11, smooth muscle serine (or cysteine) proteinase inhibitor, clade B, member 6 histidine decarboxylase glia maturation factor, gamma p75 tnf receptor dna CD34 antigen

Change 0.00 0.01 0.01 0.02 0.02 0.02 0.04 0.05 0.05 0.06 0.07 0.09 0.12 0.12 0.13 0.13 0.13 0.16 0.20 0.20 0.23 0.23 0.23 0.24 0.25 0.26 0.27 0.28 0.28 0.31 0.32 0.32 0.34 0.34 0.35 0.35 0.36 0.37 0.37

Rosi Avg 90 110 90 101 88 1068 97 274 180 427 53 526 255 144 408 3569 391 253 225 851 719 526 1178 581 142 433 550 496 611 1066 686 786 755 1624 2482 1171 679 1015 1161

Vehicle Avg 19455 16650 14184 4518 3603 50448 2619 5056 3909 7354 760 5704 2156 1161 3255 27179 3060 1623 1106 4181 3159 2324 5082 2457 573 1661 2052 1777 2193 3408 2152 2442 2195 4737 7057 3322 1890 2767 3106

Unigene Mm.222830 Mm.4979 Mm.321 Mm.2128 Mm.154144 Mm.4662 Mm.1360 Mm.1282 Mm.3506 Mm.24920 Mm.4406 Mm.3460 Mm.1349 Mm.2436 Mm.3732 Mm.22650 Mm.177539 Mm.196484 Mm.4258 Mm.1349 Mm.21013 Mm.1360 Mm.2692 Mm.147387 Mm.42124 Mm.123225 Mm.3459 Mm.22194 Mm.3153 Mm.2623 Mm.18603 Mm.29766 Mm.29798

fbj osteosarcoma oncogene annexin A1 chemokine (C-C motif) ligand 9 formyl peptide receptor 1 reticulocalbin 2 testis derived transcript annexin A3 matrix gamma-carboxyglutamate (gla) protein expressed sequence C79715 cysteine rich intestinal protein insulin-like growth factor binding protein 5 carboxypeptidase E lymphocyte cytosolic protein 1 FXYD domain-containing ion transport regulator 5 cytotoxic T lymphocyte-associated protein 2 alpha calponin 3, acidic protein phosphatase 1, catalytic subunit, beta isoform lipocortin 1 myocyte enhancer factor 2A hypoxia inducible factor 1, alpha subunit hypoxia inducible factor 1 alpha subunit phosphatase and tensin homolog actin, alpha 2, smooth muscle, aorta destrin prostaglandin I2 (prostacyclin) synthase fibrinogen-like protein 2 retinol binding protein 1, cellular activating transcription factor 3 B-cell translocation gene 3 i kappa b alpha gene exons 2-6 partial cds S100 calcium binding protein A6 (calcyclin) clusterin cathepsin S retinoic acid-inducible e3 protein mrna complete nephronectin tropomyosin 1, alpha cytochrome b-245, alpha polypeptide 2,3-bisphosphoglycerate mutase paraoxonase 1 procollagen, type I, alpha 2 regulator of G-protein signaling 2 expressed sequence AA409223 rhob gene

0.38 0.38 0.38 0.39 0.40 0.40 0.40 0.40 0.42 0.42 0.42 0.42 0.42 0.42 0.42 0.43 0.43 0.44 0.44 0.44 0.45 0.45 0.45 0.45 0.45 0.45 0.46 0.46 0.46 0.47 0.48 0.48 0.48 0.48 0.49 0.50 0.51 0.52 0.52 0.52 0.53 0.53 0.54

400 2887 2362 711 552 507 1331 6486 2329 5478 8061 3185 2169 1104 3555 2460 2935 5368 1585 3556 2684 1430 30948 31906 2993 412 3858 1046 1080 5914 12484 4527 3608 3455 1152 4486 1965 5461 3722 6364 2966 3209 3160

1061 7508 6180 1826 1373 1263 3289 16243 5574 12919 19406 7514 5105 2626 8431 5771 6857 12081 3626 8026 5937 3182 68339 70920 6716 913 8456 2282 2341 12558 25924 9421 7554 7157 2366 8886 3884 10472 7127 12304 5601 6094 5809

Mm.14860 Mm.2271 Mm.1782 Mm.88645 Mm.7214 Mm.193459 Mm.119265 Mm.22049 Mm.578 Mm.31395 Mm.153911 Mm.1870 Mm.30144 Mm.22171 Mm.4572 Mm.87279 Mm.3879 Mm.3879 Mm.1591 Mm.16537 Mm.28919 Mm.2339 Mm.2450 Mm.2706 Mm.2823 Mm.100144 Mm.196344 Mm.3619 Mm.205021 Mm.121878 Mm.448 Mm.197824 Mm.30107 Mm.4482 Mm.28262 Mm.200776

telomerase binding protein, p23 biglycan CD9 antigen enabled homolog (Drosophila) interleukin 10 receptor, beta actin, gamma, cytoplasmic secreted frizzled-related sequence protein 2 proline rich protein expressed in brain myosin, light polypeptide kinase LPS-induced TN factor capping protein beta 1 matrin 3 ESTs, Moderately similar to S11390 tropomyosin 5 - mouse [M.musculus] guanine nucleotide binding protein, alpha inhibiting 2 DnaJ (Hsp40) homolog, subfamily B, member 10 actin, beta, cytoplasmic complement component 1, q subcomponent, beta polypeptide stearoyl-coenzyme a desaturase 1 Mus musculus, clone IMAGE:5037053, mRNA, partial cds RIKEN cDNA 5031422I09 gene procollagen type iii alpha 1 lysozyme m gene aminolevulinic acid synthase 2, erythroid aldehyde dehydrogenase family 1, subfamily A1 interleukin 11 receptor, alpha chain 2 Ia-associated invariant chain elastin spermidine/spermine N1-acetyl transferase open reading frame 18 peripheral myelin protein, 22 kDa expressed sequence AI182287 ADP-ribosylation-like factor 6 interacting protein 2 epithelial membrane protein 3 dna for type iib intracisternal a-particle element dynein, cytoplasmic, light chain 1 BCL2/adenovirus E1B 19 kDa-interacting protein 1, NIP2 nuclear protein 1 amyloid precursor protein (app) gene partial cds dual specificity phosphatase 1 stearoyl-coenzyme a desaturase 1 S100 calcium binding protein A11 (calizzarin) hypothetical protein AF506821 glutathione peroxidase 3

0.54 0.55 0.55 0.55 0.55 0.55 0.55 0.56 0.56 0.56 0.57 0.57 0.57 0.57 0.58 0.58 0.59 0.59 0.60 0.60 0.60 0.60 0.61 0.61 0.61 0.61 0.61 0.62 0.63 0.63 0.63 0.63 0.64 0.64 0.64 0.65 0.65 0.65 0.66 0.66 0.66 0.67 0.67

1620 18154 2521 709 2800 20417 1956 3560 4680 1335 2044 8003 5620 11818 1116 47964 3457 159778 2520 2201 3343 3044 13869 1344 1627 4404 14287 3454 1612 1351 2961 1624 10095 3058 19548 2801 5322 15379 4077 186444 7983 819 37902

3025 33146 4546 1287 5061 37048 3582 6372 8410 2373 3584 14140 9794 20857 1909 82259 5884 271681 4226 3666 5568 5100 22665 2196 2686 7195 23562 5591 2576 2159 4666 2593 15681 4809 30760 4316 8169 23640 6173 280404 12024 1224 56415

Mm.22421 Mm.2608 Mm.2956 Mm.87759 Mm.4154 Mm.196173 Mm.19155 Mm.28797 Mm.27680 Mm.21119 Mm.2945 Mm.25575 Mm.27685 Mm.196464 Mm.103605 Mm.297 Mm.2570 Mm.10160 Mm.153748

Mm.140509 Mm.4514 Mm.193451 Mm.7043 Mm.111845 Mm.2734 Mm.220858 Mm.1237 Mm.28848 Mm.24719 Mm.20829 Mm.29908 Mm.1561 Mm.18742 Mm.2404 Mm.175848 Mm.211477 Mm.200916

LIM and senescent cell antigen-like domains 1-like thioredoxin 1 selenoprotein R synapsin I chloride channel 3 apolipoprotein e serine (or cysteine) proteinase inhibitor, clade G, member 1 RIKEN cDNA 1300019P08 gene capping protein alpha 1 orosomucoid 1

0.67 0.68 0.70 0.71 0.71 0.71 0.72 0.74 0.80 0.85

1758 31398 8592 4953 2163 77700 11330 11357 3661 10340

2633 45997 12347 6983 3040 108872 15675 15312 4561 12231

Mm.29097 Mm.1275 Mm.28212 Mm.196611 Mm.28842 Mm.38888 Mm.229291 Mm.19142 Mm.4777

Supplemental Table 4: Sequence and Location of PPRE in Differentially Regulated Genes Accession X13586 X81627 X13297 M12481 Y12577 M15268 AW125480 AJ001633 AV212934 D00466 AB010100 U51805 D83745 Y07836 U16740 U10407 AW226939 AJ223208 X13333 X97227 L08115 J04491 X53798 D14077 M22531 M13018 D88793 AW120896 D50834 AB025406 X61940 AF020185 U87948 AI835858 AI845935 AW120950 X14961 V00727 L22181 AW122897 U72680 L32838 X54149 X53584 X57437 AW012588 AI841484 Y09085 AI323667 L12447 X59769 U53696

Description 2,3-bisphosphoglycerate mutase 24p3 gene actin, alpha 2, smooth muscle, aorta actin, beta, cytoplasmic adp-ribosylation-like 4 aminolevulinic acid synthase 2, erythroid amyotrophic lateral sclerosis 2 (juvenile) homolog annexin A3 apolipoprotein A-I binding protein apolipoprotein e aquaporin 7 arginase 1, liver B-cell translocation gene 3 basic helix-loop-helix domain containing, class B2 capping protein alpha 1 capping protein beta 1 carboxylesterase 3 cathepsin S CD14 antigen CD53 antigen CD9 antigen chemokine (C-C motif) ligand 3 chemokine (C-X-C motif) ligand 2 clusterin complement component 1, q subcomponent, beta polyp cysteine rich intestinal protein cysteine rich protein 1 cysteine sulfinic acid decarboxylase cytochrome P450, subfamily IV B, polypeptide 1 destrin dual specificity phosphatase 1 dynein, cytoplasmic, light chain 1 epithelial membrane protein 3 ESTs, Moderately similar to S11390 tropomyosin 5 expressed sequence AA409223 expressed sequence AI182287 fatty acid binding protein 3, muscle and heart fbj osteosarcoma oncogene formyl peptide receptor 1 frizzled homolog 4 (Drosophila) FXYD domain-containing ion transport regulator 5 germline interleukin 1 receptor antagonist (il-1rn growth arrest and DNA-damage-inducible 45 beta heat shock protein, 60 kDa histidine decarboxylase hypothetical protein MGC29978 hypothetical protein, clone 1-82 hypoxia inducible factor 1 alpha subunit immunoresponsive gene 1 insulin-like growth factor binding protein 5 interleukin 1 receptor, type II interleukin 10 receptor, beta

Location 4452 1959 4830 2615 4339 3432 1162 3517 868 1923 1930 1034 4787 1739 3200 2549 4564 1479 420 1718 2885 4421 4032 689 1590 3731 2554 1764 551 2165 4094 1415 4598 3752 3620 1103 867 4078 754 878 3632 659 877 1143 1170 4711 2953 2829 1179 3296 2713 1482

Sequence GGTTCAAAGGTCA AAGTCAAAGGTCT TGGGCAAAAGTCA AGGGCAAAGTTCT AGGGCAGAGGTCT AAGTTATAGGTCA TGGCCAAAGGTTA ATGGCAAATGTCA AGGGCAGAGGTCC AGGTCAGAGGTCT GGGGCAACTGTCA AGGTCAGAGATCA AGGGCAGAGGTGT AGCGCAGAGGTCA GGTCCAAAGGTCA AGGGTAGAGGGCA AGGTCAGATGTCA AGGTTAGATGTCA GGCGCATAGGTCA AGGAGAAAGTGCA GGGTCAGAGGTTA AGGTCCAAGGTGA AGGTCCAGGGTCA GGGTCCTAGGTCC AGGTCAAGGCTCA AGGACATAGGGCA AGGGGAAAGGCCA GGGTTAAAGGTGA TGGGTATAGTTCA GGGTTAAAGTTCG AGGGCCTGGGTCA TGAGCAGAGGTCA AGGGCAAAAGTCA AGCCCAAAGTTCA AGGCCCAAGGTGT AGGGTAAATTTCA AGCTCAGAGGTCA AGGTCAGAGGTCA GGGGGAAGGGTGA AGTGCACAGGTCA GGTGCAAAGGTCC AGGGCAGAGGTCA GGGGGCAGGGTCA AGGTCAAAACTGA GGGTGAAGGGTGA AGATCAAAAGTCA AGGTGACAGGTGA AGGTCAAAGGTCA AGGACACAGGTCA GGGGAAAAAGTCA AGGGCAAAGGGTA GGGTCAGAGGTAA

AJ223066 U69543 M69260 AI852632 D37837 M21050 X72795 AI847546 AI117835 AW123802 AI852641 AW125185 U32684 Z38110 X57638 AF009605 AF064748 AI120844 X52046 AB001607 U67187 AF049125 U29539 X99963 AI848871 AW121217 AI853864 AW049373 AI837021 U41341 X66449 M83219 AF099973 U88567 X13986 AF002719 AI840996 M64086 U25844 AF010254 AW121865 U58883 L10244 X87685 AF085809 AB024935 X77585 AI839138 U43836

keratinocyte lipid binding protein lipase hormone sensitive lipocortin 1 LPS-induced TN factor lymphocyte cytosolic protein 1 lysozyme m gene matrix metalloproteinase 9 mitochondrial ribosomal protein L4 myosin, light polypeptide kinase NADH dehydrogenase (ubiquinone) Fe-S protein 3 nuclear protein 1 open reading frame 18 paraoxonase 1 peripheral myelin protein, 22 kDa peroxisome proliferator activated receptor alpha phosphoenolpyruvate carboxykinase (pepck) gene co plasma membrane associated protein, S3-12 pleckstrin homology, Sec7 and coiled/coil domains, procollagen type iii alpha 1 prostaglandin I2 (prostacyclin) synthase regulator of G-protein signaling 2 reticulocalbin 2 retinoic acid-inducible e3 protein mrna complete rhob gene RIKEN cDNA 0610011B04 gene RIKEN cDNA 0610039C21 gene RIKEN cDNA 2010100O12 gene RIKEN cDNA 2310016A09 gene RIKEN cDNA 5031422I09 gene S100 calcium binding protein A11 (calizzarin) S100 calcium binding protein A6 (calcyclin) S100 calcium binding protein A9 (calgranulin B) schlafen 2 secreted frizzled-related sequence protein 2 secreted phosphoprotein 1 secretory leukoprotease inhibitor gene complete c selenoprotein R serine (or cysteine) proteinase inhibitor, clade A serine (or cysteine) proteinase inhibitor, clade B serine (or cysteine) proteinase inhibitor, clade G similar to peroxisomal biogenesis factor 6 sorbin and SH3 domain containing 1 spermidine/spermine N1-acetyl transferase sterol carrier protein 2-pseudogene synapsin I telomerase binding protein, p23 thioredoxin 1 thioredoxin interacting protein vascular endothelial growth factor B

4728 1996 1472 1549 1966 4009 919 3685 727 3471 806 3213 3580 404 1400 269 1494 2795 2559 1635 1601 176 4447 4757 3485 3594 784 2149 4082 2272 2845 3522 3609 3868 3453 563 4882 1954 4481 3147 3354 2065 2317 3454 4920 2015 2924 3014 4644

CGGAAAAAGTTCA GGGACACAGGTGA TGTGCAAAGTTCA AGGCCAGAGGCCA GGGTCAAGGTTCA AGGTTAGAGGTCA AGGGGAAGGGTCC TGGGCAATGGTGA AGGGCAGAGTGCA GGGGCTAAGGGCA GTGTCCAAGGTCA GGAGGAAAAGTCA AGTTCAAAGGACA TGGGCACAGTTCA GGGGCAGAGTTCA GGGTCAAAGTTTA AGGGCAAAGGCCA AGATCAAAGTGCA CGTGCAAAGTTCA GGGGCACAGCTCA GTGTCAAGGGTCA GCGGCAAAGGTGA AGGTCAGAGGTCC GGGAGAGAGGTCA AGGTCAAATGACA GGTTCATAGGTCA AGGTCCAGTGTCA AGTTCAAAGTTCA AGGACAAAGTTGA GGGCACAAGGTCA AGGTCCTAAGTGA AGGTCAAATTTCA AGGCCAAAGGGCA GGGCCAAAGGCCA AGGGAAAATGTCA AAGTCAAAGGACA AGGTCATGAGTGA TGGTATAAGGTCA GGGACATAGGTCC GGGGTAAAGGTTA AGGTTATAGTTGA AGGGAAGAGGGCA AGGGTATAGGGGA AGGGGAGATGTCA TGGGGCAAGGTGA AGGGCATATGTCA AGGTCAAGGTTGA GGGCCAGAGGTGA AGGGCAGCGGTGA

500

400

400

Frequency

Frequency

500

Vehicle-3

300 200 100

300 Vehicle-4 200 100

0

0 0

25

50

75

100

0

25

500

500

400

400 Rosiglitazone-1

300

75

100

Relative Signal

Frequency

Frequency

Relative Signal

50

200

Rosiglitazone-2

300 200 100

100 0

0 0

25

50 75 Relative Signal

100

0

25

50

75

100

Relative Signal

Supplemental Figure 1. Histogram of signal intensity values (log 2) of transcripts from each of the 4 arrays. Upper Panels: Vehicle treated control mice. Lower Panels: PPAR gamma agonist (rosiglitazone) treated mice.

None-expressing transcripts removed

20

20

18

18

16

16

14

14

Vehicle-4

Vehicle-4

All Samples

12 10 8

12 10 8

6

6

4

4 2

2 2

4

6

8

10

12

14

16

18

2

20

4

6

8

12

14

16

18

20

14

16

18

20

Vehicle-3

Vehicle-3 20

20

18

18

16

16

Rosiglitazone-2

Rosiglitazone-2

10

14 12 10 8

14 12 10 8

6

6

4

4 2

2 2

4

6

8

10

12

14

Rosiglitazone-1

16

18

20

2

4

6

8

10

12

Rosiglitazone-1

Supplemental Figure 2. Scatterplot of signal intensity values (log 2) from replicate array experiments. Upper Panels: Vehicle treated control mice. Lower Panels: PPAR gamma agonist (rosiglitazone) treated mice. Transcripts determined to be absent using Affymetrix software have been removed from the panels on the right.

Treatment Rosiglitazone

Vehicle

Microarray Results from Microarray Screening Step Assess Gene Expression Levels

•Real Time RT-PCR

Informatics •Map to Unigene •Map to Gene Ontology •Map to Blood Pressure QTLs •PPRE Binding Site Model •Sequence Conservation •Map to SAGE database

•Identification of Novel PPARJ Target Genes in Mouse Aorta •Identification of Major Pathways Activated/Inhibited by PPARJ •Identification of Potentially Important Regulatory Sequences Figure 1

Expression Differential via QPCR

6 5

R = 0.93 R2= 0.86 P