1). Chemicals, Cell Culture and Medium

2). Serial Analysis of Gene Expression (SAGE)

3). In Vitro DNA-Binding Site-Selection for PPARd & RXRa

4). Gel Electrophoretic Mobility Shift Assays (GEMSA)

5). Construction of a PPARd-Responsive Reporter

6). Construction of PPARd Promoter Reporters

7). Tansfections and Reporter Assays

8). In Vitro Transcription and Translation Assays

9). Generation of Recombinant Adenovirus Expressing PPARd or c-MYC
 
 

Chemicals, Cell Culture and Medium

Human colorectal cancer cells HT29, HCT116, SW480, and DLD1 were maintained in McCoy’s 5A medium (Life Technologies, MD) supplemented with 10% fetal bovine serum (HyClone, UT), 100 units / ml penicillin, and 100 mg/ml of streptomycin. Human embryonic kidney cells 293 were maintained in DMEM (Life Technologies) supplemented with 10% fetal bovine serum, 100 units / ml penicillin, and 100 mg/ml of streptomycin. BRL49653 and cPGI (Carbaprostacyclin, cat#18210) were purchased from American Radiolabeled Chemicals and Cayman Chemical Company, respectively. Sulindac derivatives and indomethacin were purchased from BIOMOL. Unless otherwise indicated, all chemicals were purchased from Sigma (St. Louis, MO).
 

Serial Analysis of Gene Expression (SAGE)

As previously described , SAGE was performed on mRNA harvested from exponentially growing HT29-APC and HT29-b-Gal cells 9 hours after zinc induction. A total of 55,233 and 59,752 tags were obtained from APC-expressing and control cells. Analysis of internal linker controls revealed a sequencing error rate of 0.065 per tag, corresponding to a sequencing error rate of 0.0067 per base. This was in good agreement with instrument specifications and previous estimates of SAGE tag errors based on the analysis of the complete yeast genome . These tags represented 14,346 unique transcripts, of which 7,811 transcripts appeared at least twice. Expression differences were considered significant if they had a P_false < 0.1 as determined by Monte Carlo simulations and they were at least fourfold in magnitude.
 

In Vitro DNA-Binding Site-Selection for PPARd and RXRa

GST fusion proteins containing the N-terminal DNA-binding domains of human PPARd and human RXRa were constructed by PCR amplifying the cDNA coding sequences of residues 1-249 of PPARd and residues 1-224 of RXRa and cloning them into pGEX-2TK vector. As controls, GST fusion proteins containing the DNA-binding domains of human PPARa (AA 1-249) and PPARg (AA 1-248) were also constructed. The fusion proteins were produced and purified according to the manufacturer’s protocol.

To identify the potential consensus DNA sequence motifs recognized by PPARd and RXRa, a previously described in vitro site selection procedure was utilized . Briefly, for binding to the PPARd and RXR proteins, the following oligonucleotide was synthesized: 5’-TAGTAAACACTCTATCAATTGG(N)₂₀TCTAG AAAGCTTGTCGACGC-3’, where "N" represents an equimolar mixture of each nucleotide. By using this oligonucleotide as template, a random duplex pool was generated by PCR amplification with primers that hybridized to the flanking sequences. The fusion proteins were mixed with the random duplex pool and subjected to GEMSA (see below). A broad region of the gel predicted to contain DNA-protein complexes (from control binding experiment) was excised. Gel slices were homogenized, incubated at 65⁰C for 30 min, and passed through Spin-X column (Costar). Eluted DNA was extracted with phenol-chloroform, precipitated with ethanol, re-amplified by PCR, and subjected to the next round of binding. Following completion of the third round of selection-amplification, PCR products were cloned into pZero 2.1 (Invitrogen). The 60-bp probes corresponding to single clones were generated for GEMSA by direct colony PCR using the following ³²P- labeled primers: 5’-TAGTAAACACTCTATCAATTGG-3’ and 5’-GTCCAGTATCGTTTACAGCC-3’. To determine the DNA sequences contained within single clones, inserts were PCR amplified by using M13 forward and reverse primers and the PCR products were sequenced with Amershan’s Thermosequenase kit and an SP6 primer.
 

Gel Electrophoretic Mobility Shift Assays (GEMSA)

DNA-binding assays supplemented with Poly dIdC (6mg/ml) were performed essentially as described . For binding to PCR products derived from in vitro site selections, 1.0-1.5 mg of protein and 50 ng of DNA were used. For competitions, a 100-fold excess of unlabeled probe was used. For GEMSA with GST fusion proteins, 0.3-0.5 mg of fusion protein and 0.5 ng of ³²P kinase labeled (~10⁶ dpm) DNA were used. The probes for Tcf-4 binding was as previously reported . For GEMSA with in vitro translated proteins, 0.1 to 0.2 ml of programmed lysate and ³²P-labeled probe (~10⁶ dpm) was used. The DRE probe was formed by annealing 5’-GCGTGAGCGCTCACAGGTCAATTCG-3’ and 5’-CCGAATTGACCTGTGAGCGCTCACG-3’. The ACO probe was formed by annealing 5’-GCGGACCAGGACAAAGGTCACGTTC-3’ and 5’-CGAACGTGACCTTTGTCCTGGTCCG-3’.
 

Construction of a PPARd -Responsive Reporter

The following oligonucleotides containing PPARd and RXR recognition motifs that were identified from in vitro site-selection approach were synthesized: 5’-CTAGCGTG AGCGCTCACAGGTCAATTCGGTGAGCGCTCACAGGTCAATTCG-3’ and 5’-CTAGCGAATTGACCTGTGAGCGCTCACCGAATTGACCTGTGAGCGCTCACG-3’. As a control, the following oligonucleotides containing a PPARa and PPARg responsive element from the acyl-CoA oxidase promotor were also synthesized: 5’-CTAGCGGACCAGGACAAAGGTCACGTTCGGACCAGGACAAAGGTCACGTTCG-3’ and 5’-CTAGCGAACGTGACCTTTGTCCTGGTCCGAACGTGACCTTTGTC CTGGTCCG-3’. The oligonucleotide cassettes were dimerized and cloned into pBV-Luc, a luciferase reporter plasmid with very low basal activity (He et al., 1998). All constructs were verified by DNA sequencing.
 

Constructions of PPARd Promotor Reporters

To identify the genomic sequence of the human PPARd promotor, the following PCR primers were used to screen a BAC library (Research Genetics): 5’-CCTGTA GAGGTCCATCTGCGTTC-3’ and 5’-CATGCTGTGGTCCCCCATTGAGC-3’. Three independent BAC clones containing the PPARd promotor sequence were obtained. Upon subcloning and sequencing, the genomic sequence of 3.1-kb immediately upstream of the first exon was determined (GenBank accession # 187850).

For the construction of PPARd promotor reporters, corresponding restriction fragments (illustrated in Figure 2A) were subcloned into pBV-Luc. The following primer pair was used to PCR amplify the mutant NP fragment: 5’-CTAGCTAGCGAGGGTG CATCGTCAATGTTTTGTGTGGGAAG-3’ and 5’-CCGGAATTCTAGGGACGATGA CGATGAACAAAGCTTGACTC-3’. The following oligonucleotide pairs were used for dimerization to construct corresponding reporters in pBV-Luc: 5’-CTAGCATGTCTT TGTACTCGATGTCTTTGTACTCG-3’ and 5’-CTAGCGAGTACAAAGACATCGAG TACAAAGACATG-3’ for p4XTRE1-Luc; 5’-CTAGCATGTC TTTGGCCTCGATGT CTTTGGCCTCG-3’ and 5’-CTAGCGAGGCCAAAGACATCGAGGCCAAAGACA TG-3’ for p4XmTRE1-Luc; 5’-CTAGCTTGGCTTTCATCTGATTGGCTTTCATCTG AG-3’ and 5’-CTAGCTCAGATGAAAGCCAATCAGATGAAAGCCAAG-3’ for p4XTRE2-Luc; and 5’-CTAGCTTGGCTTTCGCCTGATTGGCTTTCGCCTGAG-3’ and 5’-CTAGCTCAGGCGAAAGCCAATCAGGCGAAAGCCAAG-3’ for p4XmTRE2-Luc.
 

Transfections and Reporter Assays

Exponentially growing cells were seeded to 12-well tissue culture plates and each assay was carried out in triplicate. Reporter plasmid, effector plasmid and b -gal control plasmid were transfected into cells using LipofectAmine (Life Technologies). Twenty-four hours after transfection, cells were lysed and collected for assays of luciferase activity using Promega’s Luciferase Assay System.
 

In Vitro Transcription and Translation Assays

The full-length proteins of PPARd , PPARg , and RXRa were generated by in vitro transcription-coupled translation using the TNT T7 Quick-Coupled Transcription/Translation System (Promega). Briefly, the following primer pairs were used to amplify the coding sequences of PPARd , PPARg, and RXR: 5’-GGATCCTAATACGACTCACTATAGGGAGACCACCATGGAGCAGCCACAGGAGGAAGCC-3’ and 5’-TTTTTTTTAGTACATGTCCTTGTAGATCTC-3’ for PPARd; 5’-GGATCCTAATACGACTCACTATAGGGAGACCACCATGGCAACCATGGTTGACACAGAGATC-3’ and 5’-TTTTTTTTAGTACAAGTCCTTGTAGATCTCC-3’ for PPARg ; and 5’-GGATCCTAATACGACTCACTATAGGGAGACCACCATGGACACCAAACATTTCCTGCCGC-3’ and 5’-TTTTTTTTAAGTCATTTGGTGCGGCGCCTCC-3’ for RXRa . The full-length proteins were produced according to the manufacturer’s protocol.
 

Generation of Recombinant Adenovirus Expressing PPARd or c-MYC

The following PCR primer pair was used to amplify human PPARd coding sequence: 5’-AGAATGCGGCCGCTGCTCGAGGAATGGAGCAGCCACAGGAG GAAGCC-3’ and 5’-CGCGGATCCTCTAGATTAGTACATGTCCTTGTAGATCTC-3’. The PCR product was cloned into pCMV-HAHA, which contained a double HA-tag driven by a CMV promotor. Authentic PPARd coding sequence was verified by DNA sequencing and its expression was confirmed by Western blot using an anti-HA antibody. The expression cassette of HA-tagged PPARd was further subcloned into pAdTrack vector, which also expressed green fluorescent protein. The AdPPARd recombinant virus was subsequently generated and purified by using the AdEasy system as previously described. AdMYC was generated in a similar fashion and a gift of Heiko Hermeking.
 
 

pBV-Luc Vector Sequence