Mouse and human DNA sequences were obtained from the University California Santa Cruz Genome Browser (mouse genome, release Feb 2007; human genome, release June 2007). From this database, a region was selected spanning 275 kb of the entire Pax8 genomic locus in Homo sapiens and Mus musculus. The first known gene at the 3' and the first known gene at 5' of the Pax8 gene were taken as border limits of the genomic fragment selected. The genomic fragment was first chosen from the mouse genome. Then, the corresponding genomic fragment from the human genome containing the same size of DNA at the 5' and 3' flanking sides of the PAX8 gene (77.653 bp at the 3' and 140.982 at the 5') was taken. Comparative genomic analysis was carried out using the PipMaker http://bio.cse.psu.edu/pipmaker 51 and VISTA http://www.gsd.lbl.gov/vista/ 50 programs. The conserved sequences identified in this way were analyzed and compared with the TRANSFAC 4.0 database using Mat Inspector vertebrate matrix database to search for potential transcription factor binding sites. Sum of both minimized false positive and false negative errors were considered to ensure proper interpretation of the survey results.
PCR Amplification and subcloning of the Conserved Sequences
32 CNS positioned at the 5' of the Pax8 transcription start site or immediately downstream of it were selected for experimental test. These fragments were amplified by PCR using Platinum Taq DNA Polymerase High Fidelity (Invitrogen) and the PCR products were separated on a 2% agarose gel electrophoresis stained with ethidium bromide.
The conserved DNA sequences amplified by PCR were cloned in the pGL3basic vector (Promega) containing an E1b TATA box upstream the Luciferase reporter gene (pGL3-LUC), or into pGL3-TK vector containing the thymidine-kinase promoter .
The constructs CNS87-FT1m, CNS87-FT6m and CNS87-FT1-6m containing the mutated sequences of the CNS87 were generated by recombinant PCR.
Preparation of Cell Nuclear Extract and DNaseI Footprinting Assay
Total cell extracts were prepared from PC Cl3 cells grown in one hundred plates (diameter, 150 mm) as previously described . The footprinting probes were generated by digestion of the p87-E1b-LUC plasmid. The plasmid was linearized with the restriction enzyme XhoI, dephosphorylated with calf intestinal alkaline phosphatase (Roche Diagnostics) and extracted from agarose gel using the Qiaquick gel extraction Kit (Qiagen). 1.24 μg of linearized and purified plasmid DNA were end-labelled with γ-ATP-32P using T4 DNA polynucleotide kinase (BioLabs). The reverse strand probe was prepared in a similar way using as restriction enzyme Mlu1. The end-labelled probes were released from the plasmid with a second digestion and purified on a 5% acrylamide gel. 7 kcpm of the eluted probes were digested for 1 min at 20°C with 1:5000 and 1:2000 serial dilutions of a 3 mg/ml DNaseI (Roche Diagnostics) stock solution. The eluted probes were incubated with 30 μg of PC Cl3 nuclear extract and digested for 1 min at 20°C with 1:100, 1:50 and 1:25 serial dilutions of a 3 mg/ml DNaseI (Roche) stock solution.
Cells and Transient Transfection Assay
PC Cl3 and FRTL-5 cells were grown in Coon's modified F-12 medium (Euroclone) supplemented with 5% v/v calf serum and a six-hormone mixture (6H), as described .
HeLa cells were grown in Dulbecco's modified Eagle's medium (Euroclone) supplemented with 10% v/v fetal calf serum (Hyclone). For transient transfection experiments, cells were plated at 3 × 105 cells/60-mm tissue culture dish 5 to 8 h prior to transfection, whereas PC Cl3 cells were plated at a density of 5 × 105 cells/60-mm tissue culture dish 18 h prior to transfection. Transfections were carried out with the FuGENE6 reagent (Roche Diagnostics) according to the manufacturer's directions. The DNA/FuGENE ratio was 1:2 in all the experiments. The plasmid pRL-TK (Promega) was used as internal control in the transfection assays. Cells extracts were prepared 48 h after transfection to determine the levels of the firefly and renilla luciferase with the Dual Luciferase Reporter Assay System (Promega). Transfection experiments were done in duplicate and repeated at least three times.
Protein extracts and Gel Mobility Shift Assay
Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and lysed in a buffer containing 10 mM Hepes pH 7.9, 400 mM NaCl, 0.1 mM EGTA pH 7.8, 5% v/v glycerol, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF).
TTF-1 purified protein (bTTF1) was produced as previously described . For EMSA assays, double-stranded oligonucleotides were labeled with γ-32P ATP and T4 polynucleotide kinase (New England BioLabs) and used as probes. The binding reactions were carried out in a buffer containing 10 mM HEPES (pH 7.9), 10% glycerol, 0.1 mM EDTA, 8 mM MgCl2, 1 mM dithiothreitol, 0.15 μg/ml of poly (dI-dC) for 30 min at room temperature. DNA-protein complexes were resolved on a 6% nondenaturing polyacrylamide gel and visualized by autoradiography.
The antibodies, αTTF-1  and αtubulin (sc5286, Santa Cruz), used in the supershift experiments were incubated with the protein extract for 20 min before adding the probe.
Oligonucleotides were derived from the protected sequences in the footprinting assays on the CNS87 and were as follows: FT-1: CGCACAAGAGCCCTTCTCAAGGGAT; FT-2: CTGGCTAAAGCCCAACGACACAGGT; FT-3: AACACTTGGGTGATCTACGTGAAGC; FT-4: GGGGGCAGGTTGGACAAAAGCCCCA; FT-5: CCTCAACAGCTTCTGACCTTCCTCT; FT-6: GAGAACGTTTATAAGTGTCTGGCTG.
RNA Extraction, cDNA Synthesis, and Real time-PCR
Total RNA was prepared using TRIZOL Reagent (Invitrogen) according to the manufacturer's directions. Total RNA (1 μg) was retrotranscribed using the iScript cDNA Synthesis kit (Bio-Rad). Real-time PCR analysis was performed using an iCycler-iQ real-time detection system and SYBR green chemistry (Bio-Rad, Hercules, CA). Reactions were carried out in duplicate in four independent experiments. The specific primers sets used for this analysis were for amplification of β-actin (Fwd: GGCAATGAGCGGTTCCGATG; Rev: ATGGTGGTGCCACCAGACAG), of Pax8 (Fwd: CAGCTATGCCTCTTCCGCTATT; Rev: TGTGGCTGTAGGCATTGCC), for TTF-1 (Fwd: AGGACACCATGCGGAACAGC; Rev: GGCCGCCCATGCCGCTCATA) and thyroglobulin (Fwd: TGTGGAATCTAATGCCAAGAACTG; Rev: TCCCTGAGAGCTTTTGGAATG).
For each gene, values are means ± SD of four independent experiments, normalized by the expression of β-actin, and expressed as a percentage of the value measured in parental FRTL-5 cells.
The cross-linking solution, containing 1% formaldehyde, was added directly to cell culture media. The fixation proceeded for 10 min and was stopped by the addition of glycine to a final concentration of 125 mM. PC Cl3 cells were rinsed twice with cold PBS plus 1 mM PMSF, and then scraped. Cells were collected by centrifugation at 800 × g for 5 min at 4 C. Cells were swelled in cold cell lysis buffer containing 5 mM piperazine-N, N'-bis(2-ethanesulfonic acid) (pH 8.0), 85 mM KCl, 0.5% Nonidet P-40, 1 mM PMSF, and inhibitors cocktail (Sigma) and incubated on ice for 10 min. Nuclei were spun down by microcentrifugation at 2000 × g for 5 min at 4 C, resuspended in nuclear lysis buffer containing 50 mM Tris-HCl (pH 8), 10 mM EDTA, 0.8% sodium dodecyl sulfate (SDS), 1 mM PMSF and inhibitors cocktail (Sigma), and then incubated on ice for 10 min. Samples were broken by sonication into chromatin fragments of an average length of 500/1000 bp and then microcentrifuged at 16,000 × g. The sonicated cell supernatant was diluted 8-fold in ChIP Dilution Buffer containing 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1), and 167 mM NaCl, and precleared by adding Salmon Sperm DNA/Protein A Agarose (Upstate Biotechnology, Inc., Lake Placid, NY) for 30 min at 4 C. Precleared chromatin from 1 × 106 cells was incubated with 1 μg of affinity-purified rabbit polyclonal antibody, αTTF-1  and an unrelated one, rotated at 4 C for 16 h. Immunoprecipitates were washed five times with RIPA buffer containing 10 mM Tris-HCl (pH 8), 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS, 140 mM NaCl, and 1 mM PMSF; twice with LiCl buffer containing 0.25 M LiCl, 1% Nonidet P-40, 1% Na-deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.0), and then three times with TE (10 mM Tris-HCl, pH 8; 1 mM EDTA). Before the first wash, the supernatant from the reaction lacking primary antibody was saved as total input of chromatin and was processed with the eluted immunoprecipitates beginning at the cross-link reversal step. Immunoprecipitates were eluted by adding 1% SDS, 0.1 M NaHCO3 and reverse cross-linked by addition of NaCl to a final concentration of 200 mM and by heating at 65 C for 16 h. Recovered material was treated with proteinase K, extracted with phenol-chloroform-isoamyl alcohol (25:24:1) and precipitated. The pellets were resuspended in 30 μl of TE and analyzed by PCR using specific primers for the CNS87. The input sample was resuspended in 30 μl of TE and diluted 1:10 before PCR.
Mouse BAC clone bMQ-241M1 was obtained for the bMQ mouse BAC library made at the Wellcome Trust Sanger Institute constructed from the 129SvEv/AB2.2 mouse strain. Engineered versions were produced via recombinogenic targeting in Escherichia coli by the method of Lee et al. , also know as recombineering. Briefly, a luciferase expression cassette with a neo resistance cassette was inserted in the exon 2 of Pax8 into the ATG start codon. To obtain this construct luciferase cDNA form PGL3 was cloned upstream PGK-EM7-Neo cassette from pl452.
The recombination cassette was constructed by subcloning a 50-bp 5' recombination arm overlapping part of pax8 intron 1 and part of exon 2 upstream ATG start codon and a 50-bp 3'arm recombination arm containing part of exon 2 and intron 2 downstream ATG start codon such that the recombination arms flanked the Luc-PGK-EM7-Neo cassette. The forward strand sequences of the 50-bp homology arms were as follows, for the 5'arm: TGCGTAGGAAAGCTGCGAGTGTCCCTCAGTCTGTGAGCGACTCCCCGGCG; for the 3' arm: ATGCCTCACAACTCGATAGATCCGGTAAGGACCGCGGAGGGGCCAGGAC.
The final cassette with recombination arms was digested from the vector, gel purified and then recombined with pax8 BACs as described previously. Successful recombinants BAC Luc-Neo were selected by plating the electroporated cells on LB plates containing 20 μg/ml Kanamycin, 20 μg/ml chloroamphenicol. Correct modified BACs were verified by PCR analysis. For BAC 87- (lacking 87 region) the BAC luc-Neo was modified by insertion of Amp-resistance cassette into the 87 sequence. Amp-resistance cassette was amplified from pBS vector using primer 5' flanked by recombination arm overlapping the sequence upstream 87 region and using 3' primer flanked by recombination arm overlapping sequence downstream 87 region.
The forward strand sequences of the 50-bp Homology arms were as follows, for the 5'arm: AAAGAGAGGCAAAGAAAGCTAGGGGTCTGCAGTCTCCAAACCTGCAGGGCTGGCTAAAG; for the 3'arm: TTTCTGAATCTAAATCCAAAACTTTACCCTCTTCTGATTGGTAATGAGTC.
The PCR product was purified and recombined with BAC Luc-Neo. Recombinants were selected by plating the electroporated cells on LB plates containing 20 μg/ml Kanamycin, 20 μg/ml chloroamphenicol and 50 μg/ml ampicillin and the correct modified BACs were verified by PCR analysis.
BAC DNA for transfection was prepared with Qiagen Large-Construct kit.
Stable transfection were carried out with PEI22 (MBI Fermentas, St.Leon-Rot, Germany) as described previously . PC Cl3 cells were seeded in 6-well dishes (2 × 105) the day before transfection. For each construct 1 μg of BAC DNA and 2 μg of BAC DNA were transfected with an amount of PEI giving an N/P ratio of 7.5. The medium was replaced on day 3 with fresh medium containing 150 μg/ml G418. Stably transfected cell pools were grown to confluence. Cells were then washed twice with phosphate-buffered saline and incubate for 30 min at room temperature with passive lysis buffer (Promega) with vigorous shaking. Firefly luciferase activity was determined using "Luciferase assay System" (Promega). Luciferase activity was then normalized with protein concentration of cleared lysates.