COMT was cut with SpeI and NotI and ligated into pBSK- COMT cut with XbaI and NotI. pBSK-COMT was digested with with XbaI and SpeI and cloned into pGL3-MCSII digested with SpeI to generate pGL3-COMT and again cloned as a BamH1 fill in NheI fragment into pREP4-ss digested with NotI fill in and NheI. All other newly identified CTCF and TR binding sites were cloned in the same manner. To generate enhancer-less episomes the corresponding pGL3-vector was cut with EcoRV and NheI and cloned into pREP4-ss digested with NotI fill in and NheI. Cell culture and transfection The clonal cell line Neuro-2a stably transfected with the b1 isoform of the human thyroid hormone receptor [57] (N2a-b cells), HepG2 (ATCC HB-8065TM) and HEK293T (ATCC CRL- 11268TM) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) charcoal stripped hormone-depleted serum and 1% PenStrep. N2ab and 293T cells were transfected using the calcium phosphate method essentially as described [58]. In detail, cells were transfected with 1 mg reporter plasmid per 6-well dish or as indicated. 24 h after transfection T3 (1026 M) was added to the medium for 48 h before harvesting cells. For TSA experiments, TSA (BIOMOL) was added 8 h after transfection at a concentration of 10 ng/ml. Cells were harvested after 40 h of TSA treatment. 3-ABA (Sigma) was added 28 h after transfection at a final concentration of 8 mM. and cells were collected 14 h later. Electrophoretic mobility shift assay Radiolabeled DNA probes (Table S1) were generated by phosphorylation with gamma 32P ATP and subsequently an- nealed. The probes were incubated with 0.5–2 mg of purified GST, GST-CTCF-ZF and human GST-TR. Recombinant proteins were prepared as described previously [59]. The binding reaction was performed in PBS ([pH 7.4], supplemented with 5 mM MgCl2, 1 mM ZnCl2, 1 mM DTT, 0.1% NP-40 and 10% glycerol) for 20 min at room temperature in the presence of 200 ng/ml pdIdC and 25–100 ng/ml salmon sperm DNA. Protein- DNA complexes were analyzed on nondenaturing polyacrylamide gels (5% acrylamide [w/v]) in TAE-buffer. Electrophoresis was performed at 4uC with a field strength of 12 V/cm for 3–4 h. Chromatin immunoprecipitation ChIP assays were performed as previously described [60] with following modifications: 16107 HeLa-cells (ATCC CCL-2TM) in 1 ml SDS lysis buffer were sonicated 7 times on ice with a Branson 250 sonifier on setting 1, constant for 10 secs to an average length of approximately 300–800 bp. Sonicated chromatin from 26106 cells was diluted 10-fold in ChIP dilution buffer and precleared with 30 ml of salmon sperm DNA/protein A agarose solution (Santa Cruz #16-157) and 5 mg preimmune serum for 2 hrs before overnight incubation at 4uC with antibody or controls. For immunoprecipitation we used antibodies specific for CTCF [61] and preimmune serum. Gene specific PCR mixtures contained 1 ml of DNA, 0.5 mM of each primer (Table S1); 1.5 mM MgCl2, 0.2 mM dNTPs, and 1.25 U of Taq DNA polymerase (Panscript) in a total volume of 35 ml. Following 32 to 37 cycles of amplification, PCR products were run on a 3% agarose gel and analyzed by ethidium bromide staining. Annealing References Composite CTCF Binding Sites temperatures and cycling conditions were determined empirically for each primer set. GST-pulldown GST and GST fusion proteins were expressed in Escherichia coli BL21. Bacteria were induced with 0.5 mM isopropyl-D-thiogalac- topyranoside for 5 h at RT. Recombinant proteins were purified with glutathione–sepharose beads (Amersham Pharmacia Biotech AB) and analyzed on SDS–PAGE to normalize protein amounts (Fig. S4). Equivalent amounts of GST fusion proteins were incubated with [35S]methionine-labeled proteins, produced by the T7\T3 TNT-coupled transcription/translation system (Promega) in 200 ml of binding buffer. After 2 h incubation at 4uC the beads were washed 3 times with 1 ml of binding buffer without BSA. The bound proteins were eluted with SDS sample buffer, fractionated on SDS–PAGE visualized by fluorography. Supporting Information Figure S1 ChIP-assay demonstrates in vivo binding of CTCF. ChIP was performed using chromatin from HeLa cells and immunoprecipitated using antibodies against CTCF. Specific primers (see Table S1) for the CTCF target sites (CTS) were used in the PCR-reaction. Negative controls: nonspecific antibody (IGG) and a nonbinding sequence (ESRRa-control). Found at: doi:10.1371/journal.pone.0010119.s001 (0.26 MB TIF) Figure S2 Specificity of in vitro binding of TRb to predicted target sites. EMSA experiments were performed using E.coli expressed GST and GST-TR with the indicated radioactively labeled probe. For competition experiments a 0.5 or 50-fold molar excess of non-labeled TR binding site (F2) or unspecific probe (N) were used. Arrow marks the TR specific shift. Found at: doi:10.1371/journal.pone.0010119.s002 (0.24 MB TIF) Figure S3 Tetracycline inducible shRNA-mediated knock-down of CTCF. Protein levels were measured by western blotting using an anti-CTCF antibody or GAPDH as control. 293T cells of each transfected sample (see Fig. 5B) were collected and analyzed by 7.5% SDS PAGE. The CTCF knockdown results in a reduction of about 50%. Found at: doi:10.1371/journal.pone.0010119.s003 (0.25 MB TIF) Figure S4 Coomassie stained gel shows expression levels of GST-fusion proteins. Asterisks mark the corresponding GST- fusion proteins. Found at: doi:10.1371/journal.pone.0010119.s004 (0.75 MB TIF) Table S1 List of oligonucleotides used in different applications. Found at: doi:10.1371/journal.pone.0010119.s005 (0.12 MB DOC) Acknowledgments We would like to thank Helena Klenova for the CTCF antibody and Helmut Dotzlaw for careful reading of the manuscript. Additionally we would like to thank Peter Seum for technical assistance. Author Contributions Conceived and designed the experiments: OW CW MB JL RR. Performed the experiments: OW CW FAU. Analyzed the data: OW CW MB FAU. Wrote the paper: OW MB RR. 1. Vostrov AA, Quitschke WW (1997) The zinc finger protein CTCF binds to the APBbeta domain of the amyloid beta-protein precursor promoter. Evidence for a role in transcriptional activation. J Biol Chem 272: 33353–33359. 2. Baniahmad A, Steiner C, Kohne AC, Renkawitz R (1990) Modular structure of a chicken lysozyme silencer: involvement of an unusual thyroid hormone receptor binding site. Cell 61: 505–514. PLoS ONE | www.plosone.org 11 April 2010 | Volume 5 | Issue 4 | e10119