depicted in a Venn diagram (Fig. 1B). A total of 2430 CTCF regions were detected that harbored at least one of the four different TRE sequences. As a positive control for our analysis we tested the number of cases where the CTCF consensus was found in the CTCF binding regions (Fig. 1C). In about 70% of the CTCF binding regions a consensus was detected, which is in agreement with published data [20]. As expected, we found 10- fold fewer instances in random sequences or using the scrambled versions of CTCF PSSM (Fig. 1C). Taken together these data indicate that TR binding motifs mark a fraction of CTCF binding regions. The frequency of composite CTCF/TR elements is not higher than that detected with scrambled PSSMs. This finding does not support a general role for the TR. Despite of this, individual composite elements could be functional as exemplified with the CTCF/Oct4/Sox2 cases [25,26]. From these data we reasoned that although individual examples for composite CTCT/TR binding elements are not a general feature of CTCF binding regions, a number of functional TRE motifs in the vicinity of CTCF binding sites might exist. In order to functionally verify the predicted composite CTCF/ TR binding sites, we concentrated on those genes which have been shown to be regulated by T3 in the mouse or in the rat [27–30] (Fig. 2A). From these we screened 94 genes conserved in human for the presence of a CTCF binding site (CTS) within or outside of the transcribed region including 40 kb of flanking sequence upstream and downstream. For CTCF we again used the dataset of genome wide CTCF binding [20]. We identified 31 T3 responsive genes with one or more CTCF binding sites, whereas 63 did not contain a known CTCF target site. In order to predict TR binding sites within the T3 responsive genes we used the NubiScan algorithm, which allows the prediction of nuclear receptor response elements [23]. We focused on the direct repeats DR0 and DR4, as well as on the palindromes IR4 and ER6, which are known to bind either TR homo or TR/RXR heterodimers [21]. We divided each of the gene loci into the 59region and the intronic plus 39region. Of the 31 gene loci with a CTCF site, 23 harbor CTCF sites in the 59region. For these a TR binding site in a range of +/2 1 kb flanking a CTS could be identified in 13 cases. Within the intronic and 39 CTS group, TREs could be identified in the vicinity of a CTS in 4 cases. Thus, a total of 17 gene loci harbor a composite TRE/CTS. Based on the score of binding site predictions, we chose 10 (Fig. 2) for experimental analysis. Composite elements are bound by TR and CTCF In vivo binding of CTCF to the gene regions shown in Figure 2 has been documented by genome wide binding analysis [20]. The in vivo response to T3 for the selected genes has been shown as well [27–30]. In order to identify the precise binding sites and to validate the TRE predictions, we tested TR and CTCF binding in vitro using the electrophoretic mobility shift assay (EMSA). For CTCF analysis we used a fusion of GST with the zinc finger domain of CTCF (GST-CTCF-ZF) expressed in E.coli. The DNA probes were selected by CTCF binding site prediction for each of the 10 genes chosen (Fig. 2B). In all cases, GST-CTCF-ZF resulted in a retarded band (Fig. 3A). For the Catechol-o-methyl-transferase gene (COMT) two CTS are predicted at the downstream promoter, both of which are bound as well. The bound complex could be competed by an unrelated CTCF binding site (F1), whereas the negative control, GST protein expressed in E.coli, resulted in no specifically retarded complex. In order to confirm that these CTS elements are indeed the same sequences identified by genome wide CTCF binding analyses [20,31], we precipitated HeLa cell chromatin with an Composite CTCF Binding Sites Figure 2. Schematic representation of genes with a composite element. (A) 94 T3 responsive genes conserved in mice and man searched for CTCF composite elements (see main text). (B) Each diagram presents a region of the indicated gene. Putative binding sites for CTCF (CTS), binding sites for TR (IR4, DR4, DR5, ER6 or DR0) and the transcriptional start site (arrow) are shown. Elements being shown to bind TR or CTCF (see Fig. 3) are indicated in green. doi:10.1371/journal.pone.0010119.g002 antibody directed against CTCF. Exemplarily, we analyzed the precipitate for the presence of five of the sequences shifted by CTCF and found all to be bound in vivo as well (Fig. S1). PLoS ONE | www.plosone.org 3 April 2010 | Volume 5 | Issue 4 | e10119