Composite CTCF Binding Sites Figure 3. In vitro binding assays (EMSA) show direct binding of CTCF and TRb to predicted target sites. EMSA experiments were performed using E.coli expressed GST, GST-CTCF-ZF (A) or GST-TR (B) with the indicated radioactively labeled probes. For competition experiments a 50-fold molar excess of non-labeled probes were used. These were either an unrelated CTCF binding site (F1) or an unrelated TR binding site (F2). Arrows mark the CTCF or TR specific shift. doi:10.1371/journal.pone.0010119.g003 Similarly, we analyzed binding of a GST-TR fusion to the predicted binding sites of these T3 responsive genes. Binding site prediction in several cases identified two or more potential TRE elements. These have half site arrangements of the type DR0, DR4, IR4, ER6 or an atypical [32] DR5 site (Fig. 2B). The DR4 element shows a clear shift in mobility when incubated with GST- TR at the COMT downstream promoter, but is not shifted with GST alone (Fig. 3B). The binding is specific as competition by a known TR binding site (F2) abolished protein binding, and competition with a mutated F2 site did not (Fig. S2). No shift is visible when the IR4 element of the COMT gene is incubated with GST-TR, indicating that this sequence is not bound by TR in vitro (Fig. 3B). We examined all predicted TR binding sites and could identify several new TR target sites. A detailed summary of these results is shown in Table 1 and the confirmed binding sites are indicated by green lettering (Fig. 2B). In general, all of these 10 identified regions at T3 responsive genes harbor a functional TR binding element, validating the PSSM procedure to predict TR binding sites as described above. Chromatin precipitation for TR could not PLoS ONE | www.plosone.org 4 April 2010 | Volume 5 | Issue 4 | e10119