or RXR. The pulldown experiments clearly showed that both TR and RXR interact with CTCF (Fig. 6A). To identify domains of the receptor required for interaction we generated GST fusions with the individual DNA binding domains (DBD) of TR and RXR or with the ligand binding domain (LBD). The TR-DBD binds very efficiently and seems to harbor the main interaction interface. The N-terminal domain of RXR binds to CTCF to a lesser extent,the ligand binding domain shows no CTCF binding in the case of RXR, and only weak binding in the case of TR (Fig. 6A). Thus the DBD of TR harbors two potentially important functions for enhancer blocking, such as DNA binding and an interface for the physical interaction with CTCF. Therefore we functionally tested the TR-DBD for enhancer blocking. We used cells that do not express endogenous TR (N2a cells) and expressed either full length TR or the DNA binding domain of TR. In contrast to the TR-DBD, only full length TR results in strong enhancer blocking of the F1F2 episome (Fig. 6B). This indicates that the TR-DBD is not sufficient for enhancer blocking and that other domains outside the DNA binding domain of TR are needed as well to mediate enhancer blocking of the CTCF bound module and the unliganded TR. Discussion CTCF binding sites have been found next to thyroid or steroid receptor binding sites in several cases [8,39,40]. For a subset of these sites we could demonstrate that enhancer blocking is regulated by thyroid hormone [8]. In other cases, clustered binding sites for steroid receptors next to highly conserved CTCF sites in the Igf2/ H19 locus, a function could not be demonstrated [19]. Therefore, we wanted to know the frequency of combined binding sites for CTCF and the thyroid hormone receptor in the genome. Of 13720 CTCF bound regions we found about 18% to harbor a potential TRE. This ratio is slightly higher than random sequences associated with CTCF bound regions. Clearly, the positive control to search for the CTCF consensus within these regions revealed a significant 22 fold higher frequency as compared to random controls. We checked the PSSMs for different half-site models against whole genome datasets of the estrogen receptor (ER) and the retinoic acid receptor alpha Composite CTCF Binding Sites (RARa) [40,41] in order to validate our approach in identifying such elements next to CTCF sites. In these calculations we found that the respective half-site models (IR3 and DR5) showed no significant enrichment when we compared the genome-wide binding datasets with control sequences (data not shown). Previous analysis of estrogen receptor binding sites in compar- ison with CTCF binding revealed that CTCF site distribution strongly correlated with gene density in contrast to ER sites [20]. Based on the CTCF distribution, the genome can be dissected into sequence blocks framed by CTCF sites. About 3000 such CTCF blocks have been found to contain ER binding sites [42]. The authors concluded that CTCF confines the distal action of the estrogen receptor. This would argue for a clear separation of CTCF and ER sites in function and space for most of the binding sites. This is in line with the finding that of 1600 ER sites analyzed, about 100 colocalized with the CTCF motif [40]. Overall, the ratio of composite CTCF and TR binding sites relative to the total number of TR or CTCF sites is within the same magnitude as found for ER and CTCF. Despite the low frequency of composite CTCF/TR binding elements, these can be important and functional. The importance of low frequency composite elements has been illustrated by the recent finding that binding of Oct4 and CTCF to a composite element of the Xist gene is required for the regulation of X chromosome inactivation [25] (Fig. 7). Nevertheless, there is no overall correlation of combined binding of CTCF and Oct4 in the genome [26]. Likewise, we do not observe an enrichment of the Oct4 motif in the vicinity of CTCF binding regions as compared to random genomic regions (see Fig. 1A). For the functional characterization of the TR/CTCF composite elements we used the episomal system. Episomal constructs are able to mimic endogenous chromatin and can be used for insulator analysis [43–46]. We found that for the episomal vectors that we constructed, the analysis of enhancer blocking is also possible. Addition of T3 relieves enhancer blocking, an important control that demonstrates that the episomal system can be used to study T3 regulated enhancer blocking. Thereby we identified a new composite element at the ESRRA gene, which indeed mediates T3 regulation of enhancer blocking. Furthermore, CTCF and Figure 6. TR requires more than just the CTCF interaction domain to mediate enhancer blocking. (A) GST-pulldown experiments were carried out using the indicated E.coli expressed GST-fusion proteins. (B) N2a cells were transfected with the indicated episomes and expression vector for full length TRb or for TRb DBD and incubated in the absence or presence of T3. doi:10.1371/journal.pone.0010119.g006 PLoS ONE | www.plosone.org 9 April 2010 | Volume 5 | Issue 4 | e10119