blocking assay is able to fully simulate enhancer blocking of endogenous genomic insulators, PAR modification should affect the episomal assay. Therefore we used the inhibitor of PAR polymerases, 3-Aminobenzamide (3-ABA), for the transfection experiments. The CTCF-mediated enhancer blocking is decreased to about 70% upon the addition of 3-ABA (Fig. 4C). Thus, the episomal system used in these experiments fully mimics the enhancer blocking function of insulators integrated in the genome. T3 responsive and non-responsive composite CTS elements Since the episomal system proved to be suitable for the analysis of composite CTCF/TR binding sites, we generated enhancer blocking episomes with the newly identified composite elements. HepG2 cells were chosen as a test system because most T3 responsive genes found are expressed in liver. Single insertions of LMNA, PCK1, ESSRA, COMT, NR2F1, BCL3 and PTPRF mediated weak enhancer blocking (Fig. 4D). The arrangement of the LMNA composite element relative to the promoter is, in contrast to the other genes, downstream of the promoter. Therefore, we also tested the antisense orientation in the episome (pR LMNA(as) E), which did not show any enhancer blocking activity. Two of the elements, the positive control F1F2 and ESRRA, mediated enhancer blocking in the absence of T3, whereas T3 fully abolished this activity. In order to detect possible weak effects in enhancer blocking and T3 mediated relief from enhancer blocking, we generated four or five times multimerized composite CTCF/TR binding sites. In six cases, F1F2, ESRRA, COMT, NR2F1, BCL3 and PTPRF a significant increase in enhancer blocking activity could be seen (Fig. 4E). In contrast to these new enhancer blocking elements, the composite CTS of LRP1 and LMNA in either orientation did not confer enhancer blocking. In all cases we challenged the luciferase activity by incubation with T3. The composite elements of PCK1, COMT, NR2F1, BCL3 and PTPRF mediated constitutive enhancer blocking, which could not be relieved by T3 (Fig. 4D, E). In contrast, the ESRRA element as a single insertion or when multimerized four times resulted in a T3 mediated complete release of enhancer blocking (Fig. 4D, E). We could not detect any repressive/silencing effects of the ESRRA element on the luciferase promoter using the enhancer-less episomal vectors (Fig. 4F). Thus, the effects seen are dependent on an enhancer and can therefore be defined as enhancer blocking. Taken together the ESRRA element is a new composite element mediating hormone sensitive enhancer blocking and together with the F1F2 element is suitable for further studies. Functional CTS composite elements depend on both, TR and CTCF Since T3 relieves the enhancer blocking activity from the F1F2 and the ESRRA composite elements, we wanted to address a possible contribution of the TR to enhancer blocking in the absence of T3. As a control we tested the contribution of CTCF to enhancer blocking by utilizing 293T cells, which allow for high level expression. Since CTCF is expressed in all cell types, we used transfection conditions resulting in a low level of enhancer blocking. Co-transfection of an expression vector for CTCF leads to a significant increase in enhancer blocking on the F1F2 and ESRRA elements, indicating that CTCF is at least contributing to enhancer blocking (Fig. 5A). In a complementary approach we reduced the amount of CTCF using a tetracycline inducible shRNA-mediated knock-down of CTCF. After 48 hours this Composite CTCF Binding Sites antisense approach resulted in a down regulation of CTCF protein to about 50% (Fig. S3). This decreased expression of CTCF led to a 50% decrease in enhancer blocking (Fig. 5B), again confirming that CTCF is involved in the observed enhancer blocking. The addition of T3 completely abolishes enhancer blocking both when the blocking is increased after CTCF overexpression, and is decreased after CTCF knockdown. We then tested the influence of TR on enhancer blocking. We transfected F1F2-containing episomes into N2a cells, that is a cell line which, in contrast to N2ab cells, does not express TR. This allows for the testing of TR mediated effects upon TR expression. We observed that CTCF alone is not sufficient to mediate enhancer blocking, whereas the cotransfection of a TR expression vector restores enhancer blocking (Fig. 5C). The same is true for the ESRRA element containing episomes when transfected into HepG2 cells. These cells have a reduced amount of TR resulting in a reduced T3 induction of transfected DR4 luc reporter DNA. Full DR4 reporter activity can only be achieved upon overex- pression of TRb (data not shown). Therefore, we cotransfected the TRb expression vector and the episomal reporter construct. This resulted in about a 5-fold enhancer blocking using the ESRRA or the F1F2 elements (Fig. 5C). Likewise, the enhancer blocking activity of both elements, F1F2 and ESRRA, was dependent on the expression of TRb when using 293T recipient cells. This strong impact of the unliganded TR on enhancer blocking was unexpected. Therefore we wanted to understand the individual contribution of CTCF and TR to enhancer blocking and mutated the individual binding sites within the F1F2 element. To abolish CTCF binding to its cognate binding site, 2 nucleotides within the F1 binding site of CTCF were changed (AA – GG) and tested in band shift assays using E.coli expressed CTCF-zinc finger domain fused to GST (GST-CTCF ZF). The F1wt binding site is shifted by GST-CTCF ZF. This shift can be competed with an excess of unlabelled F1wt site but not by the same excess of unlabelled mutated F1 site (Fig. 5D). To test the functional consequence on enhancer blocking by the mutated binding site, we performed transfection experiments with episomes containing the F1F2 or the F1mutF2 binding sites. These experiments revealed that in contrast to the wt F1F2 element, the F1mutF2 binding site is not capable of mediating significant enhancer blocking (Fig. 5E). To test the effects of TR on CTCF mediated enhancer blocking, we mutated the two half sites of the F2 element, which is characterized by an everted palindrome spaced by 6 nucleotides. We performed band shift assays with the wt F2 sequence as a probe, and bound GST-TR could be fully competed by the addition of unlabelled wild type probe. In contrast, the mutated unlabelled F2 element could not compete this shift even at a high molar excess (Fig. 5D). To test the functional consequences of the mutated F2 in enhancer blocking we introduced this mutation into the F1F2 enhancer blocking construct. Transfection experiments with constructs containing F1F2wt or F1F2mut showed that mutation of the F2 binding site did not only abrogate any T3 effect, but also impaired enhancer blocking (Fig. 5E). These results demonstrate that enhancer blocking depends on the presence of two factors, CTCF and TR. CTCF alone is not able to mediate enhancer blocking on these composite elements, an unliganded TR synergizes with CTCF and this synergism can then be abolished by the presence of T3. TR interaction with CTCF has been shown [38]. The physiological TR/DNA complex may be a TR homodimer or a TR and RXR heterodimer [21]. Therefore we tested whether CTCF interacts with TR, RXR, or with both. We performed GST pull-down assays with GST fused CTCF and 35S-radiolabeled TR PLoS ONE | www.plosone.org 7 April 2010 | Volume 5 | Issue 4 | e10119