Figure 7. Modular CTCF binding sites. Enhancer blocking elements may require multiple CTCF sites for optimal function, as exemplified at the H19 locus or the Xist and Tsix locus [15,50,51] (top). The composite elements of the lysozyme and ESRRA genes require binding of RXR and/ or TR for enhancer blocking (second row). Long range chromatin interaction is not only required for enhancer blocking, but for X chromosomal pairing and HLA locus interaction as well. In addition to CTCF, this involves Yy1 and Oct4 [25,53,62] (third row) or CIITA at the CTCF site and RFX at the interacting site [63,64] (bottom row). doi:10.1371/journal.pone.0010119.g007 inhibition of histone deacetylation showed similar effects in this system as has been demonstrated for endogenous genes. Both the enhancer blocking activity of CTCF as well as the transcriptional repression of rDNA genes by CTCF require poly(ADP)ribosyla- tion [36,47]. Tumor suppressor silencing has been recently shown to be caused by a deficiency in poly(ADP)ribosylation [37]. In line with these results we could demonstrate that episomal enhancer blocking mediated by a composite binding site is sensitive to a poly(ADP)ribosylation inhibitor as well. Furthermore, changes in histone acetylation in the context of enhancer blocking or insulation have been shown. For CTCF binding sites, an increase in acetylation of histone H3 as well as of H4 has been reported [48,49]. In contrast, CTCF binding to histone deacetylase activity has been found as well [35], and functional episomal tests of the b globin insulator demonstrated a general histone deacetylation in the region extending from the enhancer to the gene [43]. Why is the ESRRA gene down-regulated after T3 treatment, whereas the assay shown above indicates a loss of enhancer blocking by T3? We can only speculate that from the different promoters of the gene one may be induced, which may cause other promoters to be turned of. This assay revealed for the first time that in the case of two composite elements a functional synergy between CTCF and the unliganded TR confers enhancer blocking. This shows that, similar to enhancer elements which are comprised of functional modules (binding sites for enhancer factors), enhancer blockers are generated from functional and synergizing modules as well (Fig. 7). Again, in analogy to enhancer elements, functional modules can be multimers of identical factors (CTCF) as in the case of the H19 locus [50,51] or in the X inactivation locus of the active mammalian X chromosome [15], or different factors such as combinations of CTCF with Oct4 [25], Kaiso [52], Yy1 [53] or Composite CTCF Binding Sites TR, as shown here. In these cases (Fig. 7), binding of both factors involved is required to mediate the biological function. Materials and Methods Motif scanning Half site models for nuclear receptor were downloaded from TRANSFAC database. Next we built PSSMs with the appropriate spacing (ER6, DR4, DR0, IR4). The PSSM for CTCF was derived from published genomewide CTCF binding data [20] by scanning the top 1000 binding regions with the MEME tool [54]. The PSSM for the combined Oct4/Sox2 sites was taken from Li et al. [55]. PSSMs were used to scan 13720 described CTCF binding regions from 2500 bp to +500 bp relative to individual peak centers using the Patser tool of the RSAT suite [22]. In case of the NR half site models we compared the performance of our prediction method with the NHR-Scan and NUBIscan tools [23,24] on random 1 Mb regions and found identical motif prediction in .80% of the cases. Random control datasets were generated with custom BioPerl scripts from the repeat masked human genome downloaded from UCSC (http://hgdownload.cse.ucsc.edu/goldenPath/hg18/bigZips/). 2000 repetitions of such control experiments were conducted. To control for biases in the sequence composition of the CTCF binding regions we shuffled the base positions of the PSSMs and used these to scan the 13720 CTCF binding regions. In case of the NR half sites models we tested all possible permutations (720) using the same permutation in both half sites. For the CTCF and Oct4/Sox2 PSSMs we tested the same number of random permutations. Empirical p-values were calculated as the fraction of simulations that produced a number of mapped features as extreme as observed in the real data [56]. Plasmid construction A second MCS (PstI, EcoRI, BglII, EcoRV, Spe) was inserted into the ClaI site of the pGL3-control vector (Promega). pR-E was generated by digesting the pGL3-MCSII vector with BamHI fill in and NheI and cloned in NotI fill in and NheI cut pREP4-ss. pREP4- ss is a pREP4 vector (Invitrogen), where the RSV LTR promoter was removed. The F1F2 element of the chicken lysozyme gene was amplified with primer pairs containing SpeI and BglII restriction sites. The PCR product was cloned into the BglII and SpeI sites of the MCSII of the pGL3-MCSII vector generating pGL3F1F2. pRF1F2E was generated by digesting the pGL3F1F2 vector with BamHI and XhoI, and was cloned blunt end into pREP4-ss NotI blunt ended. The F1F2 element was multimerized by ligating PCR products amplified from the pGL3F1F2 vector with primers containing SpeI and XbaI restriction sites. The 5xF1F2 was subcloned blunt into pBK-CMV (pBK-CMV5xF1F2), further cloned as a XbaI and BamH1 fragment into pGL3-MCSII digested with SpeI and BglII (pGL3-5xF1F2) and again cloned as a BamH1/XhoI fragment into pREP4-ss NotI blunt to generate pR5xF1F2E. pRF1mutF2E was generated as follows: Primer containing the F1mut binding site were annealed and directly cloned into the XbaI, HindIII site of pBK- CMV (Stratagene). Primer containing the F2 element were annealed and directly cloned into the HindIII, BamHI site of pBK-CMV F1mut. pGL3F1mutF2 was generated by digested pBK- CMVF1mutF2 with XbaI and SpeI and cloning the fragment into the SpeI site of pGL3-MCSII. pRF1mutF2E was generated by digesting pGL3-F1mutF2 with SalI fill in and NheI and cloned into pREP4-ss cut with NotI fill in and NheI. pRF1F2mutE was cloned in the same manner with F1 and F2mut oligos. pRCOMTE was generated by PCR amplification of the genomic locus with corresponding primer pairs containing SpeI and XbaI restriction sites and subcloned into pBSK (Stratagene) generating pBSK-COMT. To multimerize binding sites pBSK- PLoS ONE | www.plosone.org 10 April 2010 | Volume 5 | Issue 4 | e10119