genome, and whether such sites are functional in regulating enhancer blocking. Results Genome wide search for binding sites for TR and for CTCF In order to analyze whether the occurrence of composite CTCF and TR binding sites is a common feature for many or most CTCF binding sites, or whether this is limited to a subset of CTCF sites, we examined the distribution of CTCF and thyroid hormone receptor binding elements in a genome-wide fashion. CTCF binding has been mapped in a number of experimental approaches. A consensus binding sequence was derived from these studies, which was found in a large number of the identified in vivo binding regions [20]. In contrast to CTCF, no genome-wide binding data are available for the thyroid hormone receptor. Nevertheless it is known that the thyroid hormone receptor binds to DNA through conserved dimers of the hexanucleotide sequence AGGTCA (thyroid hormone response elements: TRE). The latter can be found in different arrangements, such as direct repeats with 4 or 0 nucleotides spacing (DR4 or DR0), inverted repeats spaced by 4 nucleotides (IR4) and everted repeats with a 6 nucleotide spacing (ER6) [21]. We constructed position specific scoring matrices (PSSM) based on half- site models derived from the TRANSFAC-database and used the Patser tool [22] to identify potential TR binding sites throughout the repeat-masked human genome. Performance of our PSSM-based approach was tested against two well-known motif prediction tools that are especially well suited for the detection of occurrences of nuclear hormone receptor binding sites: NHR-scan and NUBIscan [23,24]. In order to compare the different detection methods we ran comparisons on randomly selected 1 Mb genomic regions with the following setting: raw score .0.7 and p,0.05 (NUBIscan), score .6.6, p,10213 (Patser), default parameter (NHR-scan). Despite of Composite CTCF Binding Sites the different scoring algorithms we detected identical motifs in .80% of the cases. Furthermore we were able to detect a number of previously identified TREs such as the DR4 element in the myc N site ([8] data not shown). To test whether the candidate motifs might be enriched in CTCF binding regions as compared to random genomic sequences, we scanned 13720 CTCF bound regions of 1000 bp length ([20]; +/2 500 bp from annotated binding region center) and compared them to 3500 times 13720 random genomic control regions. As shown in Figure 1A we could detect various half- site arrangements in a subset of CTCF binding regions, ranging from 2.9% (400 ER6 sites) to 8.7% (1200 DR0 sites) of the total number of CTCF binding regions. We included the binding sequence for Oct4/Sox2 as a control, which has been shown to occur in functionally relevant combinations with CTCF [25], but for which no significant correlation of CTCF and Oct4/Sox2 binding was observed [26]. Here we also find that the number of CTCF regions with Oct4/Sox2 binding sequences is similar to random sequences (Fig. 1A). In contrast, the occurrence of TRE typical half site arrangements (DR4, DR0, ER6 and IR4; Fig. 1A) was slightly increased as compared to random sequences. In order to control whether this observation indeed represents an enrichment of NR motifs next to CTCF binding sites or a bias in the underlying base composition, we applied an additional control experiment: we shuffled the base positions of the individual TRE motifs and used the scrambled PSSMs to scan the 13720 CTCF binding sites. In contrast to our previous experiment, we did not find a significantly better performance of the real PSSM over scrambled ones (p-values: 0.17; 0.3; 0.44; 0.44 for DR4, IR4, DR0 and ER6 respectively). Therefore we conclude that there is no significant enrichment of TREs next to CTCF binding sites throughout the genome. This does not rule out the principal presence of functional combinations of CTCF/TR binding elements. Often, several TREs occurred simultaneously in CTCF regions, with up to three different TRE types in one CTCF region as Figure 1. Frequency of TREs at CTCF binding regions. 13720 CTCF binding regions [20] from 2500 to +500 bp relative to the respective peak centers were scanned with the PSSMs (DR4, DR0, IR4, ER6 and Oct4/Sox2) downloaded from TRANSFAC and Li et al. [55]. (A) Depicted is the number of 1000 bp regions that were marked by one or more of the respective motifs (found). Those results were compared to 3500 sets of 13720 random 1000 bp sequences from the repeat masked human genome (random). Shown is the mean number of sites marked by the respective motif. To control for potential sequence bias in the CTCF bound regions we shuffled the matrices keeping the information content constant. Shuffled matrices were used to scan the 13720 CTCF binding sequences (yellow). Error bars indicate the maximum deviation from the mean. (B) Venn diagram presentation of all TREs identified in CTCF regions, indicating the frequency of single, double and triple TRE occurrences at CTCF regions. (C) The positive control for this procedure shows the highly significant detection of the CTCF consensus (found) in contrast to random sequences (random) as in (A). doi:10.1371/journal.pone.0010119.g001 PLoS ONE | www.plosone.org 2 April 2010 | Volume 5 | Issue 4 | e10119