D84 Nucleic Acids Research, 2008, Vol. 36, Database issue DATA SOURCES AND PROCESSING Data sources Experimentally identiﬁed and computationally predicted CTCF-binding sites are processed separately. First, 34 417 experimentally identiﬁed CTCF-binding sites are collected from four sources: (i) 110 manually curated CTCF- binding sites from biomedical literature, denoted by identiﬁers starting with ‘INSUL_MAN’, (ii) 244 mouse CTCF-binding sites identiﬁed by Ohlsson and coworkers using ChIP-chip assay (21), denoted by identiﬁers starting with ‘INSUL_OHL’, (iii) 13 801 human CTCF-binding sites identiﬁed by Ren and coworkers using ChIP-chip assay (20), denoted by identiﬁers starting with ‘INSUL_REN’ and (iv) 20 262 human CTCF-binding sites identiﬁed by Zhao and coworkers using massive direct sequencing of ChIP DNA (23), denoted by identiﬁers starting with ‘INSUL_ZHAO’. Second, we collected the conserved CTCF-speciﬁc sequence motifs ( 20 bp) in the human and mouse genomes that were predicted using motif scan (20,22). We excluded those ( 40%) overlapped with any of the experimentally determined CTCF-binding sites. The resulting 18 905 entries include 7736 human and 5504 mouse CTCF- binding sites predicted in (20) and 5665 human CTCF- binding sites predicted in (22). The computationally predicted CTCF-binding sites have identiﬁers beginning with ‘INSUL_PRE’. Annotation of the CTCF-binding sites Table 1 shows the major data ﬁelds of the database. For the 110 manually curated CTCF-binding sites, we used a set of controlled vocabularies to describe their properties. The ‘Validation Method’ ﬁeld speciﬁes whether CTCF binding was validated by in vitro and/or in vivo assays and whether this CTCF-binding sequence showed enhancer-blocking function in transgenic experi- ment (24). The ‘In situ Function’ ﬁeld annotates the biological roles of a CTCFBS in its natural genomic context (enhancer-blocking, chromatin boundary, etc.). The ‘Description’ ﬁeld contains other features of the CTCF-binding site (e.g. methylation-sensitivity of the CTCF binding). Genomic coordinates of the CTCF- binding sequences were determined using the BLAT alignment program (25). The assemblies of genomes used are hg18 for human, mm8 for mouse, rn3 for rat and galGal2 for chicken. CTCF-binding sequences without chromosome location information usually mean that they were probably mapped to unsequenced portions (e.g. heterochromatic regions) of the genome (21). Sequence features of the CTCF-binding sites The sequences of CTCF-binding sites in the database vary from 20 bp to several hundred bp. There are two reasons for this length heterogeneity. First, diﬀerent experimental methods may have diﬀerent basepair resolutions for locating CTCF-binding sites. Second, diﬀerent labora- tories often have diﬀerent research goals when they publish the original sequences. Some researchers may stop at a 500-bp region encapsulating the CTCF-binding Table 1. Description of the ﬁelds Field name IDa Speciesa Name Chromosome location Orientation 50-Flanking gene 30-Flanking gene Validation methoda In situ function Description Referencea Sequencea aA mandatory ﬁeld. Description Unique identiﬁer of an entry Species name Name used by the authors in the original paper CTCF-binding site position Forward (+) or reverse ( ) strand 50-Flanking gene of the CTCF-binding site along the genome 30-Flanking gene of the CTCF-binding site along the genome The validation methods including in vitro binding, in vivo binding, enhancer-blocking assay and sequence analysis In situ function of the CTCF-binding site Other important features of the CTCF-binding site PubMed reference DNA sequence of the CTCF-binding site sites while others may further narrow down to the sequences covered by the CTCF protein physically. Most CTCF-binding sites were found to share a 20-bp motif (20), which is highlighted using consecutive arrows. The direction of the arrows shows the genomic orienta- tion of the motif. We also highlighted all the single nucleotide polymorphisms (SNP) in the dbSNP database (26) that are located in a CTCF-binding site using vertical indicators. Mutations that disrupt CTCF-binding sites may lead to abnormal gene expression and cause diseases. Indeed, a recent study showed that inherited muta- tions that abolish CTCF-binding sites in the human H19 diﬀerentially methylated region (DMR) can cause Beckwith–Wiedemann syndrome (27,28). Thus, the natu- rally occurring mutations in CTCF-binding sites may represent new types of genetic variations that underlie phenotypes including disease status. To get more infor- mation about any of the SNPs, the user can click the SNP indicator to browse the corresponding dbSNP webpage (26). Genomic context track The genomic context of a CTCF-binding site provides clues for its in situ functions. The CTCF-binding site (red) and ﬂanking genes within 100 kb distance are displayed using the UCSC genome browser (29) (Figure 1). Other CTCF-binding sites located in this genomic region are also displayed and diﬀerent colors are used to distin- guish the sources of the CTCF-binding sites: yellow for INSUL_MAN, blue for INSUL_OHL, green for INSUL_REN, cyan for INSUL_ZHAO and black for INSUL_PRE. An important function of CTCF- bound insulators is to demarcate transcriptionally active and silent chromatin domains, which are marked by distinct histone methylation patterns. A recent study provided high-resolution maps of histone methylations (chromatin domains) in the human genome (23). H3K4 trimethylation (H3K4me3) and H3K27 trimethylation