Nucleic Acids Research, 2008, Vol. 36, Database issue D85 Figure 1. The genomic context of a few CTCF-binding sites. The CTCF-binding sites reside at the boundary between the two histone methylation domains (H3K4me3 and H3K27me3). (H3K27me3) are a pair of ‘Yin-Yang’ modiﬁcations with high level of H3K4me3 and H3K27me3, representing gene activation and silencing, respectively (23). We integrated H3K4me3 and H3K27me3 maps with our genomic context track of CTCF-binding sites using the genome browser to facilitate the utilization of this valuable information. Flanking gene expression track Another in situ function of insulators is to maintain independent expression patterns of neighboring genes. Suppose there is a tissue-speciﬁc enhancer that should control the transcription of one gene but not that of the other in a pair of neighboring genes. The CTCF-binding site located between the enhancer and the promoter of the second gene may function as enhancer-blocking insulator to protect against illegitimate transcriptional activation. In this scenario, the neighbor- ing genes may have very diﬀerent expression status in that tissue. We created a ﬂanking gene expression track to compare the expression patterns of the genes ﬂanking the CTCF-binding site. The data were obtained from The Genomics Institute of the Novartis Research Foundation (GNF) Gene Expression Atlas 2 (30), which contains genome-wide gene expression proﬁles of 61 mouse tissues and 79 human tissues. The raw data was log-transformed (base 2) and normalized to have a mean of 0 and SD of 1. The expression images were created using the Slcview software (http://slcview.stanford.edu), in which red indicates overexpression and green indicates underexpression. An example of gene expression track is shown in Figure 2. Mammalian orthologous region track Comparative genomic studies on human, mouse and rat may provide insights into the evolution of CTCF- binding sites. To this end, we created a track of mammalian orthologous regions. For any of the three genomes, the regions containing CTCF-binding sites and ﬂanking genes were used to query orthologous regions in the other two genomes from the UCSC precomputed block chains (31,32). Only the DNA blocks with the maximal alignment score against the query region were retained as orthologous regions. The aligned orthologous sequences in up to 16 vertebrate genomes can be displayed by clicking the ‘view alignment’ button (Figure 2). CTCF-binding site prediction CTCF uses diﬀerent combinations of its zinc ﬁngers to recognize divergent DNA sequences. Recent studies have identiﬁed core motifs for CTCFBS sequences (20,22). The motifs are represented by position weight matrices (PWM). Altogether, four closely related PWM have been derived to accommodate the sequence divergences in CTCF-binding sites (20,22). The database provides a simple web tool to search for the core CTCF-binding motifs in a query sequence. It uses the STORM program (33) to scan for each of the four PWM in the query sequences and reports the best hits. UTILITY AND DISCUSSION First, a web interface was developed for browsing the experimentally identiﬁed and computationally predicted CTCF-binding sites. Users can focus on entries of interest using four selection controls—Species, Validation Method, In Situ Function and Description. The in situ function of most known CTCF-binding sites is to act as boundary element. However, in some biological contexts, CTCF-binding sites may also function as elements for transcription activation/repression [reviewed in (34)]. Second, a text search interface was developed for querying the database. Users can search for CTCF-binding sites by element name or by the PubMed identiﬁer of the original literature. A useful approach is to retrieve the CTCF-binding sites contiguous to a gene of interest by entering an oﬃcial gene symbol or words used in the gene description. Third, the database provides sequence similarity search (35) for the comparison