Analysis of the vertebrate insulator protein CTCF binding sites in the human genome

Insulators are an important class of transcriptional regulatory sequences that affect gene expression by preventing the spread of heterochromatin and restricting how transcriptional enhancers select their target genes. The vertebrate insulators function by interacting with the CCCTC-binding factor (CTCF), an evolutionarily conserved DNA binding protein. Knowledge of the CTCF binding sites in the human genome is crucial for identifying potential insulators and understanding the role of these elements in regulating genome expression in the human cells. Here, we report the identification and characterization of a large number of CTCF binding sites in the human genome. Using chromatin immunoprecipitation combined with high-resolution genome-tiling arrays, we have determined the locations of CTCF throughout the non-repetitive human DNA sequences, identifying 13,804 binding sites in the genome of human fibroblasts. Computational analysis of these sequences revealed a single dominant CTCF consensus motif that is shared by a vast majority of the CTCF binding sites and is highly conserved in other vertebrate genomes. The CTCF binding segments the human genome into 5,969 domains, which contain an average of 2.5 genes each. Many large gene families, such as the olfactory receptor genes, are bounded by the CTCF sites, implicating a role of CTCF in the regulation or evolution of these genes. We demonstrate that CTCF binding can occur in a cell type dependent manner, and that binding of CTCF between two neighboring genes coincides with a loss of coordinated expression, consistent with their role as insulators. Our results provide a general resource and framework for analyzing the role of CTCF and insulator elements in the regulation of nearly every gene in the human genome. Keywords: ChIP-chip Chromatin immunoprecipitation was performed using monoclonal antibodies against CTCF and formaldehyde crosslinked chromatin from primary human fibroblast, IMR90 cells. The immunoprecipitated DNA was amplified, fluorescently labeled and hybridized to a series of 38 arrays containing a total of 14.6 million 50-mer oligonucleotides. These oligonucleotides are evenly positioned every 100 basepairs (bp) to cover the non-repeat sequence of the entire human genome.