Characterization of a cluster of CTCF-binding sites in a protocadherin regulatory region

The mammalian clustered protocadherin (Pcdh) locus contains more than 50 highly-similar genes arrayed in tandem. These Pcdh genes are organized into three closely-linked clusters (Pcdhα, Pcdhβ, and Pcdhγ). The encoded PCDH proteins play critical roles in neuronal diversity, single cell identity, and synaptic connectivity. Recent studies revealed that directional CTCF (CCCTC-binding factor) binding to CBS (CTCF-binding site) determines the specific interaction between enhancers and promoters, and the three Pcdhβγ clusters form two CCDs (CTCF/cohesin- mediated chromatin domain) which is important for gene regulation. Here, we characterized a regulatory region, which contains several HSs (DNase I hypersensitive sites), downstream of the Pcdhβ clusters. Specifically, CTCF ChIP-Seq (Chromatin immunoprecipitation-sequencing) experiments revealed several peaks in the Pcdhβγ regulatory regions. In addition, we performed comprehensive EMSA (Electrophoresis mobility shift assay) experiments and identified exact 42-bp CBS sites for each CTCF peak. Interestingly, we found that single CTCF peaks can contain two CBS sites. Finally, genome-wide computational analyses revealed many CBS sites close to chromatin marks of regulatory elements, such as enhancers and promoters. We propose that CTCF may mediate specific interactions between enhancers and promoters to form active transcription hub for gene expression.