The establishment of a CTCF activity reporter for systematic stratification of CTCF binding sites

While the elements encoding enhancers and promoters have been relatively well studied, the full spectrum of insulator elements which bind the CCCTC binding factor (CTCF), is relatively poorly characterised. In part, this is due the fact that their roles and activity is greatly influenced by their genomic context. Here we have developed an experimental system to determine the ability of consistently sized, individual CTCF elements to interpose between enhancers and promoters and thereby reduce gene expression during differentiation. Importantly each element is tested in the identical location thereby minimising the effect of genomic context. We found no correlation between the ability of CTCF elements to block enhancer-promoter activity with the amount of CTCF or cohesin bound at the natural genomic sites of these elements; the degree of evolutionary conservation; or their resemblance to the consensus core sequences. Nevertheless, we have shown that the strongest enhancer-promoter blockers include a previously described bound element lying upstream of the CTCF core motif. In addition, we found other uncharacterised DNAse1 footprints located close to the core sequence that may affect function. We have developed a high throughput assay of CTCF elements which will enable researchers to sub-classify CTCF elements in an unbiased way. Overall design: Artificial CTCF binding sequences were genetically engineered in mouse ESCs. These mESCs were subjected to an in vitro erythroid differentiation protocol (doi: 10.1371/journal.pone.0261950) and purified CD71+ erythroid cells derived from differentiated Wildtype (WT) or mutant mouse Embryonic Stem (mES) cells were analysed by CTCF ChIP-seq.