Three-dimensional genome architectural CCCTC-binding factor makes choice in duplicated enhancers at Pcdha locus. [ChIP-seq I]

By CRISPR/Cas9 DNA-fragment editing, in conjunction with chromosome conformation capture, ChIP-seq and RNA-seq technology, we studied CRISPR single cell clones of duplicated CBS-containing enhancers or promoters in the Pcdha gene cluster. We found that CTCF plays an essential role in determining promoter choice and enhancer selection. In addition, the choices mediated by CTCF is based on the chromatin loops formed between forward-reverse convergent CBS pairs with the proximal one dominant. Overall design: ChIP-seq, 4C-seq and RNA-seq experiments were performed on both wildtype and DNA-fragment editting cell lines (mE1 insertion, mE2 insertion and ac1 repeat). For ChIP-seq experiments, Binding patterns of CTCF and Cohesin complex(showed by Rad21) as well as histone modification marker H3K27ac were examined. For 4C-seq experiments, the 3D chromatin conformations of several viewpoints were captured by 4C (Circular Chromatin Conformation Capture) method, in duplicate. For RNA-seq experiments, the mRNA profiles of these cell lines were generated by deep sequencing, in duplicate.