Control of directionality of chromatin folding for the inter- and intra-domain contacts at the Tfap2c–Bmp7 locus

Taro Tsujimura, Osamu Takase, Masahiro Yoshikawa, Etsuko Sano, Matsuhiko Hayashi, Tsuyoshi Takato, Atsushi Toyoda, Hideyuki Okano, Keiichi Hishikawa

Contact domains of chromatin serve as a fundamental unit to regulate action of enhancers for target genes. Looping between a pair of CCCTC-binding factor (CTCF)-binding sites in convergent orientations underlies the formation of contact domains, while those in divergent orientations establish domain boundaries. However, every CTCF site is not necessarily engaged in loop or boundary structures, leaving functions of CTCF in varied genomic contexts still elusive. The locus containing Tfap2c and Bmp7 encompasses two contact domains separated by a region between the two genes, termed transition zone (TZ), characterized by two arrays of CTCF sites in divergent configuration. In this study, we created deletion and inversion alleles of these and other regions across the locus and investigated how they impinge on the conformation. Deletion of the whole two CTCF arrays with the CRISPR/Cas9 system resulted in impairment of blocking of chromatin contacts by the TZ, as assessed by the circular chromatin conformation capture assay (4C-seq). Deletion and inversion of either of the two arrays similarly, but less pronouncedly, led to reduction in the blocking activity. Thus, the divergent configuration provides the TZ with the strong boundary activity. Uniquely, we show the TZ harbors a 50-kb region within one of the two arrays that contacts broadly with the both flanking intervals, regardless of the presence or orientation of the other CTCF array. Further, we show the boundary CTCF array has little impact on intra-domain folding; instead, locally associating CTCF sites greatly affect it. Our results show that the TZ not only separates the two domains, but also bears a wide interval that shows isotropic behavior of chromatin folding, indicating a potentially complex nature of actual boundaries in the genome. We also show that CTCF-binding sites inside a domain greatly contribute to the intra-domain folding of chromatin. Thus, the study reveals diverse and context-dependent roles of CTCF in organizing chromatin conformation at different levels.