Consequences of TAD's borders deletion in Pdgfra/Kit/Kdr locus

It is generally accepted that topologically associated domains (TADs) participate in genes expression regulation by bringing enhancer and promoter into proximity and maintaing their interaction. Whether TADs restrict ectopic enhancer interactions, thus exhibiting an insulation function, remains unclear. To explore the question we studied the role of 3D genome organization in the regulation of the Pdgfra/Kit/Kdr locus. The locus encodes tyrosine-kinase receptors, which are involved in cell differentiation processes. We hypothesized that removal of TADs boundaries would provide ectopic enhancer interactions leading to aberrant genes expression in an unfamiliar cell type. The mouse strains, carrying CRISPR/Cas9-mediated deletions of the key boundary CTCF-binding sites, and C57BL/6 mice as a control were used to obtain cell cultures (fibroblasts, mast cells, melanocytes). In order to analyse 3D genome organisation of the locus in the cell cultures we performed capture Hi-C and CTCF ChIP-seq, as well as H3K27Ac ChIP-seq to distinguish the enhancer pattern. To analyse a possible transcriptional change we obtained RNA from the cell cultures and proceeded with RNA-seq. For the boundary encompassing Pdgfra and Kit TADs, the deletion was not enough to disrupt the TADs insulation, as well as to activate Kit expression in fibroblasts. For the boundary encompassing Kit and Kdr TADs, the deletion revealed a tissue-specific influence. In mast cells the deletion led to a gain of inter-TADs contacts, but the Kit remained insulated from Kdr and no Kdr activation was detected. Conversely, in melanocytes the deletion did lead to TADs fusion and, most interestingly, to a noticeable activation of Kdr expression. There are at least two factors for such a difference. Firstly, unlike the mast cells, the melanocytes do express Kdr slightly. Secondly, in the mast cells Kit enhancers locate upstream of the Kit, while in the melanocytes - downstream, closer to Kdr. We presume that an epigenetic status of the locus and an enhancer location might contribute to TADs fusion restriction.