Contribución cooperativa de los promotores de genes del desarrollo y de los clústeres de CTCF al aislamiento de los TADs en humanos

Abstract

Chromatin is organized in self-interacting regions called topologically associating domains (TADs), which ensure proper interactions between enhancers and target promoters found within the same domain, thanks to the presence of CTCF binding sites at their boundaries that act as insulators. In a recent study, the Rada-Iglesias lab showed that in mice the promoters of developmental genes can cooperate with CTCF clusters to robustly insulate their own regulatory domains. In the current project, using SIX3/SIX2 as representative developmental locus, we generated human induced pluripotent stem cells (hiPSC) with various genomic re-arrangements and, upon differentiation of these hiPSC into forebrain-like neural progenitors (Fb-NPC), we demonstrate that the recently discovered mechanism is conserved in humans. To address the medical relevance of this novel regulatory mechanism, we generated hiPSC with deletions identified in frontonasal dysplasia-like phenotypes (FND) patients spanning SIX2 gene and the CTCF cluster separating SIX2 and SIX3 TADs. In the future, these hiPSC will be differentiated into neural crest cells (NCC), an embryonic cell type responsible of craniofacial development, to assess whether these deletions lead to ectopic expression of SIX3 in NCC. If this is confirmed, it would reveal that by disrupting the cooperative insulation provided by SIX2 promoter competition and CTCF-dependent physical insulation, the investigated deletions could cause FND-like phenotypes through ectopic activation of SIX3 in NCC. Overall, our results suggest that CTCF binding sites and developmental gene promoters cooperate in the insulation of their domains in humans, and this regulatory mechanism may explain the pathological effects of some structural variants identified in patients.