Viviendo al límite: ¿Importa la posición de los genes dentro de los TADs?

Abstract

ABSTRACT : The genome is organized in large (Mb-scale) self-interacting domains that have termed as topological association domains (TADs). TADs are considered fundamental regulatory units that favor the interactions between regulatory elements (e.g. enhancers) and genes located within the same domain, while insulating genes from contacting ectopic regulatory elements located in neighboring domains. Previous analyses in the Rada-Iglesias laboratory revealed that developmental genes tend to be located in the borders of TADs close to CTCF binding sites (CBS), something that had not been reported to date. Since the modification of TAD organization can cause various diseases, we decided to investigate whether the positioning of genes near TAD boundaries was of any functional relevance. We performed CRISPR/Cas9-based genetic engineering of the Six3/Six2 locus. Six3 and Six2 are located at the border of two neighboring TADs separated by a large cluster of CBS. Moreover, Six3 becomes activated upon differentiation of ESC into AntNPC, while Six2 is inactive in both cell types. Deleting the CTCF boundary between both genes, we observed that, after differentiation to AntNPC, the expression of Six2 increased significantly, confirming that the boundary prevents the ectopic activation of Six2 by Six3 enhancers. More interestingly, we found that the positioning of Six3 close to the TAD boundary increases insulation, since by removing Six3 plus the CTCF boundary, we showed that the expression of Six2 increased significantly compared to the line where only the CTCF boundary was deleted. Finally, we showed that the gene itself does not benefit from being on the border of TAD, since after performing an inversion and placing Six3 in the middle of the TAD, its expression was not affected. Overall, these results demonstrate that CBS and gene positioning at TAD borders synergistically control the insulation of developmental regulatory domains.