ИСТИНА

Cohesin­driven extrusion of a chromatin fiber is one of key mechanisms of genome folding in mammalian interphase cells. Extrusion terminates at convergently orientated CTCF binding sites resulting in chromatin loop formation. This mechanism is also involved in control of local epigenetic landscape and gene expression. A number of pathologies are characterized by alterations in the 3D genome. In particular, changes in CTCF binding profile may rewrite distant regulatory contacts between enhancers and promoters, perturbing normal gene expression. Thus, targeted CTCF recruitment to pathology­associated genome loci could be an instrument for the precise engineering of the 3D genome and transcription control. Here, we fused four truncated variants of CTCF with dCas9 and GFP to construct chimeric proteins capable of extrusion termination at selected genome positions. We found that the recruitment of a chimeric protein based on the N­terminal domain and two Zn­finger domains of CTCF results in restoration of a chromatin loop, lost after deletion of an endogenous CTCF­binding site (CBS). Recruitment of this chimeric protein into internal regions of HOXD locus leads to the de novo formation of stripes and chromatin loops with surrounding cohesin/CTCF­bound regions. Interestingly, this also decreased H3K27me3 levels at the recruitment point. We also observed expression changes for several genes of the locus. Truncated CTCF variants do not bind to endogenous CBSs that makes them precise tools for manipulating chromatin loops. This work is supported by the Russian Science Foundation (grant # 24­44­00023). This work was also supported by FEBS Summer Fellowship 2020.