Abstract
While regulation of gene-enhancer interaction is intensively studied, its application remains limited. Here, we reconstituted arrays of CTCF-binding sites and devised a synthetic topological insulator with tetO for chromatin-engineering (STITCH). By coupling STITCH with tetR linked to the KRAB domain to induce heterochromatin and disable the insulation, we developed a drug-inducible system to control gene activation by enhancers. In human induced pluripotent stem cells, STITCH inserted between MYC and the enhancer down-regulated MYC. Progressive mutagenesis of STITCH led to a preferential escalation of the gene-enhancer interaction, corroborating the strong insulation ability of STITCH. STITCH also altered epigenetic states around MYC. Time-course analysis by drug induction uncovered deposition and removal of H3K27me3 repressive marks follows and reflects, but does not precede and determine, the expression change. Finally, STITCH inserted near NEUROG2 impaired the gene activation in differentiating neural progenitor cells. Thus, STITCH should be broadly useful for functional genetic studies.
Highlights
Interaction of genes and enhancers is greatly affected by architectural proteins that bind to chromatin and organize folding of the genome (Dekker et al, 2017)
We embedded the core sequence of the tetracycline operator at four different positions within the cassette. tetO is bound by the tetracycline repressor, but not in presence of doxycycline (DOX), and allows recruitment of an effector protein to the cassette in a drug-dependent manner (Gossen and Bujard, 1992)
We expect that the CTCF binding sites of the cassette would recruit CTCF and function as a topological insulator, and that the tetO/tetracycline repressor (tetR) system would enable epigenetically modifying the insulation activity
Summary
Interaction of genes and enhancers is greatly affected by architectural proteins that bind to chromatin and organize folding of the genome (Dekker et al, 2017). According to the extrusion model, the cohesin ring extrudes the chromatin fiber from a loaded site and pauses at a CTCF binding site that is oriented towards the ring (Fudenberg et al, 2016; Sanborn et al, 2015). This model is widely accepted as the underlying mechanism for the formation of the loops and contact domains. CTCF demarcates contact domains and interferes with gene-enhancer interaction when located in between, though the mechanism of the interference of the interaction is not entirely clear
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