Tiukacheva Eugenia A, Luzhin Artem V, Kruglova Natalia, Shtompel Anastasia S, Antonov Grigorii, Tvorogova Anna, Vassetzky Yegor, Ulianov Sergey V, Razin Sergey V
Institute of Gene Biology, Moscow 119334, Russia.
Koltzov Institute of Developmental Biology, Moscow 119334, Russia.
Int J Mol Sci. 2025 Aug 1;26(15):7446. doi: 10.3390/ijms26157446.
Enhancer-promoter interactions occur in the chromatin loci delineated by the CCCTC-binding zinc-finger protein CTCF. CTCF binding is frequently perturbed in genetic disorders and cancer, allowing for misregulation of genes. Here, we developed a panel of chimeric proteins consisting of either full-length or truncated CTCF fused with programmable DNA-binding module dCas9 and fluorescent tracker EGFP. We found that the recruitment of a chimeric protein based on the CTCF N-terminal domain and two zinc-finger domains to the human locus leads to the de novo formation of a spatial contact with a nearby cohesin/CTCF-bound region, anchoring several chromatin loops. This chimeric protein did not show binding to CTCF motifs and did not affect the epigenetic and transcription profile of the locus. Recruitment of this chimeric protein is also able to restore chromatin loops, lost after deletion of an endogenous CTCF-binding site. Together, our data indicate that the ectopic recruitment of the CTCF N-terminal part could be an appropriate tool for 3D genome engineering.
增强子与启动子的相互作用发生在由CCCTC结合锌指蛋白CTCF划定的染色质位点中。在遗传疾病和癌症中，CTCF的结合经常受到干扰，从而导致基因调控异常。在此，我们开发了一组嵌合蛋白，其由全长或截短的CTCF与可编程DNA结合模块dCas9和荧光追踪器EGFP融合而成。我们发现，基于CTCF N端结构域和两个锌指结构域的嵌合蛋白募集到人基因座会导致与附近黏连蛋白/CTCF结合区域形成新的空间接触，锚定多个染色质环。这种嵌合蛋白不显示与CTCF基序的结合，并且不影响该基因座的表观遗传和转录谱。这种嵌合蛋白的募集还能够恢复在内源CTCF结合位点缺失后丢失的染色质环。总之，我们的数据表明，CTCF N端部分的异位募集可能是用于三维基因组工程的合适工具。
