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Abstract：
CCCTC-binding factor (CTCF) and cohesin play critical roles in organizing mammalian genomes into topologically associating domains (TADs). Here, by combining genetic engineering with quantitative super-resolution stimulated emission depletion (STED) microscopy, we demonstrate that in living cells, CTCF forms clusters typically containing 2-8 molecules. A fraction of CTCF clusters, enriched for those with ≥3 molecules, are coupled with cohesin complexes with a characteristic physical distance suggestive of a defined molecular interaction. Acute degradation of the cohesin unloader WAPL or transcriptional inhibition (TI) result in increased CTCF clustering. Furthermore, the effect of TI on CTCF clusters is alleviated by the acute loss of the cohesin subunit SMC3. Our study provides quantitative characterization of CTCF clusters in living cells, uncovers the opposing effects of cohesin and transcription on CTCF clustering, and highlights the power of quantitative super-resolution microscopy as a tool to bridge the gap between biochemical and genomic methodologies in chromatin research.
摘要：
CCCTC结合因子（CTCF）和粘附素在将哺乳动物基因组组织成拓扑关联域（TAD）中起关键作用。这里,通过将基因工程与定量超分辨率受激发射损耗（STED）显微镜相结合，我们证明在活细胞中，CTCF形成通常含有2-8个分子的簇。CTCF集群的一小部分，富集那些≥3个分子，与具有特征性物理距离的粘附蛋白复合物相结合，暗示了定义的分子相互作用。粘附素卸载器WAPL或转录抑制（TI）的急性降解导致CTCF聚类增加。此外，粘附素亚基SMC3的急性丢失减轻了TI对CTCF簇的影响。我们的研究提供了活细胞中CTCF簇的定量表征，揭示了粘附素和转录对CTCF聚类的相反影响，并强调了定量超分辨率显微镜作为弥合染色质研究中生化和基因组方法之间差距的工具的力量。
