Walsh Erin, Stephens Andrew D
Biology department, University of Massachusetts Amherst, Amherst, MA 01003, USA.
Molecular and Cellular Biology, University of Massachusetts Amherst, Amherst, MA 01003, USA.
bioRxiv. 2025 Jun 3:2025.05.30.657091. doi: 10.1101/2025.05.30.657091.
In mitosis the duplicated genome is aligned and accurately segregated between daughter nuclei. CTCF is a chromatin looping protein in interphase with an unknown role in mitosis. We previously published data showing that CTCF constitutive knockdown causes mitotic failure, but the mechanism remains unknown. To determine the role of CTCF in mitosis, we used a CRISPR CTCF auxin inducible degron cell line for rapid degradation. CTCF degradation for 3 days resulted in increased failure of mitosis and decreased circularity in post-mitotic nuclei. Upon CTCF degradation CENP-E is still recruited to the kinetochore and there is a low incidence of polar chromosomes which occur upon CENP-E inhibition. Instead, immunofluorescence imaging of mitotic spindles reveals that CTCF degradation causes increased intercentromere distances and a wider and more disorganized metaphase plate, a disruption of key functions of the pericentromere. These results are similar to partial loss of cohesin, an established component of the pericentromere. Thus, we reveal that CTCF is a key maintenance factor of pericentromere function, successful mitosis, and post-mitotic nuclear shape.
在有丝分裂过程中，复制后的基因组会排列整齐，并精确地分配到子细胞核之间。CTCF是一种在间期发挥作用的染色质环化蛋白，其在有丝分裂中的作用尚不清楚。我们之前发表的数据表明，CTCF的组成性敲低会导致有丝分裂失败，但其机制仍不明确。为了确定CTCF在有丝分裂中的作用，我们使用了一种CRISPR CTCF生长素诱导降解细胞系来实现快速降解。CTCF降解3天后，有丝分裂失败的情况增加，有丝分裂后细胞核的圆形度降低。CTCF降解后，CENP-E仍会被招募到动粒上，并且在CENP-E受到抑制时出现极性染色体的发生率较低。相反，有丝分裂纺锤体的免疫荧光成像显示，CTCF降解会导致着丝粒间距离增加，中期板更宽且更无序，这是着丝粒周围关键功能的破坏。这些结果与黏连蛋白部分缺失的情况相似，黏连蛋白是着丝粒周围的一个既定组成部分。因此，我们揭示了CTCF是着丝粒周围功能、成功的有丝分裂和有丝分裂后细胞核形状的关键维持因子。
