CTCF sites display cell cycle-dependent dynamics in factor binding and nucleosome positioning
CTCF sites display cell cycle-dependent dynamics in factor binding and nucleosome positioning
复制标题
DOI：
10.1101/gr.241547.118
复制
发表时间：
2019-02-01
期刊：
影响因子：
7
通讯作者：
Dekker, Job
中科院分区：
文献类型：
--
作者：
Oomen, Marlies E.;Hansen, Anders S.;Dekker, Job
CCCTC结合因子(CTCF)在相间拓扑结合域(TADS)和环的形成中起着关键作用。在有丝分裂期间，TAD不存在，但TAD的形成在细胞周期中如何动态控制尚不清楚。关于CTCF与有丝分裂染色质的结合，使用基因组学和显微镜技术已经有了几个相互矛盾的观察结果。在这里，我们使用了四种不同的分析来解决这场辩论。首先，利用5C，我们证实TADS和CTCF环在间期很容易被检测到，但在前中期没有。其次，ATAC-seq分析表明，CTCF位点的可及性大大降低，在前中期失去了CTCF足迹，这表明CTCF结合的丢失和结合基序周围的核小体阵列的重排。相反，转录起始点在前中期仍然可访问，尽管相邻的核小体也可以被重新定位，并在有丝分裂期间至少占据起始点的一个子集。第三，用Cut&Run直接证明了特异性CTCF结合的丢失。组蛋白修饰和组蛋白变体在有丝分裂中保持不变，这表明在活性CTCF位点的书签中起到了作用。最后，活细胞成像、光漂白后的荧光恢复和单分子示踪表明，几乎所有的CTCF染色质结合在前中期都丢失了。综上所述，我们的结果表明，CTCF在前中期失去了与CTCF位点的结合，并在CTCF基序周围重排了染色质景观。这一点，再加上粘附素的丢失，将有助于在有丝分裂过程中观察到的TADS和CTCF环的丢失，并揭示了CTCF位点，即关键的结构顺式元件，在因子结合和核小体定位方面表现出细胞周期阶段依赖的动力学。
CCCTC-binding factor (CTCF) plays a key role in the formation of topologically associating domains (TADs) and loops in interphase. During mitosis TADs are absent, but how TAD formation is dynamically controlled during the cell cycle is not known. Several contradicting observations have been made regarding CTCF binding to mitotic chromatin using both genomics- and microscopy-based techniques. Here, we have used four different assays to address this debate. First, using 5C, we confirmed that TADs and CTCF loops are readily detected in interphase, but absent during prometaphase. Second, ATAC-seq analysis showed that CTCF sites display greatly reduced accessibility and lose the CTCF footprint in prometaphase, suggesting loss of CTCF binding and rearrangement of the nucleosomal array around the binding motif. In contrast, transcription start sites remain accessible in prometaphase, although adjacent nucleosomes can also become repositioned and occupy at least a subset of start sites during mitosis. Third, loss of site-specific CTCF binding was directly demonstrated using CUT&RUN. Histone modifications and histone variants are maintained in mitosis, suggesting a role in bookmarking of active CTCF sites. Finally, live-cell imaging, fluorescence recovery after photobleaching, and single molecule tracking showed that almost all CTCF chromatin binding is lost in prometaphase. Combined, our results demonstrate loss of CTCF binding to CTCF sites during prometaphase and rearrangement of the chromatin landscape around CTCF motifs. This, combined with loss of cohesin, would contribute to the observed loss of TADs and CTCF loops during mitosis and reveals that CTCF sites, key architectural cis-elements, display cell cycle stage-dependent dynamics in factor binding and nucleosome positioning.