Characterization of induced cohesin loop extrusion trajectories in living cells [BrU-seq]

The cohesin complex shapes chromosomes by DNA loop extrusion, but individual extrusion trajectories were so far unappreciable in vivo. Here, we developed and validated TArgeted Cohesin Loader (TACL), a system enabling the strong activation of anchored loop extrusion from dozens of defined genomic sites in living cells. Studying their individual loop extrusion trajectories revealed that extruding cohesin-STAG2 stops not only at domain boundaries but at all flanking CTCF sites, engaging them in a complex transient looping network that supports intradomain contacts. Cohesin-STAG1 cannot associate with weak CTCF binding sites and fails to similarly support intradomain interactions. NIPBL-MAU2 remains associated with cohesin when stalled at looping CTCF sites, suggesting these factors may also be required for loop stabilization. TACL induces cohesin traffic jams and illegal loops with divergent CTCF sites, demonstrating that stalled cohesin can block extruding cohesin in vivo. Genes exposed to TACL-induced loop extrusion were collectively hindered in transcription and the underlying chromatin altered its accessibility and reduced its H3K27ac marks. Thus, by enabling the study of individual loop extrusion trajectories in vivo, we could assign new functions to players, identify new looping networks and uncover an interplay between loop extrusion, gene transcription and chromatin composition. Overall design: ChIPseq of eHAP1 cells carying 27 TetO platforms expressing TetR-MAU2 (TACL) or TetR-mCherry, with and without doxycycline treatment and/or depletion of RAD21, CTCF, PDS5A, WAPL, STAG2 using auxin treatment.

黏连蛋白复合物（cohesin complex）通过DNA环挤出（DNA loop extrusion）重塑染色体，但迄今为止体内仍无法观测单个环挤出轨迹。本研究开发并验证了靶向黏连蛋白加载系统（Targeted Cohesin Loader, TACL），该系统可在活细胞中从数十个明确的基因组位点强力激活锚定的DNA环挤出过程。通过对单个环挤出轨迹的研究发现，正在执行环挤出的黏连蛋白-STAG2（cohesin-STAG2）不仅会在染色体结构域边界处停滞，还会在所有侧翼的CCCTC结合因子（CTCF）位点处停滞，并与这些位点形成复杂的瞬时环化网络，该网络可维持结构域内的染色质相互作用。黏连蛋白-STAG1（cohesin-STAG1）无法与弱结合的CTCF位点结合，因此无法像STAG2那样维持结构域内的染色质相互作用。当在发生环化的CTCF位点停滞时，NIPBL-MAU2仍与黏连蛋白保持结合，这提示这些因子可能也参与环挤出的稳定过程。TACL可诱导黏连蛋白交通拥堵以及由反向CTCF位点介导的异常环形成，证明体内停滞的黏连蛋白能够阻断正在进行环挤出的黏连蛋白。暴露于TACL诱导的环挤出过程中的基因，其转录整体受到抑制，同时其所在的染色质开放度发生改变，H3K27ac修饰水平降低。因此，通过实现体内单个环挤出轨迹的观测研究，我们可为相关调控因子赋予新的功能，鉴定出新的环化网络，并揭示环挤出、基因转录与染色质组成之间的相互调控关系。实验设计：对携带27个TetO平台的eHAP1细胞进行染色质免疫共沉淀测序（ChIP-seq），这些细胞分别表达TetR-MAU2（即TACL系统）或TetR-mCherry，并分别经/不经强力霉素处理，同时通过生长素处理（auxin treatment）分别敲除RAD21、CTCF、PDS5A、WAPL、STAG2。