A cohesin traffic pattern genetically linked to gene regulation [Hi-C]

Cohesin-mediated loop extrusion folds interphase chromosomes at the ten to hundreds kilobases scale. This process produces structural features such as loops and topologically associating domains. We identify three types of cis-elements that define the chromatin folding landscape generated by loop extrusion. First, CTCF sites form boundaries by stalling extruding cohesin, as shown before. Second, transcription termination sites form boundaries by acting as cohesin unloading sites. RNA polymerase II contributes to boundary formation at transcription termination sites. Third, transcription start sites form boundaries that are mostly independent of cohesin, but are sites where cohesin can pause. Together with cohesin loading at enhancers, and possibly other cis-elements, these loci create a dynamic pattern of cohesin traffic along the genome that guides enhancer-promoter interactions. Disturbing this traffic pattern, by removing CTCF barriers, makes cells sensitive to deletion of genes involved in transcription initiation, such as the SAGA and TFIID complexes, and RNA processing such DEAD-Box RNA helicases. In the absence of CTCF, several of these factors fail to be efficiently recruited to active promoters. We propose that the complex pattern of cohesin movement along chromatin contributes to appropriate promoter-enhancer interactions and localization of transcription and RNA processing factors to active genes. Overall design: We engineered a HAP1-derived human cell line in which CTCF or RNApolII (RPB1) could efficiently be removed using an auxin-inducible degron system. We depleted the DDX55 and TAF5L proteins in HAP1-CTCFdegron-TIR1 cells in two ways: siRNA and knock-outs. We performed Hi-C on control cells, cells depleted for CTCF or RPB1 or DDX55 or TAF5L or combinations of CTCF depletions with DDX55 or TAF5L depletion.

黏连蛋白（Cohesin）介导的环挤出（loop extrusion）可将间期染色体折叠至10至数百千碱基的尺度。该过程会形成环状结构与拓扑关联结构域（topologically associating domains）等染色质结构特征。我们鉴定出三类顺式作用元件（cis-elements），可调控环挤出过程所产生的染色质折叠景观：其一，CTCF位点可通过阻滞正在进行环挤出的黏连蛋白来形成边界，这一点此前已被证实；其二，转录终止位点可通过充当黏连蛋白的卸载位点来形成边界，RNA聚合酶II（RNA polymerase II）可助力转录终止位点处的边界形成；其三，转录起始位点所形成的边界大多不依赖于黏连蛋白，但可作为黏连蛋白的暂停位点。这些位点与增强子及其他潜在顺式作用元件处的黏连蛋白加载过程共同作用，在基因组上形成了动态的黏连蛋白流动模式，进而指导增强子-启动子相互作用。若通过移除CTCF屏障破坏这种流动模式，细胞会对转录起始相关基因（如SAGA与TFIID复合物）以及RNA加工相关基因（如DEAD盒RNA解旋酶）的缺失变得敏感。在缺乏CTCF的情况下，其中多种因子无法被有效招募至活性启动子区域。我们提出，黏连蛋白沿染色质移动的复杂模式，可助力实现恰当的增强子-启动子相互作用，并将转录与RNA加工因子精准定位至活性基因处。实验设计概述：我们构建了一株HAP1衍生的人类细胞系，该细胞系中的CTCF或RNA聚合酶II（RPB1）可通过生长素诱导降解系统（auxin-inducible degron system）被高效去除。我们通过两种方式在HAP1-CTCFdegron-TIR1细胞中敲除或敲降DDX55与TAF5L蛋白：小干扰RNA（siRNA）敲降与基因敲除。我们对对照组细胞、单独敲除CTCF、RPB1、DDX55或TAF5L的细胞，以及同时敲除CTCF与DDX55或CTCF与TAF5L的细胞均进行了Hi-C实验。