High-resolution CTCF footprinting reveals impact of chromatin state on cohesin extrusion [HiChIP]. High-resolution CTCF footprinting reveals impact of chromatin state on cohesin extrusion [HiChIP]

Cohesin-mediated DNA loop extrusion enables gene regulation by distal enhancers through the establishment of chromosome structure and long-range enhancer-promoter interactions. The best characterized cohesin-related structures, such as topologically associating domains (TADs) anchored at convergent CTCF binding sites, represent static conformations. Consequently, loop extrusion dynamics remain poorly understood. To better characterize static and dynamically extruding chromatin loop structures, we use MNase-based 3D genome assays to simultaneously determine CTCF and cohesin localization as well as the 3D contacts they mediate. Here we present CTCF Analyzer (with) Multinomial Estimation (CAMEL), a tool that identifies CTCF footprints at near base-pair resolution in CTCF MNase HiChiP. We also use Region Capture Micro-C to identify a CTCF-adjacent footprint that is attributed to cohesin occupancy. We leverage this substantial advance in resolution to determine that the fully extruded (CTCF-CTCF loop) state is rare genome-wide with locus-specific variation from ~1-10%. We further investigate the impact of chromatin state on loop extrusion dynamics and find that active regulatory elements impede cohesin extrusion. These findings support a model of topological regulation whereby the transient, partially extruded state facilitates enhancer-promoter contacts that can regulate transcription. Overall design: We employed K562 CTCF MNase HiChIP to footprint CTCF & cohesin and investigate cohesin extrusion dynamics at the fragment level. We additionally re-sequenced the mESC RCMC libraries from Goel et al. Nature Genetics 2023 to 150bp reads (WT, DMSO, and IAA conditions for Fbn2, Klf1, and Ppm1g loci) to verify the cohesin footprint we observed in CTCF HiChIP.

黏连蛋白（Cohesin）介导的DNA环挤出（DNA loop extrusion）通过塑造染色体结构并建立远距离增强子-启动子相互作用，实现远端增强子对基因表达的调控。研究最为深入的黏连蛋白相关结构，如锚定在收敛型CCCTC结合因子（CTCF）结合位点的拓扑关联结构域（topologically associating domains, TADs），均属于静态构象。因此，环挤出的动态机制仍有待深入解析。为了更好地表征静态及动态环出的染色质环结构，本研究采用基于微球菌核酸酶（MNase）的三维基因组检测技术，同时确定CTCF与黏连蛋白的基因组定位，以及二者所介导的三维染色质相互作用。本研究开发了基于多项式估计的CTCF分析工具（CTCF Analyzer with Multinomial Estimation, CAMEL），可在接近碱基对分辨率的水平上，从CTCF MNase HiChiP数据中识别CTCF足迹。本研究还采用区域捕获Micro-C技术，鉴定出一处紧邻CTCF的足迹，该足迹可归因于黏连蛋白的基因组占据。借助分辨率上的这一重大突破，本研究确定：全基因组范围内完全环出的（CTCF-CTCF环）状态极为罕见，且存在位点特异性差异，占比约为1%至10%。本研究进一步探究了染色质状态对环挤出动态过程的影响，发现活性调控元件会阻碍黏连蛋白的环挤出行为。上述研究结果支持一种拓扑调控模型：瞬时存在的部分环出状态可促进增强子-启动子相互作用，进而调控基因转录。整体实验设计：本研究采用K562细胞系的CTCF MNase HiChiP技术，实现CTCF与黏连蛋白的足迹分析，并在片段水平上探究黏连蛋白的环挤出动态过程。此外，本研究对Goel等人2023年发表于《Nature Genetics》的小鼠胚胎干细胞（mouse embryonic stem cells, mESC）区域捕获Micro-C（RCMC）文库进行了重测序，将读长延长至150bp，涵盖Fbn2、Klf1及Ppm1g基因座的野生型（Wild Type, WT）、二甲基亚砜（DMSO）处理及吲哚乙酸（IAA）处理三组实验条件，以验证我们在CTCF HiChiP中观测到的黏连蛋白足迹。