Genome-wide analyses of chromatin interactions after the loss of Pol I, Pol II and Pol III [ATAC-seq]

Genome binding/occupancy profiling by high throughput sequencing ｜ Expression profiling by high throughput sequencing | Other We analyzed Hi-C, HiChIP, Ocean-C, RNA-seq, ATAC-seq, ChIP-seq and 4C-seq data to provide the most comprehensive evidence to date to demonstrate that transcription plays a marginal role in organizing the 3D genome in mammalian cells: 1) degraded Pol I, Pol II and Pol III proteins in mESCs, and showed their loss results in little or no changes of global 3D chromatin structures for the first time; 2) selected RNA polymerases high abundance binding sites-associated interactions and found they still persist after the degradation; 3) generated higher resolution chromatin interaction maps and revealed that transcription inhibition mildly alters small loop domains; 4) identified Pol II bound but CTCF and Cohesin unbound loops and disclosed that they are largely resistant to transcription inhibition Interestingly, we found that Pol II depletion for a longer time significantly affects the chromatin accessibility and Cohesin occupancy, suggesting RNA polymerases are capable of affecting the 3D genome indirectly. So, the direct and indirect effects of transcription inhibition explain the previous confusing effects on the 3D genome. We conclude that Pol I, Pol II, and Pol III loss only mildly alter chromatin interactions in mammalian cells, suggesting the 3D chromatin structures are preestablished and relatively stable. We have finished the Hi-C, HiChIP, Ocean-C, RNA-seq, ATAC-seq, ChIP-seq and 4C-seq to investigate the roles of Pol I, Pol II and Pol III in 3D genome organization and transcription. The V6.5 mouse ES (mES) cell line was used to perform all the high-throughput analysis. Degron mES cells were treated with 1 μg/ml Doxycycline for different hours refer to individual Series.

高通量测序基因组结合/占据谱分析 | 高通量测序表达谱分析 | 其他
本研究通过分析高通量染色体构象捕获（Hi-C）、HiChIP测序（HiChIP）、Ocean-C测序（Ocean-C）、RNA测序（RNA-seq）、转座酶可及性测序（ATAC-seq）、染色质免疫共沉淀测序（ChIP-seq）及环状染色体构象捕获测序（4C-seq）数据，提供了迄今为止最为全面的证据，证明转录过程在哺乳动物细胞三维基因组（3D genome）组织中仅发挥微弱作用：
1) 首次在小鼠胚胎干细胞（mESCs）中降解RNA聚合酶I（Pol I）、RNA聚合酶II（Pol II）与RNA聚合酶III（Pol III）蛋白，并证实这些聚合酶的缺失几乎不会或仅引发全局三维染色质结构的微小改变；
2) 选取与高丰度RNA聚合酶结合位点相关的染色质相互作用进行分析，发现即便聚合酶被降解后，此类相互作用仍可稳定存在；
3) 构建了更高分辨率的染色质相互作用图谱，并揭示转录抑制仅会轻微改变小型环状染色质结构域；
4) 鉴定出结合有Pol II但未结合CCCTC结合因子（CTCF）与黏连蛋白（Cohesin）的环状结构，并发现此类结构对转录抑制整体具有抗性。

值得注意的是，本研究发现长时间降解Pol II会显著影响染色质开放程度与黏连蛋白的占据情况，提示RNA聚合酶可通过间接途径影响三维基因组结构。因此，转录抑制的直接与间接效应可解释此前学界对三维基因组所观察到的矛盾现象。

本研究最终证实，RNA聚合酶I、II、III的缺失仅会轻微改变哺乳动物细胞内的染色质相互作用，提示三维染色质结构是预先建立且相对稳定的。本研究已完成上述Hi-C、HiChIP、Ocean-C、RNA-seq、ATAC-seq、ChIP-seq及4C-seq实验，以探究RNA聚合酶I、II、III在三维基因组组织与转录过程中的功能。

本研究采用V6.5小鼠胚胎干细胞（mESCs）系开展所有高通量测序分析。降解标签型mESCs经1 μg/ml强力霉素（Doxycycline）处理不同时长，具体时长详见各数据集系列。