RNA-seq transcriptonal profiling in human K562 cells and G1E-ER4 cells with or without dCas9 and sgRNAs

The spatiotemporal control of 3D chromatin structure is fundamental for gene regulation, yet it remains challenging to obtain high-resolution chromatin interacting profiles at cis-regulatory elements (CREs) by chromatin conformation capture (3C)-based methods. Here, we describe the redesigned dCas9-based CAPTURE method for multiplexed, high-throughput and high-resolution analysis of locus-specific chromatin interactions. Using C-terminally biotinylated dCas9, endogenous biotin ligase and pooled sgRNAs, the new system enables quantitative analysis of the spatial configuration of a few to hundreds of enhancers or promoters in a single experiment, enabling systematic comparisons across CREs within and between gene clusters. We reveal the hierarchical structure of super-enhancers (SEs) and distinct modes of SE-gene interactions. Multiplexed capture of temporal dynamics of promoter-centric interactions establishes the instructive function of enhancer-promoter looping in transcriptional regulation during lineage differentiation. These applications illustrate the ability of multiplexed CAPTURE for decoding the organizational principles of genome structure and function. RNA-seq was performed to determine the effects on global gene expression in K562 or G1ER-ER4 cells stably expressing dCas9-CBio and sgRNAs, or erythroid differentiation of G1ER-ER4 cells.

三维染色质结构的时空调控是基因调控的核心基础，但基于染色质构象捕获（chromatin conformation capture，3C）的技术手段，仍难以在顺式调控元件（cis-regulatory elements，CREs）上获取高分辨率的染色质互作图谱。本文报道了一种经过重新设计的基于dCas9的CAPTURE技术，可实现多位点、高通量且高分辨率的位点特异性染色质互作分析。该系统借助C端生物素化的dCas9、内源性生物素连接酶与混合单导RNA（single guide RNA，sgRNA）池，能够在单次实验中对数十至数百个增强子或启动子的空间构象进行定量分析，从而实现基因簇内及基因簇间顺式调控元件的系统性对比。借此我们揭示了超级增强子（super-enhancers，SEs）的层级结构，以及超级增强子与基因互作的不同模式。对以启动子为中心的互作时序动态开展多位点捕获分析，证实了增强子-启动子环化在谱系分化过程中转录调控中的指导性功能。上述应用案例表明，多位点CAPTURE技术能够用于解析基因组结构与功能的组织原则。为明确该技术对全局基因表达的影响，本研究分别在稳定表达dCas9-CBio与sgRNA的K562细胞、G1ER-ER4细胞，以及经红细胞分化诱导的G1ER-ER4细胞中开展了RNA测序（RNA-seq）实验。