Looping of upstream cis-regulatory elements is required for CFTR expression in human airway epithelial cells [4C-seq]

The CFTR gene lies within an invariant topologically associated domain (TAD) demarcated by CTCF and cohesin, but shows cell-type specific control mechanisms utilizing different cis-regulatory elements (CRE) within the TAD. Within the respiratory epithelium, more than one cell type expresses CFTR and the molecular mechanism controlling its transcription are likely divergent between them. Here we determine how two extragenic CREs that are prominent in secretory epithelial cells in the lung, regulate expression of the gene. We showed earlier that these CREs, located at -44kb and -35 kb upstream of the promoter, have strong cell-type-selective enhancer function. They are also responsive to inflammatory mediators and to oxidative stress, consistent with a key role in CF lung disease. Here we use CRISPR/Cas9 technology to remove these CREs from the endogenous locus in human bronchial epithelial cells. Loss of either site extinguished CFTR expression and abolished long-range interactions between these sites and the gene promoter. The deletions also greatly reduced promoter interactions with the 5’ TAD boundary. We show substantial recruitment of RNAPII to the -35kb element and identify CEBPβ as a key activating transcription factor for airway expression of CFTR, likely through occupancy at both the CRE and the gene promoter. Analysis of the 3D chromatin architecture at the CFTR locus to determine interaction frequencies between elements which lead to changes in CFTR gene regulatory patterns.

囊性纤维化跨膜传导调节因子（CFTR）基因位于由CTCF与黏连蛋白（cohesin）界定的保守拓扑关联结构域（topologically associated domain, TAD）内，但其基因调控机制具有细胞类型特异性，依赖于该TAD内不同的顺式调控元件（cis-regulatory elements, CRE）。在呼吸道上皮组织中，存在多种表达CFTR的细胞类型，且不同细胞间调控其转录的分子机制可能存在显著差异。本研究旨在明确两种在肺分泌上皮细胞中富集的基因外顺式调控元件如何调控该基因的表达。我们此前的研究表明，这两种分别位于启动子上游-44kb与-35kb位置的顺式调控元件具备极强的细胞类型选择性增强子功能，同时可响应炎症介质与氧化应激，这与其在囊性纤维化肺部病变中的关键作用相一致。本研究利用CRISPR/Cas9技术，在人类支气管上皮细胞的内源基因组位点中移除这两种顺式调控元件。结果显示，移除任意一个位点均可完全阻断CFTR的基因表达，并消除该位点与基因启动子之间的长程染色质相互作用；同时，该缺失还大幅降低了启动子与TAD 5’端边界的染色质相互作用频率。我们发现RNA聚合酶II（RNAPII）大量招募至-35kb位点，并证实CEBPβ是调控气道中CFTR表达的关键激活型转录因子，其作用机制可能是同时结合该顺式调控元件与基因启动子。本研究还通过分析CFTR基因座的三维染色质结构，明确各调控元件间的相互作用频率，以此揭示CFTR基因调控模式的改变机制。