PDF(Pubmed)
Abstract：
Cis-regulatory elements (CREs) interact with trans regulators to orchestrate gene expression, but how transcriptional regulation is coordinated in multi-gene loci has not been experimentally defined. We sought to characterize the CREs controlling dynamic expression of the adjacent costimulatory genes CD28, CTLA4 and ICOS, encoding regulators of T cell-mediated immunity. Tiling CRISPR interference (CRISPRi) screens in primary human T cells, both conventional and regulatory subsets, uncovered gene-, cell subset- and stimulation-specific CREs. Integration with CRISPR knockout screens and assay for transposase-accessible chromatin with sequencing (ATAC-seq) profiling identified trans regulators influencing chromatin states at specific CRISPRi-responsive elements to control costimulatory gene expression. We then discovered a critical CCCTC-binding factor (CTCF) boundary that reinforces CRE interaction with CTLA4 while also preventing promiscuous activation of CD28. By systematically mapping CREs and associated trans regulators directly in primary human T cell subsets, this work overcomes longstanding experimental limitations to decode context-dependent gene regulatory programs in a complex, multi-gene locus critical to immune homeostasis.
摘要：
顺式调节元件（CREs）与反式调节因子相互作用以协调基因表达，但是，在多基因位点中如何协调转录调控还没有实验定义。我们试图表征控制相邻共刺激基因CD28，CTLA4和ICOS的动态表达的CRE，编码T细胞介导免疫的调节因子。在原代人T细胞中进行平铺CRISPR干扰（CRISPRi）筛选，传统和监管子集，未发现的基因-，细胞亚群和刺激特异性CREs。与CRISPR敲除筛选和转座酶可接近的染色质测序分析（ATAC-seq）分析的整合确定了反式调节因子在特定CRISPRi响应元件处影响染色质状态以控制共刺激基因表达。然后，我们发现了关键的CCCTC结合因子（CTCF）边界，该边界增强了CRE与CTLA4的相互作用，同时还防止了CD28的混杂激活。通过直接在原代人T细胞亚群中系统地映射CRE和相关的反式调节因子，这项工作克服了长期的实验限制，以解码复杂的上下文相关的基因调控程序，对免疫稳态至关重要的多基因位点。
