Cis Regulatory Element Scan by Tiling-deletion and Sequencing

Here we report a highly scalable strategy for unbiased discovery and functional characterization of cis regulatory elements in the genome. These results demonstrate the utility of our cis element discovery strategy and reveal the dual function of gene promoters: they not only initiate transcription of their own genes, but could also control transcription of nearby genes. Overall design: As proof of principle, we focused on the 2Mbp POU5F1 locus (hg19 chr6:30120000-32150000) in hESCs with the goal to identify cis-regulatory elements involved in the transcriptional control of POU5F1. We first designed 11,570 sgRNA pairs that would introduce ~2kb-deletions in a highly nested manner with ~100bp step sizes. As negative controls, we included 424 ineffective oligos lacking the PAM sequence necessary for effective dsDNA breaks. Additionally, we also included 6 sgRNA pairs that target the eGFP coding sequence as positive controls. The dual CRISPR expressing cassettes were cloned into lentiCRISPRv2 vector, and the resulting lentiviral libraries were verified to be of high quality and capable of generating genomic deletions. ATAC-seq is a widely used method to mapping the open chromatin landscape of the cells. In this study, we perfomed ATAC-seq to compare the open chromain patters in wild-type and two mutant clones with PRRC2A and MSH5 core promoter region deletions. The core promoters of PRRC2A and MSH5 have been identified as distal enhancer of POU5F1 and we want to test if and how the deletion of distal enhancer affect the open chromaitn patterns.

本研究报道了一种可高度扩展的策略，用于无偏发现基因组中的顺式调控元件（cis regulatory elements）并对其进行功能表征。本研究结果验证了所开发的顺式元件发现策略的实用性，并揭示了基因启动子的双重功能：其不仅可启动自身基因的转录，还可调控邻近基因的转录。实验设计概况：为验证该策略的原理可行性，本研究以人类胚胎干细胞（human embryonic stem cells, hESCs）中2兆碱基对（Mbp）的POU5F1基因座（hg19 chr6:30120000-32150000）为研究对象，旨在鉴定参与POU5F1转录调控的顺式调控元件。本研究首先设计了11570条向导RNA（single-guide RNA, sgRNA）对，以步长约100碱基对（bp）的高度嵌套方式，引入约2千碱基对（kb）的基因组缺失。作为阴性对照，本研究纳入了424条无效寡核苷酸序列，此类寡核苷酸缺少介导有效双链DNA断裂所必需的前间区序列邻近基序（protospacer adjacent motif, PAM）。此外，还纳入了6条靶向增强型绿色荧光蛋白（enhanced green fluorescent protein, eGFP）编码序列的sgRNA对作为阳性对照。将双CRISPR表达盒克隆至慢病毒CRISPRv2（lentiCRISPRv2）载体中，经验证，所得慢病毒文库质量优良，可有效产生基因组缺失突变。转座酶可及性测序（assay for transposase-accessible chromatin using sequencing, ATAC-seq）是一种广泛用于绘制细胞开放染色质图谱的技术。本研究通过ATAC-seq技术，比较了野生型细胞与两个携带PRRC2A和MSH5核心启动子区域缺失的突变克隆之间的开放染色质模式差异。此前研究已证实PRRC2A与MSH5的核心启动子是POU5F1的远端增强子，本研究旨在探究该远端增强子的缺失是否以及如何影响细胞的开放染色质模式。