ATAC-STARR-seq reveals transcription factor-bound activators and silencers across the chromatin accessible human genome. ATAC-STARR-seq reveals transcription factor-bound activators and silencers across the chromatin accessible human genome

Massively parallel reporter assays (MPRAs) test the capacity of putative gene regulatory elements to drive transcription on a genome-wide scale. Most gene regulatory activity occurs within accessible chromatin, and recently described methods have combined assays that capture these regions—such as assay for transposase-accessible chromatin using sequencing (ATAC-seq)—with self-transcribing active regulatory region sequencing (STARR-seq) to selectively assay the regulatory potential of accessible DNA (ATAC-STARR-seq). Here, we report an integrated approach that quantifies activating and silencing regulatory activity, chromatin accessibility, and transcription factor (TF) occupancy with one assay using ATAC-STARR-seq. Our strategy, including important updates to the ATAC-STARR-seq assay and workflow, enabled high-resolution testing of ~50 million unique DNA fragments tiling ~101,000 accessible chromatin regions in human lymphoblastoid cells. We discovered that 30% of all accessible regions contain an activator, a silencer or both. Although few MPRA studies have explored silencing activity, we demonstrate silencers occur at similar frequencies to activators, and they represent a distinct functional group enriched for unique TF motifs and repressive histone modifications. We further show that Tn5 cut-site frequencies are retained in the ATAC-STARR plasmid library compared to standard ATAC-seq, enabling TF occupancy to be ascertained from ATAC-STARR data. With this approach, we found that activators and silencers cluster by distinct TF footprint combinations and these groups of activity represent different gene regulatory networks of immune cell function. Altogether, these data highlight the multi-layered capabilities of ATAC-STARR-seq to comprehensively investigate the regulatory landscape of the human genome all from a single DNA fragment source. Overall design: We examined regulatory activity, chromatin accessibility, and TF binding in GM12878 B cells using ATAC-STARR-seq. For all replicates, we sequenced reisolated plasmid DNA and reporter RNA. We also sequenced the ATAC-STARR plasmid library to check library complexity.

大规模平行报告基因检测（Massively Parallel Reporter Assays, MPRAs）用于在全基因组规模下检测推定基因调控元件驱动转录的能力。大多数基因调控活性发生在开放染色质区域，近期报道的方法将捕获开放染色质区域的检测技术——例如转座酶可及性测序检测法（Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq）——与自转录活性调控区域测序（Self-Transcribing Active Regulatory Region Sequencing, STARR-seq）相结合，以选择性检测开放染色质DNA的调控潜力（即ATAC-STARR-seq）。本研究报道了一种整合型检测方法，可通过单次ATAC-STARR-seq实验同时量化激活型与沉默型调控活性、染色质可及性以及转录因子（Transcription Factor, TF）的结合情况。我们的策略包含对ATAC-STARR-seq检测方法与实验流程的重要改进，实现了对人类淋巴母细胞系中约101000个开放染色质区域平铺覆盖的约5000万个独特DNA片段的高分辨率检测。我们发现，30%的开放染色质区域包含激活子、沉默子或二者兼具。尽管少有MPRA研究探讨沉默子活性，但我们证实沉默子的出现频率与激活子相当，且它们是一类独特的功能类群，富集有特定的转录因子基序与抑制性组蛋白修饰。我们进一步证明，与标准ATAC-seq相比，ATAC-STARR质粒文库可保留Tn5切割位点的频率信息，从而可从ATAC-STARR数据中推断转录因子结合足迹。通过该方法，我们发现激活子与沉默子可通过不同的转录因子足迹组合进行聚类，且这类活性分组代表了免疫细胞功能的不同基因调控网络。综上，这些数据凸显了ATAC-STARR-seq的多层级研究能力，可仅通过单一DNA片段来源全面解析人类基因组的调控景观。总体实验设计：我们采用ATAC-STARR-seq检测了GM12878 B淋巴细胞中的调控活性、染色质可及性与转录因子结合情况。所有重复实验均对回收的质粒DNA与报告RNA进行了测序。我们还对ATAC-STARR质粒文库进行了测序，以评估文库复杂度。