Systematic Dissection of Sequence Features Affecting the Binding Specificity of a Pioneer Factor Reveals Binding Synergy Between FOXA1 and AP-1 [ChIP-ISO]

Pioneer factors (PFs) are a subset of transcription factors (TFs) that can bind to nucleosomal DNA and invade closed chromatin. Despite that nucleosomes do not present a strong barrier to PF binding, PF can only bind to a small fraction of motifs in the genome. The underlying mechanism of such binding selectivity is not well understood. Here, we design a high-throughput assay named Chromatin Immunoprecipitation over Integrated Synthetic Oligonucleotides (ChIP-ISO) to systematically dissect the sequence features affecting the binding specificity of a classic PF, FOXA1, in A549 human lung carcinoma cells. This method involves integrating thousands of synthetic sequences containing FOXA1 motifs into a fixed genomic locus, followed by FOXA1 chromatin immunoprecipitation (ChIP) and amplicon sequencing. We find that 1) FOXA1 binding is affected by motif strength and co-binding TFs AP-1 and CEBPB, with AP-1 playing a major role in promoting FOXA1 binding, 2) FOXA1 binding in vivo and in vitro are poorly correlated, and FOXA1 and AP-1 show binding cooperativity in vitro, 3) FoxA1’s binding specificity is mostly determined by the local sequences, whereas chromatin context, including heterochromatin marks, only plays a minor role, and 4) AP-1 is at least partially responsible for differential binding of FOXA1 in different cell types. Finally, neural network analysis shows that AP-1 and CEBPB motifs are predictive of FOXA1 ChIP-seq peaks in A549, but not in some other cell types. In summary, combining ChIP-ISO with in vitro and in silico analyses, our study provides insights into the genetic rules underlying PF binding specificity and reveals a mechanism for its regulation during cell differentiation. Chromatin Immunoprecipitation with Integrated Synthetic Oligonucleotides (ChIP-ISO) for TF FOXA1 and histone modifications H3K9me3 and H3K27me3 in a mixed population of WT A549 human lung carcinoma cells with 3,203 synthetic oligonucleotides integrated individually into the AAVS1 locus.

先锋转录因子（Pioneer Factors, PFs）是转录因子（Transcription Factors, TFs）的一个子集，能够结合核小体DNA并侵入封闭染色质。尽管核小体并未对PF的结合构成强屏障，但PF仅能结合基因组中极小部分的基序，此类结合选择性的潜在机制尚未得到充分阐明。本研究设计了一种名为整合合成寡核苷酸染色质免疫沉淀（Chromatin Immunoprecipitation over Integrated Synthetic Oligonucleotides, ChIP-ISO）的高通量检测技术，以系统解析经典先锋转录因子FOXA1（叉头框蛋白A1，Forkhead Box A1）在人肺癌A549细胞中结合特异性的序列影响特征。该方法将包含FOXA1基序的数千条合成序列整合至固定的基因组位点，随后开展FOXA1染色质免疫沉淀（Chromatin Immunoprecipitation, ChIP）与扩增子测序。本研究获得以下发现：1）FOXA1的结合受到基序强度以及共结合转录因子AP-1（激活蛋白1，Activator Protein 1）与CEBPB（CCAAT增强子结合蛋白β，CCAAT/Enhancer Binding Protein Beta）的调控，其中AP-1在促进FOXA1结合过程中发挥核心作用；2）FOXA1在体内与体外的结合相关性较弱，且FOXA1与AP-1在体外表现出结合协同性；3）FOXA1的结合特异性主要由局部序列决定，而异染色质标记等染色质背景仅发挥次要作用；4）AP-1至少部分介导了不同细胞类型中FOXA1结合的差异性。最后，神经网络分析显示，AP-1与CEBPB基序可有效预测A549细胞中的FOXA1 ChIP-seq峰，但在部分其他细胞类型中无法实现该预测。综上，本研究将ChIP-ISO技术与体外、计算机模拟分析相结合，揭示了先锋转录因子结合特异性背后的遗传调控规律，并阐明了其在细胞分化过程中的调控机制。本研究的数据集为：在分别将3203条合成寡核苷酸整合至AAVS1位点的野生型人肺癌A549细胞混合群体中，针对转录因子FOXA1以及组蛋白修饰H3K9me3与H3K27me3的ChIP-ISO检测数据。