Information on ITNs.

Gene expression requires the targeting of transcription factors (TFs) to regulatory sequences often occluded within nucleosomes. To comprehensively examine TF nucleosome binding, we developed Pioneer-Seq. In Pioneer-seq a library of thousands of nucleosomes are formed from sequences containing a TF binding site (TFBS) variant in all possible nucleosome orientations and within the linker regions. Pioneer-seq has the unique ability to simultaneously examine nucleosomes created with various nucleosome positioning sequences and examine binding to in vivo targeted nucleosomes (ITNs). Pioneer-seq can be applied to address various mechanistic models for TF-nucleosome binding directly and can be used to uncover inherent TF-interaction differences. To demonstrate Pioneer-seq, we examined nucleosome binding by OCT4, SOX2, KLF4, and c-MYC. Our results demonstrate that all studied TFs can bind at nucleosome edges and nucleosome sequence is the primary factor regulating TF binding. In addition, KLF4 can bind to a non-canonical TFBS located 20 bp from the nucleosome dyad. Examination of ITNs showed binding differences between the TFs, with KLF4 and SOX2 binding more often near nucleosome centers. Overall, our results demonstrate differences in how TF recognizes their TFBS within a nucleosome and begins to define the mechanistic requirements for pioneer factor binding.基因表达过程中，需将转录因子（Transcription Factors, TFs）靶向结合至常被核小体（nucleosome）遮蔽的调控序列。为全面探究转录因子与核小体的结合特性，我们开发了Pioneer-Seq技术。在Pioneer-Seq实验中，我们以包含转录因子结合位点（Transcription Factor Binding Site, TFBS）变体的序列为模板，构建得到数千个核小体的文库，这些核小体可呈现所有可能的取向，且变体位点处于接头区域（linker regions）内。Pioneer-Seq具备独特能力，可同时探究使用不同核小体定位序列构建的核小体，并可检测其与体内靶向核小体（in vivo targeted nucleosomes, ITNs）的结合。Pioneer-Seq可直接用于验证多种转录因子-核小体结合的机制模型，还可用于揭示转录因子间固有的结合差异。为验证Pioneer-Seq的应用效果，我们探究了OCT4、SOX2、KLF4及c-MYC介导的核小体结合特性。研究结果表明，所有被检测的转录因子均可结合于核小体边缘，且核小体序列是调控转录因子结合的核心因素。此外，KLF4可结合位于核小体二分体（nucleosome dyad）20 bp处的非典型转录因子结合位点。对体内靶向核小体的分析显示，不同转录因子的结合存在差异：KLF4与SOX2更常结合于核小体中心附近区域。综上，本研究揭示了转录因子在核小体内部识别其结合位点的方式差异，并初步明确了先锋因子（Pioneer Factor）结合的机制要求。