Measuring transcription factor binding and gene expression using barcoded self-reporting transposon calling cards and transcriptomes

Calling cards technology using self-reporting transposons enables the identification of DNA-protein interactions through RNA sequencing . Here, we have drastically reduced the cost and labor requirements of calling card experiments in bulk populations of cells by introducing a DNA barcode into the calling card itself. An additional barcode incorporated during reverse transcription enables simultaneous transcriptome measurement in a facile and affordable protocol. We demonstrate that barcoded self-reporting transposons recover in vitro binding sites for four basic helix-loop-helix transcription factors with important roles in cell fate specification: ASCL1, MYOD1, NEUROD2, and NGN1. Further, simultaneous calling cards and transcriptional profiling during transcription factor overexpression identified both binding sites and gene expression changes for two of these factors. In sum, RNA-based identification of transcription factor binding sites and gene expression through barcoded self-reporting transposon calling cards and transcriptomes is an efficient and powerful method to infer gene regulatory networks in a population of cells. Overall design: We performed mutagenesis of the terminal repeat region of the piggyBac transposon to identify sites that could accommodate or serve as a barcode for self-reporting transposon calling card expeirments. We identified 4 mutable nucleotides and used these sites as barcodes. We constructed barcoded self-reporting transposons encoding either puromycin resistance or tdTomato. We then used these barcoded constructs to identify binding sites for four transcription factors involved in cell fate decisions.

基于自报告转座子的呼叫卡技术（calling cards technology）可借助RNA测序（RNA sequencing）实现DNA-蛋白质相互作用的鉴定。本研究通过在呼叫卡自身序列中引入DNA条形码（DNA barcode），大幅降低了细胞群体样本中呼叫卡实验的成本与人力投入。在逆转录（reverse transcription）过程中引入额外条形码，可通过简便且经济的实验方案同步完成转录组检测。本研究验证，带条形码的自报告转座子可回收四种在细胞命运特化中发挥关键作用的碱性螺旋-环-螺旋转录因子（basic helix-loop-helix transcription factors）的体外结合位点，即ASCL1、MYOD1、NEUROD2与NGN1。此外，在转录因子过表达过程中同步开展呼叫卡实验与转录谱分析，可鉴定出其中两种转录因子的结合位点与基因表达变化情况。综上，借助带条形码的自报告转座子呼叫卡与转录组实现转录因子结合位点及基因表达的RNA层面鉴定，是在细胞群体中推断基因调控网络（gene regulatory networks）的高效且强大的研究方法。

实验设计：本研究对piggyBac转座子的末端重复区域开展诱变筛选，以寻找可容纳条形码或可作为自报告转座子呼叫卡实验条形码的位点。最终筛选得到4个可突变的核苷酸位点，并将其用作实验条形码。本研究构建了携带嘌呤霉素抗性（puromycin resistance）或tdTomato标记的带条形码自报告转座子。随后利用这些带条形码的实验构建体，鉴定出四种参与细胞命运决策的转录因子的结合位点。