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

采用自报告转座子（self-reporting transposons）的呼叫卡（calling cards）技术，可通过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本体中引入DNA条形码（DNA barcode），大幅降低了批量细胞群体中呼叫卡实验的成本与人力投入需求。在逆转录阶段整合的额外条形码，可通过一种简便且经济的实验流程，实现转录组的同步检测。本研究证实，携带条形码的自报告转座子可在体外捕获四种在细胞命运特化中发挥关键作用的碱性螺旋-环-螺旋（basic helix-loop-helix）转录因子的结合位点，分别为ASCL1、MYOD1、NEUROD2及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组分析，可同时鉴定出其中两种转录因子的结合位点与基因表达变化。综上，通过携带条形码的自报告转座子呼叫卡与转录组开展基于RNA的转录因子结合位点鉴定与基因表达分析，是一种可在细胞群体中高效且可靠地推断基因调控网络的有力方法。实验整体设计：我们对piggyBac转座子的末端重复序列区域开展诱变实验，以筛选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；最终鉴定出4个可突变核苷酸位点，并将其用作条形码。我们构建了分别编码嘌呤霉素抗性（puromycin resistance）或tdTomato荧光蛋白的携带条形码的自报告转座子。随后利用这些携带条形码的载体，鉴定出四种参与细胞命运决策的转录因子的结合位点。