Quantitative-enhancer-FACS-seq (QeFS) reveals epistatic interactions among motifs within transcriptional enhancers in developing Drosophila tissue [Drosophila melanogaster]

Activity of enhancers in Drosophila embryos was measured by highly parallel reporter assay. We examined the results of mutating binding sites for 4 poorly studied TFs individually or in combination, and characterized complex genetic interactions among the different classes of motif mutant. We generated a population of Drosophila embryos in each of which reporter gene expression is driven by one genomically integrated reporter element from a pool of wild type enhancers or enhancers in which all binding sites for one or more selected TFs have been mutated. Each tested enhancer was tagged with several distinct 20-base degenerate sequence tags in the 3' UTR of the reporter gene. We dissociated embryonic cells and FACS-sorted informative populations, and reporter gene molecules were counted by a Unique Molecular Identifier (UMI) amplification strategy. We detected RNA and DNA levels by high-throughput sequencing of tagged reporter gene amplicons and normalized gene expression (RNA UMI counts) by dividing by DNA UMI counts to generate a measure of enhancer activity.

本研究采用高通量平行报告基因检测（highly parallel reporter assay），定量测定果蝇胚胎中增强子的活性。我们针对4个研究较少的转录因子（Transcription Factor, TF）的结合位点，分别或联合进行突变，并解析了不同类别基序突变体间的复杂遗传互作。我们构建了一组果蝇胚胎群体，每枚胚胎的报告基因表达均由基因组整合型报告元件驱动；该元件取自野生型增强子池，或是对一个或多个目标转录因子结合位点进行了全部突变的增强子。每个待测增强子均在报告基因的3'非翻译区（3' untranslated region, 3' UTR）携带数个不同的20碱基简并序列标签。我们解离胚胎细胞，并通过荧光激活细胞分选（Fluorescence-Activated Cell Sorting, FACS）筛选有效细胞群体，随后采用唯一分子标识符（Unique Molecular Identifier, UMI）扩增策略对报告基因分子进行计数。我们通过对带标签的报告基因扩增子进行高通量测序，检测RNA与DNA的水平；将基因表达标准化值（RNA的UMI计数）除以DNA的UMI计数，即可得到增强子活性的量化指标。