Self-Reporting Transposons Enable Simultaneous Readout of Gene Expression and Transcription Factor Binding in Single Cells

Abstract

In situ assays of transcription factor (TF) binding are confounded by cellular heterogeneity and represent averaged profiles in complex tissues. Single cell RNA-seq (scRNA-seq) is capable of resolving different cell types based on gene expression profiles, but no technology exists to directly link specific cell types to the binding pattern of TFs in those cell types. Here, we present self-reporting transposons (SRTs) and their use in single cell calling cards (scCC), a novel assay for simultaneously capturing gene expression profiles and mapping TF binding sites in single cells. First, we show how the genomic locations of SRTs can be recovered from mRNA. Next, we demonstrate that SRTs deposited by the piggyBac transposase can be used to map the genome-wide localization of the TFs SP1, through a direct fusion of the two proteins, and BRD4, through its native affinity for piggyBac. We then present the scCC method, which maps SRTs from scRNA-seq libraries, thus enabling concomitant identification of cell types and TF binding sites in those same cells. As a proof-of-concept, we show recovery of cell type-specific BRD4 and SP1 binding sites from cultured cells. Finally, we map Brd4 binding sites in the mouse cortex at single cell resolution, thus establishing a new technique for studying TF biology in situ..

Novel CHD8 genomic targets identified in fetal mouse brain byin vivoTargeted DamID

“…TaDa-seq thus joins the few published methods for resolving protein-DNA interactions genome-wide that can be deployed in vivo and do not require cross-linking or immunoprecipitation. Recently, another such method mapped transcription factor interactions at single cell resolution by fusing transcription factors to transposase domains and locating transposition events through direct DNA sequencing (47). Application of TaDa-seq offers the opportunity to characterize the neurodevelopmental function of chromatin remodeler proteins implicated in NDDs, as we did here for CHD8 , that might be difficult to interrogate using ChIP-seq.…”

Heart Enhancers: Development and Disease Control at a Distance

“…Furthermore, with single-cell multimodal omics being selected as the Methods of the Year 2019 ( Nature Methods, 2020 ), techniques for simultaneous measuring multiple modalities in the same single cells are blooming rapidly. Related to epigenomics, it has become possible to simultaneous profile accessible chromatin and transcriptome ( Cao et al, 2018 ; Chen et al, 2019 ; Li et al, 2019 ; Moudgil et al, 2020 ), methylome and transcriptome ( Angermueller et al, 2016 ), methylome and chromatin conformation ( Li et al, 2019 ), or even three modalities altogether ( Pott, 2017 ; Clark et al, 2018 ) within the same cells. The combinatorial use of single-cell epigenomics techniques on cardiac samples will potentially provide a holistic view of enhancer activities in all subtypes of cardiac cells across all stages in heart development.…”

Measuring Transcription Factor Binding and Gene Expression using Barcoded Self-Reporting Transposon Calling Cards and Transcriptomes

“…Repeat piggyBac and other transposases recognize and bind their cognate terminal repeat sequences that flank the transposon on both sides, and this interaction is necessary for transposition 8 . The SRT calling card captures the junction of the piggyBac TR and the adjacent genome to map insertion sites (Figure 1A) 3 . To maximize compatibility with calling cards library preparation and shortread next generation sequencing, an ideal barcode would be incorporated as close to the genomic insertion site as possible.…”

“…Devising a barcoding strategy for the SRT is challenging in several technical and biological ways. The single-cell calling card method relies on cell barcodes introduced during droplet-based reverse transcription 3 , but this strategy is incompatible with bulk experiments. The SRT consists of a promoter driving a selectable reporter flanked by the transposon terminal repeat sequences (TR).…”