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Calling Cards Technology Improved by Barcoded, Self-Reporting Transposons

By Stuart P. Atkinson, Ph.D.

May 13, 2024

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Introduction: Calling Cards Technology - Powerful, Yet Limited

Recent versions of Calling Cards technology employ a protein of interest - such as a transcription factor – fused to a hyper-active piggyBac transposase (Yusa et al.) to induce the insertion of a self-reporting transposon - the "Calling Card" - into the genome at sites of DNA-protein interaction. Subsequent RNA sequencing assays can then map transcription factor binding sites throughout the genome while also creating gene expression profiles (Moudgil et al., 2020); the subsequent combination of these datasets can then help to illustrate how various transcription factors orchestrate gene expression under a wide range of conditions. Unfortunately, this approach suffers from technical problems related to transposase requirements that necessitate a large number of replicates to accurately identify independent insertions into the same genomic locus (Moudgil et al., 2019); therefore, greater accuracy requires higher experimental and labor costs.

Researchers headed by Matthew Lalli and Robi D. Mitra (Washington University in St. Louis School of Medicine) sought to work around these problems and "evolve" Calling Cards technology into a more powerful means of jointly recording transcription factor binding sites and gene expression by implementing barcoded self-reporting transposons and barcoded transcriptomes. This alternative approach includes embedding a unique barcode within the terminal repeat of the self-reporting transposon - representing the authors' first challenge - to drastically reduce the cost and labor requirements of Calling Cards experiments. Let's hear more about barcoded self-reporting transposon Calling Cards!

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DNA Barcoding – The Way Forward for Improved Calling Cards Technology?

As described in their NAR Genomics and Bioinformatics article, Lalli et al. first performed targeted mutagenesis of the piggyBac terminal repeat sequence to identify potential sites to accommodate their unique barcodes; the team discovered at least four consecutive nucleotides within the terminal repeat that tolerated a range of mutations without prompting significant reductions in transposition activity. Of note, the authors reported a set of barcoded piggyBac self-reporting transposon plasmids and a modified analysis software package to utilize these barcodes as a resource available to those wishing to apply this evolved version of Calling Cards technology.

After successfully taking this initial step, the authors next reported that barcoded self-reporting transposon Calling Cards supported the in vitro mapping of the genomic binding sites of basic helix-loop-helix transcription factors involved in cell fate specification and trans differentiation (ASCL1, MYOD1, NEUROD2, and NGN1) (Webb et al.) in HEK293T human embryonic kidney cells. They identified both shared and unique binding sites for each transcription factor and characterized known binding motifs for these factors; furthermore, the authors revealed that the genes encountered close to the detected transcription factor binding sites displayed enrichment for the known functions of the assayed transcription factors, suggesting the validity of the results.

The team next combined barcoded self-reporting transposon Calling Cards with bulk RNA barcoding and sequencing (Alpern et al.) to successfully simultaneously identify pioneering transcription factor (ASCL1 and MYOD1) binding sites and accompanying transcriptional alterations in a quick and affordable manner via the introduction of an additional barcode during reverse-transcription for barcoding mRNA. Even more excitingly, the authors reported the ability of their evolved approach to record transcription factor binding in vivo in the mouse brain after delivery of the required components to the cortex, which entailed a 10-fold reduction in labor costs.

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Will Barcoded Self-Reporting Transposon Calling Cards Technology be Calling at Your Laboratory?

Overall, the authors report their evolved barcoded self-reporting transposon Calling Cards technology as an efficient and robust method to simultaneously measure transcription factor binding and gene expression changes, which can support the exploration of the gene regulatory networks for critical transcription factors that control development, cellular reprogramming, and disease onset.

Additionally, researchers guided by Robi D. Mitra and Joseph D. Dougherty (Washington University in St. Louis) recently published a Current Protocols paper (Yen et al.) in which they present a comprehensive guide for experimental design, reagent selection, and optional customization of barcoded self-reporting transposon Calling Cards technology to study the transcription factor of your choice and provide an updated protocol that improves throughput and decrease costs, including an overview of a newly deployed computational pipeline.

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About the author

Stuart P. Atkinson, Ph.D.

Stuart was born and grew up in the idyllic town of Lanark (Scotland). He later studied biochemistry at the University of Strathclyde in Glasgow (Scotland) before gaining his Ph.D. in medical oncology; his thesis described the epigenetic regulation of the telomerase gene promoters in cancer cells. Following Post-doctoral stays in Newcastle (England) and Valencia (Spain) where his varied research aims included the exploration of epigenetics in embryonic and induced pluripotent stem cells, Stuart moved into project management and scientific writing/editing where his current interests include polymer chemistry, cancer research, regenerative medicine, and epigenetics. While not glued to his laptop, Stuart enjoys exploring the Spanish mountains and coastlines (and everywhere in between) and the food and drink that it provides!

Contact Stuart on Twitter with any questions

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