This document describes a new technique called DAP-seq that can be used to study interactions between transcription factors (TFs) and DNA. DAP-seq is more economical and scalable than traditional Chromatin Immunoprecipitation (ChIP) techniques. The document outlines how DAP-seq works by producing TF-Halo fusion proteins in vitro that bind genomic DNA, which is then isolated using magnetic beads coated with Halo tags. Results are shown for analyzing the binding motifs of the transcription factor ABI3 in maize using DAP-seq.

1. High Throughput Analysis of DNA-Transcription Factor Interactions by DAP-seq
Ivan Sotelo, Christina Lee Ethridge, Kitra Cates, Robert Schmitz
Department of Genetics | University of Georgia
Transcription factors (TFs) are proteins that control the
expression of specific genes. Investigating the interactions
between TFs and their target DNA sequences benefits our
understanding of how regulatory pathways are modulated. In
humans, the improper regulation of transcription factors can
lead to a number of diseases including diabetes and certain
forms of cancer such as leukemia1. Traditionally, transcription
factor binding motifs are determined using Chromatin
Immunoprecipitation (ChIP)2. ChIP requires the use of specific
antibodies to target DNA-TF interactions, and can be both costly
and inefficient. Here, we utilize DNA Affinity Purification
sequencing (DAP-seq), a novel in-vitro assay that is both
economical and scalable3. This technique circumvents the
traditional limitations of ChIP-seq and provides a feasible
alternative to studying DNA-TF interactions.
Abstract Motif Analysis of DAP-seq
Acknowledgements
References
Next Steps
We would like to thank the National Science Foundation and start-up
funds from the University of Georgia for supporting this study, along
with Michael Gonzales (UGA Outreach Program Coordinator).
1. Villard, Jean. "Transcription regulation and human diseases." Swiss medical weekly
134 (2004): 571-579.
2. Schmidt, D., Wilson, M. D., Spyrou, C., Brown, G. D., Hadfield, J., & Odom, D. T.
(2009). ChIP-seq: using high-throughput sequencing to discover protein-DNA
interactions. Methods (San Diego, Calif.), 48(3), 240–248.
3. O’Malley, R., Huang, S., Song, L., Lewsey, M., Bartlett, A., Nery, J., . . . Ecker, J.
(2016). Cistrome and Epicistrome Features Shape the Regulatory DNA Landscape.
Cell, 165(5), 1280-1292.
4. Mönke G, Seifert M, Keilwagen J, et al. Toward the identification and regulation of
the Arabidopsis thaliana ABI3 regulon. Nucleic Acids Research. 2012;40(17):8240-
8254.
Methods
• Scale DAP-seq up to a 96-well plate format
• Analyze all TFs in the maize and soybean genomes
• Determine all currently unknown TF binding motifs in the
cistrome of Arabidopsis thaliana, including those of TFs that
operate during heat shock, drought, and other abiotic stressors
• Map out gene regulatory networks controlled by TFs.
• Alter and manipulate properties in gene regulatory networks
using techniques such as CRISPR
Figure 1: A plasmid containing the transcription factor sequence and
the Halo affinity tag is transcribed and translated in vitro. This
produces a fusion protein consisting of the transcription factor and the
Halo tag.
DAP-seq Results for ABI3
Figure 2: Interaction between genomic DNA, a TF-Halo fusion protein,
and a magnetic bead. The transcription factor binds sonicated genomic
DNA. The Halo tag is then covalently bound to the magnetic bead. This
interaction allows for the pull down of TF-bound DNA for subsequent
sequencing.
Figure 5 (Above): A magnified image of
an ABI3 transcription factor peak from
Figure 3C. In this region, we identified
the binding motif for ABI3 to be CATGCA
(outlined in yellow).
Figure 4 (Below): Sequence logo of the
ABI3 transcription factor binding motif
representing a genome-wide
assessment of ABI3 binding domains
from ChIP-seq4.
3A
Figure 3: Visualization of DAP-seq results for Zea mays transcription factor ABI3.
The orange peaks with black tips represent regions of the genome where this
transcription factor bound. In accordance with our expectation, these peaks align
with the promoter regions of various Z. mays genes.
3B
3C