Abstract
Identification of the protein complexes associated with defined DNA sequence elements is essential to understand the numerous transactions in which DNA is involved, such as replication, repair, transcription, and chromatin dynamics. Here we describe two protocols, IDAP (Isolation of DNA Associated Proteins) and CoIFI (Chromatin-of-Interest Fragment Isolation), that allow for isolating DNA/protein complexes (i.e., nucleoprotein elements) by means of a DNA capture tool based on DNA triple helix (triplex) formation. Typically, IDAP is used to capture proteins that bind to a given DNA element of interest (e.g., a specific DNA sequence, an unusual DNA structure, a DNA lesion) that can be introduced at will into plasmids. The plasmids are immobilized by means of a triplex-forming probe on magnetic beads and incubated in nuclear extracts; by using in parallel a control plasmid (that lacks the DNA element of interest), proteins that preferentially bind to the DNA element of interest are captured and identified by mass spectrometry. Similarly, CoIFI also uses a triplex-forming probe to capture a specific chromatin fragment from a cultured cell line that has been engineered to contain multiple copies of the DNA element of interest.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Kim T-K, Shiekhattar R (2015) Architectural and functional commonalities between enhancers and promoters. Cell 162:948–959. https://doi.org/10.1016/j.cell.2015.08.008
Sirbu BM, Couch FB, Feigerle JT et al (2011) Analysis of protein dynamics at active, stalled, and collapsed replication forks. Genes Dev 25:1320–1327. https://doi.org/10.1101/gad.2053211
Kliszczak AE, Rainey MD, Harhen B et al (2011) DNA mediated chromatin pull-down for the study of chromatin replication. Sci Rep 1:95. https://doi.org/10.1038/srep00095
Antão JM, Mason JM, Déjardin J, Kingston RE (2012) Protein landscape at drosophila melanogaster telomere-associated sequence repeats. Mol Cell Biol 32:2170–2182. https://doi.org/10.1128/MCB.00010-12
Ide S, Déjardin J (2015) End-targeting proteomics of isolated chromatin segments of a mammalian ribosomal RNA gene promoter. Nat Commun 6:6674. https://doi.org/10.1038/ncomms7674
Liu X, Zhang Y, Chen Y et al (2017) In situ capture of chromatin interactions by biotinylated dCas9. Cell 170:1028–1043.e19. https://doi.org/10.1016/j.cell.2017.08.003
Jain A, Wang G, Vasquez KM (2008) DNA triple helices: biological consequences and therapeutic potential. Biochimie 90:1117–1130. https://doi.org/10.1016/j.biochi.2008.02.011
Duca M, Vekhoff P, Oussedik K et al (2008) The triple helix: 50 years later, the outcome. Nucleic Acids Res 36:5123–5138. https://doi.org/10.1093/nar/gkn493
Nielsen PE, Egholm M (2001) Strand displacement recognition of mixed adenine-cytosine sequences in double stranded DNA by thymine-guanine PNA (peptide nucleic acid). Bioorg Med Chem 9:2429–2434
Sørensen MD, Meldgaard M, Raunkjaer M et al (2000) Branched oligonucleotides containing bicyclic nucleotides as branching points and DNA or LNA as triplex forming branch. Bioorg Med Chem Lett 10:1853–1856
Sun JS, François JC, Montenay-Garestier T et al (1989) Sequence-specific intercalating agents: intercalation at specific sequences on duplex DNA via major groove recognition by oligonucleotide-intercalator conjugates. Proc Natl Acad Sci U S A 86:9198–9202
Takasugi M, Guendouz A, Chassignol M et al (1991) Sequence-specific photo-induced cross-linking of the two strands of double-helical DNA by a psoralen covalently linked to a triple helix-forming oligonucleotide. Proc Natl Acad Sci U S A 88:5602–5606
Brunet E, Corgnali M, Perrouault L et al (2005) Intercalator conjugates of pyrimidine locked nucleic acid-modified triplex-forming oligonucleotides: improving DNA binding properties and reaching cellular activities. Nucleic Acids Res 33:4223–4234. https://doi.org/10.1093/nar/gki726
Isogawa A, Fuchs RP, Fujii S (2018) Versatile and efficient chromatin pull-down methodology based on DNA triple helix formation. Sci Rep 8:5925. https://doi.org/10.1038/s41598-018-24417-9
Gartel AL, Tyner AL (1999) Transcriptional regulation of the p21((WAF1/CIP1)) gene. Exp Cell Res 246:280–289. https://doi.org/10.1006/excr.1998.4319
Georgakilas AG, Martin OA, Bonner WM (2017) p21: a two-faced genome guardian. Trends Mol Med 23:310–319. https://doi.org/10.1016/j.molmed.2017.02.001
Iyer RR, Pluciennik A, Napierala M, Wells RD (2015) DNA triplet repeat expansion and mismatch repair. Annu Rev Biochem 84:199–226. https://doi.org/10.1146/annurev-biochem-060614-034010
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Isogawa, A., Fuchs, R.P., Fujii, S. (2020). Chromatin Pull-Down Methodology Based on DNA Triple Helix Formation. In: Hanada, K. (eds) DNA Electrophoresis. Methods in Molecular Biology, vol 2119. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0323-9_16
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0323-9_16
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0322-2
Online ISBN: 978-1-0716-0323-9
eBook Packages: Springer Protocols