Abstract
Characterizing the three-dimensional organization of chromosomes is a fundamental goal in molecular biology and will be critical to understand how gene expression is regulated by distal regulatory sequences such as enhancers. Chromosome conformation capture (3C) techniques have recently revealed that the interactions between regulatory elements appear to occur in the context of topologically associating domains (TADs), each spanning few hundreds kilobases, within which the chromatin fiber preferentially interacts. However, 3C-based data represent average interaction probabilities of the chromatin fiber over millions of cells. To understand how variable chromatin conformation is within each TAD, one needs to employ single-cell techniques such as 3D DNA FISH. Given the small size of TADs however (typically <1 Mb), classical DNA FISH design needs to be adapted to achieve high genomic and spatial resolution. Here, we describe a high-resolution 3D DNA FISH approach we recently developed, based on a combination of short plasmid probes and computational correction of optical aberrations. We describe probe design and generation and the 3D DNA FISH procedure. We further discuss how to optimize microscope settings and to implement calibration-bead-assisted computational corrections in order to achieve 50 nm resolution in two-color distance measurements between probes that can be as close as 50 kb along the genome.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
de Laat W, Duboule D (2013) Topology of mammalian developmental enhancers and their regulatory landscapes. Nature 502(7472):499–506
Gibcus JH, Dekker J (2013) The hierarchy of the 3D genome. Mol Cell 49(5):773–782
Shen Y, Yue F, McCleary DF, Ye Z, Edsall L, Kuan S, Wagner U, Dixon J, Lee L, Lobanenkov VV, Ren B (2012) A map of the cis-regulatory sequences in the mouse genome. Nature 488(7409):116–120
Nora EP, Lajoie BR, Schulz EG, Giorgetti L, Okamoto I, Servant N, Piolot T, van Berkum NL, Meisig J, Sedat J, Gribnau J, Barillot E, Bluthgen N, Dekker J, Heard E (2012) Spatial partitioning of the regulatory landscape of the X-inactivation centre. Nature 485(7398):381–385
Giorgetti L, Galupa R, Nora EP, Piolot T, Lam F, Dekker J, Tiana G, Heard E (2014) Predictive polymer modeling reveals coupled fluctuations in chromosome conformation and transcription. Cell 157(4):950–963
Beliveau BJ, Joyce EF, Apostolopoulos N, Yilmaz F, Fonseka CY, McCole RB, Chang Y, Li JB, Senaratne TN, Williams BR, Rouillard JM, Wu CT (2012) Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes. Proc Natl Acad Sci U S A 109(52):21301–21306
Boyle S, Rodesch MJ, Halvensleben HA, Jeddeloh JA, Bickmore WA (2011) Fluorescence in situ hybridization with high-complexity repeat-free oligonucleotide probes generated by massively parallel synthesis. Chromosome Res 19(7):901–909
Bienko M, Crosetto N, Teytelman L, Klemm S, Itzkovitz S, van Oudenaarden A (2013) A versatile genome-scale PCR-based pipeline for high-definition DNA FISH. Nat Methods 10(2):122–124
Sorzano CO, Thevenaz P, Unser M (2005) Elastic registration of biological images using vector-spline regularization. IEEE Trans Biomed Eng 52(4):652–663
Bolte S, Cordelieres FP (2006) A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 224(Pt 3):213–232
Sengupta P, Van Engelenburg S, Lippincott-Schwartz J (2012) Visualizing cell structure and function with point-localization superresolution imaging. Dev Cell 23(6):1092–1102
Mlynarczyk-Evans S, Royce-Tolland M, Alexander MK, Andersen AA, Kalantry S, Gribnau J, Panning B (2006) X chromosomes alternate between two states prior to random X-inactivation. PLoS Biol 4(6):e159
Acknowledgements
Research in the Heard lab is supported by the ‘‘Ligue Nationale contre le cancer,’’ the Fondation pour la Recherche Medical, Labex DEEP (ANR-11-LBX-0044) part of the IDEX Idex PSL (ANR-10-IDEX-0001-02 PSL), the EpiGeneSys FP7 257082 Network of Excellence, ERC Advanced Investigator award 250367, and EU FP7 MODHEP EU grant 259743.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Giorgetti, L., Piolot, T., Heard, E. (2015). High-Resolution 3D DNA FISH Using Plasmid Probes and Computational Correction of Optical Aberrations to Study Chromatin Structure at the Sub-megabase Scale. In: Nakagawa, S., Hirose, T. (eds) Nuclear Bodies and Noncoding RNAs. Methods in Molecular Biology, vol 1262. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2253-6_3
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2253-6_3
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2252-9
Online ISBN: 978-1-4939-2253-6
eBook Packages: Springer Protocols