title: High-resolution 3D DNA FISH using plasmid probes and computational correction of optical aberrations to study chromatin structure at the sub-megabase scale.
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.
authors.affiliation: Institut Curie, 26 Rue d'Ulm, 75248, Paris Cedex 05, France.
authors.forename: Luca
authors.initials: L
authors.lastname: Giorgetti
authors.forename: Tristan
authors.initials: T
authors.lastname: Piolot
authors.forename: Edith
authors.initials: E
authors.lastname: Heard
mesh_terms: D002478:Cells, Cultured; D002843:Chromatin / Q000235:genetics*; D015342:DNA Probes; D053595:Embryonic Stem Cells; D006801:Humans; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D021621:Imaging, Three-Dimensional; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D010957:Plasmids / Q000235:genetics*
chemical_list: D002843:Chromatin; D015342:DNA Probes