Developing novel methods to image and visualize 3D genomes

Abstract

To investigate three-dimensional (3D) genome organization in prokaryotic and eukaryotic cells, three main strategies are employed, namely nuclear proximity ligation-based methods, imaging tools (such as fluorescence in situ hybridization (FISH) and its derivatives), and computational/visualization methods. Proximity ligation-based methods are based on digestion and re-ligation of physically proximal cross-linked chromatin fragments accompanied by massively parallel DNA sequencing to measure the relative spatial proximity between genomic loci. Imaging tools enable direct visualization and quantification of spatial distances between genomic loci, and advanced implementation of (super-resolution) microscopy helps to significantly improve the resolution of images. Computational methods are used to map global 3D genome structures at various scales driven by experimental data, and visualization methods are used to visualize genome 3D structures in virtual 3D space-based on algorithms. In this review, we focus on the introduction of novel imaging and visualization methods to study 3D genomes. First, we introduce the progress made recently in 3D genome imaging in both fixed cell and live cells based on long-probe labeling, short-probe labeling, RNA FISH, and the CRISPR system. As the fluorescence-capturing capability of a particular microscope is very important for the sensitivity of bioimaging experiments, we also introduce two novel super-resolution microscopy methods, SDOM and low-power super-resolution STED, which have potential for time-lapse super-resolution live-cell imaging of chromatin. Finally, we review some software tools developed recently to visualize proximity ligation-based data. The imaging and visualization methods are complementary to each other, and all three strategies are not mutually exclusive. These methods provide powerful tools to explore the mechanisms of gene regulation and transcription in cell nuclei.

Keywords: 3D genomes; Chromatins; FISH method.

Fig. 1

3D genome visualization based on DNA-FISH methods. a False color representation of the 24 differently labeled chromosome types (1–22, X and Y) with DNA-FISH demonstrating how each chromosome accommodates its respective chromosome territory (image modified from (Bolzer et al. 2005). b Three-color DNA-FISH validation of the Haplotype-specific super-long interactions mediated by CTCF connecting three loci: DXZ4, FIRRE, G6PD in ChrX (top). Loci in the paternal-origin chromosome showed specific localization (lower left) which is absent in the maternal-origin chromosome (lower right) (image modified from Tang et al. 2015). c 3D-STORM images of three distinct epigenetic domains (left: active, middle: inactive, right: repressed) labeled by “Oligopaint” FISH probes with photoswitchable dye Alexa-647, shown with their corresponding conventional images in the inset (image modified from Boettiger et al. 2016). d Mapping the spatial organization of the central 100-kb regions of 34 TADs in Chr21. The position of each TAD is plotted as red dot in the microscopy image (left) and in 3D (right) (image modified from Wang et al. 2016b). e 3D-STORM images of a 2.5-kb exogenous viral sequence integrated in the cell (left) and an endogenous sequence (right) labeled by MB probes with the photoswitchable dye Alexa 647 (image modified from Ni et al. 2017).

Fig. 2

CRISPR-cas9 derived DNA labeling and imaging. a The system design (left) and image of labeling telomeres in live cells (right) (image modified from Chen et al. 2013). b The system design of orthogonal CRISPR/dCas9 systems from two bacterial sources allowed labeling genomic loci with multi-color (left). In an illustrative image of such a system, three loci (MUC4, 5S rDNA, and Ch17R) from chromosomes 1, 3, and 17, were simultaneously labeled, respectively, without interference (right) (image modified from Chen et al. 2016). c The system design of the CRISPR/dCas9 system utilizing MS2-MCP and PP7-PCP RNA aptamer-protein interaction pair (left) to allow labeling two different DNA loci with dual-color (right) (image modified from Wang et al. 2016a)

Fig. 3

Imaging DNA in vitro using bis-intercalating or intercalating dyes. a A schematic diagram to show the binding modes of bis-intercalator and intercalator. b Fluorescence image of λ phage DNA stained with 50 nM TOTO-1 with orientations of polarized fluorescence of detected particles shown by magenta lines. Image modified from Mehta et al. . (Scale bars: magenta, polarization factor = 1; white, 1 μm). c The arrowhead points to a location of the double stranded DNA fiber whose molecular order departs from the average behavior of the imaged region (images b and c modified from Valades Cruz et al. 2016). (Scale bar 100 nm). d Super-resolution image of a λ-DNA strand containing multiple “bends” (see arrows). e Super-resolution image of a λ-DNA strand exhibiting “tangles” (see arrow) (images d and e modified from Backer et al. 2016). (Scale bar 1um)

Fig. 4

Visualization tools developed recently to visualize proteins and genome in 3D space. a A DNA-protein complex (PDB-ID: 1BPX) is shown in Web3DMol. Schematic diagram produced by the software in Shi et al. . b HiC-3DViewer can highlight genomic regions of interest in a Hi-C frequency matrix interactively and visualize them in 3D space with 1D-to-2D-to-3D mapping (image modified from Djekidel et al. 2016).