3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells

Federica Marasca; Alice Cortesi; Lara Manganaro; Beatrice Bodega

doi:10.3791/60712

JoVE Journal > Genetics

Genética

3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells

Published: January 25, 2020

doi:

10.3791/60712

Federica Marasca¹, Alice Cortesi¹, Lara Manganaro¹, Beatrice Bodega¹

¹Istituto Nazionale di Genetica Molecolare “Romeo ed Enrica Invernizzi”, INGM

Summary

3D multicolor DNA FISH represents a tool to visualize multiple genomic loci within 3D preserved nuclei, unambiguously defining their reciprocal interactions and localization within the nuclear space at a single cell level. Here, a step by step protocol is described for a wide spectrum of human primary cells.

Abstract

A major question in cell biology is genomic organization within the nuclear space and how chromatin architecture can influence processes such as gene expression, cell identity and differentiation. Many approaches developed to study the 3D architecture of the genome can be divided into two complementary categories: chromosome conformation capture based technologies (C-technologies) and imaging. While the former is based on capturing the chromosome conformation and proximal DNA interactions in a population of fixed cells, the latter, based on DNA fluorescence in situ hybridization (FISH) on 3D-preserved nuclei, allows contemporary visualization of multiple loci at a single cell level (multicolor), examining their interactions and distribution within the nucleus (3D multicolor DNA FISH). The technique of 3D multicolor DNA FISH has a limitation of visualizing only a few predetermined loci, not permitting a comprehensive analysis of the nuclear architecture. However, given the robustness of its results, 3D multicolor DNA FISH in combination with 3D-microscopy and image reconstruction is a possible method to validate C-technology based results and to unambiguously study the position and organization of specific loci at a single cell level. Here, we propose a step by step method of 3D multicolor DNA FISH suitable for a wide range of human primary cells and discuss all the practical actions, crucial steps, notions of 3D imaging and data analysis needed to obtained a successful and informative 3D multicolor DNA FISH within different biological contexts.

Introduction

Higher eukaryotes need to systematically condense and compact a huge amount of genetic information in the minute 3D space of the nucleus¹^,²^,³^,⁴. Today, we know that the genome is spatially ordered in compartments and topologically associated domains⁵ and that the multiple levels of DNA folding generate contacts between different genomic regions that may involve chromatin loop formation⁶^,⁷. The 3D dynamic looping of chromatin can influence many different biological processes such as transcription⁸^,⁹, differentiation and development¹⁰^,¹¹, DNA repair¹²^,¹³, while its perturbations are involved in various diseases¹⁴^,¹⁵^,¹⁶ and developmental defects¹⁵^,¹⁷^,¹⁸.

Many approaches have been developed to study the 3D genome organization. Chromosome conformation capture-based technologies (C-technologies, 3C, 4C, 5C, Hi-C and derivatives) have been developed to study genome organization in fixed cells³^,⁴^,¹⁹^,²⁰. Such approaches are based on the ability to capture the contact frequencies between genomic loci in physical proximity. C-technologies, depending on their complexity, catch the global 3D genome organization and nuclear topology of a cell population³^,⁴^,¹⁹^,²⁰. Nevertheless, 3D interactions are dynamic in time and space, highly variable between individual cells consisting of multiplex interactions, and are extensively heterogenous²¹^,²².

3D multicolor DNA fluorescence in situ hybridization (FISH) is a technique that allows the visualization of specific genomic loci at a single cell level, enabling direct investigation of the 3D nuclear architecture in a complementary manner to C-technologies. It represents a technology currently used to unambiguously validate C-results. 3D multicolor DNA FISH uses fluorescently labeled probes complementary to the genomic loci of interests. The use of different fluorophores and suitable microscopy equipment allow contemporary visualization of multiple targets within the nuclear space²³^,²⁴. In recent years, FISH has been combined with technological advances in microscopy to obtain the visualization of fine-scale structures at high resolution²⁵^,²⁶ or with CRISPR-Cas approaches for the visualization of the nucleic acids in live imaging²⁷^,²⁸. Despite wide adoption, the 3D multicolor DNA FISH approach is still considered difficult in many laboratories because the biological material used must be adapted.

Here, we provide a comprehensive protocol for 3D multicolor DNA FISH (from cell/probe preparation to data analysis) applicable to a wide range of human primary cells, enabling the visualization of multiple genomic loci and preserving the 3D structure of nuclei. In order to study nuclear architecture, the 3D structure of nuclei must be preserved. For this reason, contrasting from other existing protocols²⁹^,³⁰^,³¹, we avoid the use of an alcohol gradient and the storage of the coverslips in alcohol that can affect chromatin structure³². The method is adapted from preserved 3D DNA FISH protocols²⁴^,³³ to be applied to a wide range of human primary cells, both isolated ex vivo or cultured in vitro. There are permeabilization and deproteinization parameters for different nuclear morphology and cytological characteristics (e.g., different degrees of nuclear compaction, cytoskeleton abundance)³⁴. These parameters are often generally described in other protocols²⁴^,³³, without providing a clear discrimination of the procedure within different cell types. Furthermore, we developed a specific tool named NuCLεD (nuclear contacts locator in 3D)¹⁶, providing principles for data analysis that will improve the 3D proximity between different loci and their nuclear topological distribution within the nuclear space in an automated way.

Protocol

1. DNA probe preparation and labelling procedures with nick translation

Purify and clean bacterial artificial chromosomes (BACs), plasmids or PCR products with specific kits (Table of Materials), resuspend in ddH₂O, check by electrophoresis on an agarose gel and quantify.
Perform nick translation on 1.5−2 µg of DNA from step 1.1 in a final volume of 50 µL by mixing all the reagents in a 0.5 mL low binding DNA tube according to Table 1.
NOTE: Directly labelled probes can improve the signal to noise ratio. Kits to produce indirectly and directly labelled probes with various Alexa fluorochromes by nick translation are commercially available.
Incubate the nick translation mix in a thermal mixer at 16 °C for a time depending on the length of the starting DNA material: 45 min for PCR products (a pool of PCR products of 2,000 bp each) and up to 4 h for BAC DNA and plasmids.
Check the size of the probes produced in step 1.3 by electrophoresis on a 2.2% agarose gel.
NOTE: The optimal probe size is <200 bp (Figure 1A).
1. If the DNA is not digested enough, add 5 U of DNA polymerase I and 0.05 U of DNase I to the reaction and incubate for 1−2 h at 16 °C and subsequently re-check. Stop the reaction with 0.5 mM EDTA (final concentration). Store probes at -20 °C.
For each DNA FISH experiment, precipitate the following quantities of probes depending on the starting DNA material from which the probes are produced: 200 ng from nick translated PCR products, 100 ng from nick translated BACs, or 300 ng from nick translated plasmid. Add ddH₂O up to 150 µL, 20 µg of unlabelled salmon sperm DNA, 3.5 µg of species-specific Cot-1 DNA, 3 volumes of 100% EtOH, and 1/10 volume of 3 M sodium acetate pH 5.2. Precipitate at -80 °C for 1 h.
Centrifuge at maximum speed for 1 h at 4 °C and discard the supernatant. Wash the pellet twice with 70% EtOH.
Resuspend the pellet in 2 µL of 100% formamide pH 7.0, shake at 40 °C for 30 min (can take up to a few hours) and then add an equal volume of 4x saline-sodium citrate (SSC)/20% dextran sulfate.
1. Prepare 20x SSC by mixing 175.3 g of NaCl and 88.2 g of sodium citrate in a final volume of 1 L of H₂O. Autoclave, filter and prepare aliquots.
  NOTE: For multicolor DNA FISH, the different probes can be precipitated together except for probes that can anneal with one another. In these specific cases, treat them separately, precipitate and resuspend the probes dividing the reagents mentioned in steps 1.5−1.7 with respect to the number of probes. Pool the probes only after resuspension in formamide. If glass coverslips greater than 10 mm or less than 10 mm are used, scale up/down the volume of the reagents used in steps 1.5−1.7 proportionally to the slide size. Hybridization probes can be stored at -20 °C for a long period of time (up to two months).

2. Cell fixation, pre-treatment and permeabilization

NOTE: Permeabilization and deproteinization passages are crucial steps. The time of reaction and concentration of the reagents strongly depend on the cell type, the cytoplasm abundance, and the nuclear morphology.

Cell fixation, pre-treatment and permeabilization for human primary T lymphocytes
NOTE: This protocol is suitable for small cells, with small nuclei and a low amount of cytoplasm.
1. Use glass coverslips (10 mm, thickness No. 1.5H).
2. Wash the glass with ddH₂O, then with 70% EtOH and let them dry. Use one glass for each well of a 24-well plate.
3. Add 200 µL of 0.1% poly-L-lysine (w/v) (Table of Materials) directly on the glass to form a drop. Pay attention as the drop must remain on the glass surface without touching the well for 2−5 min. Leave out the drop carefully, and let the glass dry for 30 min.
4. Perform step 2.1.3 twice more.
5. Put 200 µL of suspension cells (2 x 10⁶/mL, PBS suspension of ex vivo primary T lymphocytes) directly on the glass, allow the cells to seed at room temperature (RT) for 30 min.
6. Quickly remove the drop. Add freshly made 4% PFA (prepared in PBS/0.1% TWEEN 20, pH 7.0, filtered) for 10 min and fix the seeded cells at RT.
7. Wash three times for 5 min each with 0.05% Triton X-100/PBS (TPBS) at RT. Permeabilize with 0.5% TPBS for 10 min at RT.
8. Perform RNase treatment by adding 2.5 µL of RNase cocktail (Table of Materials) in 250 µL of PBS/well in a 24-well plate for 1 h at 37 °C.
9. Rinse in PBS, add 20% glycerol/PBS, and incubate overnight (ON) at 4 °C.
  NOTE: This step can range from 1 h to ON. Slides can be kept in 20% glycerol/PBS at 4 °C up to 7 days.
10. Freeze on dry ice (15−30 s), thaw gradually at RT, and soak in 20% glycerol/PBS. Repeat step 2.1.9 another three times.
11. Wash in 0.5% TPBS for 5 min at RT. Wash in 0.05% TPBS, twice for 5 min at RT. Incubate in 0.1 N HCl for 12 min at RT. Rinse in 2x SSC.
12. Incubate in 50% formamide pH 7.0/2x SSC ON at RT.
  NOTE: The incubation time can be optimized and eventually reduced. Slides can be kept in 50% formamide/2x SSC for several days.
Cell fixation, pre-treatment and permeabilization for human primary myoblasts
NOTE: This protocol is suitable for large cells, with a high amount of cytoplasm.
1. Grow human primary myoblasts directly on the coverslip glasses in 24-well plates in growth medium (Dulbecco's modified Eagle medium (DMEM), 20% fetal bovine serum (FBS), 25 ng/mL fibroblast growth factor (FGF), 10 ng/mL epidermal growth factor (EGF), 10 µg/mL human insulin, 1x glutamine, 1x penicillin/streptomycin) for at least 24 h (reaching 50−70% of confluence).
  NOTE: To facilitate the adhesion, gelatin or collagen or poly-L-lysine can be used to coat the coverslip prior the seeding.
2. Rinse cells in 2−3 changes of PBS. Add freshly made 4% PFA and fix the cells for 10 min at RT.
3. Wash three times for 3 min each in 0.01% TPBS at RT. Permeabilize in 0.5% TPBS for 10 min at RT.
4. Perform RNase treatment by adding 2.5 µL of RNase cocktail (Table of Materials) in 250 µL of PBS/well in a 24-well plate for 1 h at 37 °C.
5. Rinse in PBS, add 20% glycerol/PBS, and incubate ON at RT.
  NOTE: This step can range from 1 h to ON. Slides can be kept in 20% glycerol/PBS at 4 °C up to 7 days.
6. Freeze on dry ice (15−30 s), thaw gradually at RT, and soak in 20% glycerol/PBS. Repeat this step three times.
7. Wash three times for 10 min each in PBS. Incubate in 0.1 N HCl for 5 min at RT. Rinse in 2x SSC.
8. Incubate in 50% formamide pH 7.0/2x SSC ON at RT.
  NOTE: The time of incubation can be optimized and eventually reduced. Slides can be kept in 50% formamide/2x SSC for several days.
9. Equilibrate slides (kept in 50% formamide/2x SSC) in 2x SSC for 2 min. Then, equilibrate in PBS for 3 min.
10. Treat with 0.01 N HCl/0.0025% pepsin from a few seconds up to 5 min, depending on the cell type. During this step, observe the cells under an optical microscope and stop the reaction (step 2.2.11) as soon as the nuclei are free from the cytoplasm, while maintaining their structure intact (e.g., nucleoli are still visible and intact).
11. Inactivate the pepsin by washing twice for 5 min each in 50 mM MgCl₂/PBS.
12. Post-fix in 1% PFA/PBS for 1 min. Wash for 5 min in PBS. Wash twice for 5 min each in 2x SSC, and then add 50% formamide/2x SSC for at least 30 min.

3. 3D multicolor DNA FISH hybridization

Denature the probes at 80 °C for 5 min, and then put quickly on ice.
Load the hybridization probes on a clean microscope slide. Turn the coverslip with cells upside down on the drop of hybridization probes.
Seal the coverslip with rubber cement. Let the rubber cement dry completely. Then place slides on a heating block and denature at 75 °C for 4 min.
NOTE: The timing of denaturation and temperature of denaturation can be augmented, up to 80 °C.
Hybridize at 37 °C ON in a metallic box floating in a water bath.
NOTE: To improve the signal to noise ratio, the hybridization temperature can go up to 42 °C.
Peel off rubber cement, immerse the slides in 2x SCC, strip off the glass coverslip and transfer it to 2x SSC in 6-well plate.
Wash in 2x SSC three times for 5 min each at 37 °C, shaking at 90 rpm in an incubator shaker. Wash in 0.1x SSC three times for 5 min each at 60 °C, shaking at 90 rpm in an incubator shaker. Rinse briefly in 4x SSC/0.2% TWEEN 20.

4. 3D multicolor DNA FISH detection

NOTE: For directly labelled probes, skip steps 4.1 and 4.2.

Block in 4x SSC/0.2% TWEEN 20/4% bovine serum albumin (BSA) for 20 min at 37 °C in a 24-well plate, shaking at 20 rpm in an incubator shaker.
Incubate with the appropriate concentration of anti-digoxygenin (1:150), and/or streptavidin (1:1,000) (Table of Materials) diluted in 4x SSC/0.2% TWEEN 20/4% BSA for 35 min in a dark and wet chamber at 37 °C.
Wash in 4x SSC/0.2% TWEEN 20 three times for 5 min each at 37 °C, shaking at 90 rpm in a 6-well plate in an incubator shaker.
Equilibrate in PBS and post-fix in 2% formaldehyde/PBS for 2 min at RT in a 24-well plate.
NOTE: Directly labelled probes do not need post-fixation.
Wash 5x briefly in PBS. Stain with 1 ng/mL DAPI (4,6-diamidino-2-phenylindole)/PBS for 5 min at RT.
Wash 5x briefly in PBS. Mount with antifade solution (Table of Materials).

5. 3D multicolor DNA FISH microscopy and analysis

Acquire 3D images with a microscope system.
NOTE: Here, a widefield microscope with an axial distance of 0.2−0.25 μm between consecutive sections (Figure 1B) is used.
Analyze 3D image stacks using different software and tools (here, NuCLεD or Nuclear Contacts Locator in 3D).
NOTE: The tool NuCLεD has been developed in order to automatically analyze 3D multicolor DNA FISH in fluorescence cell image z-stacks. NuCLεD reconstructs the nuclei from cell image stacks in 3D as well as detects and localizes fluorescent 3D spots. It measures the relative positioning of spots in the nucleus (e.g., distance from the centroid of the nuclei and/or periphery of the nuclei) and the maximum radius for each nucleus and inter-spots distances. The tool and the algorithm used are fully described in Cortesi et al.¹⁶.
Analyze the data (e.g., 3D distances between specified genomic loci and the nuclear centroid and contact frequencies) retrieved by NuCLεD.
NOTE: For 3D distances between specified genomic loci and the nuclear centroid, normalize distances on the maximum radius for each nucleus and represent these data as frequency distributions of normalized distances from the nuclear centroid. For long range interactions studies, contact frequencies are supposed to be in the range of 10−20%²¹ with a threshold inter-distance that can vary and can be put around 2 µm³⁵.
1. To perform statistical analysis, analyze approximately 100 nuclei per biological replicate. Represent 3D distances between specified genomic loci as cumulative frequency distributions of distances that are below the threshold inter-distance selected and use a t-test to assess the significance of differences in the distributions. Also, calculate the percentage of nuclei positive for the interactions that are below the threshold inter-distance selected, and use Fisher's exact test to assess the significance of differences in the percentages.

Representative Results

The method of 3D multicolor DNA FISH described in this article allows contemporary visualization of different genomic loci within preserved 3D nuclei (Figure 1B). This protocol permits the measurement of distances between alleles, and different genomic loci in order to evaluate their spatial proximity, and to assess their location within the nuclear space (e.g., loci distance from the centroid or the periphery of the nuclei)¹⁶. However, there are many crucial steps that must be accurately and specifically set up for each cell type used; it is highly recommended to pay particular attention to the following steps for success of 3D multicolor DNA FISH.

For DNA probe preparation, check that probe size is <200 bp (Figure 1A). This size ensures a successful procedure of 3D multicolor DNA FISH (Figure 1B). Suboptimal DNA FISH probes produced by nick translation can be partially digested (Figure 2A) or over digested (Figure 2B). With partially digested probes, the procedure will have no signal in the cells, due to the inability of the probe to enter the nuclei and properly hybridize to the complementary genomic loci. Over digested probes will result in a nonspecific signal, due to a loss of specificity in the hybridization and a consequent increase of the background. A representative example of over digested probes is shown in Figure 3A in comparison to an optimal digested probe in Figure 3B.

For deproteinization and pepsinization, follow these steps according to the cell type. In particular, take into consideration nuclear size and cytoplasm abundance. For human primary ex vivo isolated T lymphocytes and cells with small, highly compacted nuclei and low abundant cytoplasm, HCl deproteinization is crucial. Treatment with 0.1 N HCl for 5 min is not sufficient for DNA FISH visualization. 0.1 N HCl treatment for 12 min is recommended to promote nuclei accessibility to DNA probes and preserve nuclear integrity (Figure 4A). Pepsin digestion of the cytoplasm is not needed to obtain a good signal of DNA FISH (Figure 4B).

For human primary myoblasts and cells that have large nuclei and abundant cytoplasm, the pepsinization step is fundamental. A short and suboptimal pepsinization of the cytoskeleton will hamper the entry of the probe in the nuclei (Figure 4C), ending in the absence of a DNA FISH signal. However, if the cells are over pepsinized, nuclei will not remain intact (Figure 4D), losing their 3D structure. An example of successful 3D multicolor DNA FISH is provided in Figure 4E.

During hybridization, seal the coverslip accurately; otherwise, the probe will disperse and dry. Denaturation and hybridization steps must be performed rapidly such that the probe and genomic DNA will not reanneal. The duration of the denaturation can be increased.

Figure 1: Representative DNA probes and 3D multicolor DNA FISH. (A) Nick translated DNA probes of optimal size run on an 2.2% agarose gel (lane 1, 2), 50 bp marker (M). (B) Representative 3D multicolor DNA FISH nucleus using probes mapping to 3q11.2 region (green), 10q26.3 region (red) and 8q24.13 region (magenta) in human primary myoblasts. Nuclei are counterstained with DAPI (blue). 63x magnification. Scale bar = 5 µm. Please click here to view a larger version of this figure.

Figure 2: Examples of not optimally digested DNA FISH probes. (A) Not digested (lane 1, 2) or partially digested (lane 3) nick translated DNA probes run on an 2.2% agarose gel, 2log marker (M). (B) Over digested nick translated DNA probes run on an 2.2% agarose gel, 2log marker (M). Please click here to view a larger version of this figure.

Figure 3: Comparison of 3D multicolor DNA FISH using suboptimal or optimal DNA FISH probes. (A) Representative 3D multicolor DNA FISH nuclei using over digested probe mapping to 8q24.13 region (magenta) in human primary myoblasts. Nuclei are counterstained with DAPI (blue). 63x magnification. Scale bar = 10 µm. (B) Representative 3D multicolor DNA FISH nuclei using optimally digested probe mapping to 8q24.13 region (magenta) in human primary myoblasts. Nuclei are counterstained with DAPI (blue). 63x magnification. Scale bar = 10 µm. Please click here to view a larger version of this figure.

Figure 4: Possible outcomes of suboptimal deproteinization and pepsinization steps on 3D multicolor DNA FISH results. (A) Representative 3D multicolor DNA FISH nuclei of human primary T lymphocytes treated for 5 min (left) or 12 min (right) with 0.1 N HCl, using probe mapping to 8q24.13 region (green). Nuclei are counterstained with DAPI (blue). 100x magnification. Scale bar = 10 µm. (B) Representative 3D multicolor DNA FISH nuclei of human primary T lymphocytes treated for 12 min with 0.1 N HCl (left) or coupled with 0.01 N HCl/0.0025% pepsin for 2 min (right), using probe mapping to 8q24.13 region (green). Nuclei are counterstained with DAPI (blue). 100x magnification. Scale bar = 10 µm. (C) Representative 3D multicolor DNA FISH nuclei of human primary myoblasts treated with short and suboptimal pepsinization, using probe mapping to 8q24.13 region (magenta). Nuclei are counterstained with DAPI (blue). 63x magnification. Scale bar = 10 µm. (D) Representative 3D multicolor DNA FISH nuclei of human primary myoblasts treated with prolonged pepsinization step, using probe mapping to 8q24.13 region (magenta). Nuclei are counterstained with DAPI (blue). 63x magnification. Scale bar = 5 µm. (E) Representative 3D multicolor DNA FISH nuclei of human primary myoblasts treated with optimal HCl/pepsin conditions using probe mapping to 8q24.13 region (magenta). Nuclei are counterstained with DAPI (blue). 63x magnification. Scale bar = 25 µm. Please click here to view a larger version of this figure.

Nick translation reagents	Initial concentration	Final concentration
dNTPs (C-G-A)	0.5 mM	0.05 mM
dTTP	0.1 mM	0.01 mM
Biotin/Dig/Cy3 dUTP	1 mM	0.02 mM
Tris HCl pH 7.8	1 M	50 mM
MgCl₂	100 mM	5 mM
β-mercaptoethanol	100 mM	10 mM
BSA	100 ng/µL	10 ng/µL
DNA Pol I	10 U/µL	0.1 U/µL
DNase I	1 U/µL	0.002 U/µL
DNA 2 µg	x	x
ddH₂O		Up to 50 µL

Table 1: Nick Translation. Table describing all the reagents, their concentration and suggested timing for nick translation reaction.

Discussion

The current method describes a step by step protocol to perform 3D multicolor DNA FISH on a wide range of human primary cells. Although DNA FISH is a technology in wide use, 3D multicolor DNA FISH on preserved 3D interphase nuclei is still difficult to perform in many laboratories, mainly due to the characteristics of the samples used²³^,²⁴.

Probe nick translation is a fundamental step for successful 3D multicolor DNA FISH; many different substrates (BAC, fosmid, plasmid, PCR products) can be used for this reaction, and the timing of the reaction and enzyme concentration can be accordingly adjusted with respect to the length of the substrate. A proper probe digestion is fundamental (Figure 1), as nonoptimal probes (Figure 2) will result in no signal or a nonspecific signal (Figure 3A). Permeabilization, deproteinization, and pepsinization steps are crucial passages that strongly depend on the cell type used. Cells with small nuclei and low cytoskeleton abundance, such as ex vivo isolated T lymphocytes, require deproteinization with a prolonged 0.1 N HCl treatment. Also, washes in PBS with higher percentages of Triton X-100 can help the probe entry in the nuclei of these cells. On the contrary, in vitro cultured human primary myoblasts that present larger nuclei, with a high content of cytoskeleton, need digestion of the cytosolic structures with pepsin. These general roles can be applied to a wide range of cells, eventually combining the different steps depending on the specific cellular characteristics.

The use of freshly prepared biological material, fresh solutions (in particular solutions with detergent), and fluorescent reagents are strongly suggested: filtered PFA at pH 7.0; autoclaved and filtered 20x SSC at pH 7.0; filtered formamide at pH 7.0; nuclease free water; and disposable aliquots of modified UTP. Prolonged incubation with 20% glycerol/PBS, or 50% formamide/2x SSC can facilitate the hybridization. HCl and/or pepsin treatment can be further increased. The timing of hybridization, the quantity of probes, the concentration and the timing of incubation of anti-digoxigenin and streptavidin can all be further adjusted to improve the signal to noise ratio.

3D multicolor DNA FISH represents a complementary tool to C-technologies, the standard method to validate C-based results. If combined with 3D microscopy and analysis, 3D multicolor DNA FISH can monitor the proximity between genomic loci and their topological distribution within the nuclear space at single cell level. 3D multicolor DNA FISH can be further integrated with other methodologies such as RNA FISH and immunofluorescence for a comprehensive overview of the dynamics and interactions between genomic loci, RNAs (messenger RNA or regulatory non coding RNA) and a wide range of proteins, providing a unique opportunity to visualize the nuclear structure and investigate the epigenetic mechanisms that subtend cellular identity.

Despite the huge improvement of FISH technologies with super resolution²⁵^,²⁶, live cell imaging²⁷^,²⁸^,³⁶, single molecule detection³⁷, and contemporary visualization of multiple targets with oligonucleotide arrays such as Oligopaint³⁷^,³⁸ with 3D high-throughput approaches³⁹, a limitation of the technology remains the discrete number of predetermined genomic loci that can be visualized. This prevents a wide-ranging analysis of nuclear architecture. Several studies have recently described sequential methods of hybridization to address genome organization in single cells such as barcode DNA FISH⁴⁰^,⁴¹^,⁴²^,⁴³. Further efforts will be needed to couple the single cell nature of 3D multicolor DNA FISH to genome wide features to broadly visualize nuclear architecture heterogeneity with imaging technologies, as the number of loci that can be tested at a time will increase.

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

The authors acknowledge the technical assistance of the INGM Imaging Facility (Istituto Nazionale di Genetica Molecolare "Romeo ed Enrica Invernizzi" (INGM), Milan, Italy), in particular C. Cordiglieri, for assistance during 3D multicolor DNA FISH images acquisition. This work has been supported by the following grants to B.B.: EPIGEN Italian flagship program, Association Française contre les Myopathies (AFM-Telethon, grant nr 18754) and Giovani Ricercatori, Italian Ministry of Health (GR-2011-02349383). This work has been supported by the following grant to F.M.: Fondazione Cariplo (Bando Giovani, grant nr 2018-0321).

Materials

24-well plates	Thermo Fisher Scientific	142475
6-well plates	Thermo Fisher Scientific	140675
Anti-Digoxigenin 488	DBA	DI7488
b-Mercaptoethanol	Sigma	M3148
bFGF	PeproTech	100-18B
Biotin 11 d-UTP	Thermo Fisher Scientific	R0081
BSA (bovine serum albumine)	Sigma	A7030
Coverlsips	Marienfeld	117500
CY3 d-UTP	GE Healthcare	PA53022
DAPI (4,6-diamidino-2-phenylindole)	Thermo Fisher Scientific	D21490
Deoxyribonucleic acids single strand from salmon testes	Sigma	D7656
Dextran sulfate (powder)	Santa Cruz	sc-203917A
Digoxigenin 11 d-UTP	Roche	11093088910
DMEM	Thermo Fisher Scientific	21969-035 500mL
DNA polymerase I	Thermo Fisher Scientific	18010-017
DNase I	Sigma	AMPD1
dNTPs (C-G-A-T)	Euroclone	BL0423A/C/G
EGF	Sigma	E9644.2MG
Ethanol	Sigma	02860-1L
FBS Hyclone	Thermo Fisher Scientific	SH30109
Formaldehyde solution	Sigma	F8775-25mL
Formamide	Sigma	F9037
Glutammine	Thermo Fisher Scientific	25030-024 100mL
Glycerol	Sigma	G5516-100mL
Glycogen	Thermo Fisher Scientific	AM9510
HCl	Sigma	30721
Human Cot-1 DNA	Thermo Fisher Scientific	15279-001
Insulin Human	Sigma	I9278-5 mL
MgCl₂	Sigma	63069
NaAc (Sodium Acetate, pH 5.2, 3 M)	Sigma	S2889
NaCl	Sigma	S9888
Paraformaldehyde	Sigma	158127-25G
PBS (phosphate-buffered saline)	Sigma	P4417
Pennycillin/Streptavidin	Thermo Fisher Scientific	15070-063 100mL
Pepsin	Biorad	P6887
PhasePrep BAC DNA Kit	Sigma	NA0100-1KT
Poly-L-lysine solution	Sigma	P8920
ProLong Diamond Antifade Mountant	Thermo Fisher Scientific	P36970
PureLink Quick Gel Extraction & PCR Purification Combo Kit	Thermo Fisher Scientific	K220001
PureLink Quick Plasmid Miniprep Kit	Thermo Fisher Scientific	K210010
RNAse cocktail	Thermo Fisher Scientific	AM2288
Rubbercement	Bostik
Slides	VWR	631-0114
Streptavidina Alexa fluor 647	Thermo Fisher Scientific	S21374
Tri-Sodium Citrate	Sigma	1110379026
Tris-HCl	Sigma	T3253-500g
Triton X-100	Sigma	T8787-250mL
TWEEN 20	Sigma	P9416-100mL

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Marasca, F., Cortesi, A., Manganaro, L., Bodega, B. 3D Multicolor DNA FISH Tool to Study Nuclear Architecture in Human Primary Cells. J. Vis. Exp. (155), e60712, doi:10.3791/60712 (2020).