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W tym Artykule

  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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.

Protokół

1. DNA probe preparation and labelling procedures with nick translation

  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. Centrifuge at maximum speed for 1 h at 4 °C and discard the supernatant. Wash the pellet twice with 70% EtOH.
  7. 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.

  1. 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.
  2. 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

  1. Denature the probes at 80 °C for 5 min, and then put quickly on ice.
  2. Load the hybridization probes on a clean microscope slide. Turn the coverslip with cells upside down on the drop of hybridization probes.
  3. 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.
  4. 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.
  5. 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.
  6. 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.

  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. Wash 5x briefly in PBS. Stain with 1 ng/mL DAPI (4,6-diamidino-2-phenylindole)/PBS for 5 min at RT.
  6. Wash 5x briefly in PBS. Mount with antifade solution (Table of Materials).

5. 3D multicolor DNA FISH microscopy and analysis

  1. 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.
  2. 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.
  3. 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.

Wyniki

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...

Dyskusje

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 differe...

Ujawnienia

The authors have nothing to disclose.

Podziękowania

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).

Materiały

NameCompanyCatalog NumberComments
24-well platesThermo Fisher Scientific142475
6-well platesThermo Fisher Scientific140675
Anti-Digoxigenin 488DBADI7488
b-MercaptoethanolSigmaM3148
bFGFPeproTech100-18B
Biotin 11 d-UTPThermo Fisher ScientificR0081
BSA (bovine serum albumine)SigmaA7030
CoverlsipsMarienfeld117500
CY3 d-UTPGE HealthcarePA53022
DAPI (4,6-diamidino-2-phenylindole)Thermo Fisher ScientificD21490
Deoxyribonucleic acids single strand from salmon testesSigmaD7656
Dextran sulfate (powder)Santa Cruzsc-203917A
Digoxigenin 11 d-UTPRoche11093088910
DMEMThermo Fisher Scientific21969-035 500mL
DNA polymerase IThermo Fisher Scientific18010-017
DNase ISigmaAMPD1
dNTPs (C-G-A-T)EurocloneBL0423A/C/G
EGFSigmaE9644.2MG
EthanolSigma02860-1L
FBS HycloneThermo Fisher ScientificSH30109
Formaldehyde solutionSigmaF8775-25mL
FormamideSigmaF9037
GlutammineThermo Fisher Scientific25030-024 100mL
GlycerolSigmaG5516-100mL
GlycogenThermo Fisher ScientificAM9510
HClSigma30721
Human Cot-1 DNAThermo Fisher Scientific15279-001
Insulin HumanSigmaI9278-5 mL
MgCl2Sigma63069
NaAc (Sodium Acetate, pH 5.2, 3 M)SigmaS2889
NaClSigmaS9888
ParaformaldehydeSigma158127-25G
PBS (phosphate-buffered saline)SigmaP4417
Pennycillin/StreptavidinThermo Fisher Scientific15070-063 100mL
PepsinBioradP6887
PhasePrep BAC DNA KitSigmaNA0100-1KT
Poly-L-lysine solutionSigmaP8920
ProLong Diamond Antifade MountantThermo Fisher ScientificP36970
PureLink Quick Gel Extraction & PCR Purification Combo KitThermo Fisher ScientificK220001
PureLink Quick Plasmid Miniprep KitThermo Fisher ScientificK210010
RNAse cocktailThermo Fisher ScientificAM2288
RubbercementBostik
SlidesVWR631-0114
Streptavidina Alexa fluor 647Thermo Fisher ScientificS21374
Tri-Sodium CitrateSigma1110379026
Tris-HClSigmaT3253-500g
Triton X-100SigmaT8787-250mL
TWEEN 20SigmaP9416-100mL

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