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Biology

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Published: February 3rd, 2013

DOI:

10.3791/50087

1Department of Pathology, New York University School of Medicine, 2New York University Center for Health Informatics and Bioinformatics, 3NYU Cancer Institute, 4Department of Pathology and Yale Cancer Center, Yale University School of Medicine

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Epigenetic mechanisms trigger establishment and inheritance of developmental and cell-type specific transcriptional profiles. At one level this involves modulation of chromatin packaging that defines active or silent genomic regions. On a larger scale, global 3D organization of the genome and nuclear architecture also play a role in the control of transcriptional patterns. Thus, dissection of these epigenomic features is essential for a full understanding of how genes are regulated 6-11.

Combined immunofluorescence and 3D DNA FISH provide a unique opportunity to complement molecular and biochemical analyses by assessing ....

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1. DNA Probe Labeling with Fluorophores: Nick Translation (~ 6 hr)

  1. Clean BAC DNA (prepared by maxi-prep) or plasmids or PCR products, all resuspended in H2O, can be used for labeling. Note that for a robust FISH signal, probes should span at least 10 kb.
  2. Incubate DNA in RNase A for 30 min at 37 °C (All reagents are listed in Table 1).
  3. Incubate the nick translation reaction for 2 hr at 16 °C (see Tables 2 and 3

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DNA and immuno-FISH are used in the Skok lab to study the changes in nuclear organization associated with the process of V(D)J recombination of antigen receptor loci during B and T lymphocyte development. The techniques detailed above enable us to i) measure distances between the two ends of a locus (contraction) ii) measure distances between alleles or loci (pairing), iii) analyze the DNA damage occurring within loci, iv) assess the location of alleles and loci relative to nuclear sub-compartments (repressive per.......

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The techniques detailed above were used in our lab to analyze the regulation of V(D)J recombination of the Immunoglobulin and Tcra/d loci in developing lymphocytes 30,31 . We are confident that this technique can be adapted for detection of various nuclear proteins, nuclear compartments and loci, in different cell-types. Modifications of the protocol may be necessary, and in this case the major steps to focus on are the following. First, the length of permeabilization can be adjusted de.......

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We would like to thank the members of the Skok lab, especially Susannah Hewitt, for discussions and comments. This work is supported by the National Institute of Health grants R01GM086852, RC1CA145746 (J.A.S.). J.A.S. is a Leukemia & Lymphoma Society scholar. J.C. is an Irvington Institute Fellow of the Cancer Research Institute. M.M. is supported by a National Science Foundation Grant Integrative Graduate Education and Research Traineeship (NSF IGERT 0333389).

....

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Name Company Catalog Number Comments
Name of Reagent/Material Company Catalogue Number Comments
H2O Fisher # BP2470
RNase A Sigma # R4642
dNTP Sigma # DNTP100
Alexa dUTP Invitrogen # C11397 to C-11401
Cy3 or Cy5 dUTP Fisher # 45-001-xxx
DNase I Roche # 04536282001
DNA Pol I Biolabs # M0209
0.025 μm filters Millipore # VSWP02500
Cot-1 DNA 1 mg/ml Invitrogen # 18440
Hybloc DNA 1 mg/ml Applied Genetics # MHB
Salmon sperm Sigma # D1626 powder to be resuspended at 10 mg/ml in H2O
NaAc (Sodium Acetate, pH 5.2, buffer solution) Sigma # S7899
Ficoll 400 (Mol Biol grade) Fisher # 525
Polyvinylpyrrolidone (Mol Biol grade) Fisher # BP431
Dextran sulfate powder Sigma # D8906
SSPE (Saline-Sodium Phosphate-EDTA) 20x solution Fisher # BP1328
Formamide Fisher # BP227
Coverslips Fisher # 12-548-B
Slides Fisher # 12-550
6-well plates Fisher # 0720080
PBS, 10x Fisher # MT-46-013-CM
Poly-L-lysine solution Sigma # P8920
Paraformaldehyde, prills, 95% Sigma # 441244
Triton-X-100, Mol Biol grade Sigma # T8787
BSA (Bovine Serum Albumin) Fraction V Fisher # BP 1600
Normal goat serum Vector Labs # S-1000
Tween-20, Mol Biol grade Sigma # P9416
SSC (Saline Sodium Citrate) 20x solution Fisher # BP1325
ProLong Gold antifade reagent Invitrogen # P36930
DAPI (4',6-diamidino-2-phenylindole) Sigma # D9542
Best test one coat rubber cement Art or office supply stores
Table 1. Specific reagents and small equipment.

  1. Chaumeil, J., Okamoto, I., Heard, E. X-chromosome inactivation in mouse embryonic stem cells: analysis of histone modifications and transcriptional activity using immunofluorescence and FISH. Methods in enzymology. , 376-405 (2004).
  2. Cremer, M., et al. Multicolor 3D fluorescence in situ hybridization for imaging interphase chromosomes. Methods Mol. Biol. 463, 205-239 (2008).
  3. Chaumeil, J., Augui, S., Chow, J. C., Heard, E. Combined immunofluorescence, RNA fluorescent in situ hybridization, and DNA fluorescent in situ hybridization to study chromatin changes, transcriptional activity, nuclear organization, and X-chromosome inactivation. Methods Mol. Biol. 463, 297-308 (2008).
  4. Solovei, I., Cremer, M. 3D-FISH on cultured cells combined with immunostaining. Methods Mol. Biol. 659, 117-126 (2010).
  5. Markaki, Y. The potential of 3D-FISH and super-resolution structured illumination microscopy for studies of 3D nuclear architecture: 3D structured illumination microscopy of defined chromosomal structures visualized by 3D (immuno)-FISH opens new perspectives for studies of nuclear architecture. BioEssays : news and reviews in molecular, cellular and developmental biology. 34, 412-426 (2012).
  6. Heard, E., Bickmore, W. The ins and outs of gene regulation and chromosome territory organisation. Current opinion in cell biology. 19, 311-316 (2007).
  7. Misteli, T. Beyond the sequence: cellular organization of genome function. Cell. 128, 787-800 (1016).
  8. Fraser, P., Bickmore, W. Nuclear organization of the genome and the potential for gene regulation. Nature. 447, 413-417 (2007).
  9. Cremer, T., et al. Chromosome territories--a functional nuclear landscape. Current opinion in cell biology. 18, 307-316 (2006).
  10. Mao, Y. S., Zhang, B., Spector, D. L. Biogenesis and function of nuclear bodies. Trends in genetics : TIG. 27, 295-306 (2011).
  11. Dostie, J., Bickmore, W. A. Chromosome organization in the nucleus - charting new territory across the Hi-Cs. Current opinion in genetics & development. 22, 125-131 (2012).
  12. van Steensel, B., Dekker, J. Genomics tools for unraveling chromosome architecture. Nature. 28, 1089-1095 (2010).
  13. Massey, F. J. The Kolmogorov-Smirnov Test for Goodness of Fit. Journal of the American Statistical Association. 253, 1951 (1951).
  14. Collins, A., et al. RUNX transcription factor-mediated association of Cd4 and Cd8 enables coordinate gene regulation. Immunity. 34, 303-314 (2011).
  15. Fisher, R. A. On the interpretation of χ2 from contingency tables, and the calculation of P. Journal of the Royal Statistical Society. 85, 87-94 (1922).
  16. Benjamini, Y. H., Yosef, Controlling the false discovery rate: a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society, Series B (Methodological). 57, 125-133 (1995).
  17. Fitzsimmons, S. P., Bernstein, R. M., Max, E. E., Skok, J. A., Shapiro, M. A. Dynamic changes in accessibility, nuclear positioning, recombination, and transcription at the Igkappa locus. J. Immunol. 179, 5264-5273 (2007).
  18. Fuxa, M., et al. Pax5 induces V-to-DJ rearrangements and locus contraction of the immunoglobulin heavy-chain gene. Genes Dev. 18, 411-422 (2004).
  19. Goldmit, M. Epigenetic ontogeny of the Igk locus during B cell development. Nature. 6, 198-203 (2005).
  20. Hewitt, S. L. Association between the Igk and Igh immunoglobulin loci mediated by the 3' Igk enhancer induces 'decontraction' of the Igh locus in pre-B cells. Nature. 9, 396-404 (2008).
  21. Johnson, K. IL-7 Functionally Segregates the Pro-B Cell Stage by Regulating Transcription of Recombination Mediators across Cell Cycle. Journal of Immunology. , (2012).
  22. Karnowski, A., et al. Silencing and nuclear repositioning of the lambda5 gene locus at the pre-b cell stage requires Aiolos and OBF-1. PLoS ONE. 3, e3568 (2008).
  23. Kosak, S. T. Subnuclear compartmentalization of immunoglobulin loci during lymphocyte development. Science. 296, 158-162 (2002).
  24. Liu, H., et al. Yin Yang 1 is a critical regulator of B-cell development. Genes Dev. 21, 1179-1189 (2007).
  25. Parker, M. J. The pre-B-cell receptor induces silencing of VpreB and lambda5 transcription. Embo J. 24, 3895-3905 (2005).
  26. Roldan, E., et al. Locus 'decontraction' and centromeric recruitment contribute to allelic exclusion of the immunoglobulin heavy-chain gene. Nature immunology. 6, 31-41 (2005).
  27. Skok, J. A. Nonequivalent nuclear location of immunoglobulin alleles in B lymphocytes. Nature. 2, 848-854 (2001).
  28. Skok, J. A. Reversible contraction by looping of the Tcra and Tcrb loci in rearranging thymocytes. Nature immunology. 8, 378-387 (2007).
  29. Xiang, Y., Zhou, X., Hewitt, S. L., Skok, J. A., Garrard, W. T. A multifunctional element in the mouse Igkappa locus that specifies repertoire and Ig loci subnuclear location. Journal of Immunology. 186, 5356-5366 (2011).
  30. Hewitt, S. L. RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci. Nature immunology. 10, 655-664 (2009).
  31. Deriano, L., et al. The RAG2 C terminus suppresses genomic instability and lymphomagenesis. Nature. 471, 119-123 (2011).
  32. Brown, K. E., Baxter, J., Graf, D., Merkenschlager, M., Fisher, A. G. Dynamic repositioning of genes in the nucleus of lymphocytes preparing for cell division. Molecular cell. 3, 207-217 (1999).
  33. Fernandez-Capetillo, O., Lee, A., Nussenzweig, M., Nussenzweig, A. H2AX: the histone guardian of the genome. DNA repair. 3, 959-967 (2004).
  34. Croft, J. A., et al. Differences in the localization and morphology of chromosomes in the human nucleus. The Journal of cell biology. 145, 1119-1131 (1999).
  35. Chaumeil, J., Le Baccon, P., Wutz, A., Heard, E. A novel role for Xist RNA in the formation of a repressive nuclear compartment into which genes are recruited when silenced. Genes Dev. 20, 2223-2237 (2006).
  36. Walter, J., et al. Towards many colors in FISH on 3D-preserved interphase nuclei. Cytogenetic and genome research. 114, 367-378 (2006).
  37. Toomre, D., Bewersdorf, J. A new wave of cellular imaging. Annual review of cell and developmental biology. 26, 285-314 (2010).
  38. Schermelleh, L., Heintzmann, R., Leonhardt, H. A guide to super-resolution fluorescence microscopy. The Journal of cell biology. 190, 165-175 (2010).
  39. Dobbie, I. M. OMX: a new platform for multimodal, multichannel wide-field imaging. Cold Spring Harbor protocols. , 899-909 (2011).
  40. Boyle, S., Rodesch, M. J., Halvensleben, H. A., Jeddeloh, J. A., Bickmore, W. A. Fluorescence in situ hybridization with high-complexity repeat-free oligonucleotide probes generated by massively parallel synthesis. Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology. 19, 901-909 (2011).

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