Summary

Kombineret Immunofluorescens og DNA FISH på 3D-bevaret Interfasen Kerner til Uddannelse Ændringer i 3D Nuclear Organization

Published: February 03, 2013
doi:

Summary

Her beskriver vi en protokol til samtidig påvisning af histon ændringer ved immunofluorescens og DNA sekvenser ved DNA FISK efterfulgt af 3D mikroskopi og analyser (3D immuno-DNA FISH).

Abstract

Fluorescerende in situ hybridisering med DNA-prober på 3-dimensionelt bevarede kerner efterfulgt af 3D konfokal mikroskopi (3D DNA FISH) repræsenterer den mest direkte måde at visualisere placeringen af gen loci, kromosomale subregioner eller hele områder i de enkelte celler. Denne type analyse giver indblik i den globale arkitektur af kernen samt opførsel af specifikke genomiske loci og regioner i det nukleare område. Immunofluorescens på den anden side tillader påvisning af kerneproteiner (modificerede histoner, histon varianter og modifikatorer, transkriptionsmekanisme og faktorer, nukleare delrum osv.). Den største udfordring i at kombinere immunofluorescens og 3D DNA FISH er, på den ene side at bevare epitopen detekteres af antistoffet såvel som 3D arkitektur af kernen, og på den anden side, for at tillade gennemtrængning af DNA-probe til påvisning gen loci eller kromosom territorier 1-5. Her giver vi en protokol, der kombinerer visualisering af kromatin modifikationer med genomiske loci i 3D konserveret kerner.

Introduction

Epigenetiske mekanismer udløser etablering og arv af udviklings-og celletype specifikke transkriptionelle profiler. På et niveau dette indebærer modulering af kromatin emballage, der definerer aktive eller stille genomiske regioner. I en større målestok, også global 3D organisering af genomet og nuklear struktur spiller en rolle i kontrollen af ​​transkriptionelle mønstre. Således dissektion af disse epigenomic funktioner er afgørende for en fuld forståelse af, hvordan generne reguleres 6-11.

Kombineret immunofluorescens og 3D DNA FISH giver en unik mulighed for at supplere molekylære og biokemiske analyser ved at vurdere specifikke interaktioner / sammenslutninger af DNA-sekvenser og / eller proteiner i kernen. Og selv om genom-dækkende high throughput-teknikker, såsom chromatin immunoprecipitation (chip-seq) eller kromosom capture konformation kombineret med dyb sekventering (4C-seq, 5C, Hi-C) giver global daten på cellepopulationer 12, immunofluorescens / DNA FISH teknikker muliggøre analyser på enkelt celle niveau.

Her beskriver vi en protokol til samtidig påvisning af histon ændringer ved immunofluorescens og DNA sekvenser ved DNA FISK efterfulgt af 3D mikroskopi og analyser (3D immuno-FISH). Fordelen ved denne protokol er den kombinerede visualisering af DNA og bevarelse af proteinstrukturer. Vores erfaring på dette område har gjort det muligt for os at forbedre og forenkle eksisterende protokoller. Selv om vi har anvendt denne protokol til påvisning af DNA dobbeltstrengede brud i lymfocytter undergår rekombination, kan denne metode anvendes på andre proteiner og andre celletyper.

Protocol

1. DNA Probe Mærkning med fluoroforer: Nick Translation (~ 6 timer) Ren BAC DNA (fremstillet ved maxi-prep) eller plasmider eller PCR-produkter, alle resuspenderet i H2O, kan anvendes til mærkning. Bemærk, at for en robust FISH signal, bør prober spænde mindst 10 kb. Inkuber DNA i RNase A i 30 minutter ved 37 ° C (Alle reagenser er anført i tabel 1). Inkubér nick-translation reaktion i 2 timer ved 16 ° C (se tabel 2 og 3).</stro…

Representative Results

DNA og immuno-FISH anvendes i Skok laboratoriet at studere ændringer i nuklear organisering i forbindelse med fremgangsmåden ifølge V (D) J rekombination af antigen-receptor loci i B-og T-lymfocyt-udvikling. De teknikker beskrevet ovenfor giver os mulighed for i) måle afstande mellem de to ender af et locus (sammentrækning) ii) måle afstande mellem alleler eller loci (parring), iii) at analysere DNA-skader forekommer inden loci, iv) vurdere placeringen af ​​alleler og loci i forhold til nukleare delrum (repres…

Discussion

Teknikkerne beskrevet ovenfor blev anvendt i vores laboratorium til analyse reguleringen af V (D) J rekombination af immunoglobulinet og Tcra / d loci i udviklingen af lymfocytter 30,31. Vi tror på, at denne teknik kan tilpasses til påvisning af forskellige nukleare proteiner, nukleare rum og loci, i forskellige celletyper. Ændringer af protokollen kan være nødvendig, og i dette tilfælde de vigtigste trin til at fokusere på, er følgende. For det første kan længden af ​​permeabi…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Vi vil gerne takke medlemmerne af Skok lab, især Susannah Hewitt, for diskussioner og kommentarer. Dette arbejde er støttet af National Institute of Health tilskud R01GM086852, RC1CA145746 (JAS). JAS er en Leukemia & Lymphoma Society lærd. JC er en Irvington Institute Fellow af Cancer Research Institute. MM er støttet af en National Science Foundation Grant Integrativ Graduate Uddannelse og Forskning praktik (NSF IGERT 0.333.389).

Materials

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.

References

  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).

Play Video

Cite This Article
Chaumeil, J., Micsinai, M., Skok, J. A. Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization. J. Vis. Exp. (72), e50087, doi:10.3791/50087 (2013).

View Video