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. 1994 Jul 2;126(2):289–304. doi: 10.1083/jcb.126.2.289

Dynamic changes in the higher-level chromatin organization of specific sequences revealed by in situ hybridization to nuclear halos

PMCID: PMC2200020  PMID: 8034736

Abstract

A novel approach to study the higher level packaging of specific DNA sequences has been developed by coupling high-resolution fluorescence hybridization with biochemical fractionation to remove histones and distend DNA loops to form morphologically reproducible nuclear "halos." Results demonstrate consistent differences in the organization of specific sequences, and further suggest a relationship to functional activity. Pulse-incorporated bromodeoxyuridine representing nascent replicating DNA localized with the base of the chromatin loops in discrete clustered patterns characteristic of intact cells, whereas at increasing chase times, the replicated DNA was consistently found further out on the extended region of the halo. Fluorescence hybridization to unique loci for four transcriptionally inactive sequences produced long strings of signal extending out onto the DNA halo or "loop," whereas four transcriptionally active sequences remained tightly condensed as single spots within the residual nucleus. In contrast, in non-extracted cells, all sequences studied typically remained condensed as single spots of fluorescence signal. Interestingly, two transcriptionally active, tandemly repeated gene clusters exhibited strikingly different packaging by this assay. Analysis of specific genes in single cells during the cell cycle revealed changes in packaging between S-phase and non S-phase cells, and further suggested a dramatic difference in the structural associations in mitotic and interphase chromatin. These results are consistent with and suggestive of a loop domain organization of chromatin packaging involving both stable and transient structural associations, and provide precedent for an approach whereby different biochemical fractionation methods may be used to unravel various aspects of the complex higher-level organization of the genome.

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Selected References

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  1. Berezney R., Coffey D. S. Nuclear protein matrix: association with newly synthesized DNA. Science. 1975 Jul 25;189(4199):291–293. doi: 10.1126/science.1145202. [DOI] [PubMed] [Google Scholar]
  2. Berezney R. The nuclear matrix: a heuristic model for investigating genomic organization and function in the cell nucleus. J Cell Biochem. 1991 Oct;47(2):109–123. doi: 10.1002/jcb.240470204. [DOI] [PubMed] [Google Scholar]
  3. Bickmore W. A., Sumner A. T. Mammalian chromosome banding--an expression of genome organization. Trends Genet. 1989 May;5(5):144–148. doi: 10.1016/0168-9525(89)90055-3. [DOI] [PubMed] [Google Scholar]
  4. Bodnar J. W. A domain model for eukaryotic DNA organization: a molecular basis for cell differentiation and chromosome evolution. J Theor Biol. 1988 Jun 22;132(4):479–507. doi: 10.1016/s0022-5193(88)80086-9. [DOI] [PubMed] [Google Scholar]
  5. Bodnar J. W., Hanson P. I., Polvino-Bodnar M., Zempsky W., Ward D. C. The terminal regions of adenovirus and minute virus of mice DNAs are preferentially associated with the nuclear matrix in infected cells. J Virol. 1989 Oct;63(10):4344–4353. doi: 10.1128/jvi.63.10.4344-4353.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bodnar J. W., Jones G. S., Ellis C. H., Jr The domain model for eukaryotic DNA organization. 2: A molecular basis for constraints on development and evolution. J Theor Biol. 1989 Apr 6;137(3):281–320. doi: 10.1016/s0022-5193(89)80074-8. [DOI] [PubMed] [Google Scholar]
  7. Carter K. C., Bowman D., Carrington W., Fogarty K., McNeil J. A., Fay F. S., Lawrence J. B. A three-dimensional view of precursor messenger RNA metabolism within the mammalian nucleus. Science. 1993 Feb 26;259(5099):1330–1335. doi: 10.1126/science.8446902. [DOI] [PubMed] [Google Scholar]
  8. Carter K. C., Taneja K. L., Lawrence J. B. Discrete nuclear domains of poly(A) RNA and their relationship to the functional organization of the nucleus. J Cell Biol. 1991 Dec;115(5):1191–1202. doi: 10.1083/jcb.115.5.1191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chelly J., Kaplan J. C., Maire P., Gautron S., Kahn A. Transcription of the dystrophin gene in human muscle and non-muscle tissue. Nature. 1988 Jun 30;333(6176):858–860. doi: 10.1038/333858a0. [DOI] [PubMed] [Google Scholar]
  10. Chesa P. G., Rettig W. J., Thomson T. M., Old L. J., Melamed M. R. Immunohistochemical analysis of nerve growth factor receptor expression in normal and malignant human tissues. J Histochem Cytochem. 1988 Apr;36(4):383–389. doi: 10.1177/36.4.2831267. [DOI] [PubMed] [Google Scholar]
  11. Ciejek E. M., Tsai M. J., O'Malley B. W. Actively transcribed genes are associated with the nuclear matrix. Nature. 1983 Dec 8;306(5943):607–609. doi: 10.1038/306607a0. [DOI] [PubMed] [Google Scholar]
  12. Cockerill P. N., Garrard W. T. Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. Cell. 1986 Jan 31;44(2):273–282. doi: 10.1016/0092-8674(86)90761-0. [DOI] [PubMed] [Google Scholar]
  13. Cook P. R., Brazell I. A., Jost E. Characterization of nuclear structures containing superhelical DNA. J Cell Sci. 1976 Nov;22(2):303–324. doi: 10.1242/jcs.22.2.303. [DOI] [PubMed] [Google Scholar]
  14. Cook P. R. The nucleoskeleton and the topology of replication. Cell. 1991 Aug 23;66(4):627–635. doi: 10.1016/0092-8674(91)90109-c. [DOI] [PubMed] [Google Scholar]
  15. Cook P. R. The nucleoskeleton and the topology of transcription. Eur J Biochem. 1989 Nov 20;185(3):487–501. doi: 10.1111/j.1432-1033.1989.tb15141.x. [DOI] [PubMed] [Google Scholar]
  16. Devilee P., Cremer T., Slagboom P., Bakker E., Scholl H. P., Hager H. D., Stevenson A. F., Cornelisse C. J., Pearson P. L. Two subsets of human alphoid repetitive DNA show distinct preferential localization in the pericentric regions of chromosomes 13, 18, and 21. Cytogenet Cell Genet. 1986;41(4):193–201. doi: 10.1159/000132229. [DOI] [PubMed] [Google Scholar]
  17. Dijkwel P. A., Hamlin J. L. Matrix attachment regions are positioned near replication initiation sites, genes, and an interamplicon junction in the amplified dihydrofolate reductase domain of Chinese hamster ovary cells. Mol Cell Biol. 1988 Dec;8(12):5398–5409. doi: 10.1128/mcb.8.12.5398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fey E. G., Bangs P., Sparks C., Odgren P. The nuclear matrix: defining structural and functional roles. Crit Rev Eukaryot Gene Expr. 1991;1(2):127–143. [PubMed] [Google Scholar]
  19. Fey E. G., Ornelles D. A., Penman S. Association of RNA with the cytoskeleton and the nuclear matrix. J Cell Sci Suppl. 1986;5:99–119. doi: 10.1242/jcs.1986.supplement_5.6. [DOI] [PubMed] [Google Scholar]
  20. Fisher E. M., Beer-Romero P., Brown L. G., Ridley A., McNeil J. A., Lawrence J. B., Willard H. F., Bieber F. R., Page D. C. Homologous ribosomal protein genes on the human X and Y chromosomes: escape from X inactivation and possible implications for Turner syndrome. Cell. 1990 Dec 21;63(6):1205–1218. doi: 10.1016/0092-8674(90)90416-c. [DOI] [PubMed] [Google Scholar]
  21. Fu X. D., Maniatis T. Factor required for mammalian spliceosome assembly is localized to discrete regions in the nucleus. Nature. 1990 Feb 1;343(6257):437–441. doi: 10.1038/343437a0. [DOI] [PubMed] [Google Scholar]
  22. Ganner E., Evans H. J. The relationship between patterns of DNA replication and of quinacrine fluorescence in the human chromosome complement. Chromosoma. 1971;35(3):326–341. doi: 10.1007/BF00326282. [DOI] [PubMed] [Google Scholar]
  23. Gasser S. M., Laemmli U. K. Cohabitation of scaffold binding regions with upstream/enhancer elements of three developmentally regulated genes of D. melanogaster. Cell. 1986 Aug 15;46(4):521–530. doi: 10.1016/0092-8674(86)90877-9. [DOI] [PubMed] [Google Scholar]
  24. Gazit B., Cedar H., Lerer I., Voss R. Active genes are sensitive to deoxyribonuclease I during metaphase. Science. 1982 Aug 13;217(4560):648–650. doi: 10.1126/science.6283640. [DOI] [PubMed] [Google Scholar]
  25. Henderson A., Ripley S., Heller M., Kieff E. Chromosome site for Epstein-Barr virus DNA in a Burkitt tumor cell line and in lymphocytes growth-transformed in vitro. Proc Natl Acad Sci U S A. 1983 Apr;80(7):1987–1991. doi: 10.1073/pnas.80.7.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Heng H. H., Squire J., Tsui L. C. High-resolution mapping of mammalian genes by in situ hybridization to free chromatin. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9509–9513. doi: 10.1073/pnas.89.20.9509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Holmquist G., Gray M., Porter T., Jordan J. Characterization of Giemsa dark- and light-band DNA. Cell. 1982 Nov;31(1):121–129. doi: 10.1016/0092-8674(82)90411-1. [DOI] [PubMed] [Google Scholar]
  28. Jackson D. A., Cook P. R. Transcriptionally active minichromosomes are attached transiently in nuclei through transcription units. J Cell Sci. 1993 Aug;105(Pt 4):1143–1150. doi: 10.1242/jcs.105.4.1143. [DOI] [PubMed] [Google Scholar]
  29. Jackson D. A., Dickinson P., Cook P. R. The size of chromatin loops in HeLa cells. EMBO J. 1990 Feb;9(2):567–571. doi: 10.1002/j.1460-2075.1990.tb08144.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Jankelevich S., Kolman J. L., Bodnar J. W., Miller G. A nuclear matrix attachment region organizes the Epstein-Barr viral plasmid in Raji cells into a single DNA domain. EMBO J. 1992 Mar;11(3):1165–1176. doi: 10.1002/j.1460-2075.1992.tb05157.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Johnson C. V., McNeil J. A., Carter K. C., Lawrence J. B. A simple, rapid technique for precise mapping of multiple sequences in two colors using a single optical filter set. Genet Anal Tech Appl. 1991 Apr;8(2):75–76. doi: 10.1016/1050-3862(91)90052-s. [DOI] [PubMed] [Google Scholar]
  32. Johnson C. V., Singer R. H., Lawrence J. B. Fluorescent detection of nuclear RNA and DNA: implications for genome organization. Methods Cell Biol. 1991;35:73–99. [PubMed] [Google Scholar]
  33. Kerem B. S., Goitein R., Diamond G., Cedar H., Marcus M. Mapping of DNAase I sensitive regions on mitotic chromosomes. Cell. 1984 Sep;38(2):493–499. doi: 10.1016/0092-8674(84)90504-x. [DOI] [PubMed] [Google Scholar]
  34. Korenberg J. R., Rykowski M. C. Human genome organization: Alu, lines, and the molecular structure of metaphase chromosome bands. Cell. 1988 May 6;53(3):391–400. doi: 10.1016/0092-8674(88)90159-6. [DOI] [PubMed] [Google Scholar]
  35. Lawrence J. B., Carter K. C., Gerdes M. J. Extending the capabilities of interphase chromatin mapping. Nat Genet. 1992 Nov;2(3):171–172. doi: 10.1038/ng1192-171. [DOI] [PubMed] [Google Scholar]
  36. Lawrence J. B., Singer R. H., Marselle L. M. Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybridization. Cell. 1989 May 5;57(3):493–502. doi: 10.1016/0092-8674(89)90924-0. [DOI] [PubMed] [Google Scholar]
  37. Lawrence J. B., Singer R. H., McNeil J. A. Interphase and metaphase resolution of different distances within the human dystrophin gene. Science. 1990 Aug 24;249(4971):928–932. doi: 10.1126/science.2203143. [DOI] [PubMed] [Google Scholar]
  38. Lawrence J. B., Villnave C. A., Singer R. H. Sensitive, high-resolution chromatin and chromosome mapping in situ: presence and orientation of two closely integrated copies of EBV in a lymphoma line. Cell. 1988 Jan 15;52(1):51–61. doi: 10.1016/0092-8674(88)90530-2. [DOI] [PubMed] [Google Scholar]
  39. Lewis C. D., Laemmli U. K. Higher order metaphase chromosome structure: evidence for metalloprotein interactions. Cell. 1982 May;29(1):171–181. doi: 10.1016/0092-8674(82)90101-5. [DOI] [PubMed] [Google Scholar]
  40. Little R. D., Braaten D. C. Genomic organization of human 5 S rDNA and sequence of one tandem repeat. Genomics. 1989 Apr;4(3):376–383. doi: 10.1016/0888-7543(89)90345-5. [DOI] [PubMed] [Google Scholar]
  41. Lydersen B. K., Pettijohn D. E. Human-specific nuclear protein that associates with the polar region of the mitotic apparatus: distribution in a human/hamster hybrid cell. Cell. 1980 Nov;22(2 Pt 2):489–499. doi: 10.1016/0092-8674(80)90359-1. [DOI] [PubMed] [Google Scholar]
  42. Manuelidis L., Ward D. C. Chromosomal and nuclear distribution of the HindIII 1.9-kb human DNA repeat segment. Chromosoma. 1984;91(1):28–38. doi: 10.1007/BF00286482. [DOI] [PubMed] [Google Scholar]
  43. McCready S. J., Godwin J., Mason D. W., Brazell I. A., Cook P. R. DNA is replicated at the nuclear cage. J Cell Sci. 1980 Dec;46:365–386. doi: 10.1242/jcs.46.1.365. [DOI] [PubMed] [Google Scholar]
  44. Mirkovitch J., Mirault M. E., Laemmli U. K. Organization of the higher-order chromatin loop: specific DNA attachment sites on nuclear scaffold. Cell. 1984 Nov;39(1):223–232. doi: 10.1016/0092-8674(84)90208-3. [DOI] [PubMed] [Google Scholar]
  45. Nakayasu H., Berezney R. Mapping replicational sites in the eucaryotic cell nucleus. J Cell Biol. 1989 Jan;108(1):1–11. doi: 10.1083/jcb.108.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Nelson W. G., Pienta K. J., Barrack E. R., Coffey D. S. The role of the nuclear matrix in the organization and function of DNA. Annu Rev Biophys Biophys Chem. 1986;15:457–475. doi: 10.1146/annurev.bb.15.060186.002325. [DOI] [PubMed] [Google Scholar]
  47. Pardoll D. M., Vogelstein B., Coffey D. S. A fixed site of DNA replication in eucaryotic cells. Cell. 1980 Feb;19(2):527–536. doi: 10.1016/0092-8674(80)90527-9. [DOI] [PubMed] [Google Scholar]
  48. Paulson J. R., Laemmli U. K. The structure of histone-depleted metaphase chromosomes. Cell. 1977 Nov;12(3):817–828. doi: 10.1016/0092-8674(77)90280-x. [DOI] [PubMed] [Google Scholar]
  49. Phi-Van L., von Kries J. P., Ostertag W., Strätling W. H. The chicken lysozyme 5' matrix attachment region increases transcription from a heterologous promoter in heterologous cells and dampens position effects on the expression of transfected genes. Mol Cell Biol. 1990 May;10(5):2302–2307. doi: 10.1128/mcb.10.5.2302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Robinson S. I., Small D., Idzerda R., McKnight G. S., Vogelstein B. The association of transcriptionally active genes with the nuclear matrix of the chicken oviduct. Nucleic Acids Res. 1983 Aug 11;11(15):5113–5130. doi: 10.1093/nar/11.15.5113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Saez L. J., Gianola K. M., McNally E. M., Feghali R., Eddy R., Shows T. B., Leinwand L. A. Human cardiac myosin heavy chain genes and their linkage in the genome. Nucleic Acids Res. 1987 Jul 10;15(13):5443–5459. doi: 10.1093/nar/15.13.5443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Spector D. L., Fu X. D., Maniatis T. Associations between distinct pre-mRNA splicing components and the cell nucleus. EMBO J. 1991 Nov;10(11):3467–3481. doi: 10.1002/j.1460-2075.1991.tb04911.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Stuurman N., de Jong L., van Driel R. Nuclear frameworks: concepts and operational definitions. Cell Biol Int Rep. 1992 Aug;16(8):837–852. doi: 10.1016/s0309-1651(05)80026-8. [DOI] [PubMed] [Google Scholar]
  54. Sørensen P. D., Frederiksen S. Characterization of human 5S rRNA genes. Nucleic Acids Res. 1991 Aug 11;19(15):4147–4151. doi: 10.1093/nar/19.15.4147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Trask B. J., Massa H., Kenwrick S., Gitschier J. Mapping of human chromosome Xq28 by two-color fluorescence in situ hybridization of DNA sequences to interphase cell nuclei. Am J Hum Genet. 1991 Jan;48(1):1–15. [PMC free article] [PubMed] [Google Scholar]
  56. Trask B., Pinkel D., van den Engh G. The proximity of DNA sequences in interphase cell nuclei is correlated to genomic distance and permits ordering of cosmids spanning 250 kilobase pairs. Genomics. 1989 Nov;5(4):710–717. doi: 10.1016/0888-7543(89)90112-2. [DOI] [PubMed] [Google Scholar]
  57. Van Arsdell S. W., Weiner A. M. Human genes for U2 small nuclear RNA are tandemly repeated. Mol Cell Biol. 1984 Mar;4(3):492–499. doi: 10.1128/mcb.4.3.492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Vaughn J. P., Dijkwel P. A., Mullenders L. H., Hamlin J. L. Replication forks are associated with the nuclear matrix. Nucleic Acids Res. 1990 Apr 25;18(8):1965–1969. doi: 10.1093/nar/18.8.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Verheijen R., van Venrooij W., Ramaekers F. The nuclear matrix: structure and composition. J Cell Sci. 1988 May;90(Pt 1):11–36. doi: 10.1242/jcs.90.1.11. [DOI] [PubMed] [Google Scholar]
  60. Villarreal L. P. Relationship of eukaryotic DNA replication to committed gene expression: general theory for gene control. Microbiol Rev. 1991 Sep;55(3):512–542. doi: 10.1128/mr.55.3.512-542.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Vogelstein B., Pardoll D. M., Coffey D. S. Supercoiled loops and eucaryotic DNA replicaton. Cell. 1980 Nov;22(1 Pt 1):79–85. doi: 10.1016/0092-8674(80)90156-7. [DOI] [PubMed] [Google Scholar]
  62. Waye J. S., Willard H. F. Structure, organization, and sequence of alpha satellite DNA from human chromosome 17: evidence for evolution by unequal crossing-over and an ancestral pentamer repeat shared with the human X chromosome. Mol Cell Biol. 1986 Sep;6(9):3156–3165. doi: 10.1128/mcb.6.9.3156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Weintraub H., Groudine M. Chromosomal subunits in active genes have an altered conformation. Science. 1976 Sep 3;193(4256):848–856. doi: 10.1126/science.948749. [DOI] [PubMed] [Google Scholar]
  64. Weisbrod S. Active chromatin. Nature. 1982 May 27;297(5864):289–295. doi: 10.1038/297289a0. [DOI] [PubMed] [Google Scholar]
  65. Westin G., Zabielski J., Hammarström K., Monstein H. J., Bark C., Pettersson U. Clustered genes for human U2 RNA. Proc Natl Acad Sci U S A. 1984 Jun;81(12):3811–3815. doi: 10.1073/pnas.81.12.3811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Wiegant J., Kalle W., Mullenders L., Brookes S., Hoovers J. M., Dauwerse J. G., van Ommen G. J., Raap A. K. High-resolution in situ hybridization using DNA halo preparations. Hum Mol Genet. 1992 Nov;1(8):587–591. doi: 10.1093/hmg/1.8.587. [DOI] [PubMed] [Google Scholar]
  67. Xing Y. G., Lawrence J. B. Preservation of specific RNA distribution within the chromatin-depleted nuclear substructure demonstrated by in situ hybridization coupled with biochemical fractionation. J Cell Biol. 1991 Mar;112(6):1055–1063. doi: 10.1083/jcb.112.6.1055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Zlatanova J. S., van Holde K. E. Chromatin loops and transcriptional regulation. Crit Rev Eukaryot Gene Expr. 1992;2(3):211–224. [PubMed] [Google Scholar]
  69. de Lara J., Wydner K. L., Hyland K. M., Ward W. S. Fluorescent in situ hybridization of the telomere repeat sequence in hamster sperm nuclear structures. J Cell Biochem. 1993 Nov;53(3):213–221. doi: 10.1002/jcb.240530306. [DOI] [PubMed] [Google Scholar]

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