A visualization method that combines simultaneous fluorescence immunostaining and FISH represents a powerful means to monitor and visualize DNA–protein interactions in situ. This method has been extensively used in basic chromosomal biology and in medical diagnostics related analysis due to its versatility; it is capable of analyzing the structures, functions, behaviors and abnormalities of chromatin domains, individual chromosomes or specific regions, and entire genomes or various cell populations. In this chapter, detailed protocols that can be applied to study both mitotic and meiotic chromosomes and cells are presented, along with a brief introduction of the methodology and its popular uses in the field of chromosome and genome research. Some technical considerations and possible future directions are also covered.
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References
Azzalin CM, Reichenbach P, Khoriauli L, Giulotto E, Lingner J (2007) Telomeric repeat containing RNA and RNA surveillance factors at mammalian chromosome ends. Science 318:798 – 801
Barlow AL, Hulten MA (1996) Combined immunocytogenetic and molecular cytogenetic analysis of meiosis I human spermatocytes. Chromosome Res 4:562 – 573
Baurmann H, Cherif D, Berger R (1993) Interphase cytogenetics by fluorescent in situ hybridization (FISH) for characterization of monosomy-7-associated myeloid disorders. Leukemia 7: 384 – 391
Beatty B, Heng HH (2004) Gene mapping by fluorescence in situ hybridization. In: Meyer R (ed) Encyclopedia of molecular cell biology and molecular medicine, vol 5. Wiley-VCH, Weinheim, pp. 137 – 171
Beatty B, Mai S, Squire J (2002) FISH: a practical approach. Oxford University Press, Oxford
Buchwalow IB, Minin EA, Boecker W (2005) A multicolor fluorescence immunostaining technique for simultaneous antigen targeting. Acta Histochem 107:143 – 148
Craig JM, Earle E, Canham P, Wong LH, Anderson M, Choo KH (2003) Analysis of mammalian proteins involved in chromatin modification reveals new metaphase centromeric proteins and distinct chromosomal distribution patterns. Hum Mol Genet 12:3109 – 3121
Cremer T, Cremer C (2001) Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nat Rev Genet 2:292 – 301
Csankovszki G, McDonel P, Meyer BJ (2004) Recruitment and spreading of the C. elegans dosage compensation complex along X chromosomes. Science 303:1182 – 1185
Froenicke L, Anderson LK, Wienberg J, Ashley T (2002) Male mouse recombination maps for each autosome identified by chromosome painting. Am J Hum Genet 1:1353 – 1368
Fukagawa T, Pendon C, Morris J, Brown W (1999) CENP-C is necessary but not sufficient to induce formation of a functional centromere. EMBO J 18:4196 – 4209
Haaf T, Ward DC (1994) Structural analysis of alpha-satellite DNA and centromere proteins using extended chromatin and chromosomes. Hum Mol Genet 3:697 – 709
Haaf T, Warburton PE, Willard HF (1992) Integration of human alpha-satellite DNA into simian chromosomes: Centromere protein binding and disruption of normal chromosome segregation. Cell 70:681 – 696
Hassold T, Judis L, Chan ER, Schwartz S, Seftel A, Lynn A (2004) Cytological studies of meiotic recombination in human males. Cytogenet Genome Res 107:249 – 255
Heng HH (2007a) Elimination of altered karyotypes by sexual reproduction preserves species identity. Genome 50:517 – 24
Heng HH (2007b) Cancer genome sequencing: The challenges ahead. Bioessays 29:783 – 794
Heng HH (2008) The gene-centric concept: A new liability? Bioessays 30:196 – 197
Heng HH, Tsui L-C (1993) Modes of DAPI banding and simultaneous in situ hybridization. Chromosoma 102:325 – 332
Heng HH, Squire J, Tsui LC (1992) High-resolution mapping of mammalian genes by in situ hybridization to free chromatin. Proc Natl Acad Sci USA 89:9509 – 9513
Heng HH, Tsui LC, Moens PB (1994) Organization of heterologous DNA inserts on the mouse meiotic chromosome core. Chromosoma 103:401 – 407
Heng HH, Chamberlain J, Shi XM, Spyropoulos B, Tsui L-C, Moens PB (1996) Regulation of meiotic chromatin loop size by chromosomal position. Proc Natl Acad Sci USA 93: 2795 – 2800
Heng HH, Spyropoulos B, Moens PB (1997) FISH technology in chromosome and genome research. Bioessays 19:75 – 84
Heng HH, Shi XM, Tsui L-C, Moens PB. (1998) Visualization of mitotic and meiotic chromatin loop domains. Presented at the International Genetic conference, Beijing
Heng HH, Spyropoulos B, Moens P (2000) DNA — protein in situ covisualization for chromosomal analysis. Meth Mol Biol 123:15 – 27
Heng HH, Liu G, Lu W, Bremer S, Ye CJ, Hughes M, Moens P (2001a) Spectral karyotyping (SKY) of mouse meiotic chromosomes. Genome 44:293 – 298
Heng HH, Krawetz SK, Lu W, Bremer S, Liu G, Ye CJ (2001b) Re-defining the chromatin loop domain. Cytogenet Cell Genet 93:155 – 161
Heng HH, Goetze S, Ye CJ, Liu G, Stevens JB, Bremer SW, Wykes SM, Bode J, Krawetz SA (2004a) Chromatin loops are selectively anchored using scaffold/matrix-attachment regions. J Cell Sci 117:999 – 1008
Heng HH, Stevens J, Liu G, Bremer SB, Ye CJ (2004b) Imaging genome abnormalities in cancer research. Cell Chromosomes 3:1
Heng HH, Stevens J, Yang F, Liu G, Bremer SW, Chen X, Korenberg J, Spyropolos B, Moens P, Ye C (2004c) Packaging of meiotic chromosomes correlated to GC-content, loop size, and recombination rates. Annual Meeting of American Society of Human Genetics, Toronto, Program No. 161
Heng HH, Liu G, Bremer S, Ye KJ, Stevens J, Ye CJ (2006a) Clonal and non-clonal chromosome aberrations and genome variation and aberration. Genome 49:195 – 204
Heng HH, Bremer SW, Stevens J, Ye KJ, Miller F, Liu G, Ye CJ (2006b) Cancer progression by non-clonal chromosome aberrations. J Cell Biochem 98:1424 – 1235
Heng HH, Stevens JB, Liu G, Bremer SW, Ye KJ, Reddy PV, Wu GS, Wang YA, Tainsky MA, Ye CJ (2006c) Stochastic cancer progression driven by non-clonal chromosome aberrations. J Cell Physiol 208:461 – 472
Hiatt EN, Kentner EK, Dawe RK (2002) Independently regulated neocentromere activity of two classes of tandem repeat arrays. Plant Cell 14:407 – 420
Hudson DF, Vagnarelli P, Gassmann R, Earnshaw WC (2003) Condensin is required for nonhis-tone protein assembly and structural integrity of vertebrate mitotic chromosomes. Dev Cell 5:323 – 336
Hunt P, LeMaire R, Embury P, Sheean L, Mroz K (1995) Analysis of chromosome behavior in intact mammalian oocytes: Monitoring the segregation of a univalent chromosome during female meiosis. Hum Mol Genet 4:2007 – 2012
Kolas NK, Yuan L, Hoog C, Heng HH, Marcon E, Moens PB (2004) Male mouse meiotic chromosome cores deficient in structural proteins SYCP3 and SYCP2 align by homology but fail to synapse and have possible impaired specificity of chromatin loop attachment. Cytogenet Genome Res 105:182 – 188
Korenberg JR, Chen XN (1995) Human cDNA mapping using a high-resolution R-banding technique and fluorescence in situ hybridization. Cytogenet Cell Genet 69:196 – 200
Lewis JT, Ketterling RP, Halling KC, Reynolds C, Jenkins RB, Visscher DW (2005) Analysis of intratumoral heterogeneity and amplification status in breast carcinomas with equivocal (2 +) HER-2. Am J Clin Pathol. 124:273 – 281
Liehr T, Claussen U (2002) Current developments in human molecular cytogenetic techniques. Curr Mol Med 2:283 – 297
Lottner C, Schwarz S, Diermeier S, Hartmann A, Knuechel R, Hofstaedter F, Brockhoff G (2005) Simultaneous detection of HER2/neu gene amplification and protein overexpression in paraffin-embedded breast cancer. J Pathol 205:577 – 584
Lynn A, Koehler KE, Judis L, Chan ER, Cherry JP, Schwartz S, Seftel A, Hunt PA, Hassold TJ (2002) Covariation of synaptonemal complex length and mammalian meiotic exchange rates. Science 296:2222 – 2225
Martin-Subero JI, Chudoba I, Harder L, Gesk S, Grote W, Novo FJ, Calasanz MJ, Siebert R (2002) Multicolor-FICTION: Expanding the possibilities of combined morphologic, immunopheno-typic, and genetic single cell analyses. Am J Pathol 161:413 – 420
Masumoto H, Sugimoto K, Okazaki T (1989) Alphoid satellite DNA is tightly associated with centromere antigens in human chromosomes throughout the cell cycle. Exp Cell Res 181:181 – 196
Moens PB, Heddle JAM, Spyropoulos B, Heng HH (1997) Identical megabase transgenes on mouse chromosomes 3 and 4 do not promote ectopic pairing or synapsis at meiosis Genome 40:770 – 773
Osborne CS, Chakalova L, Brown KE, Carter D, Horton A, Debrand E, Goyenechea B, Mitchell JA, Lopes S, Reik W, Fraser P (2004) Active genes dynamically colocalize to shared sites of ongoing transcription. Nat Genet 36:1065 – 1071
Page SL, Earnshaw WC, Choo KHA, Shaffer L (1995) Further evidence that CENP-C is a necessary component of active centromeres: Studies of a dic(X;15) with simultaneous immunofluores-cence and FISH. Hum Mol Genet 4:289 – 294
Parada L, Misteli T (2002) Chromosome positioning in the interphase nucleus. Trends Cell Biol 12:425 – 432
Parra I, Windle B (1993) High resolution visual mapping of stretched DNA by fluorescent hybridization. Nat Genet 5:17 – 21
Ried T, Baldini A, Rand TC, Ward DC (1992) Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy. Proc Natl Acad Sci USA 89:1388 – 1392
Scherthan H, Weich S, Schwegler H, Heyting C, Harle M, Cremer T (1996) Centromere and telomere movements during early meiotic prophase of mouse and man are associated with the onset of chromosome pairing. J Cell Biol 134:1109 – 1125
Schrö ck E, du Manoir S, Veldman T, Schoell B, Wienberg J, Ferguson-Smith MA, Ning Y, Ledbetter DH, Bar-Am I, Soenksen D, Garini Y, Ried T (1996) Multicolor spectral karyotyp-ing of human chromosomes. Science 273:494 – 497
Schnedl W, Dev VG, Tantravahi R, Miller DA, Erlanger BF, Miller OJ (1975) 5-Methylcytosine in heterochromatic regions of chromosomes: Chimpanzee and gorilla compared to the human.Chromosoma 52:59 – 66
Selim AG, El-Ayat G, Naase M, Wells CA (2002) C-myc oncoprotein expression and gene amplification in apocrine metaplasia and apocrine change within sclerosing adenosis of the breast.Breast 11:466 – 472
Speicher MR, Carter NP (2005) The new cytogenetics: Blurring the boundaries with molecular biology. Nat Rev Genet 6:782 – 792
Speicher MR, Gwyn Ballard S, Ward DC (1996) Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat Genet 12:368 – 375
Stevens JB, Liu G, Bremer SW, Ye KJ, Xu W, Xu J, Sun Y, Wu GS, Savasan S, Krawetz SA, Ye CJ, Heng HH (2007) Mitotic cell death by chromosome fragmentation. Cancer Res 67:7686 – 7694
Sun F, Oliver-Bonet M, Liehr T, Starke H, Ko E, Rademaker A, Navarro J, Benet J, Martin RH (2004) Human male recombination maps for individual chromosomes. Am J Hum Genet 74:521 – 531
Tan EM. (1983) autoantibodies to nuclear antigens (ANA): Their immunobiology and medicine. Adv Immunol 33:167 – 240
Tease C, Hartshorne GM, Hulten MA (2002) Patterns of meiotic recombination in human fetal oocytes. Am J Hum Genet 70:1469 – 1479
Trelles-Sticken E, Loidl J, Scherthan H (2003) Increased ploidy and KAR3 and SIR3 disruption alter the dynamics of meiotic chromosomes and telomeres. J Cell Sci 116:2431 – 2442
Van den Berg H, Vossen JM, Langlois van den Bergh R, Bayer J, van Tol MJ (1991) Detection of Y chromosome by in situ hybridization in combination with membrane antigens by two-color immunofluorescence. Lab Invest 64:623 – 6288
Wang J, Shiels C, Sasieni P, Wu PJ, Islam SA, Freemont PS, Sheer D (2004) Promyelocytic leukemia nuclear bodies associate with transcriptionally active genomic regions. J Cell Biol 164: 515 – 526
Weber-Matthiesen K, Deerberg J, Muller-Hermelink A, Schlegelberger B, Grote W (1993) Rapid immunophenotypic characterization of chromosomally aberrant cells by the new FICTION method. Cytogenet Cell Genet 63:123 – 125
Ye CJ, Lu W, Liu G, Bremer SW, Wang YA, Moens P, Hughes M, Krawetz SA, Heng HH (2001) The combination of SKY and specific loci detection with FISH or Immunostaining. Cytogenet Cell Genet 93:195 – 202
Ye CJ, Stevens JB, Liu G, Ye KJ, Yang F, Bremer SW, Heng HH (2006) Combined multicolor-FISH and immunostaining. Cytogenet Genome Res 114:227 – 234
Ye CJ, Liu G, Bremer SW, Heng HH (2007) The dynamics of cancer chromosomes and genomes. Cytogenet Genome Res 118:237 – 246
Acknowledgments
This work was supported by the fund from the Wayne State University Office of the Vice President for Research and Center for Molecular Medicine and Genetics, as well as the Karmanos Cancer Institute. Additional support came from the Susan Komen Breast Cancer Foundation and the research and development fund from SeeDNA Biotech Inc. We would like to thank SeeDNA Inc. for sharing their experience with us. Special thanks go to Peter Moens and Barbara Spyropoulos for their decades of support. Due to space limitations, we regret that we were unable to cite all of the notable references deserving acknowledgement.
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Ye, C.J. et al. (2009). Simultaneous Fluorescence Immunostaining and FISH. In: Liehr, T. (eds) Fluorescence In Situ Hybridization (FISH) — Application Guide. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-540-70581-9_19
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