Name: fluorescence in situ hybridization on dna halo preparations to reveal whole chromosomes, telomeres and gene loci
Uploaded: 2021-03-04T13:30:00
Duration: 9 min 7 s
Description: Watch this Scientific Journal Video about Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci at JoVE.com

We would like to thank Prof Michael Bittner for the kind gift of chromosome arm painting probes. LG was supported by EU funded EURO-Laminopathies project and the Brunel Progeria Research Fund.

<ol>
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H., Coverley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+DNA+replication+and+the+nuclear+matrix.">Relationship between DNA replication and the nuclear matrix.</a> Genes Cells. 18 (1), 17-31 (2013).</li><li>Wilson, R. H. C., Coverley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation-induced+changes+in+the+DNA-nuclear+matrix+interface,+revealed+by+high-throughput+analysis+of+DNA+halos.">Transformation-induced changes in the DNA-nuclear matrix interface, revealed by high-throughput analysis of DNA halos.</a> Science Reports. 7 (1), 6475 (2017).</li><li>Iarovaia, O. V., Akopov, S. B., Nikolaev, L. G., Sverdlov, E. D., Razin, S. 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U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Farnesyltransferase+inhibitor+and+rapamycin+correct+aberrant+genome+organisation+and+decrease+DNA+damage+respectively,+in+Hutchinson-Gilford+progeria+syndrome+fibroblasts.">Farnesyltransferase inhibitor and rapamycin correct aberrant genome organisation and decrease DNA damage respectively, in Hutchinson-Gilford progeria syndrome fibroblasts.</a> Biogerontology. 19 (6), 579-602 (2018).</li><li>Telenius, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degenerate+oligonucleotide-primed+PCR:+general+amplification+of+target+DNA+by+a+single+degenerate+primer.">Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer.</a> Genomics. 13 (3), 718-725 (1992).</li><li>Bridger, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+pKi-67+with+satellite+DNA+of+the+human+genome+in+early+G1+cells.">Association of pKi-67 with satellite DNA of the human genome in early G1 cells.</a> Chromosome Research. 6, 13-24 (1998).</li><li>Sales Gil, R., Vagnarelli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ki-67:+More+Hidden+behind+a+'Classic+Proliferation+Marker'.">Ki-67: More Hidden behind a 'Classic Proliferation Marker'.</a> Trends in Biochemical Sciences. 43 (10), 747-748 (2018).</li><li>Bikkul, M. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomere+elongation+through+hTERT+immortalization+leads+to+chromosome+repositioning+in+control+cells+and+genomic+instability+in+Hutchinson-Gilford+progeria+syndrome+fibroblasts,+expressing+a+novel+SUN1+isoform.">Telomere elongation through hTERT immortalization leads to chromosome repositioning in control cells and genomic instability in Hutchinson-Gilford progeria syndrome fibroblasts, expressing a novel SUN1 isoform.</a> Genes Chromosomes Cancer. 58 (6), 341-356 (2019).</li><li>Jackson, D. A., Cook, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+a+filamentous+nucleoskeleton+with+a+23+nm+axial+repeat.">Visualization of a filamentous nucleoskeleton with a 23 nm axial repeat.</a> EMBO Journal. 7, 3667-3677 (1988).</li><li>Albrethsen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unravelling+the+nuclear+matrix+proteome.">Unravelling the nuclear matrix proteome.</a> Journal of Proteomics. 72, 71-81 (2009).</li><li>Mika, S., Rost, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMPdb:+Database+of+nuclear+matrix+proteins.">NMPdb: Database of nuclear matrix proteins.</a> Nucleic Acids Research. 33, 160-163 (2005).</li><li>Gavrilov, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=of+the+nuclear+matrix-bound+chromatin+hubs+by+a+new+M3C+experimental+procedure.">of the nuclear matrix-bound chromatin hubs by a new M3C experimental procedure.</a> Nucleic Acids Research. 38, 8051-8060 (2010).</li><li>Sjakste, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+nuclear+matrix+proteins+in+malignant+transformation+and+cancer+diagnosis.">Role of the nuclear matrix proteins in malignant transformation and cancer diagnosis.</a> Experimental Oncology. 26 (3), 170-178 (2004).</li><li>Leman, E. S., Getzenberg, R. 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The authors have nothing to disclose.

The DNA halo method is an excellent method of choice when analyzing interactions between the nucleoskeleton and genome, however, there are some critical steps that must be adhered too. One of the most important parameters is the optimization of the cell seeding density. If cells become over confluent, then the DNA halos will overlap with neighboring cells making it impossible to perform the analysis. The CSK and extraction buffers must always be made fresh on the day of use with spermine, spermidine and digitonin being added to the extraction buffer at the end of the preparation process to maintain their biological activity. If performing Halo-FISH it is extremely important to use the correct denaturation temperature of the DNA halos to enable the probe or paint to subsequently hybridize.
Electron microscopy has been used to visualize the nuclear matrix, with filamentous structures being identified20. However, electron microscopy is limited as matrix associations with chromatin cannot easily be deduced. Indeed, the DNA Halo method is more versatile compared with electron microscopy as specific genes, chromosomes and cell states can all be examined. Furthermore, proteomic analysis of nuclear matrix proteins is being studied21,22. This method is good for comparing nuclear matrix components, particularly when comparing diseased cells, however, it doesn&#39;t provide the spatial distribution and attachments highlighted by the standard DNA Halo technique.
DNA Halo assays do have limitations. Firstly, as the matrix is extracted, this can only be performed on fixed cells so live imaging is not possible. Although the DNA Halo method is relatively quick and easy to perform, the overall process may be time consuming when cell culture, probe generation, Halo-FISH and analysis is all taken into account.
Image capture of DNA Halos and HALO-FISH using super-resolution microscopy would greatly improve the resolution of DNA specific probes and antibodies. In addition, as fluorochromes can be more easily spectrally resolved, it may be possible to use a number of DNA probes in a single experiment, providing even more information. Improvements in molecular biology techniques such as chromosome conformation capture (3C) have been used to determine interactions of gene loci and analyze the spatial organization on chromatin in the cell. DNA Halo assays and 3C can be combined, a term known as M3C23, again demonstrating the adaptability of the DNA Halo technique.
The original data presented here are to demonstrate the possibilities for genome behavior interrogation and how to present those data. With these data we have demonstrated that it is possible to determine significant differences in genome attachment using (1) chromosome painting probes, in this study revealing chromosome 18 being the least attached chromosome out of those analysed (Figure 3); (2) Gene loci with significant differences between two gene loci and (Figure 4) (3) Telomeres, which are less strongly attached in quiescent cells compared to proliferating and senescent cells (Figure 5). We are able to differentiate between proliferating and non-proliferating cells via the presence of the proliferation marker Ki67 antigen which is an insoluble protein so remains with the residual nuclei or using the incorporation of nucleotides to highlight cells that have been through S-phase within a specific time period (Figure 2). This technique has also enabled us to analyze genome behavior in cells that are compromised in their nucleoskeletons i.e. laminopathy cells and here and in Bikkul et al., 2018 we reveal that the genome can be less tightly attached when compared to control cells and can be restored when treating with specific drugs that ameliorate the effect of the lamin A mutation in classical HGPS cells15. However, we show new data here for the atypical HGPS AGO8466 cells, lacking a lamin A mutation but containing an unusual form of the LINC complex protein SUN119 that chromosome 13 is less tightly attached in (Figure 6).
HALO-FISH is a unique method by enabling the study of genomic interactions with the nucleoskeleton in combination with indirect immunofluorescence to resolve proteins not removed from the extraction procedure. It has been demonstrated that the nucleoskeleton is modified in various diseases such as certain cancer types19 and the importance of some nucleoskeleton-associated proteins as diagnostic biomarkers24,25. Thus, this technique has an important role in examining the effect of the nucleoskeleton on chromatin organization/disorganization in disease15,24,25,27 and is not restricted to human cells, with chromosomal painting probes from other animals, the same DNA-halo protocol could be employed28.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62017">Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci</a>. The Authors section was updated from:
Lauren S. Godwin1 
Joanna M. Bridger1 
Helen A. Foster2 
1Laboratory of Nuclear and Genomic Health, Centre for Genome Engineering and Maintenance, Division of Biosciences, Department of Life Sciences, College of Health, Medicine and Life Sciences, Brunel University London 
2Biosciences, Department of Clinical, Pharmaceutical and Biological Science, School of Life and Medical Sciences, University of Hertfordshire
to:
Lauren S. Godwin1 
Emily Roberts2 
Joanna M. Bridger1 
Helen A. Foster2 
1Laboratory of Nuclear and Genomic Health, Centre for Genome Engineering and Maintenance, Division of Biosciences, Department of Life Sciences, College of Health, Medicine and Life Sciences, Brunel University London 
2Biosciences, Department of Clinical, Pharmaceutical and Biological Science, School of Life and Medical Sciences, University of Hertfordshire

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62017">Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci</a>. The Authors section was updated.

Erratum: Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci

The &#34;nuclear&#160;matrix&#34; was first described by Berezney and Coffey in 19741. After performing extractions with high salt molarities and nuclease treatment on rat liver nuclei, they identified a proteinaceous structural framework. The DNA halo procedure was subsequently adapted from this method and involves the removal of soluble proteins so that only the nuclear matrix (NM) and NM-associated proteins and chromosomes persist. DNA attachment regions are located at the base of DNA loops and are called matrix attached regions (MARs) or scaffold attachment regions (SARs), which are resistant to extraction with high salt concentrations and ionic detergent lithium-3,5-diiodosalicylate (LIS) respectively. In DNA halos, DNA associated with MARs/SARs are bound within the residual nucleus whereas the DNA loops extend away from these sites and form the DNA halo. We know now that the genome is anchored via lamina associated domains (LADs) to the nuclear lamina and through nucleolar associated regions (NADs) and possibly through other nuclear structures such as specific nuclear bodies.
The DNA halo method can be used for physical mapping of DNA, genes, and chromosomal regions as the extended DNA and chromatin provides a greater resolution because the chromatin is stripped of histones and the DNA is stretched out2,3,4,5,6. However, there are some limitations when using DNA halos for this application. For instance, DNA tightly associated with residual nuclei of DNA halos can be inaccessible to probes thus precluding it from analysis and physical mapping6. Other techniques such as fiber-FISH2,4,5,7 and molecular combing8 also enable physical mapping and have the advantage of being relatively quick and easy to perform. Both are preferentially used for DNA mapping of genes over DNA halos. These methods extract chromatin fibers via the use of solvent or salt extractions from the nucleus, however, molecular combing tends to have better reproducibility8,9.
There is an increasing evidence that the nucleoskeleton has a role in supporting key nuclear processes, such as attachment sites for DNA, chromatin remodeling, DNA transcription, DNA repair and DNA replication11,12. As such, the DNA halo technique was developed to investigate the interactions between the nucleoskeleton and genome during these cellular activities and has been routinely used and reported in research. This technique has also been used to investigate interactions between the genome and nucleoskeleton in relation to disease progression with malignancy-associated changes in nuclear structure being identified11.
The DNA halo technique has also been used to investigate the relationship between the genome and nucleoskeleton during development and differentiation12. A number of studies have used a variation of the DNA halo technique known as halosperm13 or SpermHalo-FISH if coupled with FISH14. Spermatozoa chromatin is tightly bound to proteins known as protamines and this technique was developed to improve access to the sperm DNA. Halosperm has been used to investigate the integrity of spermatozoa DNA and determine if DNA damage is present. Spermatozoa with less DNA damage correlate to a larger DNA halo size, whereas spermatozoa with increased levels of fragmented and damaged DNA had either small halos or none at all. Thus, halosperm can be used as a potential prognostic marker of embryo quality and successful pregnancy with IVF13. This example emphasizes the potential clinical applications of this technique. In our work we have used HALO-FISH to assess changes in genome behavior and the effect of specific drug treatments in the premature ageing disease Hutchinson-Gilford Progeria Syndrome (HGPS)15.
Together these, and other studies, highlight the breadth of processes/applications that the DNA halo technique can be used to study and utility of the technique.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10X PBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>10388739</td>
			<td>Used to create DNA halos</td>
		</tr>
		<tr>
			<td>5-bromo-2&#8242;-deoxy-uridine&#160;&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>B5002-100MG</td>
			<td>Labelled nucleotide</td>
		</tr>
		<tr>
			<td>5-Fluoro-2&#8242;-deoxyuridine</td>
			<td>Sigma-Aldrich</td>
			<td>F0503-100MG</td>
			<td>Labelled nucleotide</td>
		</tr>
		<tr>
			<td>Agar Technical</td>
			<td>Thermo Fisher Scientific</td>
			<td>15562141</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A939-50G</td>
			<td>Check product size of DOP-PCR and nick translation</td>
		</tr>
		<tr>
			<td>Atypical type 2 HGPS fibroblasts (AG08466)</td>
			<td>Coriell Institute</td>
			<td>AG08466</td>
			<td>Cell line</td>
		</tr>
		<tr>
			<td>Bacto tryptone</td>
			<td>Thermo Fisher Scientific</td>
			<td>16269751</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>Biotin-16-dUTP</td>
			<td>Roche Diagnostics&#160;</td>
			<td>11093711103</td>
			<td>Labelled nucleotides</td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>C0378-25G</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>Classical Hutchinson-Gilford progeria syndrome (HGPS) fibroblasts (AG06297)&#160;</td>
			<td>Coriell Institute</td>
			<td>AG0297</td>
			<td>Cell line</td>
		</tr>
		<tr>
			<td>Coplin jar</td>
			<td>Thermo Fisher Scientific</td>
			<td>12608596</td>
			<td>Holds 5 slides or 8 slides back to back</td>
		</tr>
		<tr>
			<td>Cot-1 DNA</td>
			<td>Thermo Fisher Scientific</td>
			<td>15279011</td>
			<td>Block nonspecific hybridization in HALO FISH</td>
		</tr>
		<tr>
			<td>DEPC-treated water</td>
			<td>Sigma-Aldrich</td>
			<td>693520-1L</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>Dextran sulphate</td>
			<td>Sigma-Aldrich</td>
			<td>S4030</td>
			<td>Hybridisation mixture</td>
		</tr>
		<tr>
			<td>Digitonin</td>
			<td>Sigma-Aldrich</td>
			<td>D141</td>
			<td>Component of extraction buffer</td>
		</tr>
		<tr>
			<td>Digoxigenin-11-dUTP&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>11093088910</td>
			<td>Labelled nucleotides</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse Cy3</td>
			<td>Jackson Laboratory</td>
			<td>715-165-150</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E6758</td>
			<td>Component of extraction buffer</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td>Component of extraction buffer</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>443611</td>
			<td>Probe precipitation and HALO FISH</td>
		</tr>
		<tr>
			<td>Fetal bovine system</td>
			<td>Thermo Fisher Scientific</td>
			<td>26140079</td>
			<td>Cell culture serum</td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Thermo Fisher Scientific</td>
			<td>10523525</td>
			<td>2D FISH of DNA halos</td>
		</tr>
		<tr>
			<td>Glass wool</td>
			<td>Sigma-Aldrich</td>
			<td>18421</td>
			<td>Spin column</td>
		</tr>
		<tr>
			<td>Herring sperm</td>
			<td>Sigma-Aldrich</td>
			<td>D7290</td>
			<td>Probe precipitation</td>
		</tr>
		<tr>
			<td>HXP&#8482; Lamp (metal halide microscope lamp)</td>
			<td>OSRAM</td>
			<td>HXP-R120W45C VIS</td>
			<td>Image capture of DNA halos</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Thermo Fisher Scientific</td>
			<td>10313680</td>
			<td>Cleaning microscope slides</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma-Aldrich</td>
			<td>I9516-25ML</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>KAPA HiFi PCR Kit</td>
			<td>KAPA Biosystems</td>
			<td>KK2103</td>
			<td>PCR Kit</td>
		</tr>
		<tr>
			<td>Leica DM4000 fluorescent microscope with DFC365 FX camera and LAS AF (Version: 4.5.0) image acquisition software.</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td>Image capture of DNA halos</td>
		</tr>
		<tr>
			<td>Luria-Bertani agar&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>13274843</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td>Component of CSK buffer</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Thermo Fisher Scientific</td>
			<td>10284580</td>
			<td>Cleaning and sterilizing microscope slides</td>
		</tr>
		<tr>
			<td>Mouse anti-BrdU antibody</td>
			<td>BD Pharmingen</td>
			<td>B2531-100UL</td>
			<td>BrdU visualisation</td>
		</tr>
		<tr>
			<td>Newborn calf serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>16010159</td>
			<td>Cell culture serum and blocking reagent</td>
		</tr>
		<tr>
			<td>Nick translation kit</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR grade water</td>
			<td>Sigma-Aldrich</td>
			<td>693520-1L</td>
			<td>PCR and DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>PCR Primers</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma-Aldrich</td>
			<td>P1851</td>
			<td>Component of CSK and extraction buffers</td>
		</tr>
		<tr>
			<td>Potassium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>P1190-100G</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>QuadriPERM&#174; 4 X 12</td>
			<td>SARSTEDT</td>
			<td>94.6077.307</td>
			<td>Square cell culture dish, polysterene with four compartments.&#160; This has hydrophobic surface, is sterile, non-pyrogenic/endotoxin-fee and non-cytotoxic.</td>
		</tr>
		<tr>
			<td>Rabbit Anti-Ki67 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>ZRB1007-25UL</td>
			<td>Proliferation marker</td>
		</tr>
		<tr>
			<td>Rnase A</td>
			<td>Sigma-Aldrich</td>
			<td>R6513</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>Rubber cement</td>
			<td>Halford&#39;s</td>
			<td>101836</td>
			<td>2D FISH of DNA halos</td>
		</tr>
		<tr>
			<td>Sephadex G-50</td>
			<td>Sigma-Aldrich</td>
			<td>S6022-25G</td>
			<td>Spin column</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S2889</td>
			<td>Probe precipitation</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td>Component of CSK, extraction and SSC buffers</td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td>Sigma-Aldrich</td>
			<td>C8532</td>
			<td>Component of SSC buffer</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulphate&#160;</td>
			<td></td>
			<td>L3771-100G</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S8045-500G</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma-Aldrich</td>
			<td>S2626</td>
			<td>Component of extraction buffer</td>
		</tr>
		<tr>
			<td>Spermine</td>
			<td>Sigma-Aldrich</td>
			<td>S4264</td>
			<td>Component of extraction buffer</td>
		</tr>
		<tr>
			<td>Streptavidin-Cy3&#160;</td>
			<td>Amersham Life Sciences Ltd, Scientific Laboratory Supplies</td>
			<td>pa43001</td>
			<td>Probe antibody</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>Component of CSK buffer</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>CSK buffer+A66:D68</td>
		</tr>
		<tr>
			<td>SuperFrost&#8482; microscope slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>12372098</td>
			<td>Microscope slides: 1 mm thickness, 76 mm length, 26 mm width. Uncoated.</td>
		</tr>
		<tr>
			<td>Swine anti-rabbit TRITC&#160;</td>
			<td>Dako</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TELO-PNA FISH KIT</td>
			<td>Agilent Dako</td>
			<td>K532511-8</td>
			<td>Delineation of telomeres</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>T3253-100G</td>
			<td>Column buffer</td>
		</tr>
		<tr>
			<td>Triton&#8482; X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td>Component of CSK buffer</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Thermo Fisher Scientific</td>
			<td>10158962</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416- 100ML</td>
			<td>Detergent</td>
		</tr>
		<tr>
			<td>Vectashield mountant containing DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td>2D FISH of DNA halos</td>
		</tr>
		<tr>
			<td>Whole human chromosome probes</td>
			<td>Calbiochem</td>
			<td></td>
			<td>2D FISH of DNA halos</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Thermo Fisher Scientific</td>
			<td>10108202</td>
			<td>DNA isolation of BAC clones</td>
		</tr>
	</tbody>
</table>

fluorescence in situ hybridization on dna halo preparations to reveal whole chromosomes, telomeres and gene loci

The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These structures are collectively known as the nucleoskeleton and include the nuclear lamina, the nucleoli, and nuclear bodies. Although many variants of fluorescence in situ hybridization (FISH) exist to study the genome and its organization, these are often limited by resolution and provide insufficient information on the genome&#39;s association with nuclear structures. The DNA halo method uses high salt concentrations and nonionic detergents to generate DNA loops that remain anchored to structures within nuclei through attachment regions within the genome. Here, soluble nuclear proteins, such as histones, lipids, and DNA not tightly bound to the nuclear matrix, are extracted. This leads to the formation of a halo of unattached DNA surrounding a residual nucleus which itself contains DNA closely associated with internal nuclear structures and extraction-resistant proteins. These extended DNA strands enable increased resolution and can facilitate physical mapping. In combination with FISH, this method has the added advantage of studying genomic interactions with all the structures that the genome is anchored by. This technique, termed HALO-FISH, is highly versatile whereby DNA halos can be coupled with nucleic acid probes to reveal gene loci, whole chromosomes, alpha satellite, telomeres and even RNA. This technique provides an insight into nuclear organization and function in normal&#160;cells and in disease progression such as with cancer.

This method of DNA halo preparation has helped us in our endeavors to determine differences in genome behavior within young and old cells, but also in cells derived from premature ageing diseases with aberrant nucleoskeletal proteins15. Figure 1 displays examples of DNA halos where it is possible to see the edge of a residual nucleus, the DNA remaining within the residual nucleus and the unattached DNA that has spooled out into the surrounding area creating a DNA halo. It also depicts the analysis showing how the residual nucleus is obtained and the NE and CTE measurements. It is possible to differentiate between proliferating and non-proliferating cells by either incorporating a labeled nucleotide such as BrdU when cells are in S-phase or employing the diagnostic proliferation marker anti-pKi67, which reveals nucleoli, and regions of heterochromatin in G1 cells17,18. Primary cells grown in high serum without achieving confluency, that are negative for the proliferation markers, are assumed to be senescent. Primary cells grown in low serum or have become confluent i.e., contact inhibited that are negative for the proliferation markers are deemed quiescent and would be able to reenter the proliferative cell cycle given the correct nutrients and situation. Being able to differentiate between Ki67 positive and negative cells has enabled us to determine differences between proliferating, quiescent and senescent human dermal fibroblasts. Figure 2 displays DNA halos of proliferating human dermal fibroblasts created from cells where BrdU was incorporated into them during DNA replication, a mechanism that does not occur in non-proliferating cells, and subsequently stained with anti-BrdU antibody. Staining with the proliferative marker anti-pKi67 antibody is also visible in Figure 2. This is a robust antigen and survives the FISH protocol and so can be stained for post-FISH and pre-mounting. Thus, proliferating cells are positive (red) for BrdU and anti-pKi67 (red) in the left-hand column and non-proliferating cells, indeed senescent cells in Figure 2 are displayed in the right-hand column. The green signals are individual telomeres revealed with a telomere PNA FISH/FITC kit. Combining immunofluorescence with DNA halos enables analysis during different cell states, as shown in Figure 2 when investigating proliferating, quiescent and senescent cells. Depending on the antibody chosen other conditions can be examined, such as differentiation, DNA damage via irradiation etc.
Chromosome territories can also be visualized within DNA halos using FISH. Due to the preparation permitting spooling of DNA out the nuclei, the chromosome territory shape can be disturbed, with smaller or larger amounts of the chromosome found in the DNA halo, depending on the anchorage of the genome inside the residual nucleus and its structures. Figure 3 reveals a panel of DNA halos whereby individual chromosomes have been revealed with specific whole arm chromosome painting probes (red) for chromosomes 1, 13, 17 and 18. Anti-pKi67 (green) has been used to mark proliferating cells and its absence within the same culture, upon the same slide, denoting senescent cells. It is very obvious from the images and the data presented as CTE/NE that the small gene-poor chromosome 18 is a chromosome that has few attachments and spools further out into the DNA halo away from the residual nuclei and is significantly further from the center of the residual nuclei than the other chromosomes. However, this is also true for chromosome 1 as well. Using the proliferative marker anti-pKi67 it has also been possible to compare proliferating with senescent cells, within the same culture, and on the same slide, and this analysis has revealed that chromosomes within these two very different cell statuses are not significantly different from one another, with respect to attachment with the residual nuclear structures.
Interestingly, genes also are showing statistically significant differences between proliferating and senescent cells with respect to remaining within a residual nucleus or being located in the DNA Halo. Figure 4 demonstrates this with gene loci delineated by labeled BAC probes in red and anti-Ki67 in green. There are no significant differences between gene locations in the proliferating versus the senescent cells, after a DNA Halo preparation. However, there are significantly more catenin alpha 1 CTNNA1 loci within the DNA halo than cyclin D1 CNDD1 loci, where there are very few. Figure 5 displays DNA halo preparations with telomeres in green. The background is left deliberately high to enable telomere signals to be visualized within the DNA halo. In this set of data quiescent cells i.e., cells that have been serum starved for 7 days have been included and interestingly there are significantly more telomeres unattached and located within the DNA halos in quiescent cells than for proliferating and senescent cells. In Figure 5a the proportion of telomeres in the DNA halo can be observed, particularly for the image &#39;Experiment 2&#39;. This corresponds with Figure 5b where the mean percentage of telomeres in DNA halo is approximately 17% in quiescent cells. There is some evidence that not all telomeres in senescent cells can be seen as some of them maybe very short.
This method of DNA halo has been successful for us to investigate genome interaction alterations within nuclei in diseased cells15. Figure 6 demonstrates differences in chromosome attachment in primary control fibroblasts and in diseased cells with typical (lamin A mutation) and atypical Hutchinson-Gilford Progeria Syndrome, expressing a different SUN1 isoform and no lamin A mutation19. Chromosomes 1 and 13 show statistically significant differences in their attachment within the residual nuclei when compared to control DNA halos. Figure 6b correlates the position of the whole chromosome territory to the residual nucleus and DNA Halo. Values of 1 or less indicates the chromosome is located within the residual nucleus and values over 1 demonstrate chromosomes or portions of chromosomes within the DNA Halo.
Overall, this highlights the utility of HALO-FISH in investigating genomic interactions of whole chromosomes, specific genes and telomeres under a variety of conditions that affect the cell cycle (proliferation, quiescence and senescence) or within disease cells e.g., progeria and cancer cell lines. Indeed, the differences in interactions between these states implies the nucleoskeleton has an important role in regulating key processes within the nucleus.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62017/62017fig01.jpg" /> 
Figure 1:&#160;HDF extracted nucleus displaying the residual nucleus and DNA halo and overview of analysis method. (a) An HDF nucleus prepared via DNA halo assay and counterstained with DAPI. The brightly stained residual nucleus shows DNA anchored to the nucleoskeleton and this is surrounded by the non-attached DNA which forms a halo of DNA. Magnification = x 100; scale bar 10 &#181;m. (b) The blue channel captures the DAPI-stained nucleus and surrounding DNA. The residual nucleus is selected and removed using ImageJ. The arrow depicts the distance from the nuclear center to the residual nuclear edge (NE). (c) The red channel shows the probe signal. (d) The image denoted &#39;Result&#39; is the outcome of superimposing the red channel on the blue channel image; this allows the distance from the nuclear center to the furthest chromosome territory edge (CTE). <a href="https://www.jove.com/files/ftp_upload/62017/62017fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62017/62017fig02.jpg" /> 
Figure 2:&#160;DNA halo preparation with telomere PNA FISH on proliferating and senescent HDFs. Telomere PNA FISH on HDFs subjected to DNA halo assay. Telomere signals are visualized in green (FITC), residual and halo DNA was counterstained using DAPI (blue) and proliferating nuclei were detected using either anti-BrdU or anti-pKi67 antibodies via indirect immunofluorescence in red (TRITC). Magnification = x 100; scale bar 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/62017/62017fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62017/62017fig03.jpg" /> 
Figure 3:&#160;Nucleoskeleton-chromosome interactions and analysis using DNA halo assay. (a) 2D-FISH with probes specific for chromosomes 1, 13, 15, 17 and 18 was performed on HDFs subjected to DNA halo preparation. Whole chromosomes were painted in red (Cy3) and nuclei were probed with pKi67 to determine if they were proliferating or senescent. Proliferating cells (pKi67+) were delineated in green (FITC), whereas senescent cells remained unstained (pKi67-) i.e. no green signal detected. Magnification = x 100; scale bar 10 &#181;m. (b) Chromosome anchorage by the nucleoskeleton in proliferating and senescent HDFs that had undergone HALO-FISH. Measurements show the ratio of the furthest chromosome territory edge (CTE) to respective nuclear edge (NE) for chromosomes 1, 13, 15, 17 and 18 in proliferating (pKi67+) and senescent (pKi67-) cells. Error bars represent &#177; SEM. (c) Modified box plot representation of chromosome territory edge (CTE) to respective nuclear edge (NE) of specific chromosomes in pKi67+ and pKi67- nuclei. Q1 = lower quartile; Min = lowest value recorded; Med = median; Max = maximum value recorded; Q3 = upper quartile. <a href="https://www.jove.com/files/ftp_upload/62017/62017fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62017/62017fig04.jpg" /> 
Figure 4:&#160;Gene-specific interactions in HDFs using HALO-FISH. (a) DNA halo extracted nuclei were probed with gene specific probes (CCND1 and CTNNA1) to investigate their anchorage to the NM on proliferating and senescent cells. The gene signals are shown in red (Cy3) and anti-pKi67 depicts proliferating cells and signal is visualized in green (FITC). For the proliferating CCND1 image, the residual nucleus is enclosed within the white circle, and the space between the white and green circle depicts the DNA Halo. Magnification = x 100; scale bar 10 &#181;m. (b) Gene-specific signals for CCND1 and CTNNA1 are compared between the residual nucleus and DNA halo, and also, between proliferating and senescent cells. Error bars represent &#177; SEM. <a href="https://www.jove.com/files/ftp_upload/62017/62017fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62017/62017fig05.jpg" /> 
Figure 5:&#160;DNA halo assay on quiescent HDFs probed with telomere PNA-FISH. (a) Quiescence of HDFs was induced by culture in low serum medium for 7 days. The DNA halo assay was performed, and PNA-FISH enabled visualization of telomeres by FITC signal (green) and the residual nucleus and surrounding DNA halo was counterstained with DAPI (blue). Cells were also stained with anti-pKi67 antibody to ensure nuclei were non-proliferating. This was repeated on two separate occasions. Magnification = x 100; scale bar 10 &#181;m. (b) Comparison of the mean percentage of telomeres localized within the DNA halo in proliferating, senescent and quiescent HDF cells. Error bars represent &#177; SEM. <a href="https://www.jove.com/files/ftp_upload/62017/62017fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62017/62017fig06.jpg" /> 
Figure 6: Examining whole chromosome anchorage to the nucleoskeleton in HGPS cells using HALO-FISH26. (a) Control HDF (2DD), classical HGPS (AG06297) and atypical type 2 HGPS (AG08466) nuclei underwent DNA halo preparation and then 2D-FISH using whole chromosome paints for chromosome 1, 13, 15 and 17. Whole chromosomes are depicted in green (FITC) and DNA was counterstained with DAPI (blue). Magnification = x 100; scale bar 10 &#181;m. (b) Positioning of chromosomes within extracted nuclei was determined by measuring the ratio of the mean chromosome territory edge (CTE) to the nuclear edge (NE). A ratio above 1 demonstrates that the furthest CTE lies outside the corresponding NE within the DNA halo, while a ratio below 1 signifies that the furthest CTE lies within the NE within the residual nucleus. <a href="https://www.jove.com/files/ftp_upload/62017/62017fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Constituents</td>
			<td>Volume(&#956;L)</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5XDOP-PCRbuffer</td>
			<td>10</td>
		</tr>
		<tr>
			<td>dNTPmix(withoutdTTP)(2mM)</td>
			<td>5</td>
		</tr>
		<tr>
			<td>dTTP(2mM)</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Biotin-16-dUTPorDigoxigenin-11-dUTP</td>
			<td>10</td>
		</tr>
		<tr>
			<td>DOPprimer(20&#956;M)</td>
			<td>5</td>
		</tr>
		<tr>
			<td>TaqDNAPolymerase(1U/&#956;L)</td>
			<td>1</td>
		</tr>
		<tr>
			<td>PCRgradewater</td>
			<td>12</td>
		</tr>
		<tr>
			<td>Template</td>
			<td>5</td>
		</tr>
	</tbody>
</table>
Table 1: Table showing the DOP-PCR components and volumes for a 1x reaction
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step&#160;</td>
			<td>Cycles&#160;</td>
			<td>Temp (degree Centigrade)</td>
			<td>Time&#160;</td>
		</tr>
		<tr>
			<td>Initial Denaturation&#160;</td>
			<td>1</td>
			<td>95</td>
			<td>3 min</td>
		</tr>
		<tr>
			<td>Denaturation&#160;</td>
			<td rowspan="3">34</td>
			<td>98</td>
			<td>20 s</td>
		</tr>
		<tr>
			<td>Primer Annealing&#160;</td>
			<td>62</td>
			<td>1 min</td>
		</tr>
		<tr>
			<td>Extension&#160;</td>
			<td>72</td>
			<td>30 s</td>
		</tr>
		<tr>
			<td>Final Extension&#160;</td>
			<td>1</td>
			<td>72</td>
			<td>5 min&#160;</td>
		</tr>
		<tr>
			<td>Cooling&#160;</td>
			<td></td>
			<td>4</td>
			<td>Hold</td>
		</tr>
	</tbody>
</table>
Table 2: Table showing the DOP-PCR cycle, temperature, and time profile.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Constituent</td>
			<td>Volume(&#956;L)</td>
		</tr>
		<tr>
			<td>10x NT buffer (0.5M Tris-HCl pH 8,50 mM MgCl2, 0.5 mg/ml BSA)</td>
			<td>5</td>
		</tr>
		<tr>
			<td>0.1 M beta-mercaptoethanol</td>
			<td>5</td>
		</tr>
		<tr>
			<td>10X Nucleotide stock (0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.5 mM dTTP, 0.5 mg/ml biotin-16-dUTP)</td>
			<td>5</td>
		</tr>
		<tr>
			<td>Dnase I (1 ng/ml)</td>
			<td>2</td>
		</tr>
		<tr>
			<td>DNApolymerase I</td>
			<td>5U per &#956;g of DNA</td>
		</tr>
		<tr>
			<td>DNAtemplate (1 &#956;g)</td>
			<td>1</td>
		</tr>
		<tr>
			<td>DEPC-treated water</td>
			<td>To 50 &#956;L</td>
		</tr>
	</tbody>
</table>
Table 3: Table showing the nick translation components and volumes for a one probe.

Watch this Scientific Journal Video about Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci at JoVE.com

Fluorescence In Situ Hybridization on DNA Halo Preparations to Reveal Whole Chromosomes, Telomeres and Gene Loci

Combining DNA halo preparations with fluorescence in situ hybridization enables high-resolution analysis of genomic interactions with the nucleoskeleton. Attached genome leads to hybridized fluorescent signals located within the residual extracted nuclei, whereas non-attached genome is in the halo of DNA surrounding the residual nuclei.

The genome is associated with several structures inside cell nuclei, in order to regulate its activity and anchor it in specific locations. These ...

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