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Mapping Protein/DNA Interactions by Cross-Linking [Internet]. Paris: Institut national de la santé et de la recherche médicale; 2001.

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Nuclear Organization and Gene Expression: Visualization of Transcription and Higher Order Chromatin Structure

and .

Abstract

The way chromatin is organized inside the nucleus of eukaryotic cells is a key factor in gene control. It seems that the cells are able to transcriptionally regulate genomic loci by changing their local environments in the nuclei. Such environment may inhibit, permit, or boost gene activity. To unravel underlying molecular mechanisms, it is essential to develop techniques that allow the simultaneous microscopic visualization of specific DNA sequences and of components of the protein machineries in the nucleus that are involved in the synthesis, processing, and transport of RNA and in the replication and repair of DNA. Visualization of DNA sequences can be achieved by fluorescence in situ hybridization (FISH). Fluorescent labelling of specific proteins is achieved by indirect immunolabelling.

This report will concentrate on the combination of both techniques under conditions that the structure of the cell nucleus remains largely intact. Applications will be presented in the context of investigation of the functional organization of the cell nucleus. The approach that is taken and the protocol that is presented can be generalized for labelling of essentially any DNA sequence and protein antigen.

Scientific Context

It is textbook knowledge that the expression of genes is regulated via the binding of proteins (transcription factors) to cis-acting regulatory sequences, such as promoters and enhancers. However, in recent years, it has become increasingly clear that the way the genome is organized inside the nucleus of eukaryotic cells is also an important factor in gene control [for a recent review, see ]. Several studies have shown that there is a striking relationship between the position of a genomic locus in the cell nucleus and its transcriptional activity. For instance, it has been shown that active genes are localized exclusively at the surface of the compact chromosomal fibre (Cmarko et al. 1999; Verschure et al. 1999), and for a number of genes, evidence has been presented that in cell types where these genes are silent, they are associated with centromeric heterochromatin, whereas in differentiated states where they are transcriptionally active, they are located away from heterochromatin (e.g., see Brown et al. 1997 and Francastel et al. 1999). In general agreement with these studies, it has been shown that activation of genes can result in large rearrangements of chromatin in the nucleus (Tumbar et al. 1999; Volpi et al. 2000).

In the past few years, we have investigated the relationship between gene expression and nuclear structure in general and large-scale organization of chromatin in particular. Using confocal 3D light microscopy and electron microscopy (in cooperation with Dr. Stan Fakan, University of Lausanne), we have analysed and compared the spatial distribution in the cell nucleus of: (i) chromosomes and chromatin ; (ii) components of the gene expression machinery (including sites of transcription (Wansink et al. 1993), transcription factors (Grande et al. 1997; Van Steensel et al. 1995), hnRNP proteins involved in RNA processing and transport ; and (iii) a number of special nuclear domains, such as PML bodies, Cajal (coiled) bodies, and cleavage bodies.

On the basis of our results and those of others, the following picture is emerging. The interphase nucleus is highly compartmentalized, which is instrumental in the regulation of gene expression. Two major, highly convoluted compartments can be distinguished. One contains compact chromatin and little or no RNPs (ribonucleoparticles, hnRNA--protein complexes). The other compartment is the interchromatin space, i.e., the nuclear compartment that contains a high concentration of RNA and proteins but little or no chromatin. This notion is based on electron microscopy and light microscopy observations, mainly on nuclei of higher eukaryotes, and is an extension of the interchromosome domain (ICD) model first formulated by Thomas Cremer and coworkers (Cremer et al. 1993). Transcriptionally active loci are localized exclusively at the surface of the compact chromatin domains, i.e., in the perichromatin area (e.g., see Cmarko et al. 1999 and Verschure et al. 1999). In this way, components of the transcriptional machinery, present predominantly in the interchromatin compartment, have immediate access to the regulatory sequences. Newly synthesized RNA is deposited directly into the interchromatin space, which contains all factors required for processing, packaging, and transport through the interchromatin channels, for instance, to the nuclear pores. The interchromatin space contains a number of additional subnuclear compartments, most of which are dedicated to the control of specific sets of genomic loci (well-studied example, the nucleoli). Nuclear organization in general and large-scale folding of chromatin in particular seem to create specific, local micro-environments for genes that inhibit, permit, or boost expression of nearby genes.

Technical Strategies

Investigation of the structure--function relationship of the cell nucleus in the context of unravelling "higher order" mechanisms of gene control requires the study of chromosome and chromatin structure in situ, i.e., in the intact nucleus. Similarly, the spatial organization of the molecular machineries for RNA synthesis, processing, and transport and for DNA replication and repair requires the visualization of proteins inside the cell nucleus. Often light microscopic techniques are used, mainly based on fluorescent labelling of nuclear components of interest. Structural information at a higher spatial resolution can be obtained by using immunogold labels instead of fluorescent tags, followed by electron microscopy.

In these studies, it is essential to realize that, by definition, labelling of cellular components disturbs the structure that one intends to analyse. Structural changes induced by the visualization procedure cannot be avoided completely but should be kept to a minimum. Therefore, cells are generally "fixed" by chemical crosslinking (often formaldehyde or glutaraldehyde) or precipitation (e.g., by exposure to high concentrations of ethanol or acetone).

Proteins are generally visualized by using specific antibodies after the cell (nucleus) has been fixed and permeabilized. Often, commercially available secondary antibodies are used that recognize the primary antibody (recognizing the antigen of interest), to which small fluorescent molecules are covalently bound, such as FITC, rhodamine, etc. This technique is called indirect immunolabelling. Specific DNA sequences in the intact nucleus can be visualized by hybridization with fluorescently labelled DNA probes with the correct sequence, a technique called fluorescence in situ hybridization (FISH). Either the DNA used for hybridization is itself made fluorescent, e.g., by the incorporation of deoxynucleotides that carry a covalently attached fluorescent molecule, such as FITC or rhodamine, and alternatively modified nucleotides (e.g., bromodeoxyuridine) that can be recognized by fluorescence-specific antibodies. In addition, total DNA can be fluorescently labelled using a number of fluorescent, general DNA-binding molecules, such as DAPI or Hoechst. However, one should realize that many such DNA labelling components also have an affinity for RNA, and many also have certain DNA sequence preferences. A wealth of information about fluorescent labelling, antibodies, DNA stains, and techniques can be found at the web site of Molecular Probes Inc. (http://www.probes.com/).

A major problem of the FISH techniques is that it requires rather harsh conditions to denature the double-stranded DNA in chromatin in the nucleus into single-stranded DNA, allowing the (fluorescently) tagged probe DNA to bind, i.e., incubation in high concentrations of formamide and heating. This may result in major structural changes, even after fixation. Another drawback is that it has been shown that under FISH conditions, a substantial amount of double-strand breaks are introduced, resulting in a considerable loss of DNA (Rapp et al. 1986). Therefore, in all cases adequate controls should be incorporated into experimental approaches, with proofing that loss of biological structure is kept to a minimum.

Immunolabelling and FISH are techniques that are typically used on fixed (i.e., dead) cells. In the past few years, new technologies have been developed to visualize specific proteins and genomic loci in vivo. These involve tagging cellular (nuclear) components of interest with green fluorescent protein (GFP) (Tsein 1998). To this end, cells are transfected with DNA constructs that code for a chimeric protein comprising the protein of interest and GFP. Obviously, tests should be carried out to prove that the GFP tag is not disturbing the normal function of the protein of interest. GFP-labelled proteins that bind to specific DNA sequences can be used to visualize specific genomic loci in the intact nucleus. Examples are the use of the GFP-lac-repressor protein, binding to a genetically engineered array of lac-R binding sites (Belmont et al. 1999), and GFP-glucocorticoid receptor binding to a naturally occurring array of GR binding sites (McNally et al. 2000). Finally, another method to fluorescently label DNA in vivo is to incorporate fluorescent dNTPs during replication (Bornfleth et al. 1999; Manders et al. 1999).

Protocol for Simultaneous Labelling of DNA and Antigens in the Cell Nucleus

General Comments on the Procedure

a. The protocol presented below allows immunolabelling of specific nuclear components and FISH labelling of specific DNA sequences in the same cell. The method has been described in detail by Verschure et al. 1999. The FISH technique was used to visualize complete chromosomes. The probe DNA was a commercially available library of DNA sequences that is specific for one individual human chromosome. The same procedure can be used to detect, for instance, the position of specific genomic loci instead of complete chromosomes. Immunolabelling in this work was used to visualize sites of RNA synthesis in the cell nucleus. Our aim was to analyse the position of transcription sites inside the nucleus in relation to the position and structure of individual chromosomes (Verschure et al. 1999). In short, nascent RNA was fluorescently labelled by allowing the RNA polymerase to incorporate Br-UTP (instead of UTP), then the cells were fixed, with subsequently use of indirect immunofluorescent labelling, employing an antibody against bromouridine that is incorporated into the newly synthesized RNA. However, the immunolabelling procedure presented below can be used in principle to detect any nuclear antigen.

b. The result of the double labelling procedure presented here was verified by using GFP-labelled chromatin instead of FISH (Verschure et al. 1999) and by using immunogold electron microscopy in which we visualized chromatin with yet another technique, called EDTA regressive staining (Cmarko et al. 1999). All approaches gave the same result, showing that transcription is confined to the edge of compact chromatin domains.

c. The FISH procedure was optimized to allow specific immunolabelling and to minimize corruption of nuclear structure during DNA denaturation. The latter was tested by comparing in one and the same cell the 3D distribution of chromosome centromeres before and after hybridization, showing that little or no changes in 3D nuclear organization occurred (Verschure et al. 1999).

d. After fixation, the immunolabelling is carried out. Subsequently, a second fixation step is carried out to covalently link the antibodies to the antigens in the nucleus. If FISH labelling were to precede immunolabelling, it is likely that the DNA denaturation procedure would destroy the antigen, making immunolabelling impossible.

e. In this protocol, we used commercially available, chromosome-specific DNA probes that contain nucleotides with covalently attached biotin. This probe can be recognized after hybridization by fluorescently labelled streptavidin. Alternatively, DNA probes are available containing fluorescently labelled nucleotide.

f. Different fluorescent labels should be selected for immunolabelling, FISH, and visualization of total DNA. The labels should fit the capabilities of the fluorescent microscope.

g. Some antigen--antibody combinations have been found not to be resistant to the harsh FISH procedure. The reason for this is not completely known. It may be that the fixation of these antigens and/or antibodies is not complete. Alternatively, the fluorescent properties of the secondary antibody may not survive the hybridization conditions.

h. Note that IgM antibodies are bigger than IgGs. Because of accessibility problems, the use of IgMs sometimes results in a less strong immunolabelling.

i. The protocol as described here takes 2 days.

j. Manufacturers of reagents can be found in (Verschure et al. 1999).

Protocol for Combined Immunolabelling and FISH

Step #1 Fixation

1.

Cells (primary human female fibroblasts) are grown on coverslips to about 50% confluency.

2.

Cells on coverslips are washed once in PBS; 3 min, 4°C.

3.

Cells are fixed on coverslips in 2% freshly prepared formaldehyde; 15 min, 4 °C. 1 g of paraformaldehyde is dissolved in 10 ml of H2O by stirring at 65°C and adding 1 N NaOH until the solution just clarifies (about 40 μl); subsequently, add 5 ml of 10X PBS and 40 ml of H2O.

4.

Wash 2 x 3 min in PBS, room temperature.

5.

Incubate 5 min in PBS containing 0.5% (w/v) Triton X-100, room temperature. The detergent makes the cell and the nucleus accessible for antibodies.

6.

Wash 2 x 3 min in PBS, room temperature.

7.

Block free aldehyde groups of the formaldehyde by incubation with 100 mM glycine, pH 7, room temperature.

8.

Wash 2 x 3 min in PBS, room temperature.

Step #2 Immunolabelling

9.

Incubate the coverslip with primary antibody (recognizes antigen of interest); use appropriate dilution in PBS containing 0.1 μg/ml herring sperm DNA; 1.5 hours, room temperature. Optimize the antibody concentration in a separate experiment. Herring sperm DNA is used for blocking nonspecific binding of antibodies.

10.

Wash 4 x 5 min with PBS, room temperature.

11.

Incubate with fluorescent secondary antibody (recognizes first antibody; often commercially available); use appropriate dilution (see information of manufacturer); 1.5 hours, room temperature.

12.

Wash 4 x 5 min with PBS, 4°C.

Step #3 Second Fixation

13.

Immunolabelled cells on coverslips are fixed in 4% freshly prepared formaldehyde; 10 min, 4°C.

14.

Wash 3 x 5 min in PBS, room temperature.

15.

Incubate with 0.1 M HCl; 10 min, room temperature. HCl improves accessibility of the DNA probe to chromatin.

16.

Wash 3 x 5 min in PBS, room temperature.

17.

Incubate in PBS containing 0.5% (w/v) Triton X-100 and 0.5% (w/v) saponin, 10 min, room temperature. Detergents further improve accessibility for DNA probes.

18.

Wash 3 x 5 min in PBS, room temperature.

Step #4 Preparation of DNA Probe for Chromosome-specific in Situ Hybridization

19.

Denature the biotinylated DNA probe (2 to 3 μg/ml DNA) by incubation by incubation at 85°C, 5 min.

20.

Allow re-annealing for 90 min at 37°C; use immediately for hybridization (see 24.) This incubation results in re-annealing of predominantly repetitive sequences, which often are not chromosome specific. In this way, the chromosome specificity of the probe is increased. These two steps can be omitted in the case of using FISH, for instance, for labelling of individual genes.

Step #5 Preparation of Cells for in Situ Hybridization

21.

Incubate cells on a coverslip (from 18.) in pre-warmed 70% formamide in 2X SSC, 73°C, 4 min.

22.

Incubate cells in pre-warmed 50% formamide in 2X SSC, 73 °C, 1 min.

23.

Allow all liquid to flow from the coverslip by touching a Kleenex tissue; avoid complete drying of the glass surface.

24.

Add hybridization probe (from 20.) to the coverslip; incubate overnight at 42°C in a closed, wet box to avoid evaporation and condensation of liquid on the coverslip.

25.

Wash 3 x 5 min with 50% (w/v) formamide in 2X SSC, 42°C.

26.

Wash 2 x 5 min with 2X SSC containing 5% (w/v) non-fat dry milk, 30 min, room temperature. The milk powder inhibits nonspecific binding.

27.

Wash 5 min with PBS, room temperature.

28.

Incubate with commercially available, fluorescently labelled streptavidin (use dilution as indicated by manufacturer) in 4X SSC containing 5% (w/v) non-fat dry milk and 0.1 μg/ml herring sperm DNA, 20 min, room temperature.

29.

The fluorescent signal can be enhanced if necessary by incubating subsequently with a fluorescently labelled antibody to which biotin is bound covalently (commercially available), 90 min, room temperature. Streptavidin has four binding sites for biotin. One, or at most two, will bind to the biotinylated, in situ hybridized DNA. This leaves two or three binding sites for a biotinylated fluorescent antibody. For signal enhancement, the fluorescent tag to the streptavidin and the antibody should be identical (e.g., FITC or Cy3).

30.

Wash 2 x 5 min with 4X SSC containing 0.05% Tween 20, room temperature.

31.

Wash 2 x 5 min in PBS, room temperature.

32.

Incubate with PBS containing 0.4 μg/ml Hoechst 33258 or in 25 mM HEPES buffer, pH 7.4, containing 0.5 μM Sytox Green (Molecular Probes), 5 min, room temperature. Hoechst 33258 and Sytox Green fluorescently label all DNA and allow one to find nuclei in the microscope efficiently.

33.

Mount coverslip with cells in Vectashield on an object glass and seal with nail polish. Vectashield contains substances that slow down photobleaching.

34.

Analyse by fluorescent microscopy as soon as possible; store at 4°C.

Control Experiment

To estimate nonspecific binding of fluorescent probes (antibodies and streptavidin), carry out in parallel an experiment in which the primary antibody (against the nuclear antigen of interest) is omitted and an experiment in which the biotinylated DNA probe is omitted. The signal should be considerably less than after specific labelling. To reduce nonspecific background labelling, one may increase the time for washing and/or reduce the concentration or incubation time of the primary antibody and/or the DNA probe and/or the fluorescent probes. Typical images can be seen in Figure 4 of Verschure et al. 1999.

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