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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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Mapping Protein/DNA Interactions by Cross-Linking [Internet].

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Localizing Replication Sites and Nuclear Proteins

, , , , , , and .

Abstract

To investigate the mechanisms that assure the maintenance of heterochromatin regions, we took advantage of the fact that clusters of heterochromatin DNA replicate late in S phase and are processed in discrete foci with a characteristic nuclear distribution (see Figure 1). At the light microscopy level, within these entities, we followed DNA synthesis, histone H4 acetylation, heterochromatin protein 1 (Hp1α and -ß), and chromatin assembly factor 1 (CAF-1). During replication, Hp1α and -ß domains of concentration are stably maintained, whereas heterochromatin regions are enriched in both CAF-1 and replication-specific acetylated isoforms of histone H4 (H4Ac 5 and 12). We defined a time window of 20 min for the maintenance of this state. Furthermore, treatment with Trichostatin A (TSA), during and after replication, sustains the H4Ac 5 and 12 state in heterochromatin, excluding H4Ac 8 and 16. In comparison, early replication foci, at the same level, did not display any specific enrichment in H4Ac 5 and 12. These data emphasize the specific importance for heterochromatin of the replication-associated H4 isoforms. We propose that perpetuation of heterochromatin involves self-maintenance factors, including local concentration of Hp1 and -ß, and that a degree of plasticity is provided by the cycle of H4 acetylation/deacetylation assisted by CAF-1. The approach of "pulse-chase" to locate both a DNA synthesis event and a nuclear protein should help to establish how a protein of interest can be dynamically involved in complexes associated with replication foci.

Figure 1. Replication foci.

Figure 1

Replication foci. The following figure legend was translated from the French version. The French version appears below the translated version for comparison. A, sites of DNA synthesis in A6 cells (a line derived from Xenopus fibroblasts) at different (more...)

Practical Approach

Cell Culture, Synchronization, and Specific Treatments

Human HeLa and mouse L929 cells are usually grown in 9-cm-diameter glass Petri dishes (Falcon) in DME supplemented with 10% FCS, 10 μg/ml antibiotics (penicillin and streptomycin), and 2 mM l-glutamine (GIBCO BRL) at 37°C in an atmosphere of 5% CO2.

For synchronization, HeLa cells are grown to ~50% confluence and arrested in early S phase with 2 mM hydroxyurea (Sigma Chemical Co.) for 16 h. The release from HU block is obtained following several washes in PBS before a final wash in fresh medium. FACS analyses are carried out in parallel. For these cells (HeLa and L929), we usually observe that they remain in S phase for 10 h before entering G2 (3 h), and finally mitosis (1 h). For late S phase samples, cells are routinely collected 7.5–8 h after release. For immunofluorescence, cells are grown on glass coverslips in culture dishes. For Trichostatin A (TSA; Sigma Chemical Co.) treatment, exponentially growing cells are incubated in the presence of TSA (50 ng/ml, a dose compatible with cell growth) in complete medium for a chosen time.

In Situ Replication Assays

5-Bromo-2'-deoxyuridine (BrdU; Sigma Chemical Co.) incorporation is routinely performed by incubating exponentially growing cells in the presence of 40 μM BrdU for 10 min. Cells are then washed in PBS and fixed with 2% paraformaldehyde in PBS for 15 min at room temperature. For pulse–chase experiments, cells are washed twice with prewarmed PBS and once in prewarmed medium after the BrdU pulse and then cultured for different time periods. The cells are further processed for isolation of nuclei or fixed with 2% paraformaldehyde in PBS.

Biotin-16-deoxyuridine (BiodU) incorporation on isolated nuclei is performed essentially as described by Krude et al. 1997. In brief, after nuclei isolation in vitro run-on is initiated by adjusting the nuclear preparation to final concentrations: 40 mM K-HEPES, pH 7.8; 7 mM MgCl2; 3 mM ATP; 0.1 mM each of GTP, CTP, and UTP; 0.1 mM each of dATP, dGTP, and dCTP; 40 μM bio-dUTP; 20 mM creatine phosphate; 0.5 mM DTT; and 2.5 μg phosphocreatine kinase (all Boehringer Mannheim Corp.). After a 10-min incubation at 37°C, reactions were stopped by a 25X dilution in PBS, and nuclei are immediately fixed in 2% paraformaldehyde. Nuclei are then deposited onto polylysine-coated coverslips by gentle centrifugation through a 30% cushion of glycerol in PBS, rinsed twice with PBS and permeabilized in 0.2% Triton X-100 in PBS for 5 min. For the BiodU detection, FITC-conjugated streptavidin (Enzo) is used, and fluorescence analysis was carried out as described below.

Immunolabeling

Paraformaldehyde-fixed cells or nuclei were washed twice in PBS and then incubated in 0.2% Triton X-100 in PBS for 5 min and blocked in 5% BSA, 0.1% Tween 20 in PBS (blocking buffer) for 10 min. Specific antibodies are then added at the appropriate dilution in blocking buffer. Incubation was carried out for 45 min at room temperature. To visualize the staining, FITC or Texas red-conjugated goat anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc.) are used. During the final washing steps with 0.1% Tween 20 in PBS, 0.25 μg/ml 4',6-diamino-2-phenylindole (DAPI; Sigma Chemical Co.) is added for DNA staining.

In experiments involving BrdU immunodetection, a denaturation step in 4 N HCl for 10 min at room temperature is incorporated, after the permeabilization treatment, to render BrdU incorporated into DNA accessible. Neutralization is achieved by washes in PBS, and the slides are further processed as described above. Because detection of the nuclear proteins studied here is sensitive to HCl extraction, a specific protocol has to be applied for double staining with BrdU. Proteins are first revealed (first and second antibodies) on paraformaldehyde-fixed cells as described. After three washes in PBS and 0.1% Tween 20, a second fixation step is included using either 2% paraformaldehyde in PBS for 15 min or 5 mM ethylene glycol-bis(succinimidylsuccinate) (Pierce Chemical Co.) in PBS for 30 min at 37°C, followed by several washes in 0.3 M glycine in PBS. After this step, DNA is denatured with 4 N HCl, before BrdU revelation. The coverslips are mounted in Vectashield (Vector Laboratories Inc.). Despite postfixation, some loss of signal may still occur. Incorporation of BiodU (on isolated nuclei) into DNA can be used instead of BrdU as an alternative to avoid the HCl denaturation because the biotinyl residue is accessible in the double helix of DNA.

Antibodies

Rabbit polyclonal antibodies (pAbs) against the different acetylated isoforms of histones H4 and H2A have been characterized in detail (Turner et al. 1989). They recognize H4 isoforms acetylated on one of the four lysines (K) acetylated in vivo or H2A acetylated at lysine 5 (H2A Ac 5). They are used at final dilutions of 1:500 for R101/12 (K12), R14/16 (K16), and R123 (H2A Ac 5) and 1:1,000 for R232/8 (K8) and R41/5 (K5). mAbs against HP1 (2HP1H5) and HP1ß (IMOD-1A97), kindly provided by Dr. R. Losson and Dr. P. Chambon (CNRS, Strasbourg, France), are used at final dilutions of 1:400. The mAb against human CAF-1 p150 (mAb1) is provided by Dr. B. Stillman (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; Smith and Stillman 1991). The rabbit pAb1, directed against the human CAF-1 p60, was previously characterized (Marheineke and Krude 1998; Martini et al. 1998). The specificities of the antibodies are always tested by Western analysis on all sources of material studied. Monoclonal rat anti-BrdU were purchased from Harlan; Sera-Labo, Texas red goat anti-rabbit immunoglobulin (Ig), FITC goat anti-rabbit Ig, and Texas red goat anti-rabbit Ig antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc.

Image Acquisition and Quantitative Analysis

Image acquisition was performed using a Leica TCS-4D confocal scanning microscope, equipped with an Acousto-Optical Tuneable Filter (AOTF), with a 100X NA 1.4 plan--apochromat oil immersion objective. Single optical sections are usually presented. FITC and Texas red are excited by the argon--krypton laser at 488 and 568 nm, respectively. Red and green fluorescence are separated by a 580-nm long-pass, dichroic beam-splitter. A 520-nm band-pass filter and a 590-nm long-pass filter are used to minimize cross-talk and stop laser scattered light. DAPI staining is acquired with the UV laser. Before acquiring a double staining z series, the intensity of excitation wavelengths and the power of photodetectors must be adjusted to avoid cross-talk. The fluorescence signals from both fluorochromes are always recorded simultaneously in one scan and saved separately on two channels to be processed independently.

Three-dimensional, two-color images of doubly stained nuclei are recorded using the same equipment. The voxel dimensions (x, y, and z) of each recorded stack is adjusted to 0.05 x 0.05 x 0.25 μm to verify the Shannon sampling criteria (Goodman 1968). The averaging parameter is adjusted to have the best signal/noise ratio without bleaching the sample during the acquisition process. For H4Ac and H2AAc staining, we could not reach this criterion without bleaching, leading us to perform the quantitative analysis on single optical sections (two-dimensional analysis). The same set-up is systematically used for all sample acquisitions to facilitate comparisons between data. Numerical data are stored in an 8-bit tiff format series for each color and transferred for processing and analysis. Noise reduction and image quantification are developed in-house. Metamorph software (Universal Imaging) is used as a support for executing our own Dynamic Link Libraries (DLL) written in Visual Basic and C languages for displaying results and basic treatments. A nonlinear, rank-adaptive de-noising filter (Vila and Bolon 1993) is applied to better distinguish fluorescent structures from background fluorescence without loss of resolution in transition regions. Red and green volumes were thresholded separately to avoid background contributions in the quantitative analysis. To make the measurements reproducible and comparable, red and green thresholds, designated Tr and Tg, respectively, remained unchanged for each volume corresponding to each series.

Voxels are sorted in four categories: C, colocalized; R, red only; G, green only; and N, noise. In N, voxels displaying green and red signal intensities below both thresholds Tr and Tg are counted. In C, voxels displaying green and red signal intensities above both thresholds Tr and Tg are counted. In G, voxels displaying green signal intensity above Tg and red signal intensity below Tr are counted. In R, voxels displaying red signal intensity above Tr and green signal intensity below Tg are counted.

A control experiment, in which replication sites pulse-labeled with BrdU are revealed by a balanced mix of Texas red and FITC secondary antibodies, gives 90% of colocalizing BrdU within the total population of BrdU [(C)/(C + R) ratio], using the same parameters. This 90% approaches the 100% normally anticipated. It is important to note that the difference is attributable to the definition of our thresholds, which eliminates noise but also leads to systematic underestimation of our values. In addition, we find that these values are dependent on the shape of the objects revealed in each color, and this has to be taken into account for interpretation of the data. In all of the graphs, for each point several experimental measurements are collected, and the average value can be plotted in arbitrary units. For each pair of markers, plots can be obtained to represent the red signals colocalizing with the green signals in arbitrary units [(C)/(C + R) ratio] normalized to the maximal value obtained in all the experiments involving this pair of markers.

Selected References

  1. Cook P R. The organization of replication and transcription. Science. 1999;284:1790–1795. [PubMed: 10364545]
  2. Goodman, J. W. 1968. Introduction to Fourier optics. In: McGraw-Hill, New York.
  3. Jackson D A, Pombo A. Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells. J Cell Biol. 1998;140:1285–1295. [PMC free article: PMC2132671] [PubMed: 9508763]
  4. Krude T, Jackman P J, Laskey A. Cyclin/Cdk-dependent initiation of DNA replication in a human cell-free system. Cell. 1997;88:109–119. [PubMed: 9019396]
  5. Marheineke K, Krude T. Nucleosome assembly activity and intracellular localization of human CAF-1 changes during the cell division cycle. J Biol Chem. 1998;273:15279–15286. [PubMed: 9614144]
  6. Martini E, Roche D M J, Marheineke K, Verreault A, Almouzni G. Recruitment of phosphorylated chromatin assembly factor 1 to chromatin following UV irradiation of human cells. J Cell Biol. 1998;3:563–575. [PMC free article: PMC2148138] [PubMed: 9813080]
  7. Smith S, Stillman B. Immunological characterization of chromatin assembly factor I, a human cell factor required for chromatin assembly during DNA replication in vitro. J Biol Chem. 1991;266:12041–12047. [PubMed: 2050697]
  8. Taddei A, Roche D, Sibarita J B, Turner M, Almouzni G. Duplication and maintenance of heterochromatin domains. J Cell Biol. 1999;147:1153–1166. [PMC free article: PMC2168099] [PubMed: 10601331]
  9. Taddei A, Maison C, Roche D, Almouzni G. Reversible disruption of pericentric heterochromatin and centromere function by inhibiting deacetylases. Nature Cell Biol. 2001;3:114–120. [PubMed: 11175742]
  10. Vila, J.-L., and P. Bolon. 1993. Filtrage d'ordre adaptatif pour le prétraitement d'images naturelles. In: 14ème colloque Gretsi.
Copyright © 2001, Institut national de la santé et de la recherche médicale (INSERM)
Bookshelf ID: NBK7103PMID: 21413362
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