RAPID COMMUNICATION |
Correspondence to: Caroline Jolly, INSERM U309, Institut Albert Bonniot, Domaine de la Merci, 38706 La Tronche Cedex, France.
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Summary |
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Detection of specific nuclear transcripts by fluorescence in situ hybridization (FISH) has constituted a major breakthrough in the study of the organization of transcription in the cell nucleus. Using the model of heat shock genes, we present an optimized procedure for nuclear transcripts that provides high efficiency for RNA detection and good preservation of cell morphology and nuclear texture. Using this procedure, we designed an original high-efficiency methodology combining FISH and fluorescence immunocytochemistry (FICC), which is used here for the simultaneous detection of heat-shock protein (hsp) nuclear transcripts and the specific heat-shock transcription factor 1 (HSF1). We show that the nuclear accumulation sites of HSF1 in heat-shocked cells do not correspond to the sites of transcription of the hsp70 gene. (J Histochem Cytochem 45:1585-1592, 1997)
Key Words: fluorescence in situ, hybridization, fluorescence, immunocytochemistry, HSF1 transcription factor, hsp genes, nuclear transcripts, transcription factors
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Introduction |
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The development in the late 1980s of a FISH procedure that allows the detection of nucleus-specific transcripts has constituted a major breakthrough in the study of the organization of transcription in the cell nucleus (
We were interested in combining FISH and FICC to simultaneously detect nuclear transcripts and specific transcription factors. Several procedures with substantial different efficiency for the detection of nuclear transcripts have been reported thus far (
This study was performed using the model of heat-shock genes (hsp) (
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Materials and Methods |
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Cell Culture and Heat Shock
Normal primary fibroblasts (skin) were grown directly on glass slides in RPMI medium (Gibco BRL; Gaithersburg, MD) supplemented with 10% fetal calf serum. Heat shock was performed in a water bath for 1 hr at 44C.
Probes and Antibodies
hsp70 transcripts were detected using the genomic probe pH2.3 (a gift from Dr. R.I. Morimoto, Evanston, IL), covering the entire coding sequence of the intronless hsp70 gene (2.3 KB) (-transcripts (a gift from Dr. E. Hickey, Reno, NE) contains 1.3 KB of the coding region (
Fluorescence In Situ Hybridization
Four procedures differing in the type of permeabilization treatments used to improve accessibility to the target were tested for the detection of nuclear transcripts. The simplest procedure uses no permeabilization (Protocol 1). Two other procedures use different postfixation permeabilization steps (Protocols 2 and 3), and the fourth one uses pre- and postfixation permeabilizations (Protocol 4). All these procedures are summarized in Table 1. Unless otherwise noted, all incubations were performed for 5 min.
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Protocol 1
This protocol is derived from
Cell Treatment. Immediately after heat shock, cells were fixed in 4% formaldehyde/PBS (pH 7) for 10 min and dehydrated through ethanol baths. If 3D analyses have to be performed, dehydrations can be omitted and replaced by an equilibration step in 50% deionized formamide/2 x SSC.
Hybridization. A total of 100 ng of labeled probe was precipitated with 10 µg of salmon sperm DNA. The DNA pellet was resuspended in 10 µl of 50% deionized formamide/10% dextran sulfate/2 x SSC, denatured for 5 min at 75C, and applied to the dry slide. Hybridization was allowed to proceed overnight at 37C. Cells were then washed in 60% formamide/2 x SSC, once at 45C and twice at room temperature (RT), and rinsed three times in 2 x SSC at RT. After a 30-min incubation at 37C in a blocking buffer (3% BSA/0.3% Triton X-100/4 x SSC), the probe was detected using either avidin-FITC (Vector; Burlingame, CA) or an anti-digoxigenin antibody coupled to TRITC (Boehringer; Mannheim, Germany). Cells were then washed three times in 4 x SSC/0.1% Tween-20 at 42C and embedded in an anti-fading solution consisting of 90% glycerol, 20 mM Tris-Cl, 2.3% 1,4-diazobicyclo-octane (DABCO; Sigma, St Louis, MO), 0.02% NaN3, and DAPI (250 ng/ml) as a DNA counterstain.
Protocol 2
This procedure derives from
Protocol 3
This protocol combines Protocol 2 with the procedure from
Protocol 4
This protocol combines Protocol 2 with a procedure derived from
Fluorescence Immunocytochemistry
HSF1 was detected according to the procedure described by
Combined FICC and FISH
The best results were obtained when FISH was performed before FICC (see Results and Discussion). Specific nuclear transcripts were detected according to Protocol 3 as described above. Detection of transcript-specific probes labeled with biotin was performed using avidin-FITC. After postdetection washes in 4 x SSC/0.1% Tween-20, a 45-min incubation in 10% FCS/0.3% Triton X-100/PBS was performed, followed by incubation for 90 min at 37C with the rat monoclonal anti-HSF1 antibody. Detection was performed using a sheep anti-rat antibody coupled to TRITC (Sigma), and nuclei were counterstained with DAPI.
Simultaneous Detection of DNA and RNA Sequences
hsp70 transcripts were detected with the pH 2.3 probe, together with a cosmid probe specific for the 6p21.3 locus (
Digital Imaging
Preparations were observed under an epifluorescence microscope (Zeiss Axiophot; Oberkochen, Germany) equipped with a 100-W mercury lamp using appropriate filter sets. Images were acquired with a cooled CCD camera (C4880 Hamamatsu; Tokyo, Japan) using a x63, 1.25 NA oil immersion objective with an intermediate magnification of x1.25. All images were acquired with the same exposure time to allow comparison of hybridization signals.
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Results and Discussion |
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Optimization of Nuclear Transcript Detection by FISH
We present here an optimized procedure for nuclear transcript detection that was developed on the basis of a comparison of four procedures described in the literature (van den Engh 1984; and hsp70 nuclear transcripts were detected using these procedures in non-heat-shocked and heat-shocked primary fibroblasts, and the protocols were compared on the basis of the following criteria: (a) efficiency for RNA detection, (b) preservation of cytoplasmic RNAs, and (c) preservation of nuclear texture.
Efficiency of the Different Procedures for RNA Detection
In the absence of permeabilization treatment (Protocol 1), two foci corresponding to nuclear accumulations of hsp90 (Figure 1A) and hsp70 transcripts (Figure 1B) were detected in all heat-shocked cells. Although the hsp90
gene, in contrast to the hsp70 gene, is known to be constitutively expressed in these cells, no hybridization signals could be detected in unstressed cells (data not shown). As previously demonstrated by others for other transcripts, nuclear transcript foci represent the sites of transcription of the corresponding genes (
(data not shown) transcripts with a cosmid probe specific for the gene locus revealed a close spatial proximity between nuclear transcripts and their corresponding gene loci.
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When postfixation permeabilization steps with HCl and saponin/Triton were added (Protocol 2), nuclear foci corresponding to hsp90 (Figure 1D) and hsp70 transcripts (Figure 1E) were significantly larger and brighter than the signals obtained with Protocol 1 (compare with Figure 1A and Figure 1B). When an additional postfixation permeabilization step with liquid nitrogen was used (Protocol 3), hybridization signals corresponding to both hsp transcripts appeared larger than with Protocol 2 (compare Figure 3G and Figure 3H with Figure 3D and Figure 3E). In addition, hsp90
transcripts could be detected in non-heat-shocked cells using either Protocol 2 (data not shown) or Protocol 3 (Figure 1M). As expected from the lower constitutive expression of the hsp90
gene (
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When a prefixation permeabilization step in a Triton-containing buffer was added to the HCl and saponin/Triton permeabilizations (Protocol 4), hybridization signals corresponding to hsp90 (Figure 1J) and hsp70 (Figure 1K) transcripts were found to be identical in size to those obtained with Protocol 3 (compare with Figure 1G and Figure 1H). The use of Triton before fixation has been shown to significantly improve the efficiency of nuclear RNA detection (
transcripts could also be detected in non-heat-shocked cells (data not shown).
These observations clearly show that pre- and postfixation permeabilization steps greatly improve the efficiency of nuclear RNA detection.
Preservation of Cytoplasmic RNAs
In addition to the focal concentrations of hsp transcripts, diffuse labeling was also detected in the nucleoplasm of non-heat-shocked and heat-shocked cells with all procedures (Figure 1A-K). Diffuse staining of lesser intensity was also observed in the cytoplasm, but only with Protocols 1, 2, and 3 (Figure 1A and Figure 1H). In contrast, no cytoplasmic labeling was detected using Protocol 4 (compare Figure 1J and Figure 1K with Figure 1G and Figure 1H). As previously suggested (
Preservation of the Nuclear Texture
The nuclear texture was investigated on the basis of DAPI counterstaining. With all four protocols, the overall nuclear texture was similar (Figure 1C, Figure 1F, Figure 1I, Figure 1L, and Figure 1O). In particular, the nucleoli and the Barr body were always clearly visible (arrows and arrowheads, respectively), suggesting that no major rearrangements in the nuclear organization were induced by pre- and postfixation permeabilizations.
In conclusion, pre- and postfixation permeabilization steps significantly improve the efficiency of RNA detection, as assessed by measuring the size of the hybridization signals. Two procedures proved to be more efficient with respect to RNA detection, the one using liquid nitrogen after fixation (Protocol 3) and the one using a Triton extraction before fixation (Protocol 4). However, the procedure using liquid nitrogen as a postfixation permeabilization step appears to be the method of choice, because it combines maximal nuclear RNA detection and good preservation of the cellular and nuclear morphology. This procedure appears to be particularly interesting for the detection of poorly expressed RNAs and/or for investigation of the transcriptional activity of amplified genes in tumor cells, as previously described (
Co-detection of Nuclear Transcripts and Transcription Factors
Detection of HSF1 by Fluorescence Immunocytochemistry.
HSF1 was detected using a rat monoclonal anti-HSF1 antibody. As previously reported (
The optimized procedure for nuclear transcript detection using postfixation permeabilization steps with HCl, saponin/Triton, and liquid nitrogen was chosen for combination with fluorescence immunocytochemistry. The impact of these treatments on the distribution of HSF1 factor in heat-shocked cells was thus investigated. As shown in Figure 2C, no significant difference in the size and intensity of nuclear HSF1 foci was observed compared to the original FICC procedure, which uses only Triton in the blocking and detection steps (
Simultaneous Detection of HSF1 and hsp Nuclear Transcripts
Two different approaches were tested. Because FICC requires only mild permeabilization steps, it seemed a priori preferable to perform FICC first (FICC>FISH). Alternatively, because nuclear transcripts are highly sensitive to nucleases and may be digested during the FICC detection, a procedure in which FISH was performed first was also tested (FISH>FICC).
Illustrations of co-detection of HSF1 and hsp70 transcripts using either the FICC>FISH or the FISH> FICC procedure are shown in Figure 3. Signals corresponding to hsp70 nuclear transcripts (Figure 3A and Figure 3C) and transcription factors (Figure 3B and Figure 3D) were detected with both procedures. However, the intensity and size of the signals varied from one procedure to another. With the FISH>FICC procedure, fluorescent signals obtained for both transcripts and transcription factors were similar to those obtained when HSF1 or nuclear transcripts were detected independently (compare Figure 3A with Figure 1H and Figure 3B with Figure 2B). With the FICC>FISH procedure, the fluorescent signals corresponding to HSF1 foci (Figure 3D) appeared identical in shape and size to HSF1 foci observed with the FISH>FICC procedure (compare with Figure 3B). In contrast, the signals corresponding to hsp70 transcripts (arrows in Figure 3C) were significantly smaller than with the FISH> FICC procedure (compare with Figure 3A). Similar results were obtained with hsp90 transcripts (data not shown). This may be due to either a diffusion or a digestion of RNAs by nucleases during the FICC procedure.
In conclusion, we found that FISH>FICC is unambiguously the best procedure to simultaneously detect nuclear transcripts and specific transcription factors. This method was also found to be valuable for the co-detection of UBF nucleolar protein and 28S ribosomal RNAs within the nucleoli. One of the major advantages of this procedure is the absence of permeabilization steps before fixation, thus limiting the degradation of RNAs and the possible occurrence of rearrangements in the overall organization of the nucleus. In consequence, this procedure offers the best compromise between gain of sensivity and preservation of the general nuclear organization. In that respect, it is interesting to point out that both procedures reported here can be used for 3D analysis, because dehydration steps can be omitted without any significant decrease in efficiency (data not shown).
Using this original procedure, we have investigated the relative intranuclear distribution of HSF1 foci and hsp70 transcripts. As shown in Figure 3, we found that the nuclear foci enriched in HSF1 transcription factor do not co-localize with hsp70 gene transcription sites. The distribution of HSF1 foci was also investigated with regard to other active genes (submitted for publication). HSF1 nuclear foci are a new stress-induced compartment that may play a key role in cellular response to stress. Recent studies have shown that other transcription factors, such as GATA (
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Acknowledgments |
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Supported by the Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche, by the Groupement de Recherche et d'Etudes sur le Génome (GREG), the Université J. Fourier (Grenoble I), the Commission of the European Community (Human Genome Analysis contract GENO-CT91-0029 and Human Capital and Mobility CHRX-CT93-0177), and by a fellowship to C.J. from the Ligue Nationale contre le Cancer.
We thank Drs E. Hickey (Reno, NE), R.I. Morimoto (Evanston, IL), and F. Amblard (Grenoble, France) for kindly providing us with the cells, probes, and antibodies used in this study. We are very grateful to F. Zennaf for excellent technical assistance.
Received for publication August 4, 1997; accepted September 1, 1997.
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