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DNA Exposure and Condensation in the X and 21 Chromosomes

^a Eleanor Roosevelt Institute for Cancer Research, Denver, Colorado, USA;
^b Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado, USA

Key Words. X-chromosome • Chromosome 21 • Chromosome condensation • Gene exposure • Gene sequestration • Nuclear DNA positioning

Correspondence: Dr. Theodore Puck, 1899 Gaylord Street, Denver, CO 80206-1210, USA.

	Abstract

Top Abstract Introduction Materials and Methods Experimental Results Discussion Acknowledgements References

 
Fluorescence in situ hybridization (FISH) whole chromosome painting probe studies have been carried out with X and 21 chromosomes on normal human fibroblasts grown in tissue culture. The majority of the cells were in G₁ phase (including G₀). The X chromosome, which exhibits differential inactivation, displays an active form which is most commonly in the nuclear periphery, is diffused over a large area with dark regions interspersed with bright regions, and exhibits punctate bright spots at its edges. The inactive X, which contains a small fraction of active genes, is also most often at the nuclear periphery, is highly condensed and also exhibits punctate labeling around its outer edge. Occasional nuclei exhibit X chromosomal material adjacent to a nucleolus. These observations fit the pattern proposed by the genome exposure theory in which inactive gene regions are sequestered by chromosome condensation, and become exposed by decondensation into a condition invisible by the video-imaging technique employed. Such exposed genes can then be activated by appropriate molecular messengers. In accordance with this theory, the total fluorescence observed from the active X is appreciably less than that of the inactive.

The FISH pattern from chromosome 21 is very different, displaying two fluorescent bodies usually connected with the nucleoli. Both bodies contain condensed and decondensed regions, and both are much more similar in their degree of decondensation than was the case with the X chromosomes, although a small difference cannot be ruled out.

Use of DNase I treatment of nuclei reveals the existence of exposed DNA. The use of FISH as demonstrated here can indicate sequestered DNA. Together the two techniques promise elucidation of gene regions of various chromosomes which are active and inactive in particular tissues and in normal and pathologic conditions.

	Introduction

Top Abstract Introduction Materials and Methods Experimental Results Discussion Acknowledgements References

 
In earlier studies a two-level theory of genome regulation underlying mammalian cell differentiation was proposed. In each state of differentiation a specific set of genes is converted to an exposed condition in which they can react directly with signaling molecules in the medium inducing transcriptional activation, replication or other activities. Genes involved in alternative states of differentiation which are not utilized by the cell in question are maintained in a sequestered or condensed state which prevents their interaction with specific activating molecular signals in the nucleus. Gene sequestration or exposure is controlled by messenger molecules and by the fiber system of the cell which presumably includes the cytoskeleton, the nuclear fibers and the extracellular fibers which communicate with the microtubules by means of molecules like integrins. Unlike the specific differentiation genes, housekeeping genes are presumed to be fairly continuously exposed in all cells [1-11].

Exposed genes have been detected by their hypersensitivity to hydrolysis by an enzyme like DNase I [1-15]. Three different regions of exposure have been demonstrated by means of DNase I hypersensitivity in cultured mammalian cells: a region near the nuclear periphery, the vicinity of the nucleolus, and punctate regions in the interior of the nucleus [8]. Ribosomal genes, which are housekeeping genes par excellence, have been shown to hybridize to the region of the nucleoli [14]. Because of this, plus the fact that several cancer cells display reduced exposure in the nuclear periphery but undiminished exposure in the nucleolar region, we have postulated the nuclear periphery to be the seat of at least some of the active differentiation genes, and the nucleolus as the locus of at least some of the housekeeping genes [1-11]. The nature of the punctate exposed regions in the interior of the nucleus is as yet unknown, although evidence has been presented by other laboratories that transcription can occur at such loci [15]. Presumably, the sequestered state involves various degrees of DNA supercoiling together with specific protein attachments which render these particular sequences condensed and relatively inaccessible to reactive molecules in the medium. Exposed DNA would be much less involved in protected configurations than the sequestered form. Obviously, sequestered and exposed DNA should respectively correspond at least in large part to heterochromatin and euchromatin.

Exposed regions presumably represent activatable genes. The experiments described here were undertaken to determine whether the fluorescence in situ hybridization (FISH) procedure can identify exposed and sequestered DNA. The X chromosome, which reveals a regulatory phenomenon of large magnitude, would seem to offer a useful model system for testing the theory of genome exposure and the measuring of exposure by means of the FISH procedure. Only one X chromosome is active over a large amount of its length in any cell. All additional X's are inactive except for small stretches like the pseudo-autosomal genes, some of which are located on the short arm [16]. Consequently, if the X chromosome is regulated in accordance with the general exposure scheme outlined above, the following behavior using the FISH procedure with the entire X chromosome painting probe could be predicted: the largely inactive X chromosome should be highly condensed corresponding to the sequestered state, as has been long recognized. However, the small region of active genes even in the predominantly inactive X chromosomes should serve to anchor these inactive chromosomes in the nuclear periphery [11]. The large active part of the active X chromosome should also be in the nuclear periphery, but it should be in an elongated and diffused state permitting many genes to be exposed. Under conditions of our microscopic digital image capture of the fluorescent chromosomal domains, the tiny amount of fluorescent light from individual exposed DNA fibers is below the background level and therefore invisible. Thus under these conditions, the total fluorescent light detected from the inactive X chromosome should be appreciably greater than that from the active X chromosome. It should be noted that the nuclear periphery as here designated encompasses about 10%-15% of the mean nuclear diameter.

An earlier study by Dyer et al. [12] describes FISH experiments using a probe from the centromeric region of the X chromosome, revealing a dense signal for the inactive X marker and a diffuse signal for that of the active X in the great majority of the cells. The probe for the inactive X centromere was located in the nuclear periphery in the great majority of the cells, whereas it was located peripherally or centrally with equal frequency in the active X. Bischoff et al. [13] have measured the area and volume of active and inactive X chromosome domains in human cells and demonstrated quantitatively the more condensed state of the inactive form.

In our study, experiments with confocal microscopy using the whole chromosome X painting probe and the whole chromosome 21 painting probe were carried out using the FISH procedure to test the expectations of the genome exposure hypothesis. Preliminary results were presented earlier bearing out some of these predictions [11]. Here we compare the nuclear location, the presence of discontinuities in the patterns, and the total fluorescence brightness of the X and 21 chromosomes, and interpret the results in terms of the exposure theory of genome regulation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Experimental Results Discussion Acknowledgements References

 
Low to medium passage human male or female skin fibroblast cells were seeded in NUNC 8-chamber slides in Ham's F12 + 15% fetal bovine serum at a density of 500-1,000 cells per chamber. Cells were grown for 24 h in a tissue culture incubator at 37°C and 5% CO₂. To obtain cells in G₁ phase of the cell cycle, serum-containing medium was removed and the cell monolayer was washed once with F12 without serum. F12 without serum was added to the chambers and the cells were incubated for 48-72 h. Fetal bovine serum was then added to the chambers to a final concentration of 20%. Cells were incubated for an additional hour and fixed in methanol-acetic acid (20:1) at –20°C for 30 min, then in HistoChoice aqueous tissue fixative for 10 min at room temperature. Cells were dehydrated in a series of 70%, 80% and 95% ethanol baths, then air dried.

Essentially similar results were obtained with random and G₁-phased cultures, although the phased culture results were somewhat more impressive as shown in Tables 1 and 2. Flow cytometer analysis indicated the percent of G₁ cells in random cultures to be 82% and 94% in G₁-phased cultures, so that in either case we are dealing mainly with G₁ cells (including G₀).

For some experiments, FISH probes and target DNA were denatured separately. A tube containing the standard in situ hybridization mix was incubated at 70°C for 10 min then put on ice. The slide containing the target cells was incubated at 70°C for 3 min in a solution containing 70% formamide, 2x SSC (0.3 M sodium citrate) then placed into ice-cold 70% ethanol. The slide was dehydrated in 80% then 95% ethanol and air dried. The previously denatured probe was added to the target cells, and the slide was coverslipped, rubber cemented and incubated at 37°C overnight.

Nonhybridized probe was washed from the cells by a 15 min incubation at 43°C in 50% formamide, 2x SSC, followed by a 15 min incubation at 60°C in 0.1x SSC. Slides were then incubated in PN (0.1 M NaH₂PO₄/Na₂HPO₄, pH 8.0 and 0.05% nonidet P-40) for 5 min followed by a 10 min incubation in PNGG (PN + 5% normal goat serum and 5% gelatin) all at room temperature. Antidigoxigenin/fluorescein isothiocyanate (FITC) was diluted in PNGG according to the instructions of the manufacturer. Diluted antidigoxigenin/FITC was applied to the slides and incubated for 20 min at room temperature. Slides were washed in three changes of PN. The DNA stain DAPI (or propidium iodide) was added to the last wash at a concentration of 20 ng/ml. An antifade solution in 90% glycerol was applied and the slide was coverslipped. For experiments with chromosome 21 which required visualization of the nucleoli, a human autoantibody against the nucleolus was diluted 1:10 in PNGG and incubated on the cells for 20 min at room temperature. The slides were washed in three changes of PN. Texas red conjugated goat antihuman IgG was diluted 1:40 in PNGG and incubated on the cells for 20 min at room temperature. Slides were washed in three changes of PN.

X chromatin (the Barr body, representing the highly condensed, largely inactive X chromosome) was stained with 2.5% orcein in 45% acetic acid. Staining was carried out for 10-30 min at room temperature. The staining procedure was terminated by extensively rinsing the slides with a phosphate-buffered saline solution. The slides were coverslipped and inserted in the microscope where coordinates for several nuclei displaying obvious Barr bodies were recorded and images were captured and saved. The slides were destained and FISH was performed as described above. Images of the whole chromosome FISH probes were then captured and demonstrated to reside at the previously recorded coordinates in the same nuclei. This procedure confirmed that the bright, compact chromosome X FISH domain overlayed the orcein Barr body.

Fluorescence microscopy was performed on a Nikon Microphot equipped with a 60x Plan Apochromatic oil objective with a N.A. of 1.4. Laser confocal microscopy was performed on a Bio-Rad MRC500 equipped with the objective listed above. Confocal image thickness was set at 1 µM, and three to five sections were obtained for each field of cells. Signal to noise ratio was improved by averaging eight video images per section. Images were captured and stored in the "Image 1" image processing system manufactured by Universal Imaging Corp. (West Chester, PA). This is an 8-bit digital imaging system providing a gray scale of 0-255 levels per pixel and 512 x 480 pixels per image.

Whole chromosome domains were quantitated by first applying a thresholding protocol that identified the total domain area. The thresholding protocol was applied equally to the two domains within each nucleus. Total brightness, B, for each domain was calculated as follows: B = (P x N) x S; where P is the brightness level for each video pixel, N is the total number of pixels within the thresholded area and S is the number of serial sections through the nucleus.

Human skin fibroblast cell lines GM00254 and GM08447 were obtained from the NIGMS Human Genetic Mutant Cell Repository in Camden, NJ. Human skin fibroblast cell line TS92-102 was kindly provided by Dr. Loris McGavran, University of Colorado Health Sciences Center (Denver, CO). Whole chromosome painting probes Coatasome X and Coatasome 21 were obtained from Oncor Inc. (Gaithersburg, MD). Whole chromosome painting libraries pBS-X and pBS-21 were kindly provided by Drs. Joe Gray and Rick Seagraves at Lawrence Livermore National Laboratory.

Nunc glass chamber slides were obtained from Fisher Scientific Inc., (Pittsburgh, PA); HistoChoice tissue fixative from Amresco Inc. (Solon, OH); Cot-1 DNA from GIBCO-BRL (Gaithersburg, MD); sheep antidigoxigenin/FITC from Boehringer Mannheim Inc. (Indianapolis, IN); and orcein stain, human nucleolar autoantibody (ANA-N) and goat antihuman IgG/Texas red from Sigma Chemical Co. (St. Louis, MO).

	Experimental Results

Top Abstract Introduction Materials and Methods Experimental Results Discussion Acknowledgements References

 
Comparison of FISH Patterns Using Total X Chromosome Painting Probe in XX and XXX Cells
Figures 1 and 2 present the results of the FISH X chromosome procedures with typical XX and XXX cells. In the confocal figure the brightest section, which was usually the center confocal section, is presented. However, very similar geometrical relationships were displayed in all sections. In all these cases, in a majority of cells, one X chromosome is clearly different by virtue of its being elongated, diffuse and less intensely colored than its more compact and brighter counterparts. The latter were confirmed as being Barr bodies by the orcein Barr body staining procedure.

View larger version (126K): [in this window] [in a new window]   Figure 1. Confocal center section through a cultured G1-phased, normal human female fibroblast treated with X-painting probe by FISH procedure. Both chromosomes approximate the nuclear periphery. The inactive X (as confirmed by Barr body staining) is highly condensed, but the central condensed area is surrounded by bright punctate dots. The active X is diffuse, encompassing a greater overall area, is interrupted all through its area by dark spots and also displays bright punctate dots around the periphery. Sections above and below the center section display similar patterns.

View larger version (69K): [in this window] [in a new window]   Figure 2. Epifluorescence microscopy. A cultured G1-phased human female XXX fibroblast treated with X chromosome painting probe as in Figure 1. As expected, two condensed fluorescent spots and one diffuse pattern are obtained, each resembling its prototype in Figure 1.

These relationships should be even more striking in an XXX fibroblast which contains two Barr bodies. Figure 2 presents results of experiments with the XXX cell demonstrating the expected behavior in a cell containing one active and two inactive X chromosomes. Occasionally, some X chromosome signal was also obtained in some cells in association with the nucleolus. However, because of the extended, diffuse pattern of the active X, we are uncertain of the extent of the association between the X chromosome and the nucleolus. This question will be considered separately in a later study.

In Table 1 a summary of the distribution of the different types of FISH patterns obtained from hybridization of whole X chromosome painting probes on normal fibroblasts with XX chromosomal composition grown in tissue culture is presented. Both random cultures and G₁-phased cultures were tested. Results with the XXX cells, which presumably should present a clearer effect due to their possession of two essentially inactive X chromosomes, are presented in Table 2. The data of Table 1 reveal that, as expected, the most common pattern, both in random and G₁-phased cultures, involves both X chromosomes at the periphery of the nucleus with one condensed and one diffuse chromosome. This situation appears to be more definitive for the G₁-phased culture than the random culture, suggesting that a somewhat different situation may be obtained in the S and G₂ phases from that in G₁. The fact that only 50%-60% of the cell nuclei exhibit the expected behavior may mean that subpopulations of cells can have a different nuclear configuration and/or that genetic or other aberrations of cells in culture have introduced anomalies in the pattern.

In an appreciable number of cells, differences in condensation or diffuseness of the two X chromosomes could not be detected by visual inspection in contrast to the very clear difference illustrated in Figure 1. Since the frequency of such ambiguous cells appears to be greater in the random than in the phased culture, they may represent a cell cycle effect.

Measurement of Total Fluorescent Light Emission from Active and Inactive X Chromosomes
The photographs of Figures 1 and 2 have been interpreted to indicate a highly condensed configuration of most of the inactive X in contrast to the active X, which is elongated and contains many visible regions of inactive, sequestered DNA separated by regions of exposed DNA which are invisible in these preparations. To test this conclusion further, the total light emission from each X chromosome in a series of normal human cells was measured by a laser confocal microscope. All of the cells in a series of microscope fields which revealed two clearly defined X chromosomes were scored.

In these experiments, the maximum exposure latitude of the light-measuring system amounting to 256 levels of gray was set to accommodate the light from the brightest part of the inactive X chromosome, which is highly condensed DNA. As a result, fluorescent light from decondensed (presumably exposed) DNA is indistinguishable from background level. These considerations make possible a rough estimate of the relative amounts of active DNA in the two X chromosomes of the human female fibroblast.

Each chromosome was optically subdivided into three to five sections, the fluorescence intensity from each section measured and the totals summed. The inactive X was defined on the basis of possession of a higher total fluorescence yield. In a series of 36 cells all but five clearly exhibited a smaller cross-sectional area for the inactive X as well. In this set, the ratio of total brightness of the inactive to the active chromosome was found to be 2.03 ± 0.85 (SD). Moreover, the overwhelming majority of the DNA in the inactive X is concentrated in the compact central domain, whereas, in the active X, the DNA is diffused over a wide region with dark areas interrupting the bright patches as shown in Figures 1 and 2. This behavior is interpreted to mean that most of the inactive X chromosome is sequestered and that the small number of active genes that are in the inactive X are not all in one position but form islands separated by inactive regions. Similarly, in these cells the conclusion could be drawn that the active X chromosome in these fibroblastic cells has its active genes widely distributed throughout most of the chromosomal length, leading to a highly punctate pattern. In contrast to this behavior of interphase cells, the two X chromosomes of human female mitotic cells exhibited a fluorescence intensity ratio of the brighter to the dimmer chromosomes of 1.07 ± 0.045 (SD).

Comparison of X with Chromosome 21
Both X chromosomes possess equal amounts of DNA and therefore, would be expected to bind equal amounts of the X painting probe in the FISH procedure and thus emit equal amounts of fluorescent light. Sequestered or condensed DNA would be more densely distributed than the exposed form which might well be constituted of single loops. Therefore, the inactive X should emit a higher intensity of recorded light over a smaller area than the active X. Our fluorescence monitoring system has limited sensitivity of 256 levels of gray. Therefore, by setting it to register in the range of the highest light intensity emitted from the inactive X, the less intense light from the exposed DNA should fall in or below background and fail to register. Therefore, the gain on the MRC 500 was set to register 255 at the brightest region of the inactive X. With such a setting, the expectation was that the total light registering from the active X would be less than that from the Barr body. While the numbers so achieved might constitute a virtual rather than an absolute reading, they afford a means for detecting exposed DNA, and could be converted to absolute differences in studies with more sophisticated equipment with a much greater gray scale capacity. If these considerations are valid, one would expect to find a different response from the two members of chromosome 21 than from the X's, since the 21 chromosomes do not exhibit differential inactivation.

A series of experiments was conducted comparing the behavior of the X chromosome with chromosome 21, an autosome which from genetic considerations would be presumed not to display a phenomenon comparable to X inactivation. The behavior of chromosome 21 in cultured human fibroblasts demonstrated marked differences from X in positioning, in differential condensation and in total fluorescent light emission, which we interpret to reflect differences in the action of their genetic regulatory mechanism.

A representative photograph of the most common pattern found when chromosome 21 painting probe was hybridized to normal human fibroblasts is shown in Figure 3, and a comparison of the different patterns obtained from hybridization of X and 21 painting probes with normal human cells phased in G₁ is shown in Table 3. These data reveal a clearly different pattern for 21 from that of the X chromosome, even visible by epifluorescence microscopy. In no case was an almost completely diffused chromosome 21 evident: both members of the 21 pair exhibiting both diffuse and condensed areas. Virtually no cells were found with neither chromosome touching a nucleolus, and the great majority of the cells had both chromosomes in the interior of the nucleus and touching a nucleolus.

View larger version (69K): [in this window] [in a new window]   Figure 3. A cultured G1-phased normal human fibroblast treated with chromosome 21 painting probe in the FISH procedure and photographed by epifluorescence microscopy. The nucleoli have been stained red by means of nucleolus antibody fluorescent staining. Chromosome 21 is colored green and the background nucleus is dark blue due to DAPI staining. This arrangement with both 21 chromosomes adjacent to nucleoli, and both exhibiting condensed and decondensed regions, is the most common pattern. Occasional cells have one 21 chromosome in the nuclear periphery.

	Discussion

Top Abstract Introduction Materials and Methods Experimental Results Discussion Acknowledgements References

 
The present data reveal marked differences in condensation and nuclear positioning patterns between the X and chromosome 21 in cultured human fibroblasts. Most of the X chromosomes have most of their mass in or close to the nuclear periphery. One X chromosome is highly condensed whereas the other is diffuse. Both chromosomes are surrounded by punctate dots. Signals may be picked up as isolated dots as far away as approximately one-tenth of the mean nuclear diameter from the main body of the X chromosome. Finally, the total fluorescent light emitted by the condensed or largely inactive X is in the neighborhood of twice the amount of light emitted by the active X.

In contrast, chromosome 21 displays a large amount of condensation in both members. Both members are usually associated with nucleoli, although peripheral locations are also occasionally seen. The total fluorescent light emitted by the two 21 chromosomes is much more nearly equal.

The genome exposure theory presumes that differentiation is accomplished by gene regulation at two separate levels. The first involves exposure of a set of genes which is specific for each differentiation state and which involves bringing such genes into a condition where they can readily react with transcription factors and other molecules in the liquid phase. Genes not involved in the differentiation of a given cell are kept sequestered in a state which prevents interaction with molecules of the liquid phase. The second step of genome regulation involves activation of the genes in the exposed state by reaction with appropriate messenger molecules in the medium. Activated DNA forms RNA and, like all DNA, must be replicated in S. It may also perform other functions. Housekeeping genes like those required for ribosome formation are presumed to be continuously exposed. We have assumed that at least some of the differentiation exposed genes are located in the nuclear periphery and some of the housekeeping genes are located in the vicinity of the nucleolus. Gene sequestration is accomplished by high orders of supercoiling of the appropriate DNA, and protein interactions with the structures are formed so as to greatly decrease the accessibility of such DNA to molecules in the liquid phase. It is conceivable that each developmental pathway, starting from a stem cell and ending in a terminally differentiated cell, has a separate sequence of genome exposure reactions.

The behavior of the X chromosome fits this model. In the female cell, both X chromosomes are presumably anchored in the nuclear periphery by their active exposed genes. The inactive X presents a highly concentrated mass, whereas the active X forms a diffuse body with many interruptions. In the technique used here, fluorescence from exposed DNA is at background or below and so is invisible when the instrument is set so as to measure the intense fluorescence from the inactive, sequestered (or condensed) DNA. In both X chromosomes, the more extensive, highly fluorescent deposits are surrounded by punctate regions of emission which we interpret as representing small areas of sequestered genes separated from the main condensed mass by stretches of exposed, i.e., active, DNA.

The two members of chromosome 21 display a very different pattern. The overwhelming majority of these cells shows at least one chromosome associated with a nucleolus and the majority of these cells have two such chromosomes. There is no difference in their degree of condensation that is detectable by our experimental system. Some punctate emission regions are evident in the vicinity of both chromosomes, but less than those surrounding the X chromosomes.

The current results appear to be consistent with the theory of genome exposure developed on the basis of experiments using the differential sensitivity of particular genome fractions to hydrolysis by DNase I. Thus, it may be possible to use FISH probes to study genome exposure by an independent set of operations. Both methodologies require further refinement. However, by combining the two technologies, it becomes possible to measure in the same cell type the properties of exposed DNA by the DNase I method and sequestered DNA by the FISH procedure, as illustrated here. Data of the kind presented here need to be expanded to encompass the other human chromosomes, to test cells from biopsy as well as those from tissue culture, to compare exposures of different chromosomes in various differentiated tissues, to compare changes in genome exposure which may accompany growth in vitro, and to compare normal cells with cancer cells, in some of which we have already shown exposure differences [1-11]. The XIST gene which has been shown to control chromosome condensation, and therefore inactivation, may well be one of a family of genes operating widely throughout the genome, though most extensively in the X chromosome. Such genes may be involved in complex genetic syndromes [17-19]. The action of various hormones, other messenger molecules and a wide variety of toxins may yield important information when studied by means of the genome exposure approach. Finally, it would seem important to study genome exposure in virus-infected cells. Studies with different sized probes ranging from whole chromosome painting probes to single gene probes are envisaged in this program. These studies are continuing.

	Acknowledgements

Top Abstract Introduction Materials and Methods Experimental Results Discussion Acknowledgements References

 
This work was supported by the Department of Energy, Office of Health and Environmental Research, Contract ENG #0928M0013-9C from the Los Alamos National Laboratory, Cell Cycle Checkpoints and Genomic Stability program, E. Morton Bradbury, principal investigator. Also aided by grants from the Raphael Levy Memorial Foundation, the Walter Orr Roberts Memorial Fund and the Charles Brannan Memorial Fund.

	Footnotes

 
Provisionally accepted June 19, 1996.

	References

Top Abstract Introduction Materials and Methods Experimental Results Discussion Acknowledgements References

 

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