Spatial and temporal distribution of DNA replication sites localized by

Spatial and temporal distribution of DNA replication sites localized by immunofluorescence and confocal microscopy in mouse fibroblasts

M. H. FOX*, D. J. AKNDT-JOVINt, T. M. JOVIN, P. H. BAUMANN and M. R0BERT-N1C0UD Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Postfach 2841, D-3400 Gottingen, FRG •Permanent address: Department of Radiology and Radiation Biology, Colorado State University, Ft Collins, CO 80523, USA t Author for correspondence

Summary The temporal course of replication monitored by 2or 5-min pulses of bromodeoxyuridine (BrdUrd) incorporation in synchronized 3T3 cells was mapped by high-resolution light microscopy employing a charge-coupled device (CCD) camera and a confocal laser scanning microscope (CLSM). The cells were labeled simultaneously with monoclonal antibodies directed against BrdUrd and nuclear lamin, and stained with the A+T-specific dye 4',6-diamidino-2phenylindole (DAPI). Stereoscopic reconstructions of cells showing both the lamin and BrdUrd distributions demonstrate that DNA replication occurs at discrete sites in the nucleus, the locations of which progress through a programmed sequence during S phase. Replication begins in a small number of sites

in the interior of the nucleus exclusive of the nuclear membrane and proceeds rapidly in early S phase to encompass a relatively large number of small, discrete sites located throughout the nucleus, with the exception of the condensed heterochromatic regions. Replication is primarily confined to the condensed heterochromatic regions in mid-to-late S phase, and to the nuclear periphery at the end of S phase. These distinctive patterns demonstrate a programmed control of replication sites in the spatial domain in differentiated cell nuclei.

Introduction

also seems to be reflected in a bimodal distribution of the rate of DNA synthesis (Klevecz et al. 1975; Holmquist et al. 1982). Studies of DNA replication in eukaryotic organisms using autoradiography and electron microscopy suggested that replication occurs primarily at the nuclear periphery in early S phase cells (Comings and Kakefuda, 1968). Other investigations have presented conflicting evidence showing replication in early S phase distributed throughout the nucleus and perhaps bound to the nuclear matrix (Pardoll et al. 1980; Jackson and Cook, 1986), followed by sites closer to the nuclear periphery in late S phase (Williams and Ockey, 1970; Huberman et al. 1973). The earlier studies were plagued with difficulties related to poor conservation of nuclear integrity, an inherent problem of the desiccation required for electron microscope studies and radioautography. These difficulties were eliminated by the development of monoclonal antibodies for immunofluorescence detection of bromodeoxyuridine (BrdUrd) incorporated into DNA as an analog of thymidine (Gratzner, 1982). Several distinct patterns of DNA replication have been discerned after short pulses of BrdUrd incorporation (Nakamura et al. 1986; Nakayasu and Berezney, 1989; van Dierendonck et al. 1989). These patterns have also been correlated with the distribution of proliferating cell nuclear antigen, PCNA (an auxiliary protein of DNA polymerase 8), throughout S phase (Bravo and Macdonald-Bravo, 1987). However, these studies provide information based only on two-dimensional images of the cell, making it difficult to

DNA replication in eukaryotic organisms begins with the initiation of replication units followed by bidirectional elongation of replication bubbles (Hand, 1978; Stillman, 1989), albeit in a biased direction in actively transcribed genes (Linskins and Hubermann, 1988). New replication units are activated throughout S phase, apparently in a strict temporal pattern, leading to characteristic replication of specific parts of chromosomes at different times in S phase (Latt, 1973; Balazs et al. 1974; Allison et al. 1985). Previous observations correlating light Giemsa chromosome bands with early replication and dark bands with late replication (Ganner and Evans, 1971; Holmquist et al. 1982) are consistent with the fact that dark Giemsa bands include constitutive heterochromatin and suggest that G+C-rich euchromatin preferentially replicates early. The genes that have been investigated are replicated at specific times during S phase, depending upon the cell type and expression state of the gene. Most transcribed genes replicate early, whereas the majority of the inert or facultatively inert DNA replicates late, although multigene families and housekeeping genes deviate from this generalization (Goldman et al. 1984; Adolph and Hameister, 1985; Hatton et al. 1988; Dhar et al. 1989; Taljanidisz et al. 1989). Interspersed repetitive sequence families replicate as a block either early or late, whereas long interspersed repeats are exclusively late in replicating (Holmquist and Caston, 1986). The finding that particular types of chromatin replicate at specific times in S phase Journal of Cell Science 99, 247-253 (1991) Printed in Great Britain © The Company of Biologiata Limited 1991

Key words: bromodeoxyuridine, nuclear lamin, 3T3 cells, CLSM/CCD.

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localize replication sites accurately with respect to structures in the interior or at the periphery of the cell nucleus. Both the two-dimensional studies mentioned above and the body of information that has been acquired by biochemical fractionation of replicating DNA suggest that not only the initiation of replication in specific sites (Linskens and Huberman, 1988) but its termination and timing are strictly controlled in differentiated eukaryotic cells (Hatton et al. 1988). Exactly what kind of spatial regulation may be associated with the temporal regulation has not yet been investigated in detail. A number of technological advances in optical microscopy provide a basis for quantitation with high spatial resolution of very low fluorescence in cytological preparations. These include high-resolution quantitative digital imaging microscopy using charge-coupled device (CCD) cameras and three-dimensional imaging using confocal laser scanning microscopy (CLSM). Special features of these technologies have been reviewed (Jovin and Arndt-Jovin, 1989), and their specific use in studies of DNA structure and function documented (Arndt-Jovin and Jovin, 1989; Arndt-Jovin et al. 1990). Another group has attempted to look at replication complexes using CLSM (Mills et al. 1989). Their ire vitro system of sperm nuclei induced to replicate in a very fast burst by extracts from oocytes cannot be compared to normal ire vivo replication of differentiated mammalian cells. Additionally, the temporal and spatial resolution of such an ire vitro system is inadequate to address the questions that we have posed in this study. The aim of this investigation was the high-resolution three-dimensional mapping of the progression of replication sites throughout S phase in mouse fibroblast cells. We used two different monoclonal antibody classes to label simultaneously the BrdUrd incorporated during 5 or 10 min pulses and the nuclear lamin, in order to relate the location of replication sites to the nuclear periphery. We employed CCD camera-based microscopy for quantitative analysis of DNA content and immunofluorescencedetected BrdUrd incorporation and the CLSM for threedimensional imaging of the nucleus and quantitative estimates of replication complexes during replication throughout the S phase.

Materials and methods Cell culture and synchrony procedures Mouse 3T3 fibroblasts were routinely grown in Dulbecco's MEM supplemented with 10% fetal bovine serum (FBS) (Biochrom, Berlin) in a 37°C humidified incubator with 5% CO2 for pH control. Synchronized cells were prepared by two methods. (1) Serum starvation: log-phase cells were plated into Petri dishes containing 2.5 cm glass coverslips and allowed to attach for 16-20 h. They were then put into low-serum medium (0.1 % FBS), incubated for 48 h, and released into normal medium. No cells could be detected in S phase during starvation and the cells entered S phase in a semi-synchronous wave at 15-17 h after release. (2) Aphidicolin blocking: serum starved, semi-synchronous cell populations were released into normal medium containing 3 /igml" 1 aphidicolin, a drug that inhibits DNA polymerase a (Ikegami et al. 1978). Cells were incubated in this medium for 24 h to allow a maximal number of them to reach the Gi/S border. They were then washed three times with warm complete medium and incubated at 37 °C to allow synchronous progression through S phase. This latter procedure gives much more highly synchronized S phase populations (Fox et al. 1987; Tobey et al. 1988). 248

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Bromodeoxyuridine labeling and fixation At various times after synchronization, cells were labeled with BrdUrd for 2 or 5 min by replacing the medium with pre-wanned medium containing 40 /90% of the replication is restricted to the nuclear membrane at this very late stage in S phase as determined by the radial intensity distribution (Fig. 6C).

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Fig. 6. Plots of the average pixel intensity for BrdUrd and lamin labeling in concentric areal rings. The data correspond to the images seen in Fig. 5 and are derived from the central optical section of each original CLSM series. The number of pixels in each ring are indicated on the x axis. ( • •) BrdUrd intensities, left y axis; ( • • ) lamin intensities, right y axis.

matic regions, forming spiral structures or chains, with replication being reduced in other parts of the nucleus (Fig. 3C). The somewhat later pattern (Fig. 3D) shows high-intensity staining in the heterochromatic regions with individual replication sites no longer resolvable. The

Both synchronized and non-synchronized cell populations were used to characterize the temporal and spatial distributions of replication sites in S phase. Synchronization procedures are essential for achieving a large cohort of cells in a specific part of S phase. The synchronization should perturb the normal progression as little as possible. Serum starvation has been shown to result in viable cells blocked in Go, which subsequently enter S phase in a semi-synchronous wave (Tobey et al. 1988). The use of aphidicolin, an inhibitor of DNA polymerase a, after serum addition results in the cells being poised at the beginning of S phase. This results in highly synchronized cell populations throughout S phase, but may lead to an overestimation of the number of replication sites found in early S phase. We found that the general sequence of replication patterns observed with both synchronization procedures was the same. Highresolution CCD cameras can be used for quantitative DNA image analysis of cells (Arndt-Jovin and Jovin, 1989). Using this technique, we were able to sort the replication images seen in log phase cells on the basis of nuclear DNA content. A very good corroboration of the sequence of replication patterns found in the synchronized cells is found by this method, showing conclusively that the synchrony procedures do not artificially create new patterns. Thus, we are confident that the data presented here are valid examples of replication patterns in nonperturbed cells and that the temporal sequence presented is real. In the last 10 years the strict temporal control of replication in normal eukaryotic cells has been documented by a number of biochemical approaches and a large body of these data was summarized by Laskey and Temporal distribution of DNA replication sites

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co-workers (1989) recently. One of the most intriguing problems in temporal replication control is the correlation of replication with gene expression in differentiated cells (Hatton et al. 1988; Dhar et al. 1989). One model for selective replication timing suggests that the higher-order chromatin structure is an important factor in directing the replication complexes (Dhar et al. 1989). However, no attempt has heretofore been made to characterize the spatial and temporal distribution of replication complexes at high resolution. The two-dimensional replication patterns described in this paper are in general quite similar to those presented by van Dierendonck et al. (1989) and Bravo and Macdonald-Bravo (1987), who also used synchronized cell populations. This work extends these studies to describe the qualitative and quantitative three-dimensional distribution of replication sites throughout S phase in relation to the nuclear periphery. The use of nuclease treatment to expose the incorporated BrdUrd to antibody permitted very short labeling times and the simultaneous visualization of lamin. Both the CCD and CLSM used in this study have a linear response to fluorescence intensity as well as high spatial (picture element) resolution and geometric stability. Thus, we have been able to assess quantitatively the distribution of BrdUrd incorporation throughout the nucleus and to relate this at high resolution to the nuclear periphery (Figs 4-6). Attempts by ourselves and others to use AT-specific DNA dyes (e.g. DAPI) to define the nuclear boundary were found to be inadequate because of the denaturation of AT sequences required for antibody binding. The stereoscopic images (Fig. 3) and isometric projections (Fig- 5) generated from the CLSM data permit a direct perception of the three-dimensional organization of replication sites in the nucleus throughout S phase, as well as a quantitative assessment of the distribution of fluorescence intensities. The analysis of the radial distribution of the fluorescence expressed as the average pixel intensity in concentric rings throughout individual optical sections provides a quantitative description of the spatial distribution that can ultimately be related directly to a volume distribution (Fig. 6) of fluorescence. Our results, showing that early DNA replication occurs at distinct sites throughout the nucleus rather than at the nuclear periphery as reported by Comings and Kakefuda (1968), are consistent with those from a number of other studies (Nakamura et al. 1986; Nakayasu and Berezney, 1989; van Dierendonck et al. 1989; Williams and Ockey, 1970; Huberman et al. 1973). In fact, cells in very early S phase probably have very little replication occurring at the nuclear membrane (Fig. 3A and B). The DNA staining also demonstrates that condensed heterochromatin is not replicated during this early stage (Fig. 2A). Extended arrays of replication sites are frequently observed (Fig. 3A-C), which probably represent the coordinated synthesis of groups of replication units along the chromosomes (Hand, 1978; Umek et al. 1989). A number of investigators have provided evidence that early replication occurs in association with the nuclear matrix (Berezney and Buchholtz, 1981; Fernandes et al. 1988; Jackson and Cook, 1986; Pardoll et al. 1980), although the high-salt, detergent treatment used to isolate the nuclear matrix in some of these studies raises questions as to its existence in living cells. Our results do not contradict a possible association between the matrix and the replication complex that could be distributed throughout the interphase nucleus. However, the results indicate that the active replication complexes are not statically fixed in 252

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space for the duration of S phase contrary to the suggestion of Mills and co-workers (1989). In order to quantitate and visualize better spatially correlated replication units we developed techniques to enumerate the resolvable replicons in three dimensions as well as a new labeling procedure to represent contiguous units in adjacent sections (Baumann etal., unpublished data). In early S phase at least 250 separate replication sites can be distinguished at our optical resolution. Although the number of replicating units clearly increases as S phase progresses (a finding we derive from the intensity of BrdUrd fluorescence labeling; Fig. 1), these sites become more coordinated in space as indicated by progressively longer correlated chains and fewer individual sites. After mid S phase the sites condense further to less than 80, of which as few as 15 contain greater than 80% of the replication complexes and reside in the condensed heterochromatin (Fig. 3D). Late in S phase replication in the highly condensed heterochromatin subsides, leaving sites in the less-condensed chromatin and regions adjacent to the nuclear membrane (Figs 3E, 4C). We clearly demonstrate that at the end of S phase replication is restricted to sites on the nuclear periphery. Within the limits of optical resolution, BrdUrd sites colocalized with lamin label (see Figs 3F, 4D and 5C). The obviously discrete nature of the BrdUrd labeling along the nuclear membrane in late S phase may in part represent the replication of telomeres, which are thought to be attached to the nuclear membrane (Hochstrasser et al. 1986; Moyzis, 1990; Bartholdi, 1990), and are the sites where replication ceases in polytene chromosomes (Allison et al. 1985). We interpret the rings and spirals of BrdUrd sites in mid-S phase to be the result of replication occurring around the condensed heterochromatin regions (see Figs 4C and 5C), which suggests a spiralization of the heterochromatin as a means of packing. This is contrary to the interpretation of Nakamura et al. (1986), who argued that these structures are seen only after very long BrdUrd incorporation and are the result of migration of newly replicated DNA from replication centers. The rate of DNA polymerase movement is not constant throughout S phase (Housman and Hubermann, 1975) and recent evidence suggests that some polymerase complexes pause and wait for the opposing polymerase complex to reach the same point (Umek et al. 1989). However, the large differences in thymidine incorporation measured across S phase (Klevecz et al. 1975; Holmquist et al. 1982) and our observations of 15-fold differences in BrdUrd fluorescence intensities of log phase cells (Fig. 1) are most likely to be the result of differences in the number of replicating complexes at various times during S phase. Such a conclusion is borne out by the number of small replication centers resolvable in very early (fewer than 30) and early S phase (greater than 250). Clearly, the data presented here document a tightly controlled progression of spatially distinct patterns of replication sites across S phase. The factors that regulate the initiation of specific replicons in these patterns are unknown but these data argue against a unique set of fixed replication sites for the duration of S phase and spooling of the DNA through these sites (Mills et al. 1989). Recent biochemical evidence (Umek et al. 1989) shows that only selected ARS or Ori sequences are the sites of initiation of replication in vivo. The techniques described in this paper could be extended to the direct analysis of

positional and temporal changes in Ori or other unique DNA sequences using a combination of the BrdUrd labeling and additional appropriate in situ probes. In summary, the distributions of DNA replication sites we have reported show that replication is tightly controlled in time and space in the cell nucleus. The factors that regulate these patterns are still unknown. Only through characterization of these factors and their possible pleiotropic roles in the nucleus can we unravel the relationship between chromatin structure and function. One of us (M.H.F.) was partially supported by U.S. Public Health Service Grant no. CA25636. The work was supported partially by grants from the Deutsche Forschungsgemeinschaft (Jo 105/3,105/5). We thank A. von Bogen and B. Staehr for their expert technical assistance.

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(Received 29 November 1990 - Accepted, in revised form, 28 February 1991)

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