During development of the vertebrate organism, a majority of cells eventually exit the cell cycle early in G1 phase and enter an “out-of-cycle” or quiescent state often referred to as G0 (Pardee, 1989 ). Exit from the cell cycle is reversible in certain cell types; however, in others, such as terminally differentiated frog and avian erythrocytes, it is not (Leonard et al., 1982 ). The mechanisms underlying this loss of proliferative capacity have not been clearly defined; however, it seems likely that many of the changes that accompany differentiation in these cells collectively contribute to this irreversible arrest. In the adult animal, major transitions in chromatin composition and structure occur during erythrocyte differentiation, and these changes have been implicated in the generation and/or maintenance of the quiescent state. In the frog, for example, histone H1⁰ gradually accumulates during differentiation, leading to a high content of linker histones on the chromatin (Allan et al., 1981 ; Dimitrov and Wolffe, 1996 ), and it has been suggested that this high linker histone content promotes the hypercondensation and inactivation of erythrocyte chromatin (Thomas and Maclean, 1975 ; Allan et al., 1981 ; Wolffe, 1989 ). Indeed, this idea is supported by studies that demonstrate that the overexpression of chicken histone H5 (H1⁰) in proliferative somatic cells results in chromatin compaction and the inhibition of DNA replication and transcription (Sun et al., 1989 ; Aubert et al., 1991 ). Importantly, the overall level of H1 in these overexpressing cells was similar to that observed in terminally differentiated erythrocytes.

Although the proliferative arrest of nucleate Xenopus laevis erythrocytes is irreversible in vivo, reactivation of DNA replication and transcription does occur when isolated erythrocyte nuclei are introduced into an activating environment such as enucleated Xenopus eggs (for review, see Gurdon, 1986 ). These reactivated nuclei resemble embryonic nuclei both structurally and functionally, becoming pluripotent for frog development (Gurdon and Uehlinger, 1966 ; Brun, 1978 ). The reactivation of mature erythrocyte nuclei has been recapitulated in vitro using Xenopus egg extracts (Coppock et al., 1989 ; Wolffe, 1989 ), and in this system, quiescent nuclei decondense (Leno and Laskey, 1991 ; Blank et al., 1992 ; Leno and Munshi, 1997 ), regain transcriptional competence (Wolffe, 1989 ; Dimitrov and Wolffe, 1996 ), and initiate DNA replication (Coppock et al., 1989 ; Leno and Laskey, 1991 ; Blank et al., 1992 ; Wangh et al., 1995 ; Leno and Munshi, 1997 ).

A general requirement for initiation of cellular DNA replication in eukaryotes is the coordinated assembly of a pre-replication complex (pre-RC) at origin sites. In Xenopus egg extracts, pre-RC assembly on sperm chromatin and even purified DNA occurs before nuclear envelope assembly and involves the sequential binding of origin recognition complex (ORC) proteins, Cdc6, and minichromosome maintenance (MCM) proteins to DNA (see review by Romanowski and Madine, 1996 , 1997 ; Walter et al., 1998 ). S-phase–promoting factors, such as cdk2/cyclin E, subsequently accumulate within the intact nucleus and are thought to trigger initiation at sites where pre-RCs are assembled (Jackson et al., 1995 ; Hua et al., 1997 ; Walter et al., 1998 ). Initiation of replication is accompanied by the loss of Cdc6 (Coleman et al., 1996 ; Hua and Newport, 1998 ) and MCM proteins from chromatin (Chong et al., 1995 ; Kubota et al., 1995 ; Madine et al., 1995a ,b ; Romanowski et al., 1996a ), whereas ORC remains bound to DNA until chromosome condensation at mitosis (Carpenter et al., 1996 ; Romanowski et al., 1996b ; Rowles et al., 1996 ). It appears that reinitiation of replication is blocked during S and G2 phases by the nuclear envelope, which prevents the reassociation of MCM proteins with chromatin until passage through mitosis and entry into the next cell cycle (Madine et al., 1995b ).

The molecular mechanisms regulating the reactivation of replication in quiescent nuclei have not been determined. However, several observations indicate that nuclear envelope integrity plays an important role. Indeed, nuclei isolated from contact-inhibited cultured cells (Leno and Munshi, 1994 ; Fang and Benbow, 1996 ), and avian erythrocytes (Leno and Munshi, 1997 ) require nuclear envelope permeabilization for initiation in egg extract. Although the molecular basis for this requirement is unknown, the recent demonstration that restoration of replication competence in nuclei from quiescent fibroblasts requires an activity from egg extract (Munshi and Leno, 1998 ) coupled with the observation that the level of Mcm3, an essential member of the pre-RC, is reduced in quiescent cells both in vivo (Musahl et al., 1998 ) and in vitro (Stoeber et al., 1998 ) raises the interesting possibility that permeabilization of quiescent nuclei may be required for the reassembly of pre-RCs on chromatin, much like it is in G2-phase nuclei (Madine et al., 1995b ). Thus, nuclear envelope integrity could also play a role in regulating initiation within quiescent cell nuclei by modulating pre-RC assembly on chromatin.

Accompanying the reactivation of replication in erythrocyte nuclei by egg extract is the replacement of somatic H1 histones with the embryonic linker histone B4 and HMG1, another chromosomal protein found in early embryonic chromatin (Blank et al., 1992 ; Dimitrov and Wolffe, 1996 ). These transitions in chromatin composition are mediated by the molecular chaperone nucleoplasmin (NPL) and play an essential role in the reacquisition of transcriptional competence in these nuclei (Dimitrov and Wolffe, 1996 ). What role, if any, these changes in chromatin structure play in the reactivation of DNA replication is not clear. However, recent work showing that the assembly of somatic H1 on embryonic chromatin reduces the frequency of initiation in egg extract by limiting pre-RC assembly (Lu et al., 1998 ) raises the interesting possibility that efficient initiation also requires the removal of H1 from erythrocyte chromatin.

In this report we have used Xenopus egg extract to investigate the mechanisms regulating the reactivation of replication in nuclei from terminally differentiated Xenopus erythrocytes. We find that these nuclei lack essential components of the pre-RC, including XORC, XCdc6, and XMCM proteins. Pre-RC proteins from the extract form a stable association with the chromatin of permeable nuclei, which replicate in this system, but not with the chromatin of intact nuclei, which do not replicate, even though these proteins are able to cross an intact nuclear envelope. Thus, an intact nuclear envelope prevents initiation in quiescent nuclei, at least in part, by preventing the assembly of pre-RCs on chromatin. Erythrocyte nuclei contain histone H1 and H1⁰ that are removed from the chromatin by the molecular chaperone NPL during reactivation in the extract. Immunodepletion of NPL from the extract prevents the removal of H1 from chromatin, limits pre-RC assembly, and reduces the frequency of initiation within permeable nuclei, all of which are restored by readdition of NPL to the depleted extract. Furthermore, restoring the overall H1 content on erythrocyte chromatin in control (NPL-containing) extract inhibits replication to the same extent as that observed in NPL-depleted extract. Thus, a high level of somatic H1 on erythrocyte chromatin, whether the result of NPL depletion or the addition of exogenous H1 to NPL-containing extract, inhibits replication to a similar extent. Moreover, intact G1-phase tissue culture nuclei, which contain fully assembled pre-RCs, replicate to very similar levels in mock-depleted and NPL-depleted extracts, suggesting that once pre-RC assembly is complete, removal of H1 may no longer be required for replication in the extract. Taken together, these data indicate that loss of nuclear envelope integrity and the removal of somatic linker H1 from erythrocyte chromatin are required for the acquisition of essential pre-RC proteins from the extract and the reactivation of DNA replication in this system.

Lu et al., 1997

For permeabilization of cells with streptolysin-O (SLO), freshly isolated erythrocytes were resuspended in 1,4-piperazinediethanesulfonic acid (PIPES) buffer (50 mM KCl, 2 mM EGTA, 5 mM MgCl₂, 1 mM DTT, 1 μg/ml leupeptin, pepstatin, and aprotinin, 50 mM PIPES-KOH, pH 7.0) to a final concentration of 4 × 10⁵ cells/ml. An equal volume of PIPES buffer containing 1.6 IU/ml SLO (Murex Diagnostics, Norcross, GA) was added, and the cells were incubated on ice for 10 min with gentle inversion of the tubes every minute. The cells were then sedimented by centrifugation as described above, rinsed twice in PIPES buffer to remove unbound SLO, and finally resuspended at room temperature in PIPES buffer. The permeable cells (intact nuclei) were counted in a hemacytometer.

Permeable nuclei were prepared using lysophosphatidylcholine (LPC). Sedimented erythrocytes were resuspended in 5 ml of buffer P (60 mM KCl, 15 mM NaCl, 340 mM sucrose, 15 mM β-mercaptoethanol, 0.5 mM spermidine, 0.15 mM spermine, 2.5 μg/ml leupeptin, pepstatin, and aprotinin, 15 mM HEPES-KOH, pH 7.5). An equal volume of buffer P containing 2 mg/ml LPC (Sigma, St. Louis, MO) was added, and the cells were incubated for 20 min at room temperature with gentle inversion of the tubes every minute. Permeabilization was stopped by adding 5 ml of ice-cold buffer P containing 3% BSA, and the resultant permeable nuclei were sedimented by centrifugation at 2750 rpm for 10 min at 0°C in a Jouan CR4–22 centrifuge. The nuclei were then rinsed in buffer P, sedimented, resuspended, and counted in a hemacytometer. Both intact and permeable nuclei were diluted with buffer to a final concentration equivalent to 1 μg of DNA/μl, assuming a DNA mass of 6.3 pg per diploid nucleus (Dawid, 1965 ). The permeability of plasma and nuclear membranes was determined by incubating an aliquot of SLO- or LPC-treated cells with affinity-purified TRITC-labeled immunoglobulin G (IgG) for 5 min. The percentage of nuclei excluding the labeled IgG was determined by fluorescence microscopy (Leno and Munshi, 1994 ).

Munshi and Leno, 1998

Cox and Leno, 1990

Lu et al., 1997

Lu et al., 1997

1998

Lu et al., 1998

Lu et al., 1998

To determine the levels of NPL in egg extract by Western blotting, 1 μl of extract was diluted with 4 μl of extraction buffer (50 mM HEPES-KOH, pH 7.6, 50 mM KCl, 5 mM MgCl₂, 2 mM β-mercaptoethanol) and then with 5 μl of 2× SDS-PAGE sample buffer (125 mM Tris-HCl, pH 6.8, 10% SDS, 50% glycerol, 100 mM β-mercaptoethanol). Samples were run on 15% polyacrylamide gels and blotted onto nitrocellulose membranes. Membranes were processed as described above, except that PA3C5 hybridoma culture supernatant, containing 10% dried milk, was used directly as the source of primary antibody.

Dingwall et al. (1982)

Pace et al., 1995

Dilworth et al., 1987

Philpott et al., 1991

Harlow and Lane, 1988

Philpott and Leno, 1992

Chong et al., 1997

Philpott and Leno, 1992

Lu et al., 1997

Lu et al., 1998

Leno and Munshi, 1994

Fang and Benbow, 1996

Munshi and Leno, 1998

Leno and Munshi, 1997

Madine et al., 1995a

b

Romanowski et al., 1996a

b

Lu et al., 1998

Nuclei were obtained by treating Xenopus erythrocytes with the bacterial exotoxin SLO or with LPC, and nuclear envelope integrity was determined by incubating treated cells with TRITC-labeled IgG (Leno et al., 1992 ; Leno and Munshi, 1997 ). IgG is excluded from nuclei with intact nuclear envelopes but not from nuclei with detergent-damaged (permeable) envelopes. We consistently found that >90% of SLO-prepared nuclei excluded IgG (Figure 1A, SLO), whereas >99% of the LPC-prepared nuclei did not (Figure 1A, LPC). To determine the replication competence of SLO (intact) and LPC (permeable) nuclei, we incubated each at 3 ng of DNA/μl of extract supplemented with [α-³²P]dATP (Figure 1B) or biotinylated dUTP (Figure 1, C and D) for various periods as described in MATERIALS AND METHODS. As expected, permeable nuclei replicated to a much greater extent than intact nuclei in our time course experiments (Figure 1B). Furthermore, the limited replication within the intact sample was restricted to very few nuclei (<10%; Figure 1, C, INTACT, and D, Int), whereas virtually all permeable nuclei initiated replication under identical conditions (Figure 1, C, PERMEABLE and D, Perm). Density substitution experiments confirmed that incorporation of label was the result of a single round of semiconservative DNA replication (our unpublished observation). Thus, permeabilization of the nuclear envelope is required for replication of Xenopus erythrocyte nuclei by egg extract.

Figure 1 Permeabilization of the nuclear envelope is required for replication of Xenopus erythrocyte nuclei by egg extract. (A) Nuclei were prepared by treating erythrocytes with LPC or SLO, and nuclear envelope integrity was determined by incubating nuclei with (more ...)

We next examined intact and permeable erythrocyte nuclei for the presence of pre-RC proteins both before and after extract incubation. Permeable nuclei were incubated in extract for 45 min, before initiation of replication (our unpublished observation), whereas intact nuclei, which do not initiate replication in the extract (Figure 1, C and D), were incubated for up to 4 h. Nuclei were sedimented onto coverslips, treated with different concentrations of Triton X-100, before or after fixation with paraformaldehyde, and probed with antibodies that recognize the Xenopus pre-RC proteins XOrc2, XCdc6, and XMcm3. The immunofluorescence results are shown in Figure 2A. Pre-RC proteins were undetectable within erythrocyte nuclei in the absence of extract incubation (XEN), irrespective of the method of nuclear isolation. However, after incubation in the extract, all three pre-RC proteins were found to stably associate with the chromatin from permeable nuclei (PERMEABLE, 0.5% T↓FIX) but not with the chromatin from intact nuclei (INTACT, 0.5% T↓FIX), even though all were able to cross an intact nuclear envelope (INTACT, FIX↓0.5% T). Low levels of XOrc2 and XCdc6 were detected within intact nuclei after low-stringency detergent extraction (INTACT, 0.1% T↓FIX) but not after high-stringency extraction (INTACT, 0.5% T↓FIX). By contrast, an increase in stringency had no observable effect on the association of pre-RC proteins with chromatin from permeable nuclei (PERMEABLE, compare 0.1% T↓FIX with 0.5% T↓FIX). The differential association of pre-RC proteins with chromatin from intact and permeable nuclei was confirmed by Western blot (Figure 2B). In this case, nuclei incubated in egg extract (EXT) were treated under low-stringency conditions (0.1% Triton X-100) before analysis by PAGE. All five pre-RC proteins examined (XOrc1, XOrc2, XCdc6, XMcm3, and XMcm7) were stably associated with chromatin from permeable nuclei (PERMEABLE, EXT) but not with chromatin from intact nuclei (INTACT, EXT). Taken together, these data illustrate several important points. First, terminal differentiated erythrocyte nuclei lack essential components of the pre-RC. Second, reactivation of DNA replication by egg extract involves the assembly of pre-RC proteins on erythrocyte chromatin. Third, an intact nuclear envelope prevents initiation in these quiescent nuclei at least in part by preventing the assembly of pre-RCs on chromatin.

Figure 2 An intact nuclear envelope prevents the assembly of pre-RCs on erythrocyte chromatin. (A) Permeable erythrocyte nuclei were incubated in egg extract for 45 min, diluted with buffer, sedimented on coverslips, and treated with either 0.1% or 0.5% (more ...)

Dimitrov and Wolffe, 1996

Lu et al., 1997

1998

Dimitrov and Wolffe, 1996

Figure 3 NPL accumulates within intact and permeable erythrocyte nuclei incubated in egg extract. Intact (INTACT) and permeable (PERMEABLE) erythrocyte nuclei were incubated in egg extract for 2 h (2h) and 4 h (4h) and subsequently isolated, fixed, and labeled (more ...)

Intact and permeable nuclei were incubated in the extract for various times and isolated, and the acid-soluble nuclear proteins were separated by SDS-PAGE and stained with Coomassie blue (Figure 4A). The levels of H1, H1⁰, and B4 proteins were quantitated by densitometry, normalized with the core histones in each sample, and a mean value was derived from three independent experiments in which three different egg extracts were used (Figure 4B). The replacement of H1s with B4 on erythrocyte chromatin within permeable nuclei reached a plateau by 15 min, whereas replacement within intact nuclei required >2 h to reach a plateau. Furthermore, the replacement within intact nuclei was less extensive than that observed within permeable nuclei for each of the linker histones, i.e., ~40 versus ~60% for loss of H1, ~50 versus ~70% for loss of H1⁰, and ~50 versus 100% for acquisition of B4. The amounts of H1 and H1⁰ on unincubated XEN and the amount of B4 assembled on the chromatin of permeable nuclei after 60 min were designated as 100%. Virtually identical protein profiles were obtained when intact or permeable nuclei were treated with detergent after extract incubation but before acid extraction (our unpublished observation), demonstrating the stable association of these linker histones with chromatin. Thus, these data demonstrate that an intact nuclear envelope reduces both the rate and extent of H1 removal and B4 assembly in erythrocyte nuclei by egg extract. The protein observed directly above H1 in the permeable XEN sample (Figure 4A, XEN, PERMEABLE, lane 1) is most likely BSA, which is used to stop membrane permeabilization by LPC. It is important to note, however, that no experimental differences were observed between permeable nuclei with or without this protein.

Figure 4 An intact nuclear envelope reduces the remodeling of erythrocyte chromatin by egg extract. (A) Permeable and intact erythrocyte nuclei were incubated in extract for various times as indicated. Incubated and unincubated (XEN) nuclei were then diluted and (more ...)

Dimitrov and Wolffe, 1996

The monoclonal antibody PA3C5 was used to deplete virtually all of the NPL from our extracts (Figure 5A, compare lane 3 with lanes 1 and 2), consistent with earlier reports (Philpott et al., 1991 ; Philpott and Leno, 1992 ). The Western blot shown in Figure 5A was also probed with anti-XMcm3 to demonstrate equal loading among the samples and to show that at least one pre-RC protein is not codepleted with NPL (Figure 5A, ΔNPL, lane 3). Incubation of permeable erythrocyte nuclei in depleted extract prevented the loss of H1s from chromatin as well as the assembly of B4 (Figure 5B, compare lanes 2 and 3 with lane 4). Addition of purified NPL back to depleted extracts restored the levels of H1s to control and mock depletion levels (Figure 5B, compare lane 5 with lanes 2 and 3). Permeable erythrocyte nuclei were then incubated in undepleted “control” extract (CON), mock-depleted extract (MOC), NPL-depleted extract (ΔNPL), and depleted extract reconstituted with NPL to the physiological concentration (ΔNPL+NPL) and assayed for DNA replication by the incorporation of [α-³²P]dATP (Figure 6A). Shown are the mean values from three separate experiments in which three different extracts were used. In the absence of NPL, replication was reduced nearly 50% relative to the control sample (Figure 6A, compare ΔNPL with CON). Furthermore, replication was restored to control levels in depleted extract reconstituted with NPL (compare ΔNPL+NPL with CON). Replication was not inhibited in mock-depleted extract (MOC). Interestingly, >95% of nuclei initiate replication in NPL-depleted extract as judged by the incorporation of biotin-dUTP into nascent DNA (Figure 6D). However, the intensity of streptavidin fluorescence within these nuclei was markedly reduced relative to nuclei incubated in control extract, mock-depleted extract, and depleted extract reconstituted with NPL, consistent with the data shown in Figure 6A.

Figure 5 NPL mediates the removal of H1s from erythrocyte chromatin in egg extract. (A) NPL-depleted (ΔNPL) and mock-depleted (MOC) extracts were prepared as described in MATERIALS AND METHODS. Purified NPL was added to depleted extract at a final concentration (more ...)

Figure 6 Immunodepletion of NPL from egg extract inhibits initiation but not elongation in erythrocyte nuclei. (A) Permeable erythrocyte nuclei were incubated in control (CON), mock-depleted (MOC), NPL-depleted (ΔNPL), or depleted extract reconstituted (more ...)

The immunodepletion data shown in Figures 5 and 6A demonstrate two important points. First, NPL is required for the removal of somatic H1 from erythrocyte chromatin and for the deposition of the cleavage stage linker histone B4. Second, NPL facilitates the replication of erythrocyte nuclei in egg extract. To investigate whether NPL facilitates replication by removing somatic H1 from chromatin, we added increasing concentrations of purified H1 to NPL-containing (control) extract and assayed the extent of inhibiton of replication at each concentration (Figure 7). We found that addition of H1 to a final concentration of 5.68 μM restored the overall linker histone content on chromatin to the level observed in unincubated erythrocyte nuclei (Figure 7A, compare H1 with XEN). Chromatin-bound H1 levels were determined by densitometry and normalized to the core histones in each sample. Purified somatic H1 (mouse H1c) migrates between Xenopus H1 and H1⁰ in our SDS-PAGE gels. The association of H1 with erythrocyte chromatin inhibited replication in control extract in a dose-dependent manner (Figure 7B). Thus, a high level of somatic H1 on erythrocyte chromatin, whether the result of NPL depletion (Figure 5B) or the addition of exogenous H1 to NPL-containing (control) extract (Figure 7A), results in the inhibition of replication (compare Figure 6A, ΔNPL, with Figure 7B, 5.68 μM H1). Taken together, these data argue that NPL facilitates replication in egg extract by removing somatic H1 from erythrocyte chromatin.

Figure 7 Assembly of somatic H1 on erythrocyte chromatin inhibits replication in extracts containing NPL. (A) Permeable erythrocyte nuclei were incubated (140 ng of DNA/μl of extract) in extract without (CON) or with histone H1c (H1; 5.68 μM) for (more ...)

Krude et al., 1997

Mahbubani et al., 1997

The relative size of nascent DNA strands produced in NPL-depleted extracts was determined by alkaline agarose gel electrophoresis (Figure 6C). Virtually all nascent DNA was found in a high-molecular-weight form (ΔNPL) indistinguishable from that observed in mock-depleted extract (MOC). In contrast, a range of lower-molecular-weight forms was observed when replication forks were arrested with aphidicolin shortly after initiation in mock-depleted extract (MOC+APH). Results essentially identical to those described in Figure 6C were obtained when the concentration of DNA was increased 10-fold, i.e., from 3 to 30 ng/μl of extract, demonstrating that even at high DNA concentration, replication elongation occurs in NPL-depleted extract (our unpublished observations). Taken together, these data demonstrate that elongation does occur in the presence of histone H1 and suggest that the inhibition of replication we observe in NPL-depleted extract is due to a reduction in the frequency of initiation.

The assembly of somatic histone H1 on embryonic chromatin has been shown to reduce the frequency of initiation in egg extract by limiting the assembly of pre-RCs on DNA (Lu et al., 1998 ). To determine whether pre-RC assembly on erythrocyte chromatin is limited in NPL-depleted extract, a Western blot, containing the chromatin-bound proteins from permeable erythrocyte nuclei incubated for 45 min in control (CON), mock-depleted (MOC), NPL-depleted (ΔNPL), and depleted extract reconstituted with NPL (ΔNPL+NPL), was probed with antibodies to several pre-RC proteins (Figure 8A). In each case, the level of the pre-RC protein in the NPL-depleted extract was reduced relative to that observed in control and mock-depleted samples. Protein levels were restored to control levels when NPL was added back to the depleted extract. Thus, NPL facilitates the assembly of pre-RCs on erythrocyte chromatin in egg extract. Given the essential role of pre-RCs in the establishment of replication competence, the data presented here raise the interesting possibility that removal of H1 from erythrocyte chromatin by NPL facilitates pre-RC assembly on DNA, thereby increasing the frequency of initiation and the overall extent of replication in egg extract.

Figure 8 NPL facilitates pre-RC assembly on erythrocyte chromatin in egg extract. Permeable erythrocyte nuclei were incubated in control (CON), mock-depleted (MOC), NPL-depleted (ΔNPL), or depleted extract reconstituted with NPL (ΔNPL+NPL) (more ...)

Alternatively, NPL could facilitate replication of permeable erythrocyte nuclei by some other mechanism, such as a direct effect on replication proteins themselves. Our demonstration that the maintenance of somatic linker histone content on erythrocyte chromatin inhibits replication, even in the presence of NPL, argues against this possibility (Figure 7). Furthermore, intact G1-phase tissue culture nuclei, which contain fully assembled pre-RCs, replicate to similar levels in mock-depleted (MOC) and NPL-depleted (ΔNPL) extracts (Figure 8B), indicating that once pre-RC assembly is complete, NPL’s role in facilitating replication is reduced. Therefore, these results strongly support the notion that NPL increases the frequency of initiation in the extract by promoting pre-RC assembly on chromatin.

We have used Xenopus egg extract to investigate the roles of chromatin structure and nuclear envelope integrity in the reactivation of DNA replication in nuclei from terminally differentiated Xenopus erythrocytes. We find that erythrocyte nuclei lack essential components of the pre-RC, including XOrc1, XOrc2, XCdc6, XMcm3, and XMcm7 (Figure 2). These proteins stably associate with the chromatin of permeable nuclei, which initiate replication in the extract, but not with the chromatin of intact nuclei, which do not initiate under identical conditions. The failure of pre-RC proteins to bind chromatin from intact nuclei is not due to restricted nuclear access, however, because XOrc2, XCdc6, and XMcm3 are all able to cross an intact nuclear envelope (Figure 2). Therefore, given that ORC, Cdc6, and MCM proteins are all essential for DNA replication in eukaryotic cells (reviewed by Stillman, 1996 ), these data argue that an intact nuclear envelope prevents initiation in erythrocyte nuclei at least in part by preventing the assembly of pre-RCs on chromatin.

The absence of XCdc6 and XMcm3 from the chromatin of terminally differentiated erythrocytes is consistent with the observations that these proteins are virtually undetectable in cultured mammalian cells induced to exit the cell cycle by serum deprivation (Williams et al., 1997 ; Musahl et al., 1998 ; Yan et al., 1998 ). Our data are also consistent with studies in yeast that show a dramatic reduction in Mcm3, and the disappearance of the prereplicative footprint from origin DNA, after induction of a G0-like state (Diffley et al., 1994 ; Young and Tye, 1997 ). However, the presence of a postreplicative footprint in G0 yeast cells (Diffley et al., 1994 ) along with relatively high levels of Orc2 in quiescent mammalian cells (Musahl et al., 1998 ; Stoeber et al., 1998 ) indicate that ORC proteins remain associated with chromatin during reversible growth arrest. In contrast, XOrc1 and XOrc2 are undetectable within terminally differentiated erythrocyte nuclei (Figure 2), which, in vivo, are permanently withdrawn from the cell cycle (Leonard et al., 1982 ). Conceivably, the chromatin-bound ORC that remains during reversible arrest could serve to target other pre-RC proteins back to their original sites, thereby ensuring the preservation of origin specificity (Gilbert et al., 1995 ; Lawlis et al., 1996 ; Stoeber et al., 1998 ) and the replication timing program (Fangman and Brewer, 1992 ; Jackson and Pombo, 1998 ) during S-phase reentry. In the absence of cell cycle reentry, however, marking origins of replication would appear to be unnecessary.

The absence of pre-RC proteins from erythrocyte chromatin could be explained in two ways. First, these proteins may fail to assemble on erythrocyte chromatin during the final cell cycle of the differentiation program. This idea is intriguing in light of the fact that terminal differentiation of avian erythroid progenitors proceeds in precise synchrony, suggesting that the decision to enter G0 may be programmed in advance by a “master switch” rather than in response to environmental conditions in early G1 phase (Dolznig et al., 1995 ). Second, pre-RCs may be disassembled during exit from the cell cycle, as has been suggested for quiescent cultured cells (Leno and Munshi, 1994 ; Wu and Gilbert, 1997 ). Additional work is required to distinguish between these two possibilities.

Permeabilization of the nuclear envelope appears to be a general requirement for initiation of replication in nuclei from post–S-phase cells (Blow and Laskey, 1988 ; Leno et al., 1992 ; Coverley et al., 1993 ; Madine et al., 1995b ) and quiescent cells (Leno and Munshi, 1994 , 1997 ; Fang and Benbow, 1996 ; Munshi and Leno, 1998 ; this paper, Figure 1) by egg extract. The results presented here demonstrate that an intact nuclear envelope prevents replication of erythrocyte nuclei, at least in part, by preventing the assembly of functional pre-RCs on chromatin (Figures 1 and 2), the same general mechanism by which an intact envelope prevents rereplication within G2-phase nuclei (Madine et al., 1995b ; Romanowski et al., 1996b ; Hua et al., 1997 ). However, the specific requirements for generating functional pre-RCs within erythrocyte nuclei and G2-phase nuclei are different. In the latter case, XCdc6 and XMCM proteins must rebind to replicated DNA, which retains XORC, and it is the loading of XMCM proteins on chromatin that is prevented by an intact nuclear envelope (Madine et al., 1995b ; Romanowski et al., 1996b ; Hua et al., 1997 ). By contrast, many, if not all, pre-RC proteins must be assembled on erythrocyte nuclei, including XOrc1 and XOrc2 (Figure 2), all of which are prevented from stably binding to chromatin by an intact envelope. The failure of XCdc6 and XMCM proteins to bind erythrocyte chromatin in the absence of XORC is not surprising given the stepwise assembly of the pre-RC by egg extract (reviewed by Romanowski and Madine, 1996 , 1997 ). However, it is surprising that assembly of XORC requires envelope permeabilization (Figure 2).

There are at least four general ways in which an intact nuclear envelope could prevent XORC assembly on erythrocyte chromatin. First, differentiation-specific changes in the nuclear envelope, such as a reduction in the density of nuclear pore complexes or changes in the capacity for nuclear protein import (Feldherr and Akin, 1990 , 1991 , 1993 ), could prevent the localization of ORC proteins within intact nuclei. Our data demonstrating that XOrc2 accumulates within intact nuclei (Figure 2) argue against this idea. However, we cannot rule out the possibility that other XORC proteins fail to cross an intact envelope, thereby preventing complete XORC assembly. Second, unique features of chromatin or nuclear structure (Thomas and Maclean, 1975 ; Brun, 1978 ; Wolffe, 1989 ; Chen et al., 1996 ), which are preserved within intact erythrocyte nuclei, could prevent the stable binding of nucleoplasmic XORC proteins to DNA. Indeed, our results demonstrate that an intact envelope reduces both the rate and the extent of removal of H1s from erythrocyte chromatin (Figure 4); however, this reduction cannot adequately explain the absence of XORC from chromatin, because functional pre-RCs form on the chromatin from permeable nuclei even in the absence of H1 removal (Figures 5 and 6), although to a much lesser extent (Figure 8). Yet, other features of chromatin or nuclear structure, which have not been investigated here, could prevent the assembly of XORC on erythrocyte DNA. Third, an intact envelope could exclude a factor that is required for XORC binding to DNA similar to the hypothetical loading factor for MCM proteins (Madine et al., 1995b ). Fourth, an intact nuclear envelope could prevent XORC binding by concentrating, within the nucleus, an inhibitor of pre-RC assembly. This inhibitor could be a cyclin-dependent kinase, given that addition of moderate levels of cyclin A to egg extract prevents the binding of XORC to chromatin, possibly by reducing its affinity for DNA (Hua and Newport, 1998 ). If correct, the inhibitory concentration of kinase, or other factor, must be reached before XORC is assembled. This would not happen with permeable nuclei, because pre-RCs form on erythrocyte chromatin before an intact nuclear envelope is reassembled by the extract (our unpublished observations). Interestingly, we have found that H1 kinase activity is much higher within intact nuclei than within permeable nuclei after a 45-min incubation in the extract (our unpublished data). The identity of this kinase and its possible effects on XORC assembly are currently under investigation.

The hypercondensed erythrocyte chromatin (Thomas and Maclean, 1975 ; Brun, 1978 ; Wolffe, 1989 ) is decondensed and remodeled into embryonic-like chromatin by the replacement of somatic linker histones H1 and H1⁰ with the cleavage-stage linker histone B4 and HMG1 from egg extract (Dimitrov and Wolffe, 1996 ; this paper, Figure 4). These transitions in chromatin composition and structure play a dominant and essential role in the reacquisition of transcriptional competence in erythrocyte nuclei (Dimitrov and Wolffe, 1996 ). The data shown here indicate that these transitions in composition and structure of erythrocyte chromatin also facilitate the initiation of replication (Figure 6) apparently by promoting the assembly of pre-RCs on DNA (Figure 8A). The molecular mechanism(s) by which such transitions in chromatin structure promote pre-RC assembly is unknown. The association of XORC with replication origins may involve the reorganization and displacement of nucleosomes surrounding the origin DNA, which appears to be the case in yeast (Bell and Stillman, 1992 ; Diffley and Cocker, 1992 ). Accordingly, the replacement of somatic H1s with embryonic B4 would induce a global reorganization of inactive chromatin, thereby unmasking potential XORC binding sites on the DNA. Once unmasked, other regulatory factors could facilitate a more localized reorganization at specific sites (Gavin et al., 1998 ), eventually leading to XORC binding. A component of this global reorganization may be an increase in nucleosome mobility, such as that which accompanies the replacement of somatic H1s with B4 (Nightingale et al., 1996 ; Ura et al., 1996 ), generating a more dynamic and extended chromatin structure (Dimitrov et al., 1994 ; Nightingale et al., 1996 ; Ura et al., 1996 ) in which potential binding sites become accessible to ORC.

Another protein that plays an important role in the compaction of erythrocyte chromatin is the mature erythrocyte nuclear termination stage-specific protein (MENT) (Grigoryev et al., 1992 ). MENT is a non-histone protein that is associated with condensing chromatin in terminally differentiating avian erythrocytes (Grigoryev and Woodcock, 1993 ). This heterochromatin protein has recently been shown to mediate higher-order chromatin folding in avian granulocytes (Grigoryev et al., 1999 ) presumably by acting as a glue within and between nucleosome chains (Grigoryev and Woodcock, 1998 ). It seems likely that the remodeling of erythrocyte chromatin in egg extract would also involve the removal of MENT from compact chromatin, leaving a more decondensed and open conformation, which could also contribute to the availability of potential ORC binding sites.

NPL mediates the decondensation and remodeling of sperm chromatin in egg extract (Philpott et al., 1991 ; Philpott and Leno, 1992 ; Leno et al., 1996 ). Interestingly, NPL also brings about the decondensation of nuclei from cultured somatic cells, although, in this case, limited decondensation does occur even in NPL-depleted extract (Philpott et al., 1991 ). We also observed residual decondensation of erythrocyte nuclei in NPL-depleted extract (our unpublished data), and it is possible that this level of decondensation allows for limited pre-RC assembly (Figure 8A) and DNA replication (Figure 6). The factor or factors responsible for this NPL-independent decondensation of erythrocyte chromatin have not been identified; however, it is intriguing to speculate that this process may be mediated by a protein complex similar to the chromatin accessibility complex (CHRAC), isolated from Drosophila embryo extracts, that increases nucleosome mobility even in the presence of histone H1 (Varga-Weisz et al., 1995 , 1997 ). This complex has been implicated in the stimulation of SV40 replication by facilitating the binding of large T-antigen to origin DNA (Alexiadis et al., 1998 ).

In conclusion, the data presented here indicate that loss of nuclear envelope integrity and the removal of somatic linker H1 from erythrocyte chromatin are required for the acquisition of essential pre-RC proteins from the extract and the reactivation of DNA replication in this system. Furthermore, these data also suggest that reactivation is an ordered process, requiring, first, envelope permeabilization to remove a “block” to pre-RC assembly and, second, the removal of H1 to regulate the frequency of pre-RC assembly and initiation of replication. Further analysis is required for a more precise definition of these mechanisms and to determine the role(s) they may play in the establishment and/or maintenance of the quiescent state in eukaryotic cells.

We thank Ron Laskey for providing antibodies to pre-RC proteins, Steve Dilworth for the anti-NPL hybridoma cell line PA3C5, Don Sittman for purified H1c, Jennifer Johns for technical assistance, and Asmita Kumar for helpful discussions throughout this study. This work was supported by the National Science Foundation grant MCB-9506280 (to G.H.L.).