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Copyright © 1998, The American Society for Cell Biology Histone H1 Reduces the Frequency of Initiation in Xenopus Egg Extract by Limiting the Assembly of Prereplication Complexes on Sperm Chromatin Elizabeth Blackburn, Monitoring Editor ‡Corresponding author. Received December 12, 1997; Accepted February 10, 1998.  This article has been cited by other articles in PMC. | |||||||||||||
Abstract Somatic histone H1 reduces both the rate and extent of DNA replication in Xenopus egg extract. We show here that H1 inhibits replication directly by reducing the number of replication forks, but not the rate of fork progression, in Xenopus sperm nuclei. Density substitution experiments demonstrate that those forks that are active in H1 nuclei elongate to form large tracts of fully replicated DNA, indicating that inhibition is due to a reduction in the frequency of initiation and not the rate or extent of elongation. The observation that H1 dramatically reduces the number of replication foci in sperm nuclei supports this view. The establishment of replication competent DNA in egg extract requires the assembly of prereplication complexes (pre-RCs) on sperm chromatin. H1 reduces binding of the pre-RC proteins, XOrc2, XCdc6, and XMcm3, to chromatin. Replication competence can be restored in these nuclei, however, only under conditions that promote the loss of H1 from chromatin and licensing of the DNA. Thus, H1 inhibits replication in egg extract by preventing the assembly of pre-RCs on sperm chromatin, thereby reducing the frequency of initiation. These data raise the interesting possibility that H1 plays a role in regulating replication origin use during Xenopus development. | |||||||||||||
After fertilization, Xenopus laevis eggs undergo rapid and synchronous cell divisions until the early embryos reach the midblastula transition (MBT). At the MBT, slower, asynchronous cell division cycles appear accompanied by the onset of transcription (Newport and Kirschner, 1982 It is well documented that establishment of higher-order chromatin structure during development is mediated primarily by programmed changes in individual chromatin components (Dimitrov et al., 1993 What role, if any, the cleavage stage linker B4 and somatic H1s play in regulating DNA replication during Xenopus development is not clear. Differential effects of H1 variants on S-phase progression have been reported in avian and mammalian somatic cells (Bergman et al., 1988 Somatic H1 could inhibit DNA replication in egg extract in at least three ways. First, it could reduce replication competence by limiting the assembly of prereplication complexes (pre-RCs) on sperm chromatin. The pre-RC is formed by the sequential binding of origin recognition complex (ORC) proteins, XCdc6, and the minichromosome maintenance (MCM) proteins to chromatin and is required for initiation of replication (Carpenter et al., 1996 In this report we have investigated the mechanism by which somatic H1 inhibits replication of sperm nuclei in egg extract. We find that H1 reduces the number of replication forks, but not the rate of fork progression, in preassembled Xenopus sperm nuclei. H1 does not inhibit second-strand synthesis on single-strand M13 DNA, which confirms that replication elongation is unaffected by this somatic linker histone. H1 reduces the assembly of pre-RCs on sperm chromatin and the number of replication foci after initiation. Replication competence is restored in these nuclei only under conditions that promote the loss of H1 from chromatin and licensing of the DNA. Thus, H1 inhibits replication in the extract by preventing pre-RC assembly on sperm chromatin and thereby reducing the frequency of initiation. These data raise the interesting possibility that H1 may play a role in regulating replication origin use during development. | |||||||||||||
Purification of Histone H1 Histone H1c was purified from its overexpressing mouse cell line as described by Lu et al. (1997)  . Purity of isolated H1 was verified by silver staining of SDS-polyacrylamide gels. An extinction coefficient of 18.76 ml·mg−1·cm−1 at 210 nm was used to determine the protein concentration of each sample.Preparation of Xenopus Egg Extracts and Permeable Sperm Nuclei Metaphase-arrested and interphase extracts were prepared from Xenopus laevis eggs as previously described (Blow, 1993 ; Lu et al., 1997 ). For preparation of 6-dimethylaminopurine (6-DMAP)-treated interphase extract, metaphase extract was supplemented with 3 mM 6-DMAP, and then mixed with 0.3 mM CaCl2. Permeable Xenopus sperm nuclei (“sperm chromatin”) were prepared according to Lu et al. (1997) .In Vitro DNA Replication Sperm chromatin was incubated at 1–3 ng DNA/μl extract supplemented with an energy-regenerating system (Blow and Laskey, 1986 ), 100 μg/ml cycloheximide, 2 mM ATP, and 5.68 μM histone H1c or an equivalent volume of water. The amount of H1 added to extract is comparable to the estimated amount of somatic linker histone present in a Xenopus embryo at the gastrula–neurula transition, i.e., >45 ng/embryo (Dworkin-Rastl et al., 1994 ). Deoxynucleoside triphosphates were added to a final concentration of 50 μM to readjust pool sizes after dilution (Blow and Laskey, 1986 ). All incubations were performed at 22°C. The extent of extract dilution was kept constant at 30% for all experiments. M13 replication was performed essentially as described above except that 10 ng M13 mp19 phage (GIBCO/BRL, Gaithersburg, MD) was used per microliter of extract. Nascent DNA was detected by adding 100 μCi/ml [α-32P]deoxy-ATP (dATP) or 20 μM 5-biotin-16-deoxyuridine triphosphate (Biotin-dUTP; Boehringer Mannheim, Indianapolis, IN) to the extract. Quantitation of DNA replication was carried out as previously described (Lu et al., 1997 ).For nuclear transfer, samples were diluted with 50-fold ice-cold buffer A and underlayed with fresh extract. Diluted nuclei were centrifuged into fresh extract at 770 g for 10 min at 4°C in a Jouan CR4–22 swing-out centrifuge. The supernatant was carefully removed, and the remaining extract was gently mixed to promote suspension of the chromatin or nuclei. Samples were then incubated at 22°C for various times at indicated in Figure 7.
Cytosine β-d-arabinofuranoside 5′-triphosphate (Ara-CTP) was added to egg extract to a final concentration of 200 μM. Stalled forks were released by addition of deoxycytosine triphosphate (dCTP) to a final concentration of 1 mM. Density substitution experiments were performed essentially as described by Leno and Munshi (1994) Immunofluorescence Microscopy Control and H1 samples were diluted 50-fold with ice-cold buffer A (Leno and Laskey, 1991 ), spun through 30% sucrose in buffer A onto polylysine-coated coverslips, and incubated in buffer A containing 0.5% Triton X-100 for 10 min. Samples were rinsed in PBS, fixed in PBS containing 4% paraformaldehyde for 10 min, and blocked for 10 min in PF buffer (0.75×PBS, 0.1% Triton X-100, 0.02% SDS, 2% BSA) containing 10% FCS. Coverslips were incubated in primary antibody in PF buffer for 1 h. Affinity-purified antibodies recognizing XOrc2, XCdc6, and Mcm3 were used at a dilution of 1:500, 1:50, and 1:1000, respectively. The coverslips were then washed twice in PF buffer, incubated with fluorescein-conjugated donkey anti-rabbit secondary antibody (Amersham, Arlington Heights, IL) for 1 h, and finally washed twice in PF buffer. Bulk DNA was visualized with Hoechst 33258 (Sigma, St. Louis, MO). Incorporated biotin-dUTP was detected with Texas-red streptavidin (Lu et al., 1997 ). Samples were viewed with an Odyssey laser confocal microscope (Noran Instruments, Middleton, WI).Alkaline and Neutral Agarose Gel Electrophoresis Alkaline agarose gel electrophoresis was performed as described previously (Mahbubani et al., 1997 ; Walter and Newport, 1997 ) with minor modifications. Replication reactions were mixed with 500 μl of ice-cold buffer A, and nuclei were pelleted by centrifugation at 770 × g for 5 min at 4°C in a Juan CR4–22 swing-out centrifuge. Pellets were digested with stop mix N (20 mM Tris-HCl, 200 mM NaCl, 5 mM EDTA, 0.5% SDS, pH 8.0) supplemented with 2 μg/ml RNase A and 200 μg/ml Proteinase K for 1 h at 37°C. DNA was extracted twice with phenol-chloroform, then with chloroform, and then precipitated with ethanol. Dried samples were dissolved in alkaline gel loading buffer (50 mM NaOH, 1 mM EDTA, 1.25% Ficoll, 0.0125% bromocresol green) and separated on a 0.8% alkaline gel as described by Mahbubani et al. (1997) . Autoradiographs were scanned as previously described (Lu et al., 1997 ).Neutral agarose gel electrophoresis was performed as described by Jackson et al. (1995) | |||||||||||||
H1 Reduces the Rate and Extent of DNA Replication but Does Not Affect the Length of S-Phase in Egg Extract Somatic histone H1 slows assembly of the nuclear lamina and delays the initiation of DNA replication within individual sperm nuclei in Xenopus egg extract (Lu et al., 1997 ). This H1-induced delay in lamina assembly is responsible, at least in part, for the delay in initiation because preassembled H1 nuclei initiate replication at the same time as control nuclei. However, H1 inhibits replication even when lamina assembly is complete, suggesting that H1 modulates replication through multiple pathways in egg extract (Lu et al., 1997 ). As an initial step toward determining the mechanism by which H1 inhibits replication of Xenopus sperm nuclei in egg extract, we have used the elongation inhibitor Ara-CTP, a dCTP analog (Cozzarelli, 1977 ), to arrest nuclei early in S-phase and subsequently determine the effects of H1 on S-phase progression following release from inhibition. Specifically, sperm chromatin was incubated in egg extract supplemented with Ara-CTP and [α-32P]dATP either without (Control) or with histone H1 (H1) for 90 min. In parallel experiments, >95% of both control and H1 nuclei initiated replication during this initial incubation period, as judged by the incorporation of biotin-dUTP into nascent DNA (our unpublished observations). Stalled replication forks were then released by the addition of excess dCTP to the extract, and the extent of replication was measured by incorporation of radioactivity into acid-insoluble material at various times. The results from three separate experiments in which three different extracts were used are shown in Figure 1. Control nuclei completed replication 40 min after release with dCTP; however, the rate of elongation after release is approximately 2.25-fold lower than that observed in the absence of Ara-CTP (Walter and Newport, 1997 ) and, therefore, the actual duration of S-phase in our extracts, in the absence of Ara-CTP, is approximately 18 min. This is very similar to the length of S-phase in the embryo before the MBT (i.e., from 10 to 15 min; Hyrien and Mechali, 1993 ). The rate of replication in H1 nuclei is markedly reduced relative to control nuclei, and the extent of synthesis after release from elongation arrest is only 40% of the input DNA. Interestingly, the presence of H1 on sperm chromatin does not increase the length of S-phase in the extract. Thus, H1 reduces both the rate and extent of DNA replication without affecting the length of S-phase in egg extract.
The amount of H1 added to extract in these experiments is comparable to the estimated amount of somatic linker histones present in a Xenopus embryo at the gastrula–neurula transition stage, i.e., >45 ng/embryo (Dworkin-Rastl et al., 1994 H1 Reduces the Frequency of Initiation but Not the Rate of Elongation in Egg Extract H1 could reduce the rate of DNA replication by reducing the number of active replication forks or by reducing the rate at which individual forks elongate, although the latter seems less likely given the fact that H1 does not increase the length of S-phase. To determine which of these two possibilities is correct, we incubated sperm chromatin in egg extract under the conditions described in Figure 1 and collected samples, at 2-min intervals, after release with dCTP. Nascent DNA was then resolved by alkaline agarose gel electrophoresis, and the resultant gel was subjected to autoradiography (Figure 2A). The relative amount of radioactivity in the center of each lane was determined by densitometry, and these values were plotted as a function of molecular size in kilobases (kb). Data from samples collected immediately after dCTP addition to extract (0′), and after a 4-min (4′) and 8-min (8′) release are shown in Figure 2B. The amplitudes of the control samples on the graph were reduced to facilitate comparison with the H1 samples.
The rate of replication fork movement, calculated to be approximately 7 ± 1 nucleotides per second, was virtually identical in both control and H1 nuclei (Figure 2B). This value is in close agreement with the fork rates previously reported for Xenopus embryos, egg extracts, and Xenopus somatic cells, i.e., 8–10 ± 2 nucleotides per second (Callan, 1972 When nascent DNA from both control and H1 nuclei is substituted with the thymidine analog BrdUTP and labeled with [α-32P]dATP, a single peak of radioactivity is observed at a density of ~1.75 g/ml after centrifugation to equilibrium in a cesium chloride gradient (Figure 3A). The appearance of a single heavy/light (HL) peak demonstrates that the DNA in H1 nuclei undergoes a single round of semiconservative replication in the extract. Thus, those origins that do fire in the presence of H1 produce forks that elongate to form large tracts of fully replicated DNA, supporting the view that H1 inhibits initiation, but not elongation, of replication in the extract. Second-strand synthesis on single-strand (ss) M13 DNA mimics replication elongation on double-strand DNA after replication origins unwind to form single-stranded replication bubbles (Mechali and Harland, 1982
Histone H1 Reduces the Number of Replication Foci in Sperm Nuclei Replication forks are clustered in discrete sites or foci, uniformly distributed throughout nuclei replicating in Xenopus egg extract (Mills et al., 1989 ; Leno and Laskey, 1991 ; Cox and Laskey, 1991 ). It has been estimated that there are from 300 to 1000 forks clustered within each replication focus and from 100 to 300 foci within each sperm nucleus (Mills et al., 1989 ). Our data suggest that H1 may reduce the number of replication forks in sperm nuclei by as much as 70% and, therefore, we sought to determine whether H1 also reduces the number of replication foci in these nuclei. To visualize replication foci, control (Control) and H1 (H1) chromatin was incubated with biotin-dUTP for 3 h in egg extract treated with the protein kinase inhibitor 6-DMAP. Even though 6-DMAP inhibits most initiation events in unlicensed sperm nuclei (Blow, 1993 ; Mahbubani et al., 1997 ; Munshi and Leno, 1998 ), those origins that escape inhibition are distributed in approximately the same number of foci present throughout nuclei incubated in extract without kinase inhibition (Munshi and Leno, 1998 ). Furthermore, replication foci were more easily resolved, microscopically, within nuclei incubated in 6-DMAP-treated extract than in untreated extract, presumably because of the reduced number of initiations within each focus in 6-DMAP extract (Munshi and Leno, 1998 ). Figure 4 shows a confocal series of optical sections taken at 0.5-μm intervals through two control nuclei and two H1 nuclei stained with Texas-Red streptavidin. H1 nuclei are highly condensed (Lu et al., 1997 ) and consistently contained far fewer replication foci than control nuclei. Taken together, these data demonstrate that H1 reduces the number of replication forks and the number of replication foci within sperm nuclei incubated in egg extract.
Replication protein A (RP-A) is localized within foci on sperm chromatin incubated in egg extract (Adachi and Laemmli, 1992 H1 Restricts Prereplication Complex Assembly on Sperm Chromatin A reduction in the number of replication forks implies a reduction in the number of active replication origins. One way in which H1 could restrict origin use in the extract is by preventing the formation of pre-RCs on sperm chromatin. The pre-RC is formed by the sequential binding of ORC proteins, Cdc6, and the MCM proteins to chromatin (Carpenter et al., 1996 ; Coleman et al., 1996 ; Romanowski et al., 1996 ; Rowles et al., 1996 ), and it has been shown that assembly of this complex is essential for establishing replication-competent DNA ( Chong et al., 1995 ; Kubota et al., 1995 ; Madine et al., 1995a ,b ; Coleman et al., 1996 ; Romanowski et al., 1996 ). To determine whether H1 affects pre-RC assembly, sperm chromatin was incubated in egg extract supplemented with biotin-dUTP, with H1 (H1), or without H1 (Control), for up to 90 min. The nuclei from each sample were then probed with Texas-Red streptavidin, to detect nascent DNA, and with antibodies to three components of the pre-RC, namely, XOrc2 (Figure 5A), XCdc6 (Figure 5B), or XMcm3 (Figure 5C). All three associated with sperm chromatin within 15 min in the control sample (Figure 5, A–C, Control 15′). By 45 min, most control nuclei had entered S-phase (Figure 5C, Control Biotin 45′), which was accompanied by the apparent redistribution of XCdc6 to the nuclear envelope (Figure 5B, Control 45′) and the loss of XMcm3 from the chromatin (Figure 5C, Control 45′) consistent with previous reports (Carpenter et al., 1996 ; Coleman et al., 1996 ; Romanowski et al., 1996 ; Rowles et al., 1996 ). The presence of H1 dramatically reduced the association of all three pre-RC proteins with sperm chromatin after 15 min in the extract (Figure 5, A–C, H1 15′). Even after 30 min, when nuclear assembly was complete, the levels of XOrc2, XCdc6, and XMcm3 were still markedly reduced in H1 nuclei (Figure 5, A–C, H1 30′). This difference in pre-RC protein levels between control and H1 nuclei was confirmed by Western blot, although XMcm3 levels were higher than expected in both control and H1 nuclei at all time points examined (our unpublished observations). H1 did not inhibit replication completely, nor did it prevent the loss of XMcm3 from sperm chromatin after S-phase entry (Figure 5C, H1 XMcm3 45′ and 90′) demonstrating that pre-RCs assembled in the presence of H1 are functional. These data raise the interesting possibility that H1 limits origin use, and the extent of DNA replication, by restricting the assembly of pre-RCs on sperm chromatin.
Restricting Pre-RC Formation Limits the Replication Capacity of H1 Nuclei H1 restricts the assembly of pre-RCs on sperm chromatin by egg extract (Figure 5, A–C). In light of the essential role that pre-RCs play in establishing replication competence in this system, we sought to determine whether reduced pre-RC formation is responsible for the observed reduction in DNA replication in H1 nuclei (Figure 1). To answer this question, we carried out experiments as shown in Figure 6. Sperm chromatin was incubated in egg extract supplemented with Ara-CTP and [α-32P]dATP without H1 (lane a) or with H1 (lanes b–d). After 20 min (lane c) or 60 min (lanes a, b, and d) in extract, four volumes of extract containing Ara-CTP and [α-32P]dATP without H1 (lanes a, c, and d), or with H1 (lane b) were added. After a total incubation time of 90 min, dCTP was added to all samples, which were then incubated for an additional 50 min. Control nuclei replicated completely under these experimental conditions (lane a) whereas replication was reduced nearly 5-fold in nuclei containing H1 (lane b) consistent with our earlier results (Figures 1 and 2A). However, replication was restored to ~80% of control levels when H1 chromatin was exposed to fresh H1-free extract after an initial 20-min incubation (lane c). H1 is rapidly lost from sperm chromatin after dilution with extract lacking H1 (Lu et al., 1997 ), demonstrating that loss of H1 from chromatin is associated with the acquisition of replication competence (lane c). If H1 chromatin is incubated in the extract for 60 min before dilution with H1-free extract, however, replication is not restored (lane d), demonstrating that the timing of extract addition is critical for establishing replication competence in H1 nuclei.
Under the experimental conditions described here, sperm chromatin does not possess an intact nuclear envelope after 20 min in the extract; however, by 60 min, envelope assembly is complete in virtually all nuclei (our unpublished observations). Therefore, it is possible that an intact nuclear envelope prevents the restoration of replication competence in H1 nuclei. One way in which an intact nuclear envelope can prevent replication in egg extract is by preventing the licensing of unlicensed DNA, i.e., DNA that lacks essential components of the pre-RC (Blow, 1993 | |||||||||||||
We have recently developed a system to assemble somatic H1 histones onto Xenopus sperm chromatin using cell-free extracts derived from activated Xenopus eggs. Somatic H1s added to the extract are correctly assembled onto sperm chromatin, thereby preserving normal nucleosomal organization but promoting the formation of higher-order chromatin structure (Lu et al., 1997 Studies with Xenopus egg extracts have shown that assembly of pre-RCs is a highly coordinated process involving a number of essential replication proteins. The multisubunit ORC is first assembled onto sperm chromatin, thereby facilitating the sequential binding of XCdc6 and the MCM complex of proteins (Chong et al., 1995 Immunodepletion of ORC from egg extract causes a substantial reduction in the rate of DNA replication and an extended S-phase due to a reduction in the number of initiation events in sperm nuclei (Walter and Newport, 1997 H1 reduces the number of foci that contain RP-A (our unpublished observations), the precursors of replication foci (Adachi and Laemmli, 1992 The results presented here raise the interesting possibility that linker histones play a role in regulating the initiation of DNA replication during Xenopus development. Somatic H1 could modulate replication origin use by promoting changes in chromatin structure after the MBT. In theory, localized changes in chromatin structure could be part of a general mechanism that serves to coordinately regulate the timing of DNA replication between multiple adjacent replicons in somatic cells. Further investigation into the mechanisms by which H1 restricts pre-RC assembly and origin use, along with the identification of other factors that participate in this process, will help us understand how DNA replication is regulated during development. | |||||||||||||
ACKNOWLEDGMENTS We thank Rajan Munshi for providing metaphase extract, Dr. Peter Jackson for providing RP-A antiserum, Jennifer Johns for technical assistance, and Dr. Susan Wellman for helpful comments on the manuscript. This work was supported by a grant from the National Science Foundation (MCB-9506280) to G.H.L. | |||||||||||||
Footnotes Abbreviations used: Ara-CTP, Cytosine β-d-arabinofuranoside 5′-triphosphate; 6-DMAP, 6-dimethylaminopurine; MCM, minichromosome maintenance; ORC, origin recognition complex; Pre-RC, pre-replication complex; RP-A, replication protein A. | |||||||||||||
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