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© The Rockefeller University Press,
0021-9525/1998//563 $5.00
The Journal of Cell Biology, Volume 143, Number 3,
, 1998 563-575
Regular Articles |
Recruitment of Phosphorylated Chromatin Assembly Factor 1 to Chromatin after UV Irradiation of Human Cells
Wellcome CRC Institute, Cambridge CB21QR, United Kingdom; and Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724
The subcellular distribution and posttranslational modification of human chromatin assembly factor 1 (CAF-1) have been investigated after UV irradiation of HeLa cells. In an asynchronous cell population only a subfraction of the two large CAF-1 subunits, p150 and p60, were found to exist in a chromatin-associated fraction. This fraction is most abundant during S phase in nonirradiated cells and is much reduced in G2 cells. After UV irradiation, the chromatin-associated form of CAF-1 dramatically increased in all cells irrespective of their position in the cell cycle. Such chromatin recruitment resembles that seen for PCNA, a DNA replication and repair factor. The chromatin-associated fraction of p60 was predominantly hypophosphorylated in nonirradiated G2 cells. UV irradiation resulted in the rapid recruitment to chromatin of phosphorylated forms of the p60 subunit. Furthermore, the amount of the p60 and p150 subunits of CAF-1 associated with chromatin was a function of the dose of UV irradiation. Consistent with these in vivo observations, we found that the amount of CAF-1 required to stimulate nucleosome assembly during the repair of UV photoproducts in vitro depended upon both the number of lesions and the phosphorylation state of CAF-1. The recruitment of CAF-1 to chromatin in response to UV irradiation of human cells described here supports a physiological role for CAF-1 in linking chromatin assembly to DNA repair.
Key Words: assembly • CAF-1 • chromatin • repair • UV irradiation
Abbreviations used in this paper: CAF-1, chromatin assembly factor 1; CSK, cytoskeleton; DAPI, 4',6-diamidino-2-phenylindole; NER, nucleotide excision repair; NuMa, nuclear matrix-associated protein; PCNA, proliferating cell nuclear antigen; UV-C, ultraviolet C.
Address correspondence to G. Almouzni, Institut Curie, Research Section UMR 144 du CNRS, 26 rue d'Ulm, 75231 Paris Cedex 05, France. Tel.: (33) 1 42 34 64 10. Fax: (33) 1 42 34 64 21. E-mail: almouzni{at}curie.fr
IN the eukaryotic cell, genetic information is present in the form of chromatin (van Holde, 1988; Wolffe, 1995). The assembly and maintenance of this nucleoprotein complex is important to ensure regulated DNA metabolism. In proliferating cells, the bulk of histone synthesis and chromatin assembly occurs during DNA replication in S phase (Krude, 1995b; Sogo and Laskey, 1995; Kaufman, 1996). However, reassembly of chromatin may also be important after destabilizing events that may arise during transcription (Tsukiyama and Wu, 1997), recombination (Lichten and Goldman, 1995), and repair (Smerdon, 1991).
Biochemical approaches have allowed the proteins involved in assembly of the basic repeating unit of chromatin to be identified. This unit, the nucleosome core, consists of 
1.8 superhelical turns of DNA wrapped around a histone octamer (Luger et al., 1997). Nucleosome cores can be reconstituted in vitro simply by mixing the four core histones with DNA. This is based on the self-assembly properties of histones to form octamers and their transfer onto DNA which can be facilitated by salt gradient dialysis or the presence of polyanions (Laskey and Earnshaw, 1980). Several histone-binding proteins including nucleoplasmin, N1/N2, nucleosome assembly protein 1, DF 31, and Spt6p have the ability to facilitate regulated deposition of histones at physiological ionic strength. These proteins seem to act as molecular chaperones, i.e., they facilitate the formation of nucleosome cores without being a component of the final reaction product. Chromatin assembly factor 1 (CAF-1)1 is also thought to function as a molecular chaperone but, unlike the other proteins mentioned above, CAF-1 promotes assembly of nucleosomes preferentially onto replicating DNA (Smith and Stillman, 1989) and is associated with newly synthesized and modified histones H3 and H4 in cell extracts (Kaufman et al., 1995; Verreault et al., 1996). After the deposition of histones onto DNA, the formation of long arrays of regularly spaced nucleosomes was shown to be facilitated by two additional factors, recently purified from Drosophila embryo extracts, chromatin accessibility complex (CHRAC) (Varga-Weisz et al., 1997) and ATP-utilizing chromatin assembly and remodeling factor (ACF) (Ito et al., 1997). Since all these factors were isolated using in vitro biochemical approaches, additional cell biology studies should prove useful to determine the cellular events in which these proteins are involved in vivo.
The three-subunit CAF-1 protein was initially purified from human cells based upon its ability to promote chromatin assembly onto replicating DNA in the SV-40 replication system (Stillman, 1986; Smith and Stillman, 1989). The small subunit of CAF-1, known as RbAp48, or p48, is part of multiple complexes involved in different aspects of histone metabolism (Roth and Allis, 1996; Verreault et al., 1998). In contrast, the two large subunits, p150 and p60, are specific to the CAF-1 protein. The immunolocalization of the two largest subunits of CAF-1 in an asynchronous population of cells revealed that these subunits are predominantly nuclear (Smith and Stillman, 1991). In addition, analysis of S phase nuclei demonstrated colocalization of CAF-1 and replication foci (Krude, 1995a), consistent with a role for CAF-1 as an assembly factor during DNA replication. Complementation of nucleosome assembly in the human cell-free DNA replication system further allowed the identification of CAF-1 orthologues from other species, such as Drosophila, Xenopus, and Saccharomyces cerevisiae (Gaillard et al., 1996; Kamakaka et al., 1996; Kaufman et al., 1997). Although CAF-1 is not essential for S. cerevisiae viability (Enomoto et al., 1997; Kaufman et al., 1997), yeast strains lacking CAF-1 exhibit silencing defects and are sensitive to UV irradiation (Enomoto et al., 1997; Kaufman et al., 1997; Monson et al., 1997; Enomoto and Berman, 1998).
The cellular response to UV irradiation is a complex process which involves the translation of the presence of the toxic agent into cellular signaling, part of which is the detection and processing of DNA lesions (Herrlich et al., 1994). Indeed, ultraviolet C (UV-C), as a genotoxic agent (Pfeifer, 1997), produces mutagenic lesions in DNA, including cyclobutane pyrimidine dimers and 6-4 photoproducts, which are mainly repaired by nucleotide excision repair (NER) (Sancar, 1995; Wood, 1997). Importantly, studies monitoring the nuclease sensitivity of chromatin after UV irradiation revealed that NER is accompanied by nucleosomal rearrangements (Smerdon and Lieberman, 1978). Insights into the coordination between NER and chromatin assembly were obtained by in vitro studies using human cell extracts (Gaillard et al., 1996); these studies suggested a possible role for CAF-1 in linking these two events. The activity of CAF-1 during NER in vitro may account for the UV-sensitive phenotype that results from disruption of the genes encoding the CAF-1 subunits in S. cerevisiae.
To investigate the role of CAF-1 in vivo in human cells, we analyzed the subcellular distribution and posttranslational modification of CAF-1 after UV irradiation of HeLa cells. The association of the p150 and p60 subunits of CAF-1 with chromatin was found to increase substantially in irradiated cells. This occurred in parallel with the recruitment to chromatin of the DNA replication and repair factor proliferating cell nuclear antigen (PCNA). Furthermore, biochemical analysis of the chromatin-associated form of p60 revealed that it was mostly phosphorylated after UV irradiation. Association of a phosphorylated form of p60 with chromatin occurred rapidly and in a dose- dependent manner after UV irradiation. Consistent with these in vivo observations, we found that the amount of CAF-1 required to stimulate nucleosome assembly during the repair of UV photoproducts in vitro depended upon both the number of DNA lesions and the phosphorylation state of CAF-1. This parallel between the UV response in vivo and the in vitro assay are consistent with a response mediated through DNA damage processing rather than being a mere consequence of other cellular UV responses. This paper constitutes the first report of CAF-1 recruitment to chromatin in response to UV irradiation of human cells and supports a physiological role for CAF-1 in linking chromatin assembly to DNA repair.
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50% confluence were arrested in early S phase with 2 mM hydroxyurea (Sigma Chemical Co., St. Louis, MO) for 16 h then released by several washes in phosphate-buffered saline (PBS) before a final wash in fresh medium as described (Todorov et al., 1995). FACScanTM analysis revealed that these cells remained in S phase for 10 h before entering G2 phase (3 h), mitosis (1 h), and finally G1 phase 14 h after release. We routinely collected early G1 cells 15.5 h after release. The proportion of cells in the various phases of the cell cycle was determined by flow cytometry of fixed cells. For flow cytometry analysis, cells washed in PBS were collected by centrifugation after trypsinization, fixed in ethanol, washed with PBS, and then pelleted by centrifugation at 600 g to be treated with 100 mg/ml RNase A (Boehringer Mannheim, Mannheim, Germany) and 10 mg/ml propidium iodide (Sigma Chemical Co.) in PBS with 0.1% Tween. Data were collected using a FACScanTM flow cytometer (Becton Dickinson, San Jose, CA). For UV treatment, HeLa cells grown in a glass Petri dish at a density of 5 x 104 cells/cm2 (50% confluent), were rinsed with PBS twice, PBS was removed, and then the cells were exposed to UV-C light (254 nm) from a low-pressure mercury lamp. The distance between the lamp and the dish was adjusted to obtain a fluence of 1 J/m2 per second measured using a Latarjet Dosimeter (Institut Curie, Paris, France). The irradiation time was varied to obtain doses from 10 to 30 J/m2. After irradiation, growth medium was added and the cells were allowed to recover for the indicated times. Cell cycle arrest after UV irradiation was verified by flow cytometry by comparison with nonirradiated and mock-treated cells synchronized in parallel.
Cell Extractions
Extractions were performed by a method adapted from Todorov et al. (1995). For the immunofluorescence analysis, cells grown on coverslips were washed first with PBS+ which contains 0.5 mM MgCl2 and 0.5 mM CaCl2 (and then with cytoskeleton [CSK] buffer [10 mM Pipes-KOH, pH 7.0, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2 {Fey et al., 1986}]). At this stage, cells were either directly fixed with 4% paraformaldehyde in PBS+ for 20 min at 24°C or subjected to various treatments: (a) for the Triton extraction, cells were incubated in CSK buffer supplemented with 0.5% Triton X-100, 0.5 mM PMSF (Sigma Chemical Co.), and 10 mg/ml leupeptin (Boehringer Mannheim) for 5 min at 20°C; (b) for the DNase I digestion, 0.1 mg/ml DNase I (Grade II; Boehringer Mannheim) was added to the same extraction buffer and incubated 5 min at 20°C. After two washes in CSK buffer, the cells were fixed in 4% paraformaldehyde in PBS1. For biochemical analysis, the cells were trypsinized, pelleted (600 g for 10 min), and then washed with PBS+ and CSK buffer. Using the conditions described above, the cells in suspension were subjected to various extraction procedures. The Triton-extracted and DNase I–treated cells were harvested after addition of an excess of CSK buffer rapidly followed by centrifugation to collect the pellets. After a wash in CSK buffer, the pellets were resuspended in CSK buffer at a final dilution corresponding to 2.5 x 104 cells/ml. An equal volume of 2x Laemmli buffer was added to the samples before boiling for 10 min, loading on a SDS-polyacrylamide gel, and Western blotting.
Preparation of Cell-free Extracts
The procedure for preparing cytosolic extracts uses hypotonic buffer (Li and Kelly, 1984) and the properties of these extracts depends on the physiological state of the cells. In brief, the cytosolic extract was prepared from HeLa cells grown in dishes essentially as described (Krude et al., 1997). After rinsing twice in PBS buffer, cells were allowed to swell for 10 min in 20 ml of ice-cold extraction buffer E (20 mM Hepes-KOH, pH 7.8, 5 mM potassium acetate, 0.5 mM MgCl2, and 0.5 mM DTT) per dish and excess buffer was removed. All subsequent steps were carried out at 4°C. Mitotic cells that detached from the dish under the hypotonic conditions were discarded and interphase cells were then scraped off the plates and disrupted them with 25 strokes in a Dounce homogenizer (1-ml Dounce tissue grinder; Wheaton, Millville, NJ) using a loose-fitting pestle. Nuclei were pelleted at 1,500 g for 3 min and the supernatant was recentrifuged at 14,000 g for 20 min at 4°C. The cytosolic extract was then aliquoted and frozen in liquid nitrogen. For nuclear extract, pelleted nuclei were resuspended at 1.5 x 108 nuclei/ml in buffer E supplemented with NaCl to reach a final concentration of 0.6 M and incubated for 90 min at 4°C. Nuclear material was pelleted by centrifugation at 14,000 g for 20 min and the supernatant corresponding to the interphasic nuclear extract was collected. Aliquots were frozen in drops in liquid nitrogen. Mitotic extracts were prepared as previously described (Marheineke and Krude, 1998).
Antibodies
Monoclonal antibodies against the two large subunits of human CAF-1, p150 (mAb1), and p60 (mAb96) (Smith and Stillman, 1989) were kindly provided by B. Stillman (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). The rabbit polyclonal antibody, pAb1, directed against the p60 subunit was used to reveal both phosphorylated and nonphosphorylated forms of the p60 subunit as described (Marheineke and Krude, 1998). The specificity of pAb1 and mAb96 was verified by Western blotting (see Fig. 5) as well as by a double immunofluorescence labeling of fixed HeLa cells (data not shown). Double immunofluorescence assays were also carried out using pAb1 and mAb1 (Marheineke and Krude, 1998) (data not shown). The other monoclonal antibodies used were: PC10 (Dako, Carpinteria, CA) specific for PCNA, mAb 414 (Berkeley Antibody Co., Richmond, CA) detecting three nucleoporins nup62, nup175, and nup270 (Berkeley Antibody Co.) and an anti-NuMa antibody (Calbiochem-Novabiochem, La Jolla, CA) detecting the nuclear mitotic apparatus also called nuclear matrix-associated protein (NuMA).
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Repair/Chromatin Assembly Reaction In Vitro
pBS plasmid (pBluescript KS; Stratagene, La Jolla, CA) isolated using a plasmid purification kit (QIAGEN, Santa Clarita, CA) was irradiated on ice with UV-C light (254 nm) from a low-pressure mercury lamp at various fluences (Wood et al., 1995). According to Wood et al. (1995), a fluence of 100 J/m2 induces roughly one photoproduct in 1,000 bp (0.75 cyclobutane pyrimidine dimer and 0.25 [6-4] photoproduct). It can thus be estimated for our plasmids that on average approximately four photoproducts will be present for 150 J/m2 and eight for 300 J/m2. The cytosolic extracts were used in 25 µl DNA repair reactions at 37°C essentially as described (Gaillard et al., 1996). In brief, reactions contained 100 µg of proteins contributed by the extract, 150 ng of nontreated or UV-C–irradiated pBS plasmid, 5 mM MgCl2, 40 mM Hepes-KOH, pH 7.8, 0.5 mM DTT, 4 mM ATP, 20 µM each of dGTP, dATP, and dTTP, 8 µM dCTP and 2 µCi [
-32P] dCTP (3,000 Ci/mmol), 40 mM phosphocreatine, 2 µg creatine phosphokinase (Type 1; Sigma Chemical Co.), and various sources of CAF-1. Reactions were stopped by the addition of EDTA to 15 mM, SDS to 0.35%, and 100 µg/ml RNase A (Boehringer Mannheim). After treatment with 200 µg/ml proteinase K (Boehringer Mannheim), DNA was isolated and subjected to electrophoresis (1.2% agarose, in TAE buffer) for DNA topology analysis. Ethidium bromide staining of the gel allowed verification of DNA recovery. The autoradiographs revealed the migration pattern of the plasmids labeled during the reaction. Quantification was carried out using a PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
Phosphorylation Analysis
20–400 U of Lambda protein phosphatase (New England Biolabs, Beverly, MA) were used to treat 100 ng of recombinant human CAF-1 protein purified from baculovirus-infected Sf9 cells (Kaufman et al., 1995). When indicated, as a control to limit the phosphatase reaction and produce intermediate phosphorylated forms of recombinant CAF-1, 25 mM β-glycerophosphate (Sigma Chemical Co.) was added. Mock-treated samples were incubated similarly, but without Lambda phosphatase. Phosphorylation was monitored by Western blotting as shown in Fig. 8. All treated samples were adjusted to 25 mM β-glycerophosphate (Sigma Chemical Co.) and frozen before use. We reached in the repair/chromatin assembly reaction in vitro a 10 mM final concentration of β-glycerophosphate.
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A Parallel Behavior for the p60 Subunit of CAF-1 and PCNA
For the p60 subunit of CAF-1, either the monoclonal mAb96 (Fig. 1, p60) or the polyclonal antibody pAb1 (Fig. 2, p60*) allowed us to visualize a Triton-resistant nuclear pool of CAF-1 that increased in every cell upon UV irradiation. It was thus possible to perform a double staining with a monoclonal antibody raised against PCNA and the polyclonal antibody (pAb1) directed against the p60 subunit of CAF-1 (see Materials and Methods). The detection of PCNA allowed us to identify S phase cells in an asynchronous population of control cells and to ensure that the irradiated cells responded properly to UV irradiation (Bravo, 1987; Toschi, 1988).
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The Triton-resistant Nuclear Fraction of CAF-1 p150 and p60 Is Associated with Chromatin
We then investigated the nature of the nuclear retention of the p150 and p60 subunits of CAF-1. Cells were treated with DNase I in the presence of detergent. Disappearance of the p150 and p60 signals paralleled degradation of DNA as shown by DAPI staining (Fig. 3, compare p150, p60*, and DNA). The disappearance of the staining cannot be attributed to a disruption of the nuclear envelope during the DNase I treatment since staining of the nuclear pores in the envelope with a nucleoporin-specific antibody was preserved (Fig. 3 A, pore). These data demonstrate that the Triton-resistant p150 and p60 subunits of CAF-1 are associated with chromatin.
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No major cell cycle-dependent changes in p60 phosphorylation could be detected in total cell lysates either from asynchronous cells or cells enriched in various stages of the cell cycle (Fig. 6 A) (data not shown). In G2 cells, a single band was detected for p150 and multiple bands in equal proportion for p60 (Fig. 5 B, Total). In these control G2 cells, Triton extracted most of the p150 and p60 molecules (Fig. 5 B, Triton-resistant) which is consistent with our immunofluorescence data (Fig. 4 B, no UV). Strikingly though, the various phosphorylated forms of p60 were preferentially extracted by Triton in G2 control cells leading to drastic removal of the phosphorylated forms (Fig. 5 B, p60, compare Total with Triton-resistant).
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Rapid Dose-dependent Response of CAF-1 to UV Irradiation
When cells are subjected to UV irradiation, a stress response is induced (Herrlich et al., 1994), cell cycle progression is arrested (Fig. 4 A), and lesions in the DNA are repaired. To gain some insight into the role of CAF-1 in this cellular response to UV irradiation, we first examined the kinetics of appearance of the phosphorylated forms of the p60 subunit of CAF-1 in the Triton-resistant fraction of G2 cells. As shown in Fig. 7 A, a UV dose of 30 J/m2 induced a significant accumulation of phosphorylated CAF-1 p60 as early as 30 min after UV irradiation, which is also clear from the densitometric scans corresponding to the individual lanes of this Western blot. This increase in phosphorylation occurred in parallel to an increase in the Triton-resistant fraction of p150 detected by Western blotting the same samples (Fig. 7 A, top). Thus, the appearance of Triton-resistant and phosphorylated CAF-1 subunits occurred as an early event after UV irradiation, consistent with the timing of early repair events (Pfeifer, 1997). We also noticed that these changes persisted for several hours postirradiation, a period during which the cells were still arrested in G2 phase of the cell cycle (data not shown). The accumulation of Triton-resistant phosphorylated forms of the p60 subunit of CAF-1 was also detected for lower UV doses (Fig. 7 B). A dose of 10 J/m2 already induced a significant increase in the amount of p60 protein retained after Triton extraction. The appearance of Triton-resistant CAF-1 p60 proportional to the UV dose (Fig. 7 B) was consistent with a possible stoichiometric targeting of CAF-1 to sites of DNA lesion/repair.
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When added to our extract in the presence of a radiolabeled nucleotide, DNA synthesis preferentially occurred in the UV-irradiated plasmid when compared with a control nonirradiated plasmid (Fig. 8 A, compare – with + UV). Since DNA recovery at the end of these reactions was comparable (Fig. 8 A, Total DNA), these data demonstrated that our extract was competent to process UV photoducts. Under these conditions, we did not detect in the labeled/repaired plasmid DNA a significant proportion of supercoiled molecules. This could reflect either a defect in chromatin assembly (the accumulation of supercoiled molecules is proportional to the number of nucleosomes formed on a circular DNA molecule) (Germond et al., 1979), or a defect in ligation of repaired molecules. Western blotting analysis of an aliquot removed from these reaction mixtures showed that only traces of the p60 subunit of CAF-1 were provided by the extract (Fig. 8 C, p60, lanes 1 and 2), whereas the p150 was at an undetectable level (data not shown). Since the p150 and p60 subunits of CAF-1 are both essential for chromatin assembly in vitro (Kaufman et al., 1995), these data suggested that the limiting factor for supercoiling in this system was CAF-1. Nucleosome assembly and supercoiling of the repaired plasmid could indeed be complemented by the addition of purified recombinant CAF-1 (Fig. 8 A, CAF-1). Importantly, this supercoiling was preferentially observed on labeled/repaired material compared with the bulk population of plasmid DNA (Fig. 8 A, compare Labeled with Total DNA). A careful titration of the amount of CAF-1 added per reaction allowed us to define the amount of CAF-1 required to achieve maximal supercoiling (Fig. 8 A, lane 7).
To proceed further and analyze the competence of various phosphorylated forms of CAF-1, the same amounts of the recombinant protein were treated (a) with Lambda phosphatase to completely dephosphorylate p60 (Fig. 8 B, lane 1); (b) with the phosphatase in the presence of β-glycerophosphate to produce phosphorylation intermediates of p60 (Fig. 8 B, lane 2); or (c) mock-treated (Fig. 8 B, lane 3). We also compared the migration properties of the p60 subunit in our recombinant complex with respect to the p60 subunit from mitotic extracts (Fig. 8 B, lane 4) or interphasic nuclear extracts (Fig. 8 B, lane 6). These data showed that the migration properties of the recombinant form were very distinct from those of the mitotic p60 and rather could be compared with the forms with the lowest migration properties in the interphasic nuclear extract. We thus used in the in vitro assay the three different forms of recombinant CAF-1 as produced in Fig. 8 B, lanes 1, 2, and 3. Under the limiting condition defined in Fig. 8 A, we found that when CAF-1 was treated with phosphatase before its addition to the extract, it was somewhat less active in promoting nucleosome assembly on labeled/repaired DNA (Fig. 8 C, lane 4). The fact that CAF-1 remained dephosphorylated was verified by Western blotting of an aliquot removed at the end of each reaction (Fig. 8 C, p60). Interestingly, when β-glycerophosphate was initially present during the phosphatase treatment to produce phosphorylation intermediates of p60, supercoiling was comparable to the control (Fig. 8 C, compare lane 3 with 5). We can thus discard possible effects due to contaminants in the phosphatase preparation, and further conclude that even intermediate phosphorylation forms can be competent in the assay. Since the phosphorylation state of the recombinant CAF-1 protein purified from baculovirus- infected Sf9 cells may not necessarily reflect the precise modification of the protein present in UV-irradiated HeLa cells, we verified that similar observations could be made when using equivalent amounts of CAF-1 from HeLa cell nuclear extracts (data not shown). A close examination of the supercoiling obtained using plasmids irradiated at 150 and 300 J/m2 for various amounts and forms of CAF-1 (Fig. 9 A as indicated) suggested that the number of labeled/repaired sites could be a critical parameter for CAF-1 requirement (Fig. 9 A, compare 150 with 300 J/m2). Quantification of the data further evidenced the correlation between the number of labeled repaired sites and the requirement of CAF-1 as well as the importance of the form of CAF-1 provided in the reaction (Fig. 9 B).
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Comparison of the Behavior of p60 and p150 Subunits of CAF-1 to PCNA
The behavior of the p60 and p150 subunits of CAF-1 shares a number of similarities with that of PCNA, an essential protein of proliferating cells which plays crucial roles in both DNA replication and repair (Cox, 1997). Both the p150 and p60 subunits of CAF-1 rapidly become resistant to detergent extraction during DNA replication or repair of nuclei (Figs. 2, 6, and 7). Furthermore, the characteristics of the UV response (timing and dose dependence), for the binding of p150 and phosphorylated p60 to chromatin (Fig. 7) is strikingly similar to those observed for PCNA during NER (Miura et al., 1992; Prosperi et al., 1993; Aboussekhra and Wood, 1995). Interestingly, both the timing and UV dose response are somewhat distinct from those reported for the p34 subunit of replication protein A whose phosphorylation is only detected with higher doses and at a later time after UV irradiation (Liu and Weaver, 1993; Carty et al., 1994). Overall, our data provide in vivo support for a dual role of CAF-1 during S phase and DNA repair. It is intriguing that this parallel between PCNA and CAF-1 may extend beyond their dual function in DNA replication and DNA repair, since both proteins also play a role in the inheritance of transcriptionally silent heterochromatin.
In Drosophila, chromosomal translocations that bring euchromatic genes next to heterochromatin generally result in transcriptional silencing (Pirrotta, 1997). The silent state is rather stable through mitosis but, with a low frequency, the translocated gene can switch to the expressed state, which can also be inherited in a stable fashion. This form of chromosomal position effect, known as position effect variegation in Drosophila, is functionally analogous to the silencing of reporter genes introduced next to telomeric heterochromatin in S. cerevisiae (Aparicio et al., 1991). Interestingly, a PCNA mutation in Drosophila, mus 209 (mutagen-sensitive 209), results in suppression of position-effect variegation (Henderson et al., 1994; Yamamoto et al., 1997), i.e., heterochromatin-mediated silencing is impaired. This may relate to the unexpected function of CAF-1 in silencing recently uncovered through genetic studies in S. cerevisiae (Enomoto et al., 1997; Kaufman et al., 1997; Monson et al., 1997; Enomoto and Berman, 1998). These findings suggest that the role of CAF-1 during nucleotide excision repair may be critical in either restoring or setting de novo repressive chromatin structures. PCNA also plays a role in repair mechanisms other than NER such as double-strand break repair (Ayyagari et al., 1995; Henderson and Glover, 1998) and one of the base excision repair pathways (Frosina et al., 1996). Our previous work using Drosophila embryo extracts (Gaillard et al., 1997) would suggest that the DNA synthesis event is not strictly required for an assembly machinery to be recruited to specific repair sites. Thus, CAF-1 may be recruited by DNA structures and/or protein factors present during earlier stages of repair. The parallel behavior of CAF-1 with PCNA could thus imply some interaction between these molecules within such a complex. This raises the possibility that CAF-1 may also play some role in DNA repair pathways other than NER. The binding of CAF-1 to chromatin and the phosphorylation of the p60 subunit of CAF-1 described here may provide useful markers to monitor the CAF-1 response to various genotoxic agents in repair-proficient cells or cells with mutations in various DNA repair pathways (Friedberg et al., 1995).
Chromatin Association and the Phosphorylation of the p60 Subunit of CAF-1 in Response to UV Irradiation
The changes in CAF-1 after UV irradiation reported here provide a novel element to the cellular response to UV irradiation. We defined two features of this CAF-1 response to UV irradiation: (a) the chromatin retention of both the p150 and p60 subunits of CAF-1 and (b) the predominance of phosphorylated forms of p60 in this chromatin-associated fraction. The chromatin association most likely reflects an interaction with a protein component of chromatin, rather than with DNA itself, since the CAF-1 protein purified from human cells was reported to present no apparent affinity for either single- or double-stranded DNA (Smith and Stillman, 1989). The p60 subunit of CAF-1 has been previously shown to exist in various phosphorylated forms in asynchronous cells, (Smith and Stillman, 1991) and several consensus sites for protein kinase C, casein kinase 2, and tyrosine kinase phosphorylation can be found. However, no information is available concerning the possible physiological significance of the posttranslational modification of p60. Although the relative abundance of the nonphosphorylated and phosphorylated forms of p60 in the chromatin-associated fraction was found to vary during the cell cycle, a significant increase in phosphorylated p60 bound to chromatin occurred in response to UV irradiation in G1, S, or G2 phase (Fig. 6 and data not shown). Whether the chromatin-associated phosphorylated forms of p60 that are constitutively present in G1 and S phase serve similar functions to those found after UV irradation remains to be determined. It is not as yet clear whether the sites of p60 phosphorylation in normal and UV-irradiated cells are the same or, for that matter, whether phosphorylation of the p60 subunit of CAF-1 plays a similar function during normal progression through S phase and in the cellular response to UV irradiation.
In normal interphasic cells, it is remarkable that the phosphorylation of the Triton-resistant form of the p60 subunit of CAF-1 is high in G1, progressively decreases throughout S phase, and is essentially absent during G2. The significance of this observation is unclear but this progressive dephosphorylation of CAF-1 could potentially serve as a marking system to avoid DNA rereplication. Alternatively, since progression through S phase is accompanied by a decrease in the number of DNA replication foci, the level of p60 phosphorylation may simply be an indication of the amount of CAF-1 actively involved in nucleosome assembly at the DNA replication fork. This in turn would suggest that p60 phosphorylation may modulate the nucleosome assembly activity of CAF-1 in normal cells. In that respect, it is also interesting to note that a distinct modified form only observed in mitosis (see Fig. 8 B) was reported to be impaired in nucleosome depostion during complementary strand synthesis (Marheineke and Krude, 1998).
Our data do not preclude the possibility of a de novo phosphorylation of the p60 subunit of CAF-1 in response to UV irradiation. However, the increase in the phosphorylated form of p60 that is associated with chromatin after UV irradiation may be simply mediated through recruitment of a preexisting pool of phosphorylated molecules. This mechanism of recruitment could be advantageous for a rapid response to UV damage. Importantly, although both phosphorylated and nonphosphorylated forms of p60 are present in roughly equal amounts in G2 cells (Fig. 5 B, Total), phosphorylated p60 is almost completely absent from the chromatin-associated pool (Fig. 5 B and Fig. 6). Thus, phosphorylation of p60 per se is not sufficient to trigger association of CAF-1 with chromatin. Remarkably, the fraction of the p60 subunit of CAF-1 recruited to chromatin appeared to be proportional with the UV dose applied to the cells (Fig. 7). These data suggest that the UV signal triggering this recruitment could operate via the sensing of the UV lesion/repair sites into DNA which would be consistent with our data in vitro (Fig. 8).
Importance of the Phosphorylated Form of the p60 Subunit of CAF-1 in Interphase
Our data in vitro argue that phosphorylation of CAF-1 increases the ability of the factor to promote nucleosome assembly during nucleotide excision repair (Fig. 8). Although this effect is rather modest in vitro, it may become important in vivo under conditions of severe DNA damage, when the concentrations of CAF-1 and repair factors may be limiting. Potentially, the modification by phosphorylation of CAF-1 may be important for its role in nucleosome assembly activity during both DNA synthesis and DNA repair.
Phosphorylation of the p60 subunit may facilitate either binding or dissociation of CAF-1 from acetylated core histones H3 and H4. Alternatively, phosphorylation of p60 may promote recognition of the specific protein factors or DNA structures necessary to attract the CAF-1 protein to sites of DNA repair. Finally, in addition to its effect on the nucleosome assembly activity of CAF-1, the increase in phosphorylation of the p60 subunit of CAF-1 in response to UV irradiation may act as a signal to prevent progression through the cell cycle until DNA lesions have been repaired and/or the chromatin structure has been restored to its original state. This potential signaling function of phosphorylated CAF-1 would provide an interesting link between cell cycle regulation and DNA repair. These issues should be explored in the future.
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This work was supported by the Association pour la Recherche sur le Cancer (ARC), an Action Thématique et Incitative sur programme et Equipes (ATIPE) no. 7, "Biologie du Developpement" from the Centre National de la Recherche Scientifique, and the European Economic Community. E. Martini has been supported by la Ligue National Contre le Cancer and ARC fellowships, K. Marheineke is supported by a Marie Curie Research Fellowship of the European Union, and A. Verreault is a postdoctoral fellow of the Jane Coffin Childs Memorial Fund for Medical Research.
Submitted: 29 May 1998
Revised: 11 September 1998
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