Proc. Natl. Acad. Sci. USA Vol. 74, No. 1, pp. 173-177, January 1977
Biochemistry
Activation of histone gene transcription by nonhistone chromosomal proteins in WI-38 human diploid fibroblasts (chromatin/reconstitution/cell proliferation/messenger RNA/complementary DNA)
ROBERT L. JANSING, JANET L. STEIN, AND GARY S. STEIN Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Fla. 32610
Communicated by George K. Davis, November 3,1976
ABSTRACT The regulation of histone gene expression was examined after confluent, nondividing WI-38 human diploid fibroblasts were stimulated to proliferate. Histone mRNA sequences were assayed by hybrid formation with an 3H-labeled single-stranded DNA complementary to histone mRNAs. Histone mRNA became associated with polyribosomes concomitant with the activation of DNA synthesis. The ability of chromatin from WI-38 cells to serve as a template for in vitro transcription of histone mRNA sequences parallels the onset of DNA replication. A role for nonhistone chromosomal proteins in the control of histone gene readout is suggested because, when chromatin from confluent WI-38 cells was dissociated and then reconstituted in the presence of S phase nonhistone chromosomal proteins, a 500-fold activation of histone mRNA sequence transcription was observed.
The synthesis of histones in continuously dividing cells as well as in nondividing cells that have been stimulated to proliferate is restricted to the S phase of the cell cycle (1-4) and depends on concomitant DNA replication (2, 3, 5). In continuously dividing HeLa S3 cells, a functional relationship between histone synthesis and DNA replication is further suggested by results from in vitro translation (5-8) and hybridization (9) studies that indicated the presence of histone mRNAs on the polyribosomes of S phase cells. In continuously dividing cells, transcriptional regulation of histone gene expression is suggested by results from studies showing that, when chromatin from S phase and G1 phase HeLa cells is transcribed in vitro, histone mRNA sequences are detected only in transcripts of S phase chromatin (10, 11). Nonhistone chromosomal proteins have been implicated in the regulation of gene expression in general (12-19) and in the control of transcription during the cell cycle specifically (17, 20-25). Chromatin reconstitution studies from several laboratories provide evidence that, among the complex and heterogeneous nonhistone chromosomal proteins, there are macromolecules which are responsible for the tissue-specific transcription of globin genes (26-28), the steroid hormone-dependent transcription of ovalbumin genes (29), and the transient expression of histone genes during the S phase of the cell cycle in continuously dividing HeLa cells (11, 30). An important question which arises is whether the mode of histone gene regulation observed in continuously dividing cells is operative in nondividing cells stimulated to divide. Since histones are intimately involved in the structure and packaging of the genome, understanding the mechanism by which histone genes are rendered transcribable should provide important clues to the control of cell proliferation. The studies reported here were directed toward defining the mechanism by which regulation of histone gene expression occurs after nondividing WI-38 human diploid fibroblasts are Abbreviations: Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; Cot, initial RNA concentration (mol/liter) X time (sec); COt1/2, Cot value at which hybridization is half completed. 173
stimulated to proliferate. Evidence is presented which suggests that, consistent with the situation observed in continuously dividing cells, a component of the nonhistone chromosomal proteins isolated from chromatin of WI-38 cells undergoing DNA replication is responsible for the activation of histone mRNA sequence transcription.
MATERIALS AND METHODS Cell Culture. Human diploid WI-38 fibroblasts were grown to confluence in monolayer culture in Eagle's basal medium containing 10% fetal calf serum. The cells were cultivated at 370 in a moist atmosphere containing 5% CO2. Confluent cells were stimulated to proliferate by replacing the exhausted growth medium with fresh Eagle's basal medium containing 20% fetal calf serum. It has been shown that under these conditions approximately 60% of the cells are stimulated to undergo DNA replication and mitotic division (23, 31, 32). The cells used in this study ranged from passage 28 to passage 30. Isolation of Nuclei and Chromatin. Nuclei and chromatin were isolated at 40 as described previously (4, 21). Cells were harvested with a rubber policeman, washed three times with 80 volumes of Earle's balanced salt solution, and lysed with 80 volumes of 80 mM NaCI/20 mM EDTA/1% Triton X-100 (pH 7.2). Nuclei were pelleted by centrifugation at 1000 X g for 3 min and washed twice with the lysing medium. The nuclei were washed twice with 0.15 M NaCI/10 mM Tris (pH 8.0) and pelleted by centrifugation at 1000 X g for 3 min. Nuclei isolated in this manner are largely free of visible cytoplasmic contamination when examined by phase contrast microscopy. The nuclear pellet was disrupted by mechanical agitation and the nuclei were lysed in 60 volumes of distilled water. The chromatin was allowed to swell in an ice bath for 20 min and was then pelleted by centrifugation at 10,000 X g for 20 min. Isolation of Polysomal RNA. Cells were harvested with a rubber policeman, washed three times with Earle's balanced salt solution, and pelleted by centrifugation at 1000 X g for 2 min. The pellet was drained well and resuspended in 10 volumes of 10 mM KCI/10 mM Tris/1.5 mM MgCl2 (pH 7.4). The cell suspension was transferred to a Dounce homogenizer, and after 20 min the cells were lysed by homogenization with a tightly fitting pestle. The homogenate was centrifuged at 15,000 X g for 15 min to pellet nuclei and mitochondria. The supernatant was transferred to 8-ml polycarbonate tubes and centrifuged at 100,000 X g for 90 min. All glassware and solutions were treated with diethylpyrocarbonate and then autoclaved. The pelleted polyribosomes were resuspended in 1 % sodium dodecyl sulfate/0.1 M NaCl/10 mM sodium acetate/1 mM EDTA (pH 5.4). Polysomal RNA was extracted twice with 1 volume of purified phenol plus 1 volume of chloroform/isoamyl alcohol, 24:1 (vol/vol), followed by two extractions with 1
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volume of chloroform/isoamyl alcohol. The RNA was precipitated with 2 volumes of ethanol, centrifuged at 3000 X g for 15 min, and resuspended in 25 mM N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (Hepes)/0.5 M NaCl/1 mM EDTA (pH 7.0) for hybridization analysis. In Vitro Transcription. RNA was transcribed by using Fraction V Escherichia coli RNA polymerase prepared according to the method of Berg et al. (33). Transcription was carried out for 60 min at 370 in a Dounce homogenizer fitted with a wide-clearance pestle, and the reaction mixture was periodically homogenized to maintain chromatin solubility. The incubation mixture in a final volume of 3.4 ml contained 40 mM Tris (pH 8.0), 4 mM MgCl2, 1 mM MnCl2, 20 gM EDTA, 0.008% 2-mercaptoethanol, 0.4 mM each of ATP, CTP, UTP, and GTP, 150 gg of DNA as chromatin/ml, and 200 units of RNA polymerase. The reaction mixture was brought to a concentration of 1% sodium dodecyl sulfate, 0.1 M NaCl, 10 mM sodium acetate, and 1 mM EDTA (pH 5.4) and was incubated at 370 for 15 min. Nucleic acids were extracted as described for polysomal RNA and precipitated with 2 volumes of ethanol. The pellet was resuspended in 10 mM Tris/0.1 M NaCl/5 mM MgCl2 (pH 7.4) containing DNase I (Sigma) at 40,ug/ml and incubated at 370 for 60 min. The reaction mixture was deproteinized by one extraction with phenol/chloroform/isoamyl alcohol, 1:1:0.04 (vol/vol), and two extractions with chloroform/isoamyl alcohol, 24:1 (vol/vol). The aqueous phase containing the RNA transcripts was chromatographed on Sephadex G-50 (fine) and eluted with 0.1 M NaCl/10 mM sodium acetate/i mM EDTA (pH 5.4). RNA was precipitated with 2 volumes of ethanol and resuspended in 25 mM Hepes/0.5 M NaCl/1 mM ElDTA (pH 7.0) for hybridization analysis. Hybridization Analysis. Histone [3H]cDNA was synthesized as previously described (34). We isolated 7-llS RNA from the polysomes of S phase HeLa S3 cells by repeated sucrose gradient fractionations, and RNAs containing poly(A) were removed by oligo(dT)-cellulose chromatography. The remaining 7-llS RNAs (histone mRNAs) directed the synthesis of all five classes of histones in a cell-free protein synthesizing system derived from wheat germ (34). Poly(A) was added to the 3' termini of the histone mRNAs with an ATP:polynucleotidylexotransferase (E.C. 2.7.7.19) prepared from maize seedlings (35). The polyadenylylated mRNAs were then transcribed with RNA-dependent DNA polymerase from avian myeloblastosis virus (Schwarz/Mann) in the presence of actinomycin D with dT1o as a primer. The mean sedimentation coefficient of the cDNA in alkaline sucrose was 6.1 s, which corresponds to a molecule comprising 400 nucleotides. A comparison of the kinetics of the histone cDNA-histone mRNA hybridization reaction with that of the globin cDNA-globin mRNA hybridization reaction indicates that the sequence complexity of the histone cDNA probe is approximately 3000 nucleotides; the estimated genetic complexity of the mRNAs is 2800 nucleotides. Properties and specificity of the histone mRNAs and cDNA have been reported (10, 11). Specificity of the cDNA probe is supported by hybridization with S phase polysomal RNA and lack of hybridization with polysomal RNA from GI cells or S phase cells treated with cytosine arabinoside. This indicates the absence of ribosomal RNA and tRNA sequences as well as any S phase sequences that are sensitive to inhibitors of DNA replication. The absence of ribosomal RNA and tRNA is further indicated by the lack of internal methylated ribonucleotides in the histone mRNA preparation (unpublished data). [3H}cDNA and either polysomal RNA or in vitro RNA transcripts were hybridized at 520 in sealed glass capillary tubes containing in a volume of 15 sl: 50% formamide, 0.5 M NaCl,
Proc. Natl. Acad. Sci. USA 74 (1977)
25 mM Hepes (pH 7.0), 1 mM EDTA, and 0.04 ng cDNA and RNA. Reactions were carried out in conditions of RNA excess. E. colh RNA was included in reaction mixtures when necessary so that the final amount of RNA was 3.5 gg. The reaction mixtures were assayed for hybrid formation by using Fraction IV single-strand specific Si nuclease isolated from Aspergillus oryzae (36). Each sample was incubated for 20 min in 2.0 ml of 30 mM sodium acetate/0.3 M NaCl/l mM ZnSO4/5% glycerol (pH 4.6) containing Si nuclease at a concentration sufficient to degrade at least 95% of the single-stranded nucleic acids present. The amount of radioactive DNA resistant to nuclease digestion was determined by trichloroacetic acid precipitation (15%). Utilization of a histone cDNA probe templated by HeLa S3 cell histone mRNAs is justifiable for detection of histone mRNA sequences isolated from WI-38 cells or transcribed from WI-38 cell chromatin. One would not expect significant differences in the histone genetic sequences of HeLa and WI-38 cells because both these cell lines are of human origin. The similarity of the histone genes in HeLa and WI-38 cells is substantiated by indistinguishable maximal extent of hybridization and tm values for hybrids formed between HeLa histone cDNA and HeLa histone mRNAs, between HeLa histone cDNA and HeLa S phase chromatin transcripts, and between HeLa histone cDNA and WI-38 cell S phase (12 h after stimulation) chromatin transcripts (tm is temperature at midpoint of melting curve of hybrids). Chromatin Dissociation, Fractionation, and Reconstitution. Total chromosomal proteins were prepared by dissociating chromatin in 3 M NaCl/5 M urea/10 mM Tris (pH 8.3) and pelleting the DNA at 150,000 X g for 36 hr. The proteins were fractionated into histone and nonhistone chromosomal protein fractions by chromatography on QAE Sephadex (37). Chromatin was reconstituted by the gradient dialysis procedure of Bekhor et al. (38). The details of-the reconstitution procedure (20) and evidence for fidelity of chromatin reconstitution (38-40) have been reported.
RESULTS Confluent monolayers of WI-38 human diploid fibroblasts can be induced to proliferate by replacing exhausted growth medium with fresh medium containing 20% fetal calf serum. The addition of serum to such cells triggers a complex and interdependent series of biochemical events (31, 32). An activation of DNA synthesis as measured by incorporation of [3H]thymidine into DNA was evident at 10 hr after stimulation of WI-38 cells and reached a maximum at 12 hr (Fig. la). The activation of DNA synthesis in WI-38 cells was confirmed by a similar (600-fold) increase in the percentage of nuclei labeled with [3H]thymidine as determined autoradiographically (Fig. lb). An increase in mitotic activity was observed between 16 and '20 hr (Fig. ic). Concomitant with the activation of DNA synthesis there is a stimulation of histone synthesis (4). The tight coupling between histone synthesis and DNA replication in WI-38 cells is suggested by the rapid and complete shutdown of histone synthesis by inhibition of DNA replication (4). Transcription of Histone mRNA Sequences after Stimulation of WI-38 Cells. To determine the availability of histone genes for transcription as a function of time after stimulation of WI-38 cells to proliferate, we examined in vitro transcripts of chromatin from confluent WI-38 cells, from WI-38 cells during the prereplicative phase (1, 4, and 7 hr after stimulation), and from cells at 10 and 12 hr after stimulation (S phase). The presence of histone mRNA sequences was assayed by hybrid formation with histone cDNA. The kinetics of hybridization
Proc. Natl. Acad. Sci. USA 74 (1977)
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FIG. 1. (a) DNA synthesis at various times after serum stimulation of WI-38 human diploid fibroblasts. Cells were labeled with [3H]thymidine. To determine the rate of DNA synthesis (cpm/Ug of DNA), cells were harvested and nuclei were isolated. Nuclei were washed twice with cold (40) 0.3 M perchloric acid, and nucleic acids were extracted with hot (900) 1 M perchloric acid. The amount of DNA present in nucleic acid extracts was assayed by the diphenylamine reaction. Each point represents the mean of at least four determinations; the range of values did not exceed 5%. (b) Labeled nuclei per 1000 cells at various times after serum stimulation of WI-38 human diploid fibroblasts. Cells were labeled with [3Hlthymidine. To determine the percentage of cells with [3H]thymidine labeled nuclei, cells were harvested, smeared on acid-washed microscope slides, and prepared for autoradiography. Autoradiographs were exposed for 14 days and stained with hematoxylin after development. The values were obtained by counting 2000 cells. Each value represents the mean of four determinations; the range of values did not exceed 7%. (c) Mitotic figures per 1000 cells at various times after serum stimulation of WI-38 human diploid fibroblasts. Colcemide was added at 12 hr after serum stimulation and, at the indicated times, cells were harvested, smeared on acid-washed microscope slides, fixed in alcohol/acetic acid, 3:1 (vol/vol), and stained with hematoxylin. The values were obtained by counting 2000 cells. Each point represents the mean of at least four determinations; the range of values did not exceed 7%.
of the histone cDNA with RNA transcripts from chromatin of WI-38 cells at various times after serum stimulation are shown in Fig. 2. There was a significant increase in the rate of hybridization at 10 hr after stimulation (Cotl/2 = 1.0), with the maximal rate of hybridization observed at 12 hr (COtl/2 = 4.0 X 10-1). In contrast to the limited extent of hybrid formation between histone cDNA and RNA transcripts from chromatin of confluent cells and of cells 1, 4, and 7 hr after stimulation (Cotl/2 = 180), the kinetics of the hybridization reaction of histone cDNA and RNA transcripts from S phase (12 hr) chromatin reveals a 500-fold activation of histone mRNA sequence transcription after stimulation of WI-38 cells to proliferate. A comparison of the reciprocal of C0t1/2 values of hybridization reactions between histone cDNA and RNA transcripts from chromatin as a function of time after stimulation to proliferate (Fig. 2b) clearly demonstrates that activation of
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FIG. 2. (a) Kinetics of annealing of histone [3H]cDNA to in vitro transcripts of chromatin from unstimulated (X) WI-38 cells, and WI-38 cells at 1 hr (3),4 hr (m), 7 hr (&), 10 hr (O), and 12 hr (-) after serum stimulation. Histone cDNA was also annealed to endogenous RNA isolated from S phase chromatin (A). Annealing reactions were carried out in a volume of 15 pl and the extent of histone cDNA-histone mRNA hybrid formation was determined by SI nuclease digestion. Hybridization values are expressed as percent of maximal hybridization (63-65%). (b) Plot of the reciprocal of Cotl/2 values of hybridization reactions between histone cDNA and in vitro transcripts from chromatin of WI-38 cells at various times after stimulation.
histone gene transcription parallels the onset of DNA synthesis in WI-38 fibroblasts (Fig. 1). Serum stimulation of semiconfluent WI-38 cells resulted in a time course and a maximal level for activation of histone genes similar to those observed when confluent cells were stimulated (data not shown). However, an increased level of histone gene transcription from chromatin of the semiconfluent cells was detected prior to stimulation and during the prereplicative period (Cotl/2 = 15). To eliminate the possibility that endogenous RNAs associated with S phase chromatin (12 hr) were contributing to hybrid formation of RNA transcripts with histone cDNA, S phase chromatin was placed in the transcription mixture without addition of E. coli RNA polymerase. The endogenous RNAs were isolated immediately, without incubation of the sample,
by the same procedure used to isolate transcripts and were analyzed for their ability to form hybrids with histone cDNA (Fig. 2). The low level of hybrid formation with histone cDNA demonstrates that endogenous histone-specific sequences associated with S phase chromatin do not contribute significantly to the hybridization observed with S phase chromatin transcripts. Association of Histone mRNA Sequences with Polyribosomes after Stimulation of WI-38 Cells. At various times after stimulation of WI-38 cells to proliferate, polysomal RNAs were examined for the presence of histone mRNA sequences. Polysomal RNAs from WI-38 cells prior to as well as 1, 7, 10, and
176
Biochemistry: Jansing et al.
Proc. Natl. Acad. Sci. USA 74 (1977) Z0
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12 hr after stimulation were assayed for their abilities to form hybrids with histone cDNA (Fig. 3). An increase in the representation of histone mRNA sequences in polysomal RNA was evident at 10 hr after stimulation (COtI/2 = 17.8) and a further increase was observed at 12 hr (COtl,2 = 63). Comparison of the time course of appearance of histone mRNA sequences on polysomes after stimulation (Fig. 3) with the time course of DNA synthesis after stimulation (Fig. 1) clearly indicates that an increased accumulation of histone mRNA sequences on polyribosomes parallels the stimulation of DNA synthesis. Histone mRNA sequences account for 0.27% of the total polysomal RNA at 12 hr after stimulation (compare COtl/2 of the histone cDNA-histone mRNA hybridization reaction, (1.7 X 10-2), with COtl/2 of the histone cDNA-12 hr polysomal RNA reaction, 6.3), which is consistent with the in vivo situation in which approximately 10% of the S phase protein synthesis is histone synthesis (4). Nonhistone Chromosomal Proteins and Transcription of Histone mRNA Sequences in WI-38 Cells. The role of chromosomal proteins in regulating the transcription of histone genes was directly examined by a series of chromatin reconstitution experiments. To assess the involvement of nonhistone chromosomal proteins in rendering histone genes transcribable, chromatin from confluent WI-38 cells was dissociated and reconstituted in the presence of added S phase (12 hr) nonhistone chromosomal proteins. RNA transcripts from the reconstituted chromatin were tested for ability to hybridize with histone cDNA. The data in Fig. 4 indicate that the COtl/2 of the hybridization reaction between histone cDNA and RNA transcripts from this reconstituted chromatin preparation (CotI/2 = 4 X 10-') was indistinguishable from that of the hybridization reaction between histone cDNA and S phase chromatin RNA transcripts (COt1/2 = 4 X 10-1) (Fig. 2). Transcription of histone mRNA sequences from chromatin of confluent WI-38 cells was unchanged after dissociation and reconstitution in the presence of the histone fraction of S phase chromatin (data not shown). These results suggest that nonhistone chromosomal proteins are responsible for determining the availability of histone genes for transcription and that a component of the S phase nonhistone chromosomal proteins serves to activate the
transcription of histone mRNA sequences. To examine the possibility that a component of the chromosomal proteins of confluent cells specifically restricts the availability of histone genes for transcription, S phase chromatin was dissociated and then reconstituted in the presence of added total chromosomal proteins from confluent cells. Transcripts from such reconstituted chromatin preparations exhibited kinetics of hybridization
-2
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FIG. 4. Kinetics of annealing of histone cDNA to in vitro transcripts of reconstituted chromatin. Chromatin (1 mg of DNA as chromatin) from confluent WI-38 cells was dissociated in 3 M NaCl/5 M urea/10 mM Tris (pH 8.3) and 1 mg of S phase (12 hr) nonhistone chromosomal proteins was added. Chromatin was then reconstituted from the mixture. Chromatin (1 mg DNA as chromatin) from S phase (12 hr) WI-38 cells was dissociated and similarly reconstituted in the presence of 1 mg of added total chromosomal proteins from confluent WI-38 cells. RNA transcripts from each of the reconstituted chromatins were annealed with histone [3H]cDNA. Hybrid formation was assayed by using Si nuclease as described in Materials and Methods. 0, GI chromatin plus S phase nonhistone chrosomal proteins; 0, S phase chromatin plus G, total chromosomal proteins. Protein-to-DNA ratios of the native and reconstituted WI-38 cell chromatin preparations ranged from 1.86 to 1.91.
with histone cDNA (Fig. 4) similar to those of native S phase chromatin transcripts (Fig. 2). A specific repressor of histone genes associated with chromatin of confluent WI-38 cells is therefore unlikely. DISCUSSION The results presented suggest that, when confluent WI-38 cells are stimulated to proliferate, histone mRNA becomes associated with polyribosomes concomitant with the activation of DNA synthesis. A similar activation of histone synthesis in intact WI-38 cells is observed (4). The present results also indicate that the ability of chromatin from WI-38 cells to serve as a template for in vitro transcription of histone mRNA sequences parallels the onset of DNA synthesis. The constant, low levels of DNA and histone synthesis observed in confluent WI-38 cells, as well as in WI-38 cells during the first 7 hr after stimulation to proliferate, are probably attributable to the few cells that have escaped contact inhibition. The limited extent of hybridization between histone cDNA and polysomal RNA or RNA transcripts from chromatin of confluent WI-38 cells, as well as of WI-38 cells during the prereplicative phase of the cell cycle, is also reasonably explained by the minor component of the cell population undergoing DNA replication. However, to establish these points conclusively it would be necessary to demonstrate that it is the cells that continue to proliferate in confluent cultures that exclusively contain histone mRNA sequences on polysomes and transcribe histone mRNA sequences. Evidence is provided that a component of the S phase nonhistone chromosomal proteins of WI-38 cells is responsible for rendering histone genes transcribable during the period of the cell cycle when DNA replication occurs. It is not clear whether the purported activator or derepressor molecule(s) is (are) synthesized and associated with the genome at the onset of S phase, is present in the nucleoplasm or cytoplasm and becomes associated with the genome to activate histone gene readout, or is a component of the genome during the prereplicative phase of the cell cycle which is modified at the time DNA replication begins. In this regard it should be noted that the phosphate groups of nonhistone chromosomal proteins have
Proc. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: jansing et al. been implicated in the control of histone gene transcription from chromatin (41, 42). Because the present in vitro transcription studies were carried out utilizing heterologous RNA polymerase, the possibility should not be overlooked that in vivo regulation of histone gene transcription may in part be mediated at other levels-perhaps involving RNA polymerase. Resolution of this question requires isolation of homologous RNA polymerase which exhibits in vvo specificity. The suggested role of nonhistone chromosomal proteins in the activation of histone gene transcription, concomitant with the onset of DNA replication, after stimulation of confluent WI-38 human diploid fibroblasts to proliferate is consistent with previous results that suggest that the transient expression of histone genes in continuously dividing HeLa S3 cells is regulated in an analogous manner (11, 30, 43). However, caution must be exercised in assuming that a similar mode of histone gene control is operative in all biological situations. For example, during oogenesis and early stages of development, histone mRNAs exhibit increased stability, and it is likely that regulation of histone gene expression under such conditions is mediated in part at a post-transcriptional level (44, 45). These studies were supported by research grants from the National Institutes of Health (GM 20535), the National Science Foundation (BMS75-18583), and the American Cancer Society (F75UF-4). 1. Spalding, J., Kajiwara, K. & Mueller, G. (1966) Proc. Natl. Acad. Sci. USA 56, 1535-1542. 2. Robbins, E. & Borun, T. W. (1967) Proc. Natl. Acad. Sci. USA
57,409-416. 3. Stein, G. S. & Borun, T. W. (1972) J. Cell Biol. 52, 292-307. 4. Stein, G. S. & Thrall, C. L. (1973) FEBS Lett. 34, 35-39. 5. Gallwitz, D. & Mueller, G. C. (1969) J. Biol. Chem. 244, 5947-5952. 6. Borun, T. W., Scharff, M. D. & Robbins, E. (1967) Proc. Natl. Acad. Sci. USA 58, 1977-1983. 7. Gallwitz, D. & Breindl, M. (1972) Biochem. Biophys. Res. Commun. 47, 1106-1111. 8. Jacobs-Lorena, M., Baglioni, C. & Borun, T. W. (1972) Proc. Natl. Acad. Sci. USA 69,2095-2099. 9. Stein, J. L., Thrall, C. L., Park, W. D., Mans, R. J. & Stein, G. S. (1975) Science 189, 557-558. 10. Stein, G. S., Park, W.,D., Thrall, C. L., Mans, R. J. & Stein, J. L. (1975) Biochem. Biophys. Res. Commun. 63,945-949. 11. Stein, G. S., Park, W. D., Thrall, C. L., Mans, R. J. & Stein, J. L. (1975) Nature 257,764-767. 12. Paul, J. & Gilmour, R. S. (1968) J. Mol. Biol. 34,305-316. 13. Teng, C. S., Teng, C. T. & Allfrey, V. G. (1971) J. Biol. Chem.
246,3597-3609. Kostraba, N. & Wang, T. Y. (1973) Exp. Cell Res. 80, 291296. 15. Spelsberg, T. C. & Hnilica, L. S. (1970) Biochem. J. 120, 435437. 14.
177
16. Stein, G. S., Spelsberg, T. C. & Kleinsmith, L. J. (1974) Science 183, 817-824. 17. Kleinsmith, L. J., Heidema, J. & Carroll, A. (1970) Nature 226, 1025-1026. 18. Bonner, J., Dahmus, M. E., Fambrough, D., Huang, R.-C., Marushige, K. & Tuan, D. Y. H. (1968) Science 159,47-56. 19. Elgin, S. R. & Bonner, J. (1972) Biochemistry 11, 772-781. 20. Stein, G. S. & Farber, J. (1972) Proc. Natl. Acad. Sci. USA 69, 2918-2921. 21. Stein, G. S., Chaudhuri, S. C. & Baserga, R. (1972) J. Biol. Chem. 247,3918-3922. 22. Baserga, R. (1974) Life Sci. 15, 1057-1071. 23. Karn, J., Johnson, E. M., Vidali, G. & Allfrey, V. G. (1974) J. Biol. Chem. 249, 667-677. 24. Bhorjee, J. & Pederson, T. (1972) Proc. Natl. Acad. Sci. USA 69,
3345-3349. 25. Stein, G. S. & Matthews, D. (1973) Science 181, 71-73. 26. Paul, J., Gilmour, R. S., Affara, N., Birnie, G., Harrison, P., Hell, A., Humphries, S., Windass, J. & Young, B. (1973) Cold Spring Harbor Symp. Quant. Biol. 38, 885-890. 27. Barrett, T., Maryanka, D., Hamlyn, P. H. & Gould, H. J. (1974) Proc. Natl. Acad. Sci. USA 71,5057-5061. 28. Chiu, J.-F., Tsai, Y., Sakuma, K. & Hnilica, L. S. (1975) J. Biol.
Chem. 250, 9431-9433. 29. Tsai, S. Y., Harris, S. E., Tsai, M. J. & O'Malley, B. W. (1976) J. Biol. Chem. 251,6475-6478. 30. Park, W. D., Stein, J. L. & Stein, G. S. (1976) Biochemistry 15, 3296-3300. 31. Rhode, S. L. & Ellem, K. A. 0. (1968) Exp. Cell Res. 53, 184204. 32. Rovera, G. & Baserga, R. (1971) J. Cell. Physiol. 77, 201-212. 33. Berg, D., Barrett, K. & Chamberlin, M. (1971) in Methods in Enzymology, eds. Grossman, L. & Moldave, K. (Academic Press, New York), Vol. XXI, pp. 506-509. 34. Thrall, C. L, Park, W. D., Rashba, W. H., Stein, J. L., Mans, R. J. & Stein, G. S. (1974) Biochem. Biophys. Res. Commun. 61, 1443-1449. 35. Mans, R. J. & Huff, N. J. (1975) J. Biol. Chem. 250, 36723678. 36. Vogt, V. (1973) Eur. J. Biochem. 33, 192-204. 37. Gilmour, R. S. & Paul, J. (1970) FEBS Lett. 9,242-244. 38. Bekhor, I., Kung, G. M. & Bonner, J. (1969) J. Mol. Biol. 39,
351-364. 39. Stein, G. S., Mans, R. J., Gabby, E. J., Stein, J. L., Davis, J. & Adawadkar, P. D. (1975) Biochemistry 14, 1859-1866. 40. Paul, J. & More, I. (1972) Nature New Biol. 239, 134-135. 41. Kleinsmith, L. J., Stein, J. L. & Stein, G. S. (1976) Proc. Natl. Acad. Sci. USA 73, 1174-1178. 42. Thomson, J., Stein, J. L., Kleinsmith, L. J. & Stein, G. S. (1976) Science 194, 428-430. 43. Stein, J. L., Reed, K. & Stein, G. S. (1976) Biochemistry 15, 3291-3295. 44. Farquhar, M. & McCarthy, B. (1973) Biochem. Biophys. Res. Commun. 53,515-521. 45. Skoultchi, A. & Gross, P. R. (1973) Proc. Natl. Acad. Sci. USA 70,
2840-2844.
Biochemistry
Activation of histone gene transcription by nonhistone chromosomal proteins in WI-38 human diploid fibroblasts (chromatin/reconstitution/cell proliferation/messenger RNA/complementary DNA)
ROBERT L. JANSING, JANET L. STEIN, AND GARY S. STEIN Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Fla. 32610
Communicated by George K. Davis, November 3,1976
ABSTRACT The regulation of histone gene expression was examined after confluent, nondividing WI-38 human diploid fibroblasts were stimulated to proliferate. Histone mRNA sequences were assayed by hybrid formation with an 3H-labeled single-stranded DNA complementary to histone mRNAs. Histone mRNA became associated with polyribosomes concomitant with the activation of DNA synthesis. The ability of chromatin from WI-38 cells to serve as a template for in vitro transcription of histone mRNA sequences parallels the onset of DNA replication. A role for nonhistone chromosomal proteins in the control of histone gene readout is suggested because, when chromatin from confluent WI-38 cells was dissociated and then reconstituted in the presence of S phase nonhistone chromosomal proteins, a 500-fold activation of histone mRNA sequence transcription was observed.
The synthesis of histones in continuously dividing cells as well as in nondividing cells that have been stimulated to proliferate is restricted to the S phase of the cell cycle (1-4) and depends on concomitant DNA replication (2, 3, 5). In continuously dividing HeLa S3 cells, a functional relationship between histone synthesis and DNA replication is further suggested by results from in vitro translation (5-8) and hybridization (9) studies that indicated the presence of histone mRNAs on the polyribosomes of S phase cells. In continuously dividing cells, transcriptional regulation of histone gene expression is suggested by results from studies showing that, when chromatin from S phase and G1 phase HeLa cells is transcribed in vitro, histone mRNA sequences are detected only in transcripts of S phase chromatin (10, 11). Nonhistone chromosomal proteins have been implicated in the regulation of gene expression in general (12-19) and in the control of transcription during the cell cycle specifically (17, 20-25). Chromatin reconstitution studies from several laboratories provide evidence that, among the complex and heterogeneous nonhistone chromosomal proteins, there are macromolecules which are responsible for the tissue-specific transcription of globin genes (26-28), the steroid hormone-dependent transcription of ovalbumin genes (29), and the transient expression of histone genes during the S phase of the cell cycle in continuously dividing HeLa cells (11, 30). An important question which arises is whether the mode of histone gene regulation observed in continuously dividing cells is operative in nondividing cells stimulated to divide. Since histones are intimately involved in the structure and packaging of the genome, understanding the mechanism by which histone genes are rendered transcribable should provide important clues to the control of cell proliferation. The studies reported here were directed toward defining the mechanism by which regulation of histone gene expression occurs after nondividing WI-38 human diploid fibroblasts are Abbreviations: Hepes, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; Cot, initial RNA concentration (mol/liter) X time (sec); COt1/2, Cot value at which hybridization is half completed. 173
stimulated to proliferate. Evidence is presented which suggests that, consistent with the situation observed in continuously dividing cells, a component of the nonhistone chromosomal proteins isolated from chromatin of WI-38 cells undergoing DNA replication is responsible for the activation of histone mRNA sequence transcription.
MATERIALS AND METHODS Cell Culture. Human diploid WI-38 fibroblasts were grown to confluence in monolayer culture in Eagle's basal medium containing 10% fetal calf serum. The cells were cultivated at 370 in a moist atmosphere containing 5% CO2. Confluent cells were stimulated to proliferate by replacing the exhausted growth medium with fresh Eagle's basal medium containing 20% fetal calf serum. It has been shown that under these conditions approximately 60% of the cells are stimulated to undergo DNA replication and mitotic division (23, 31, 32). The cells used in this study ranged from passage 28 to passage 30. Isolation of Nuclei and Chromatin. Nuclei and chromatin were isolated at 40 as described previously (4, 21). Cells were harvested with a rubber policeman, washed three times with 80 volumes of Earle's balanced salt solution, and lysed with 80 volumes of 80 mM NaCI/20 mM EDTA/1% Triton X-100 (pH 7.2). Nuclei were pelleted by centrifugation at 1000 X g for 3 min and washed twice with the lysing medium. The nuclei were washed twice with 0.15 M NaCI/10 mM Tris (pH 8.0) and pelleted by centrifugation at 1000 X g for 3 min. Nuclei isolated in this manner are largely free of visible cytoplasmic contamination when examined by phase contrast microscopy. The nuclear pellet was disrupted by mechanical agitation and the nuclei were lysed in 60 volumes of distilled water. The chromatin was allowed to swell in an ice bath for 20 min and was then pelleted by centrifugation at 10,000 X g for 20 min. Isolation of Polysomal RNA. Cells were harvested with a rubber policeman, washed three times with Earle's balanced salt solution, and pelleted by centrifugation at 1000 X g for 2 min. The pellet was drained well and resuspended in 10 volumes of 10 mM KCI/10 mM Tris/1.5 mM MgCl2 (pH 7.4). The cell suspension was transferred to a Dounce homogenizer, and after 20 min the cells were lysed by homogenization with a tightly fitting pestle. The homogenate was centrifuged at 15,000 X g for 15 min to pellet nuclei and mitochondria. The supernatant was transferred to 8-ml polycarbonate tubes and centrifuged at 100,000 X g for 90 min. All glassware and solutions were treated with diethylpyrocarbonate and then autoclaved. The pelleted polyribosomes were resuspended in 1 % sodium dodecyl sulfate/0.1 M NaCl/10 mM sodium acetate/1 mM EDTA (pH 5.4). Polysomal RNA was extracted twice with 1 volume of purified phenol plus 1 volume of chloroform/isoamyl alcohol, 24:1 (vol/vol), followed by two extractions with 1
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volume of chloroform/isoamyl alcohol. The RNA was precipitated with 2 volumes of ethanol, centrifuged at 3000 X g for 15 min, and resuspended in 25 mM N-2-hydroxyethylpiperazine-N'-ethanesulfonic acid (Hepes)/0.5 M NaCl/1 mM EDTA (pH 7.0) for hybridization analysis. In Vitro Transcription. RNA was transcribed by using Fraction V Escherichia coli RNA polymerase prepared according to the method of Berg et al. (33). Transcription was carried out for 60 min at 370 in a Dounce homogenizer fitted with a wide-clearance pestle, and the reaction mixture was periodically homogenized to maintain chromatin solubility. The incubation mixture in a final volume of 3.4 ml contained 40 mM Tris (pH 8.0), 4 mM MgCl2, 1 mM MnCl2, 20 gM EDTA, 0.008% 2-mercaptoethanol, 0.4 mM each of ATP, CTP, UTP, and GTP, 150 gg of DNA as chromatin/ml, and 200 units of RNA polymerase. The reaction mixture was brought to a concentration of 1% sodium dodecyl sulfate, 0.1 M NaCl, 10 mM sodium acetate, and 1 mM EDTA (pH 5.4) and was incubated at 370 for 15 min. Nucleic acids were extracted as described for polysomal RNA and precipitated with 2 volumes of ethanol. The pellet was resuspended in 10 mM Tris/0.1 M NaCl/5 mM MgCl2 (pH 7.4) containing DNase I (Sigma) at 40,ug/ml and incubated at 370 for 60 min. The reaction mixture was deproteinized by one extraction with phenol/chloroform/isoamyl alcohol, 1:1:0.04 (vol/vol), and two extractions with chloroform/isoamyl alcohol, 24:1 (vol/vol). The aqueous phase containing the RNA transcripts was chromatographed on Sephadex G-50 (fine) and eluted with 0.1 M NaCl/10 mM sodium acetate/i mM EDTA (pH 5.4). RNA was precipitated with 2 volumes of ethanol and resuspended in 25 mM Hepes/0.5 M NaCl/1 mM ElDTA (pH 7.0) for hybridization analysis. Hybridization Analysis. Histone [3H]cDNA was synthesized as previously described (34). We isolated 7-llS RNA from the polysomes of S phase HeLa S3 cells by repeated sucrose gradient fractionations, and RNAs containing poly(A) were removed by oligo(dT)-cellulose chromatography. The remaining 7-llS RNAs (histone mRNAs) directed the synthesis of all five classes of histones in a cell-free protein synthesizing system derived from wheat germ (34). Poly(A) was added to the 3' termini of the histone mRNAs with an ATP:polynucleotidylexotransferase (E.C. 2.7.7.19) prepared from maize seedlings (35). The polyadenylylated mRNAs were then transcribed with RNA-dependent DNA polymerase from avian myeloblastosis virus (Schwarz/Mann) in the presence of actinomycin D with dT1o as a primer. The mean sedimentation coefficient of the cDNA in alkaline sucrose was 6.1 s, which corresponds to a molecule comprising 400 nucleotides. A comparison of the kinetics of the histone cDNA-histone mRNA hybridization reaction with that of the globin cDNA-globin mRNA hybridization reaction indicates that the sequence complexity of the histone cDNA probe is approximately 3000 nucleotides; the estimated genetic complexity of the mRNAs is 2800 nucleotides. Properties and specificity of the histone mRNAs and cDNA have been reported (10, 11). Specificity of the cDNA probe is supported by hybridization with S phase polysomal RNA and lack of hybridization with polysomal RNA from GI cells or S phase cells treated with cytosine arabinoside. This indicates the absence of ribosomal RNA and tRNA sequences as well as any S phase sequences that are sensitive to inhibitors of DNA replication. The absence of ribosomal RNA and tRNA is further indicated by the lack of internal methylated ribonucleotides in the histone mRNA preparation (unpublished data). [3H}cDNA and either polysomal RNA or in vitro RNA transcripts were hybridized at 520 in sealed glass capillary tubes containing in a volume of 15 sl: 50% formamide, 0.5 M NaCl,
Proc. Natl. Acad. Sci. USA 74 (1977)
25 mM Hepes (pH 7.0), 1 mM EDTA, and 0.04 ng cDNA and RNA. Reactions were carried out in conditions of RNA excess. E. colh RNA was included in reaction mixtures when necessary so that the final amount of RNA was 3.5 gg. The reaction mixtures were assayed for hybrid formation by using Fraction IV single-strand specific Si nuclease isolated from Aspergillus oryzae (36). Each sample was incubated for 20 min in 2.0 ml of 30 mM sodium acetate/0.3 M NaCl/l mM ZnSO4/5% glycerol (pH 4.6) containing Si nuclease at a concentration sufficient to degrade at least 95% of the single-stranded nucleic acids present. The amount of radioactive DNA resistant to nuclease digestion was determined by trichloroacetic acid precipitation (15%). Utilization of a histone cDNA probe templated by HeLa S3 cell histone mRNAs is justifiable for detection of histone mRNA sequences isolated from WI-38 cells or transcribed from WI-38 cell chromatin. One would not expect significant differences in the histone genetic sequences of HeLa and WI-38 cells because both these cell lines are of human origin. The similarity of the histone genes in HeLa and WI-38 cells is substantiated by indistinguishable maximal extent of hybridization and tm values for hybrids formed between HeLa histone cDNA and HeLa histone mRNAs, between HeLa histone cDNA and HeLa S phase chromatin transcripts, and between HeLa histone cDNA and WI-38 cell S phase (12 h after stimulation) chromatin transcripts (tm is temperature at midpoint of melting curve of hybrids). Chromatin Dissociation, Fractionation, and Reconstitution. Total chromosomal proteins were prepared by dissociating chromatin in 3 M NaCl/5 M urea/10 mM Tris (pH 8.3) and pelleting the DNA at 150,000 X g for 36 hr. The proteins were fractionated into histone and nonhistone chromosomal protein fractions by chromatography on QAE Sephadex (37). Chromatin was reconstituted by the gradient dialysis procedure of Bekhor et al. (38). The details of-the reconstitution procedure (20) and evidence for fidelity of chromatin reconstitution (38-40) have been reported.
RESULTS Confluent monolayers of WI-38 human diploid fibroblasts can be induced to proliferate by replacing exhausted growth medium with fresh medium containing 20% fetal calf serum. The addition of serum to such cells triggers a complex and interdependent series of biochemical events (31, 32). An activation of DNA synthesis as measured by incorporation of [3H]thymidine into DNA was evident at 10 hr after stimulation of WI-38 cells and reached a maximum at 12 hr (Fig. la). The activation of DNA synthesis in WI-38 cells was confirmed by a similar (600-fold) increase in the percentage of nuclei labeled with [3H]thymidine as determined autoradiographically (Fig. lb). An increase in mitotic activity was observed between 16 and '20 hr (Fig. ic). Concomitant with the activation of DNA synthesis there is a stimulation of histone synthesis (4). The tight coupling between histone synthesis and DNA replication in WI-38 cells is suggested by the rapid and complete shutdown of histone synthesis by inhibition of DNA replication (4). Transcription of Histone mRNA Sequences after Stimulation of WI-38 Cells. To determine the availability of histone genes for transcription as a function of time after stimulation of WI-38 cells to proliferate, we examined in vitro transcripts of chromatin from confluent WI-38 cells, from WI-38 cells during the prereplicative phase (1, 4, and 7 hr after stimulation), and from cells at 10 and 12 hr after stimulation (S phase). The presence of histone mRNA sequences was assayed by hybrid formation with histone cDNA. The kinetics of hybridization
Proc. Natl. Acad. Sci. USA 74 (1977)
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FIG. 1. (a) DNA synthesis at various times after serum stimulation of WI-38 human diploid fibroblasts. Cells were labeled with [3H]thymidine. To determine the rate of DNA synthesis (cpm/Ug of DNA), cells were harvested and nuclei were isolated. Nuclei were washed twice with cold (40) 0.3 M perchloric acid, and nucleic acids were extracted with hot (900) 1 M perchloric acid. The amount of DNA present in nucleic acid extracts was assayed by the diphenylamine reaction. Each point represents the mean of at least four determinations; the range of values did not exceed 5%. (b) Labeled nuclei per 1000 cells at various times after serum stimulation of WI-38 human diploid fibroblasts. Cells were labeled with [3Hlthymidine. To determine the percentage of cells with [3H]thymidine labeled nuclei, cells were harvested, smeared on acid-washed microscope slides, and prepared for autoradiography. Autoradiographs were exposed for 14 days and stained with hematoxylin after development. The values were obtained by counting 2000 cells. Each value represents the mean of four determinations; the range of values did not exceed 7%. (c) Mitotic figures per 1000 cells at various times after serum stimulation of WI-38 human diploid fibroblasts. Colcemide was added at 12 hr after serum stimulation and, at the indicated times, cells were harvested, smeared on acid-washed microscope slides, fixed in alcohol/acetic acid, 3:1 (vol/vol), and stained with hematoxylin. The values were obtained by counting 2000 cells. Each point represents the mean of at least four determinations; the range of values did not exceed 7%.
of the histone cDNA with RNA transcripts from chromatin of WI-38 cells at various times after serum stimulation are shown in Fig. 2. There was a significant increase in the rate of hybridization at 10 hr after stimulation (Cotl/2 = 1.0), with the maximal rate of hybridization observed at 12 hr (COtl/2 = 4.0 X 10-1). In contrast to the limited extent of hybrid formation between histone cDNA and RNA transcripts from chromatin of confluent cells and of cells 1, 4, and 7 hr after stimulation (Cotl/2 = 180), the kinetics of the hybridization reaction of histone cDNA and RNA transcripts from S phase (12 hr) chromatin reveals a 500-fold activation of histone mRNA sequence transcription after stimulation of WI-38 cells to proliferate. A comparison of the reciprocal of C0t1/2 values of hybridization reactions between histone cDNA and RNA transcripts from chromatin as a function of time after stimulation to proliferate (Fig. 2b) clearly demonstrates that activation of
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FIG. 2. (a) Kinetics of annealing of histone [3H]cDNA to in vitro transcripts of chromatin from unstimulated (X) WI-38 cells, and WI-38 cells at 1 hr (3),4 hr (m), 7 hr (&), 10 hr (O), and 12 hr (-) after serum stimulation. Histone cDNA was also annealed to endogenous RNA isolated from S phase chromatin (A). Annealing reactions were carried out in a volume of 15 pl and the extent of histone cDNA-histone mRNA hybrid formation was determined by SI nuclease digestion. Hybridization values are expressed as percent of maximal hybridization (63-65%). (b) Plot of the reciprocal of Cotl/2 values of hybridization reactions between histone cDNA and in vitro transcripts from chromatin of WI-38 cells at various times after stimulation.
histone gene transcription parallels the onset of DNA synthesis in WI-38 fibroblasts (Fig. 1). Serum stimulation of semiconfluent WI-38 cells resulted in a time course and a maximal level for activation of histone genes similar to those observed when confluent cells were stimulated (data not shown). However, an increased level of histone gene transcription from chromatin of the semiconfluent cells was detected prior to stimulation and during the prereplicative period (Cotl/2 = 15). To eliminate the possibility that endogenous RNAs associated with S phase chromatin (12 hr) were contributing to hybrid formation of RNA transcripts with histone cDNA, S phase chromatin was placed in the transcription mixture without addition of E. coli RNA polymerase. The endogenous RNAs were isolated immediately, without incubation of the sample,
by the same procedure used to isolate transcripts and were analyzed for their ability to form hybrids with histone cDNA (Fig. 2). The low level of hybrid formation with histone cDNA demonstrates that endogenous histone-specific sequences associated with S phase chromatin do not contribute significantly to the hybridization observed with S phase chromatin transcripts. Association of Histone mRNA Sequences with Polyribosomes after Stimulation of WI-38 Cells. At various times after stimulation of WI-38 cells to proliferate, polysomal RNAs were examined for the presence of histone mRNA sequences. Polysomal RNAs from WI-38 cells prior to as well as 1, 7, 10, and
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12 hr after stimulation were assayed for their abilities to form hybrids with histone cDNA (Fig. 3). An increase in the representation of histone mRNA sequences in polysomal RNA was evident at 10 hr after stimulation (COtI/2 = 17.8) and a further increase was observed at 12 hr (COtl,2 = 63). Comparison of the time course of appearance of histone mRNA sequences on polysomes after stimulation (Fig. 3) with the time course of DNA synthesis after stimulation (Fig. 1) clearly indicates that an increased accumulation of histone mRNA sequences on polyribosomes parallels the stimulation of DNA synthesis. Histone mRNA sequences account for 0.27% of the total polysomal RNA at 12 hr after stimulation (compare COtl/2 of the histone cDNA-histone mRNA hybridization reaction, (1.7 X 10-2), with COtl/2 of the histone cDNA-12 hr polysomal RNA reaction, 6.3), which is consistent with the in vivo situation in which approximately 10% of the S phase protein synthesis is histone synthesis (4). Nonhistone Chromosomal Proteins and Transcription of Histone mRNA Sequences in WI-38 Cells. The role of chromosomal proteins in regulating the transcription of histone genes was directly examined by a series of chromatin reconstitution experiments. To assess the involvement of nonhistone chromosomal proteins in rendering histone genes transcribable, chromatin from confluent WI-38 cells was dissociated and reconstituted in the presence of added S phase (12 hr) nonhistone chromosomal proteins. RNA transcripts from the reconstituted chromatin were tested for ability to hybridize with histone cDNA. The data in Fig. 4 indicate that the COtl/2 of the hybridization reaction between histone cDNA and RNA transcripts from this reconstituted chromatin preparation (CotI/2 = 4 X 10-') was indistinguishable from that of the hybridization reaction between histone cDNA and S phase chromatin RNA transcripts (COt1/2 = 4 X 10-1) (Fig. 2). Transcription of histone mRNA sequences from chromatin of confluent WI-38 cells was unchanged after dissociation and reconstitution in the presence of the histone fraction of S phase chromatin (data not shown). These results suggest that nonhistone chromosomal proteins are responsible for determining the availability of histone genes for transcription and that a component of the S phase nonhistone chromosomal proteins serves to activate the
transcription of histone mRNA sequences. To examine the possibility that a component of the chromosomal proteins of confluent cells specifically restricts the availability of histone genes for transcription, S phase chromatin was dissociated and then reconstituted in the presence of added total chromosomal proteins from confluent cells. Transcripts from such reconstituted chromatin preparations exhibited kinetics of hybridization
-2
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FIG. 4. Kinetics of annealing of histone cDNA to in vitro transcripts of reconstituted chromatin. Chromatin (1 mg of DNA as chromatin) from confluent WI-38 cells was dissociated in 3 M NaCl/5 M urea/10 mM Tris (pH 8.3) and 1 mg of S phase (12 hr) nonhistone chromosomal proteins was added. Chromatin was then reconstituted from the mixture. Chromatin (1 mg DNA as chromatin) from S phase (12 hr) WI-38 cells was dissociated and similarly reconstituted in the presence of 1 mg of added total chromosomal proteins from confluent WI-38 cells. RNA transcripts from each of the reconstituted chromatins were annealed with histone [3H]cDNA. Hybrid formation was assayed by using Si nuclease as described in Materials and Methods. 0, GI chromatin plus S phase nonhistone chrosomal proteins; 0, S phase chromatin plus G, total chromosomal proteins. Protein-to-DNA ratios of the native and reconstituted WI-38 cell chromatin preparations ranged from 1.86 to 1.91.
with histone cDNA (Fig. 4) similar to those of native S phase chromatin transcripts (Fig. 2). A specific repressor of histone genes associated with chromatin of confluent WI-38 cells is therefore unlikely. DISCUSSION The results presented suggest that, when confluent WI-38 cells are stimulated to proliferate, histone mRNA becomes associated with polyribosomes concomitant with the activation of DNA synthesis. A similar activation of histone synthesis in intact WI-38 cells is observed (4). The present results also indicate that the ability of chromatin from WI-38 cells to serve as a template for in vitro transcription of histone mRNA sequences parallels the onset of DNA synthesis. The constant, low levels of DNA and histone synthesis observed in confluent WI-38 cells, as well as in WI-38 cells during the first 7 hr after stimulation to proliferate, are probably attributable to the few cells that have escaped contact inhibition. The limited extent of hybridization between histone cDNA and polysomal RNA or RNA transcripts from chromatin of confluent WI-38 cells, as well as of WI-38 cells during the prereplicative phase of the cell cycle, is also reasonably explained by the minor component of the cell population undergoing DNA replication. However, to establish these points conclusively it would be necessary to demonstrate that it is the cells that continue to proliferate in confluent cultures that exclusively contain histone mRNA sequences on polysomes and transcribe histone mRNA sequences. Evidence is provided that a component of the S phase nonhistone chromosomal proteins of WI-38 cells is responsible for rendering histone genes transcribable during the period of the cell cycle when DNA replication occurs. It is not clear whether the purported activator or derepressor molecule(s) is (are) synthesized and associated with the genome at the onset of S phase, is present in the nucleoplasm or cytoplasm and becomes associated with the genome to activate histone gene readout, or is a component of the genome during the prereplicative phase of the cell cycle which is modified at the time DNA replication begins. In this regard it should be noted that the phosphate groups of nonhistone chromosomal proteins have
Proc. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: jansing et al. been implicated in the control of histone gene transcription from chromatin (41, 42). Because the present in vitro transcription studies were carried out utilizing heterologous RNA polymerase, the possibility should not be overlooked that in vivo regulation of histone gene transcription may in part be mediated at other levels-perhaps involving RNA polymerase. Resolution of this question requires isolation of homologous RNA polymerase which exhibits in vvo specificity. The suggested role of nonhistone chromosomal proteins in the activation of histone gene transcription, concomitant with the onset of DNA replication, after stimulation of confluent WI-38 human diploid fibroblasts to proliferate is consistent with previous results that suggest that the transient expression of histone genes in continuously dividing HeLa S3 cells is regulated in an analogous manner (11, 30, 43). However, caution must be exercised in assuming that a similar mode of histone gene control is operative in all biological situations. For example, during oogenesis and early stages of development, histone mRNAs exhibit increased stability, and it is likely that regulation of histone gene expression under such conditions is mediated in part at a post-transcriptional level (44, 45). These studies were supported by research grants from the National Institutes of Health (GM 20535), the National Science Foundation (BMS75-18583), and the American Cancer Society (F75UF-4). 1. Spalding, J., Kajiwara, K. & Mueller, G. (1966) Proc. Natl. Acad. Sci. USA 56, 1535-1542. 2. Robbins, E. & Borun, T. W. (1967) Proc. Natl. Acad. Sci. USA
57,409-416. 3. Stein, G. S. & Borun, T. W. (1972) J. Cell Biol. 52, 292-307. 4. Stein, G. S. & Thrall, C. L. (1973) FEBS Lett. 34, 35-39. 5. Gallwitz, D. & Mueller, G. C. (1969) J. Biol. Chem. 244, 5947-5952. 6. Borun, T. W., Scharff, M. D. & Robbins, E. (1967) Proc. Natl. Acad. Sci. USA 58, 1977-1983. 7. Gallwitz, D. & Breindl, M. (1972) Biochem. Biophys. Res. Commun. 47, 1106-1111. 8. Jacobs-Lorena, M., Baglioni, C. & Borun, T. W. (1972) Proc. Natl. Acad. Sci. USA 69,2095-2099. 9. Stein, J. L., Thrall, C. L., Park, W. D., Mans, R. J. & Stein, G. S. (1975) Science 189, 557-558. 10. Stein, G. S., Park, W.,D., Thrall, C. L., Mans, R. J. & Stein, J. L. (1975) Biochem. Biophys. Res. Commun. 63,945-949. 11. Stein, G. S., Park, W. D., Thrall, C. L., Mans, R. J. & Stein, J. L. (1975) Nature 257,764-767. 12. Paul, J. & Gilmour, R. S. (1968) J. Mol. Biol. 34,305-316. 13. Teng, C. S., Teng, C. T. & Allfrey, V. G. (1971) J. Biol. Chem.
246,3597-3609. Kostraba, N. & Wang, T. Y. (1973) Exp. Cell Res. 80, 291296. 15. Spelsberg, T. C. & Hnilica, L. S. (1970) Biochem. J. 120, 435437. 14.
177
16. Stein, G. S., Spelsberg, T. C. & Kleinsmith, L. J. (1974) Science 183, 817-824. 17. Kleinsmith, L. J., Heidema, J. & Carroll, A. (1970) Nature 226, 1025-1026. 18. Bonner, J., Dahmus, M. E., Fambrough, D., Huang, R.-C., Marushige, K. & Tuan, D. Y. H. (1968) Science 159,47-56. 19. Elgin, S. R. & Bonner, J. (1972) Biochemistry 11, 772-781. 20. Stein, G. S. & Farber, J. (1972) Proc. Natl. Acad. Sci. USA 69, 2918-2921. 21. Stein, G. S., Chaudhuri, S. C. & Baserga, R. (1972) J. Biol. Chem. 247,3918-3922. 22. Baserga, R. (1974) Life Sci. 15, 1057-1071. 23. Karn, J., Johnson, E. M., Vidali, G. & Allfrey, V. G. (1974) J. Biol. Chem. 249, 667-677. 24. Bhorjee, J. & Pederson, T. (1972) Proc. Natl. Acad. Sci. USA 69,
3345-3349. 25. Stein, G. S. & Matthews, D. (1973) Science 181, 71-73. 26. Paul, J., Gilmour, R. S., Affara, N., Birnie, G., Harrison, P., Hell, A., Humphries, S., Windass, J. & Young, B. (1973) Cold Spring Harbor Symp. Quant. Biol. 38, 885-890. 27. Barrett, T., Maryanka, D., Hamlyn, P. H. & Gould, H. J. (1974) Proc. Natl. Acad. Sci. USA 71,5057-5061. 28. Chiu, J.-F., Tsai, Y., Sakuma, K. & Hnilica, L. S. (1975) J. Biol.
Chem. 250, 9431-9433. 29. Tsai, S. Y., Harris, S. E., Tsai, M. J. & O'Malley, B. W. (1976) J. Biol. Chem. 251,6475-6478. 30. Park, W. D., Stein, J. L. & Stein, G. S. (1976) Biochemistry 15, 3296-3300. 31. Rhode, S. L. & Ellem, K. A. 0. (1968) Exp. Cell Res. 53, 184204. 32. Rovera, G. & Baserga, R. (1971) J. Cell. Physiol. 77, 201-212. 33. Berg, D., Barrett, K. & Chamberlin, M. (1971) in Methods in Enzymology, eds. Grossman, L. & Moldave, K. (Academic Press, New York), Vol. XXI, pp. 506-509. 34. Thrall, C. L, Park, W. D., Rashba, W. H., Stein, J. L., Mans, R. J. & Stein, G. S. (1974) Biochem. Biophys. Res. Commun. 61, 1443-1449. 35. Mans, R. J. & Huff, N. J. (1975) J. Biol. Chem. 250, 36723678. 36. Vogt, V. (1973) Eur. J. Biochem. 33, 192-204. 37. Gilmour, R. S. & Paul, J. (1970) FEBS Lett. 9,242-244. 38. Bekhor, I., Kung, G. M. & Bonner, J. (1969) J. Mol. Biol. 39,
351-364. 39. Stein, G. S., Mans, R. J., Gabby, E. J., Stein, J. L., Davis, J. & Adawadkar, P. D. (1975) Biochemistry 14, 1859-1866. 40. Paul, J. & More, I. (1972) Nature New Biol. 239, 134-135. 41. Kleinsmith, L. J., Stein, J. L. & Stein, G. S. (1976) Proc. Natl. Acad. Sci. USA 73, 1174-1178. 42. Thomson, J., Stein, J. L., Kleinsmith, L. J. & Stein, G. S. (1976) Science 194, 428-430. 43. Stein, J. L., Reed, K. & Stein, G. S. (1976) Biochemistry 15, 3291-3295. 44. Farquhar, M. & McCarthy, B. (1973) Biochem. Biophys. Res. Commun. 53,515-521. 45. Skoultchi, A. & Gross, P. R. (1973) Proc. Natl. Acad. Sci. USA 70,
2840-2844.
