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Selective expression of histone genes in mouse-human hybrid cells
Experimental Cell Research 159 (1985) 280-286
Selective Expression of Histone Genes in Mouse-Human Hybrid Cells FARHAD MARASHI, 1'* CARLO M. C R O C E , 2 JANET L. STEIN 1 and GARY S. STEIN 1 1University of Florida, College of Medicine, Gainesville, FL 32610, and 2The Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104, USA
Mouse-human hybrid cells preferentially segregating mouse chromosomes contain predominantly human histone mRNAs and synthesize human histone proteins. In contrast, hybrids segregating human chromosomes contain both human and murine histone mRNAs, yet synthesize only mouse histone proteins. These results suggest transcriptional control of histone gene expression in hybrids segregating mouse chromosomes and post-transcriptional regulation in hybrids segregating human chromosomes. © 1985AcademicPress, Inc.
Mammalian histone genes are represented as a family of moderately reiterated sequences with variations in the structure, organization and regulation of the different copies [1-7]. Both human and murine histone genes have been isolated as a series of clusters, most clusters containing several core or core together with H1 histone-coding sequences. The functional heterogeneity of the mammalian histone genes is reflected by expression of most [3, 8-12] but not all [13-17] histone genes in conjunction with DNA replication and by variations in the representation of histone protein and histone mRNA subspecies in cells under different biological circumstances [13-19]. Yet, beyond the localization of mRNA-coding regions [1-7], the identification of consensus sequences which putatively function as regulatory elements [I-7] and the analysis of 5' deletion mutants in vitro [20], our understanding of sequences and molecules which influence the expression of human and murine histone genes is limited. Somatic cell hybrids provide a viable system for defining levels of genetic control and for examining molecular parameters that influence the expression of specific genes. By using a series of mouse-human hybrid cell lines to examine expression of mammalian histone genes, we have observed that mouse-human hybrids preferentially segregating mouse chromosomes express only human histone genes, while hybrids segregating human chromosomes express both mouse and human histone genes. It appears that in hybrids segregating mouse chromosomes, expression of histone genes is mediated at the transcriptional level, while in cells segregating human chromosomes expression is regulated post-transcriptionally. * Present address: Room 220, CPL, Philips Petroleum, Bartlesville, OK 74004, USA.
Exp Cell Res 159 (1985)
Histone gene expression in m o u s e - h u m a n hybrid cells MATERIALS
AND
281
METHODS
Somatic Cell Hybrids Two types of human-mouse somatic cell hybrids were used in these studies: (1) hybrids obtained by fusion of HT1080 human diploid fibrosarcoma cells with mouse peritoneal macrophages (hybrids 5514F7 and 55-54F2) which retain the complete complement of human chromosomes and are segregating murine chromosomes; (2) hybrids obtained by fusion of HT1080 human fibrosarcoma cells with continuously dividing mouse TH02 cells (hybrids 56-05F1C16 and 56-05F5) which retain the complete complement of mouse chromosomes and are segrating human chromosomes. The presence of at least one copy of each murine and human chromosome in all hybrid cell lines studied permits assessment of the potential influence of all marine and human genes on both murine and human histone gene expression. Construction of the hybrid cell lines has been reported [21-25]. Representation of human and murine chromosomes has been confirmed by karyotype analysis following Giemsa staining and by isozyme analysis for markers assigned to each human and mouse chromosome. The presence of human and murine histone genes was confirmed by Southern blot analysis of restriction enzymedigested DNAs which were hybridized to 32P-labeled cloned human histone genes.
Total Cellular R N A Isolation and Electrophoretic Fractionation Approx. 5x l07 cells were lysed in 4.5 ml of a solution containing 1.3 mM Tris-HCl (pH 7.4), 0.7 mM EDTA, 1.3 I~g/ml PVS (polyvinylsulfonic acid potassium salt, Eastman Kodak Co., Rochester, N.Y.), 2.4 % (w/v) SDS and 0.9 mg/ml proteinase K. After a 15 min incubation at room temperature and the addition of 0.3 ml of a 5 M NaC1 solution, the aqueous phase was extracted twice with 2 vol of buffered phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v), and once with 1 vol of chloroform : isoamyl alcohol (24 : 1, v/v). Nucleic acids were precipitated with 3 vol of ethanol at -20°C in the presence of 100 mM sodium acetate, pH 5.5. Nucleic acids were recovered by centrifugation, resuspended in 2 ml of 10 mM Tris-HC1 (pH 7.4), 2 mM CaCI2, 10 mM MgCI2 and incubated at 37°C for 20 min in the presence of 0.1 mg/ml of DNase I (Sigma, electrophoretically pure) which had been pretreated with proteinase K for 2 h as described by Tullis & Rubin [31]. After addition of 0.05 vol of 5 M NaC1 and 0.25 vol of 10 % (w/v) SDS, the RNA solution was extracted with phenol and chloroform, and ethanol precipitated as described above. Total cellular RNAs were pelleted after precipitation with ethanol and dissolved in 20 ~tl of 50 % (w/v) urea, 0.01% (w/v) xylene cyanol FF. Samples were denatured in a boiling water bath for 2 min and quick chilled on ice prior to electrophoresis in a 6 % (w/v) polyacrylamide gel (acrylamide : bisacrylamide 29: 1, w/w) containing 50% (w/v) urea and 1x T B E (50 mM Tris/50 mM boric acid/l mM EDTA, pH 8.3). The gel was pre-electrophoresed for 30-40 min at 21 W, and the RNA samples were electrophoresed at 18-20 W for 8.5 h while maintaining the surface temperature of the gel at 50-60°C [26]. The electrophoretically fractionated RNAs were transferred from the acrylamide gel to a diazobenzyloxymethyl (DBM)-cellulose f'dter. The acrylamide gel was soaked in 50 mM NaOH for 20 rain at room temperature and neutralized by two subsequent soakings of the gel in 200 mM sodium phosphate (pH 5.5) for 20 min at room temperature. Finally the gel was equilibrated with 25 mM sodium phosphate (pH 5.5) prior to electrophoretic transfer in the same buffer at 0--4°C for 4.5 h, and the blot was allowed to cure undisturbed overnight. Aminobenzyloxy-methyl (ABM) paper was made and activated according to Alwine et al. [27].
Hybridization DBM blots were prehybridized at 50°C for 6 h in a solution containing 50% formamide/ 5xSSC(lxSSC: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.5)/1% glycine (v/v)/5xDenhardt (1 x Denhardt: 0.02 % each of Ficoll 400, Pharmacia Fine Chemicals, and polyvinylpyrrolidone, Sigma)/400 ~tg/ml E. coli nucleic acid. Hybridization was at 50°C for 48 h in 50% formamide/5×SSC/15 mM potassium phosphate, pH 7.2/5x Denhardt/200 Ixg/mlE. coli nucleic acids with nicktranslated plasmid DNAs containing cloned human histone genes, at approx. 1.5× l0 s cpm/ml. Nicktranslated DNA was denatured for 10 rain in a boiling water bath and quick-cooled before addition to the hybridization mixture. After hybridization the blots were washed sequentially in 5xSSC/1 xDenhardt at room temperature for 10 min and at 60°C for 30 min, then in 2xSSC/0.1% SDS and lxSSC/0.1% SDS, each time for 30 min at 60°C. DBM paper was blotted dry on Whatman 3MM
Exp CellRes 159 (1985)
282 M a r a s h i et al. paper and autoradiographed for an appropriate length of time using either preflashed or non-flashed XAR-5 film (Kodak) at -70°C with a Dupont high-speed screen.
RESULTS AND DISCUSSION To assess the expression of murine and human histone genes, total cellular RNA from mouse, human or hybrid cells was fractionated electrophoretically [26], transferred to diazotized cellulose [27] and histone mRNAs were identified by hybridization with 32p-labeled cloned genomic human histone DNA. Conservation of histone genes is sufficient to permit cross-species recognition. The isolation and characterization of the cloned human histone genes has been reported [1, 2, 4]. Electrophoretic fractionation of RNAs in the high resolution polyacrylamide gel system used in these studies results in separation of a series of histone mRNA subspecies which Lichtler et al. have shown by S1 nuclease protection experiments represent transcripts from different copies of the histone genes ([26] and unpublished results). The human histone mRNA subspecies represent transcripts from genes residing in several histone gene clusters which are located on at least two human chromosomes [32]. Moreover, differences in the electrophoretic mobilities of the murine and human histone mRNA subspecies permit us to distinguish between the mRNAs of mouse and human origin. Isolation of total cellular R N A yields greater than 90 % recovery, circumventing losses of RNA through nuclease activity and physical manipulations which generally occur during subcellular fractionation. Shown in fig. 1 are cellular RNAs from mouse--human hybrids which were hybridized with 32p-labelled cloned human H4 histone DNA. For comparison cellular RNAs from NP3 BALB/c mouse myeloma cells and HT1080 fibrosarcoma cells are also shown, indicating the resolution of three mouse H4 histone mRNA fractions and a single human H4 histone mRNA band. A similar distinction between the electrophoretic mobilities of murine and human H4 histone mRNAs was observed in several additional mouse and human cell lines. In 5514F7 and 55-54F2 hybrid cells which are segregating mouse chromosomes, only the human H4 histone mRNAs are present, despite retention of at least one copy of each murine chromosome. This observation is consistent with expression of only human rRNA genes in these same mouse-human hybrids (table 1 and see refs [22-24]). In contrast, both mouse and human H4 mRNAs are present in the 56-05F1C16 and 56-05F5 hybrids which are segregating human chromosomes, although only the murine ribosomal genes are expressed in these cells (table 1). It is interesting that in the hybrids in which both mouse and human histone mRNAs are present, the levels of both types of histone mRNA appear to vary in a coordinate manner with the level of DNA synthesis (data not shown). Analysis of histone gene expression in these hybrids was extended to include H3, H2A and H2B histone genes. As shown in fig. 2, in both human cells and mouse-human hybrids segregating mouse chromosomes, only the human H3 Exp Cell Res 159 (1985)
Histone gene expression in mouse-human hybrid cells
283
i
$
tD
~.. n-
4. 75
[-14
H3
Fig. 1. Detection of human and mouse H4 histone mRNA subspecies in mouse-human hybrid cells.
Total cellular RNA was fractionated electrophoretically in 6 % polyacrylamide gels, transferred to DBM paper and hybridized to nick-translated plasmid DNA (pF0108A) containing a human histone H4 gene. The blots were analyzed by fluorography. Hybrids are designated by number and the retention of all human (H) or mouse (M) chromosomes or the selective segregation of human (H ~, ) or mouse (M ~ ) chromosomes as indicated. Fig. 2. Detection of human and mouse H3 histone mRNA subspecies in mouse-human hybrid cells. Total cellular RNAs were fractionated by electrophoresis, transferred to DBM paper and hybridized to nick-translated plasmid DNA (pST519) containing human histone H3 sequences.
histone mRNA is represented while in cells segregating human chromosomes, the human H3 mRNAs as well as the three distinct murine H3 mRNA fractions are detected. Similarly, filter blots of total cellular RNAs from these same hybrid cells, hybridized with 32p-labeled cloned human H2A and H2B histone D N A (fig. 3), revealed only human H2A and H2B histone mRNAs in the cells segregating murine chromosomes, with both human and murine H2A and H2B histone
Table 1. Species-specific representation of histone mRNAs in mOuse-human hybrids Representation of histone mRNAs
Representation ol histone proteins
Cell line
Chromosome representation
Representation of rRNAs
H2A
H2B
H3
H4
H2A
H2B
H3
55-14F~ 55-54F2 56-05F1C16 56-05F5
H>M H>M M>H M>H
H H M M
H H M+H M+H
H H M+H M+H
H H M+H M+H
H H M+H M+H
H H M M
H H M M
H H M M
Exp Cell Res 159 (1985)
284 Marashi et al.
--
It)
0
Z
Fig. 3. Detection of human and mouse H2A and
H2B histone mRNA subspecies in mouse-human hybrid cells. The electrophoretically fractionated total cellular RNAs were transferred to DBM paper and hybridized to nick-translated plasmid DNA (pFF435B) containing human histone H2A and H2B sequences.
mRNAs in the cells segregating human chromosomes. In the 56-05F5 hybrid an additional RNA species is observed which is not represented in any of the mouse, human or other hybrid cell lines examined. While the identity of this RNA is not readily apparent, it may be a transcript from a histone gene expressed only under limited biological circumstances; quantitative and qualitative differences in histone gene expression have been observed in highly quiescent or terminally differentiated cells [13-19]. Alternatively, this additional histone transcript may result from the rearrangement of a human and/or mouse histone gene. Our results suggest that the presence of structurally intact histone genes is by itself insufficient to render them functional and that there are differences in the requirements for expression of the murine and human histone genes in actively proliferating cells. The transcription of both mouse and human histone genes in hybrids segregating human chromosomes, coupled with the expression of only human histone genes in hybrids segregating mouse chromosomes, may be explained by the functional representation of 'regulatory macromolecules' for murine histone gene expression only in those hybrids retaining both copies of specific mouse chromosomes. Retention of at least one copy of each mouse and human chromosome in both types of hybrids studied is compatible with this interpretation. Exp Cell Res 159 (1985)
Histone gene expression in mouse-human hybrid cells
285
Histone gene expression in mouse-human hybrids segregating human chromosomes appears to be more complex. Ajiro et al. [28] have studied the representation of mouse and human histone proteins in the same hybrids we used for analysis of histone mRNAs and have observed that the histone proteins expressed were those of the dominant parent. Because our results show that both murine and human histone mRNAs are present in hybrids segregating human chromosomes, it appears that there is a post-transcriptional or translational mechanism operative. Hsiung & Kucherlapati [29] also failed to detect human histone proteins in a systematic analysis of a series of hybrids, each containing the complete complement of mouse chromosomes and only limited numbers of specific human chromosomes. In contrast, cells segregating mouse chromosomes appear to regulate histone gene expression primarily at the transcriptional level, since only human histone mRNA subspecies are detected in these hybrids. However, the use of only peritoneal macrophage as the mouse component of hybrids segregating murine chromosomes precludes making the generalization that in all cells segregating mouse chromosomes, histone gene expression is posttranscriptionally controlled. A simple, definite interpretation is further complicated by results of Ajiro et al. [33] that in mouse-rat hybrid cells both mouse and rat H1 and H2B histone proteins are synthesized. From reports of several laboratories which have examined the expression of specific genes in mouse-human hybrids, it is becoming apparent that the minimal representation and the number of copies of chromosomes required for expression of different genes may vary. For example, in most mouse-human hybrids studied, suppression of mouse ribosomal gene expression occurs when mouse chromosomes are being segregated, while suppression of human ribosomal genes is observed in conjunction with segregation of human chromosomes, despite the presence of at least one copy of each mouse and human chromosome [22-24]. Yet, both rodent and human U1 small nuclear RNA genes remain active in hybrids containing subsets of human chromosomes [30]. In the case of histone genes in mouse-human hybrids either mouse or human histone expression can be suppressed but the level of regulation appears to depend on the dominant parent. While these results emphasize the complexity of cellular and molecular requirements for eukaryotic gene expression, they provide the basis for utilizing somatic cell hybrids to examine the interrelationships of products from genes residing in various chromosomes. These studies were supported by grants PCM80-18075 and PCM83-18177 from the NSF, GM32010 and GM20700 from the NIH, and 1-813 from the March of Dimes Birth Defects Foundation.
REFERENCES 1. Sierra, F, Lichtler, A, Marashi, F, Rickles, R, Van Dyke, T, Clark, S, Wells, J, Stein, J & Stein, G, Proc natl acad sci US 79 (1982) 1795. 2. Carozzi, N, Marashi, F, Plumb, M, Zimmerman, S, Zimmerman, A, Wells, J R E, Stein, G & Stein, J, Science 224 (1984) 1115. Exp Cell Res 159 (1985)
286 Marashi et al. 3. Plumb, M, Marashi, F, Green, L, Zimmerman, A, Zimmerman, S, Stein, J & Stein, G, Proc natl acad sci US 81 (1984) 434. 4. Marashi, F, Prokopp, K, Stein, J & Stein, G, Proc natl acad sci US 81 (1984) 1936. 5. Sittman, D B, Chiu, I-M, Pann, C-J, Cohn, R H, Kedes, L H & Marzluff, W F, Proc natl acad sci US 78 (1981) 4078. 6. Heintz, N, Zernik, M & Roeder, R G, Cell 24 (1981) 661. 7. Seiler-Tuyns, A & Birnstiel, M, J mol biol 151 (1981) 607. 8. Stein, G, Park, W D, Thrall, C L, Marts, R J & Stein, J L, Nature 257 (1975) 764. 9. Rickles, R, Marashi, F, Sierra, F, Wells, J, Stein, J & Stein, G, Proc natl acad sci US 79 (1982) 749. 10. Plumb, M, Stein, J L & Stein, G S, Nucl acids res 11 (1983) 2391. 11. Heintz, N, Sive, H L & Roeder, R G, Mol cell biol 3 (1983) 539. 12. Sittman, D, Graves, R A & Marzluff, W F, Proc natl acad sci US 80 (1983) 1849. 13. Wu, R & Bonner, W, Cell 27 (1981) 321. 14. Tarnowka, M A, Baglioni, C & Basilico, C, Cell 15 (1978) 163. 15. Waithe, W L, Renaud, J, Nadeau, P & Pallotila, D, Biochemistry 22 (1983) 1778. 16. Lennox, R W & Cohen, L W, J biol chem 258 (1983) 262. 17. Pehrson, J R & Cole, R D, Biochemistry 21 (1982) 456. 18. Stein, G S, Stein, J L, Baumbach, L, Leza, A, Lichtler, A, Marashi, F, Plumb, M, Rickles, R, Sierra, F & Van Dyke, T, Miami winter symp, vol. 19, p. 331 (1982). 19. Zweidler, A, Gene families of collagen and other structural proteins (ed D J Prockop & P C Champe) p. 47. Elsevier/North-Holland, New York (1980). 20. Sierra, F, Stein, G & Stein, J, Nucl acids res 11 (1983) 7069. 21. Croce, C M, Proc natl acad sci US 73 (1976) 3248. 22. Croce, C M, Talavera, A, Basilico, C & Miller, O J, Proc natl acad sci US 74 (1977) 694. 23. Miller, O J, Miller, D A, Dev, V G, Tantravahi, R & Croce, C M, Proc natl acad sci US 73 (1976) 4531. 24. Perry, R P, Kelly, DE, Schibler, V, Huebner, K & Croce, C M, J cell physiol 98 (1979) 553. 25. Huebner, K, Shander, M & Croce, C M, Cell 11 (1977) 25. 26. Lichtler, A C, Sierra, F, Clark, S, Wells, J R E, Stein, G S & Stein, J L, Nature 298 (1982) 195. 27. Alwine, J C, Kemp, D J, Parker, B A, Reiser, J, Renart, J, Stark, G R & Wahl, G M, Methods enzymol 68 (1979) 220. 28. Ajiro, K, Zweidler, A, Borun, T & Croce, C M, Proc natl acad sci US 75 (1978) 5599. 29. Hsiung, N & Kucherlapati, R, J cell biol 87 (1980) 227. 30. Lund, E, Bostock, C, Robertson, M, Christie, S, Mitchen, J L & Dahlberg, J E, Mol cell biol 3 (1983) 2211. 31. Tullis, R H & Rubin, H, Anal biochem 107 (1980) 260. 32. Green, L, Van Antwerpen, R, Stein, J, Stein, G, Tripputi, P, Emanuel, B, Selden, J &; Croce, C, Science 226 (1984) 838. 33. Ajiro, K, Kano-Tanaka, K, Higashida, H & Tanaka, T, Som cell genet 8 (1982) 377. Received December 18, 1984 Revised version received March 27, 1985
Exp Cell Res 159 (1985)
Selective Expression of Histone Genes in Mouse-Human Hybrid Cells FARHAD MARASHI, 1'* CARLO M. C R O C E , 2 JANET L. STEIN 1 and GARY S. STEIN 1 1University of Florida, College of Medicine, Gainesville, FL 32610, and 2The Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104, USA
Mouse-human hybrid cells preferentially segregating mouse chromosomes contain predominantly human histone mRNAs and synthesize human histone proteins. In contrast, hybrids segregating human chromosomes contain both human and murine histone mRNAs, yet synthesize only mouse histone proteins. These results suggest transcriptional control of histone gene expression in hybrids segregating mouse chromosomes and post-transcriptional regulation in hybrids segregating human chromosomes. © 1985AcademicPress, Inc.
Mammalian histone genes are represented as a family of moderately reiterated sequences with variations in the structure, organization and regulation of the different copies [1-7]. Both human and murine histone genes have been isolated as a series of clusters, most clusters containing several core or core together with H1 histone-coding sequences. The functional heterogeneity of the mammalian histone genes is reflected by expression of most [3, 8-12] but not all [13-17] histone genes in conjunction with DNA replication and by variations in the representation of histone protein and histone mRNA subspecies in cells under different biological circumstances [13-19]. Yet, beyond the localization of mRNA-coding regions [1-7], the identification of consensus sequences which putatively function as regulatory elements [I-7] and the analysis of 5' deletion mutants in vitro [20], our understanding of sequences and molecules which influence the expression of human and murine histone genes is limited. Somatic cell hybrids provide a viable system for defining levels of genetic control and for examining molecular parameters that influence the expression of specific genes. By using a series of mouse-human hybrid cell lines to examine expression of mammalian histone genes, we have observed that mouse-human hybrids preferentially segregating mouse chromosomes express only human histone genes, while hybrids segregating human chromosomes express both mouse and human histone genes. It appears that in hybrids segregating mouse chromosomes, expression of histone genes is mediated at the transcriptional level, while in cells segregating human chromosomes expression is regulated post-transcriptionally. * Present address: Room 220, CPL, Philips Petroleum, Bartlesville, OK 74004, USA.
Exp Cell Res 159 (1985)
Histone gene expression in m o u s e - h u m a n hybrid cells MATERIALS
AND
281
METHODS
Somatic Cell Hybrids Two types of human-mouse somatic cell hybrids were used in these studies: (1) hybrids obtained by fusion of HT1080 human diploid fibrosarcoma cells with mouse peritoneal macrophages (hybrids 5514F7 and 55-54F2) which retain the complete complement of human chromosomes and are segregating murine chromosomes; (2) hybrids obtained by fusion of HT1080 human fibrosarcoma cells with continuously dividing mouse TH02 cells (hybrids 56-05F1C16 and 56-05F5) which retain the complete complement of mouse chromosomes and are segrating human chromosomes. The presence of at least one copy of each murine and human chromosome in all hybrid cell lines studied permits assessment of the potential influence of all marine and human genes on both murine and human histone gene expression. Construction of the hybrid cell lines has been reported [21-25]. Representation of human and murine chromosomes has been confirmed by karyotype analysis following Giemsa staining and by isozyme analysis for markers assigned to each human and mouse chromosome. The presence of human and murine histone genes was confirmed by Southern blot analysis of restriction enzymedigested DNAs which were hybridized to 32P-labeled cloned human histone genes.
Total Cellular R N A Isolation and Electrophoretic Fractionation Approx. 5x l07 cells were lysed in 4.5 ml of a solution containing 1.3 mM Tris-HCl (pH 7.4), 0.7 mM EDTA, 1.3 I~g/ml PVS (polyvinylsulfonic acid potassium salt, Eastman Kodak Co., Rochester, N.Y.), 2.4 % (w/v) SDS and 0.9 mg/ml proteinase K. After a 15 min incubation at room temperature and the addition of 0.3 ml of a 5 M NaC1 solution, the aqueous phase was extracted twice with 2 vol of buffered phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v), and once with 1 vol of chloroform : isoamyl alcohol (24 : 1, v/v). Nucleic acids were precipitated with 3 vol of ethanol at -20°C in the presence of 100 mM sodium acetate, pH 5.5. Nucleic acids were recovered by centrifugation, resuspended in 2 ml of 10 mM Tris-HC1 (pH 7.4), 2 mM CaCI2, 10 mM MgCI2 and incubated at 37°C for 20 min in the presence of 0.1 mg/ml of DNase I (Sigma, electrophoretically pure) which had been pretreated with proteinase K for 2 h as described by Tullis & Rubin [31]. After addition of 0.05 vol of 5 M NaC1 and 0.25 vol of 10 % (w/v) SDS, the RNA solution was extracted with phenol and chloroform, and ethanol precipitated as described above. Total cellular RNAs were pelleted after precipitation with ethanol and dissolved in 20 ~tl of 50 % (w/v) urea, 0.01% (w/v) xylene cyanol FF. Samples were denatured in a boiling water bath for 2 min and quick chilled on ice prior to electrophoresis in a 6 % (w/v) polyacrylamide gel (acrylamide : bisacrylamide 29: 1, w/w) containing 50% (w/v) urea and 1x T B E (50 mM Tris/50 mM boric acid/l mM EDTA, pH 8.3). The gel was pre-electrophoresed for 30-40 min at 21 W, and the RNA samples were electrophoresed at 18-20 W for 8.5 h while maintaining the surface temperature of the gel at 50-60°C [26]. The electrophoretically fractionated RNAs were transferred from the acrylamide gel to a diazobenzyloxymethyl (DBM)-cellulose f'dter. The acrylamide gel was soaked in 50 mM NaOH for 20 rain at room temperature and neutralized by two subsequent soakings of the gel in 200 mM sodium phosphate (pH 5.5) for 20 min at room temperature. Finally the gel was equilibrated with 25 mM sodium phosphate (pH 5.5) prior to electrophoretic transfer in the same buffer at 0--4°C for 4.5 h, and the blot was allowed to cure undisturbed overnight. Aminobenzyloxy-methyl (ABM) paper was made and activated according to Alwine et al. [27].
Hybridization DBM blots were prehybridized at 50°C for 6 h in a solution containing 50% formamide/ 5xSSC(lxSSC: 0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.5)/1% glycine (v/v)/5xDenhardt (1 x Denhardt: 0.02 % each of Ficoll 400, Pharmacia Fine Chemicals, and polyvinylpyrrolidone, Sigma)/400 ~tg/ml E. coli nucleic acid. Hybridization was at 50°C for 48 h in 50% formamide/5×SSC/15 mM potassium phosphate, pH 7.2/5x Denhardt/200 Ixg/mlE. coli nucleic acids with nicktranslated plasmid DNAs containing cloned human histone genes, at approx. 1.5× l0 s cpm/ml. Nicktranslated DNA was denatured for 10 rain in a boiling water bath and quick-cooled before addition to the hybridization mixture. After hybridization the blots were washed sequentially in 5xSSC/1 xDenhardt at room temperature for 10 min and at 60°C for 30 min, then in 2xSSC/0.1% SDS and lxSSC/0.1% SDS, each time for 30 min at 60°C. DBM paper was blotted dry on Whatman 3MM
Exp CellRes 159 (1985)
282 M a r a s h i et al. paper and autoradiographed for an appropriate length of time using either preflashed or non-flashed XAR-5 film (Kodak) at -70°C with a Dupont high-speed screen.
RESULTS AND DISCUSSION To assess the expression of murine and human histone genes, total cellular RNA from mouse, human or hybrid cells was fractionated electrophoretically [26], transferred to diazotized cellulose [27] and histone mRNAs were identified by hybridization with 32p-labeled cloned genomic human histone DNA. Conservation of histone genes is sufficient to permit cross-species recognition. The isolation and characterization of the cloned human histone genes has been reported [1, 2, 4]. Electrophoretic fractionation of RNAs in the high resolution polyacrylamide gel system used in these studies results in separation of a series of histone mRNA subspecies which Lichtler et al. have shown by S1 nuclease protection experiments represent transcripts from different copies of the histone genes ([26] and unpublished results). The human histone mRNA subspecies represent transcripts from genes residing in several histone gene clusters which are located on at least two human chromosomes [32]. Moreover, differences in the electrophoretic mobilities of the murine and human histone mRNA subspecies permit us to distinguish between the mRNAs of mouse and human origin. Isolation of total cellular R N A yields greater than 90 % recovery, circumventing losses of RNA through nuclease activity and physical manipulations which generally occur during subcellular fractionation. Shown in fig. 1 are cellular RNAs from mouse--human hybrids which were hybridized with 32p-labelled cloned human H4 histone DNA. For comparison cellular RNAs from NP3 BALB/c mouse myeloma cells and HT1080 fibrosarcoma cells are also shown, indicating the resolution of three mouse H4 histone mRNA fractions and a single human H4 histone mRNA band. A similar distinction between the electrophoretic mobilities of murine and human H4 histone mRNAs was observed in several additional mouse and human cell lines. In 5514F7 and 55-54F2 hybrid cells which are segregating mouse chromosomes, only the human H4 histone mRNAs are present, despite retention of at least one copy of each murine chromosome. This observation is consistent with expression of only human rRNA genes in these same mouse-human hybrids (table 1 and see refs [22-24]). In contrast, both mouse and human H4 mRNAs are present in the 56-05F1C16 and 56-05F5 hybrids which are segregating human chromosomes, although only the murine ribosomal genes are expressed in these cells (table 1). It is interesting that in the hybrids in which both mouse and human histone mRNAs are present, the levels of both types of histone mRNA appear to vary in a coordinate manner with the level of DNA synthesis (data not shown). Analysis of histone gene expression in these hybrids was extended to include H3, H2A and H2B histone genes. As shown in fig. 2, in both human cells and mouse-human hybrids segregating mouse chromosomes, only the human H3 Exp Cell Res 159 (1985)
Histone gene expression in mouse-human hybrid cells
283
i
$
tD
~.. n-
4. 75
[-14
H3
Fig. 1. Detection of human and mouse H4 histone mRNA subspecies in mouse-human hybrid cells.
Total cellular RNA was fractionated electrophoretically in 6 % polyacrylamide gels, transferred to DBM paper and hybridized to nick-translated plasmid DNA (pF0108A) containing a human histone H4 gene. The blots were analyzed by fluorography. Hybrids are designated by number and the retention of all human (H) or mouse (M) chromosomes or the selective segregation of human (H ~, ) or mouse (M ~ ) chromosomes as indicated. Fig. 2. Detection of human and mouse H3 histone mRNA subspecies in mouse-human hybrid cells. Total cellular RNAs were fractionated by electrophoresis, transferred to DBM paper and hybridized to nick-translated plasmid DNA (pST519) containing human histone H3 sequences.
histone mRNA is represented while in cells segregating human chromosomes, the human H3 mRNAs as well as the three distinct murine H3 mRNA fractions are detected. Similarly, filter blots of total cellular RNAs from these same hybrid cells, hybridized with 32p-labeled cloned human H2A and H2B histone D N A (fig. 3), revealed only human H2A and H2B histone mRNAs in the cells segregating murine chromosomes, with both human and murine H2A and H2B histone
Table 1. Species-specific representation of histone mRNAs in mOuse-human hybrids Representation of histone mRNAs
Representation ol histone proteins
Cell line
Chromosome representation
Representation of rRNAs
H2A
H2B
H3
H4
H2A
H2B
H3
55-14F~ 55-54F2 56-05F1C16 56-05F5
H>M H>M M>H M>H
H H M M
H H M+H M+H
H H M+H M+H
H H M+H M+H
H H M+H M+H
H H M M
H H M M
H H M M
Exp Cell Res 159 (1985)
284 Marashi et al.
--
It)
0
Z
Fig. 3. Detection of human and mouse H2A and
H2B histone mRNA subspecies in mouse-human hybrid cells. The electrophoretically fractionated total cellular RNAs were transferred to DBM paper and hybridized to nick-translated plasmid DNA (pFF435B) containing human histone H2A and H2B sequences.
mRNAs in the cells segregating human chromosomes. In the 56-05F5 hybrid an additional RNA species is observed which is not represented in any of the mouse, human or other hybrid cell lines examined. While the identity of this RNA is not readily apparent, it may be a transcript from a histone gene expressed only under limited biological circumstances; quantitative and qualitative differences in histone gene expression have been observed in highly quiescent or terminally differentiated cells [13-19]. Alternatively, this additional histone transcript may result from the rearrangement of a human and/or mouse histone gene. Our results suggest that the presence of structurally intact histone genes is by itself insufficient to render them functional and that there are differences in the requirements for expression of the murine and human histone genes in actively proliferating cells. The transcription of both mouse and human histone genes in hybrids segregating human chromosomes, coupled with the expression of only human histone genes in hybrids segregating mouse chromosomes, may be explained by the functional representation of 'regulatory macromolecules' for murine histone gene expression only in those hybrids retaining both copies of specific mouse chromosomes. Retention of at least one copy of each mouse and human chromosome in both types of hybrids studied is compatible with this interpretation. Exp Cell Res 159 (1985)
Histone gene expression in mouse-human hybrid cells
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Histone gene expression in mouse-human hybrids segregating human chromosomes appears to be more complex. Ajiro et al. [28] have studied the representation of mouse and human histone proteins in the same hybrids we used for analysis of histone mRNAs and have observed that the histone proteins expressed were those of the dominant parent. Because our results show that both murine and human histone mRNAs are present in hybrids segregating human chromosomes, it appears that there is a post-transcriptional or translational mechanism operative. Hsiung & Kucherlapati [29] also failed to detect human histone proteins in a systematic analysis of a series of hybrids, each containing the complete complement of mouse chromosomes and only limited numbers of specific human chromosomes. In contrast, cells segregating mouse chromosomes appear to regulate histone gene expression primarily at the transcriptional level, since only human histone mRNA subspecies are detected in these hybrids. However, the use of only peritoneal macrophage as the mouse component of hybrids segregating murine chromosomes precludes making the generalization that in all cells segregating mouse chromosomes, histone gene expression is posttranscriptionally controlled. A simple, definite interpretation is further complicated by results of Ajiro et al. [33] that in mouse-rat hybrid cells both mouse and rat H1 and H2B histone proteins are synthesized. From reports of several laboratories which have examined the expression of specific genes in mouse-human hybrids, it is becoming apparent that the minimal representation and the number of copies of chromosomes required for expression of different genes may vary. For example, in most mouse-human hybrids studied, suppression of mouse ribosomal gene expression occurs when mouse chromosomes are being segregated, while suppression of human ribosomal genes is observed in conjunction with segregation of human chromosomes, despite the presence of at least one copy of each mouse and human chromosome [22-24]. Yet, both rodent and human U1 small nuclear RNA genes remain active in hybrids containing subsets of human chromosomes [30]. In the case of histone genes in mouse-human hybrids either mouse or human histone expression can be suppressed but the level of regulation appears to depend on the dominant parent. While these results emphasize the complexity of cellular and molecular requirements for eukaryotic gene expression, they provide the basis for utilizing somatic cell hybrids to examine the interrelationships of products from genes residing in various chromosomes. These studies were supported by grants PCM80-18075 and PCM83-18177 from the NSF, GM32010 and GM20700 from the NIH, and 1-813 from the March of Dimes Birth Defects Foundation.
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