fect COS cells. Transfection with plasmid pSVL14s, containing the HMG-14 cDNA in the sense orientation, increased the endogenous levels of HMG- 14 mRNA ...
Vol. 262, No. 20, Issue of July 15,pp. 9839-9843.1987 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Modulation ofthe Cellular Ratio of Chromosomal High Mobility Group Proteins 14 to 17 in Transfected Cells* (Received for publication, February 25, 1987)
Chandrakant GiriS,David Landsman, Nirmolini Soares, and Michael Busting From the Diuision of Virology, $Federal Drug Administration and the Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland20892
chromosomal proteinsHMG-14 and HMG-17 confer to chromatin regions containing transcribable genes, an altered conformation which is more susceptible todigestion with DNaseI than bulk chromatin (8). The correlation between the presence of these proteins and the susceptibility of active chromatin tonuclease digestion suggeststhat theymay be involved in some aspect of transcriptional regulation. Further support for this notion was obtained by experiments which indicated that microinjection of anti-HMG-17 antibodies into somatic cells inhibited transcription (9), from electrophoretic studies which indicated that nucleosomes enriched in HMG-14 and HMG-17 are somewhat enriched in transcribing sequences (IO), from immunofluorescence studies which indicated that antibodies to HMG-14 preferentially stain transcriptionally active regions of polytene chromosomes (11)and, from immunofractionation experiments which indicated that chromatin regions enriched in HMG-17 are 2 to &fold enriched in transcriptionally active genes (12, 13). However, since the resultsobtained inseveral laboratories failed to reveal a correlation between the presence of HMG-14 and -17 and the transcriptional state of a gene (14, 15), the involvement of these proteins in gene activity is stillquestionable. The availability of molecular probes to study HMG gene organization and control of expressionprovides additional avenues for studies on thecellular role of these proteins. We have recently isolated and sequenced the cDNAs for human chromosomal proteins HMG-14 (2) and HMG-17 (3). The Chromosomal proteinsHMG’-14 and HMG-17 are theonly proteins are encoded by distinct multigene families (16) which known nonhistones whose primary binding sites in the nuwhose transcription is regulated cleus are on the nucleosomal core particle (forreview, see Ref. give rise to abundant mRNAs in a cell cycle-dependent manner.2 The present paper de1). These two proteins, which structurally are very similar, are presentin nucleiof most higher eukaryotes. Their primary scribes experiments in which we transiently transfect COS structure is highly conservedfrombovine to human (2, 3) cells, with constructs containing humanHMG-14 and HMG17 cDNAs in two orientations, in order to investigate the suggesting thatthereareevolutionaryconstraintsonthe ability of cells to express additional copies of the proteins. conformation of the protein and that the protein is an imporhigh levels of endogenous tant cellular component. HMG-14 and -17 bind stronger t o Theresultsindicatethatthe nucleosomes than to the DNA obtained from these particles mRNA2 do not preclude the expression of large amounts of (4). The binding of these HMGs to nucleosomes seems t o additional, plasmid-derived mRNA, that thecellular levels of stabilize the core particle (4, 5 ) and to increase its suscepti- the two HMG mRNAs and of the proteins canbe modulated bility to DNaseI digestion (6). The amount of HMG-14 and by introduction of appropriate vectors, and suggest that the may be a usefulway -17 found in mostcells is sufficient to bind only to about 10% use of transient and stable transformants to study the cellular role of these nucleosomal binding proof the nucleosomes, yet reconstitution experiments demonstrate that each nucleosome has two binding sites for either teins. HMG-14 or HMG-17 and that the proteins bind preferentially MATERIALS ANDMETHODS tosalt-stripped nucleosomes containingtranscribablesequences (7). This finding seems to support suggestion the that Construction of Expression Vectors-Plasmid DNA manipulations
The cDNAs coding for human nonhistone chromosomal high mobility group (HMG) proteins 14 and 17 have been introduced into the eukaryotic expression vector pSVL under the transcriptional control of the SV40 late promoter and the constructs used to transfect COS cells. Transfection with plasmid pSVL14s, containing the HMG-14 cDNA in the sense orientation, increased the endogenous levels of HMG- 14 mRNA 50fold and the levels of HMG-14 protein %fold. Transfection with pSVL17s, which contains the HMG-17 mRNA in the sense orientation, resulted in a 19-fold increase in mRNA levels and a %fold increase in the protein level. Transfection with pSVLl7as, containing the HMG-17 in the antisense orientation, resulted in a noticeable decrease in theprotein levels. The overproduction of HMG mRNAs does not affect the level of other cellular mRNAs and the increase in the cellular level of either HMG-14 or -17 did not affect the level of the other HMG or that of any other cellular protein examined. The results suggest that COS cells can tolerate large excess of HMG mRNAs and protein, that the relative amounts of HMG-14 and HMG-17and their mRNAs are not constant, and that neither the transcription nor the translation of the proteins is coordinately regulated.
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* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. f To whom reprint requests should be addressed. ’ The abbreviations used are: HMG, high mobility group; HEPES, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; bp, base pair.
were carried out essentially as recommended by Maniatis et al. (17). Restriction enzymes, T4 DNAligase, and Klenow fragment were purchased from Bethesda Research Laboratories. Calf intestinal alkaline phosphatase was purchased from Boehringer Mannheim. The eukaryotic expression vector pSVL was obtained from Pharmacia P~-
* Bustin, M., Soares, N., Landsman, D., and Collins, J. M. (1987) Nucleic Acids Res.,in press.
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Transfection of HMG cDNAs
L Biochemicals. Plasmids pHl4c and pH17c containing the human cDNA coding for HMG-14 (2) and HMG-17 (3) were previously described. The 1.2-kilobase pair HMG-14 and -17 inserts were prepared from the plasmids by digestion with EcoRI. The inserts were blunt-ended using the Klenow fragment and ligated to the SmIdigested and dephosphorylated pSVL vector. The resultant recombinant plasmids, designated as pSVLl7s, pSVLl7as, and pSVL14s contain, respectively, the 1.2-kilobase pair HMG-17 cDNA insert in “sense” and “antisense” and the HMG-14 cDNA in the sense transcriptional orientations with respect to theSV40 late promotor. DNA Transfections-All DNA transfections were performed by the DEAE-dextran method essentially as described by Sompayrac and Donna (18).COS cells (passage numbers 14-28) were maintained a t 37 “C under 5% COZ in Eagle’s minimum essential medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 50 pg/ml of gentamycin sulfate, 2.5 pg/ml of Fungizone (GIBCO), and 10 mM HEPES buffer, pH 7.3. Unless otherwise mentioned, COS cells were plated onto 75-cmZflasks to be subconfluent at thetime of transfection. DEAE-dextran (average molecular weight 500,000; Sigma) was dissolved at 250pg/ml of serum-free Eagle’s minimum essential medium supplemented with 2 mM glutamine, 50 pg of gentamycin sulfate/ml, 2.5 pg of Fungizonelml, and 50 mM Tris-HC1, pH 7.3. The cells were washed two times with serum-free medium and incubated with 25 pg of twice-banded CsClEtBr gradient-purified plasmid DNAs in 5 ml of serum-free medium/ Tris/DEAE-dextran. The flasks were incubated at 37 “C in a COz incubator for 8 hr. The cells were then washed once with the serumfree medium and grown in media supplemented with 10% fetal calf serum for 48 h prior to harvest. In experiments involving butyrate treatment the media contained 10 mM sodium butyrate prepared as described elsewhere (19). Protein Synthesis-Forty-eight hours after transfection the monolayers of COS cells were washed once with lysine-free Eagle’s minimum essential medium (GIBCO) and thenincubated with the lysinefree medium for 60 min. The cells were labeled in vivo with 100 pCi of ~-[4,5-~H]lysine (75-100 Ci/mmol) (Amersham Corp.) per mlof lysine-free medium supplemented with 10% fetal calf serum for 4 h a t 37 “C. The monolayers were washed gently three times with icecold phosphate-buffered saline and the cells were scraped into 1 ml of phosphate-buffered saline containing 0.1 mM phenylmethylsulfonyl fluoride and 1%Trasylol, pelleted, suspended in 0.2 ml of 10 mM Tris-HC1, pH 7.5, 10 mM NaC1,0.1 mM phenylmethylsulfonyl fluoride, 1%Trasylol, mixed vigorously, and extracted with 5% perchloric acid. The extract was centrifuged, the supernatant made 0.3 M in HCl, and the proteins precipitated with 6 volumes of acetone. The precipitate was dissolved in sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer and electrophoresed on 18% discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fixed gel was developed for fluorography with Enlightning (New England Nuclear) and exposed to Kodak XAR-5 film at -70 “C for autoradiography. Analysis of RNA-Total cellular RNAwas extracted from the various cell lines by the guanidinium isothiocyanate method (17). For slot blot analysis, the samples were applied to Zetabind filters in33% formaldehyde, 6 X SSC. The filters were air dried and baked at 80 “C and treated as recommended by Church and Gilbert (20). For Northern analysis the RNA was electrophoresed in denaturing formamide gels (17), transferredto Zetabind filters, and treated asdescribed for slot blot analysis, The filters were probed with 32P-labeledinserts which were excised from plasmids with the appropriate restriction enzymes, purified from 1%agarose gels, and labeled by nick translation. Primer Extension-An oligonucleotide corresponding to bases 97125 of the insert of pH17c (3) was synthesized, labeled a t the 5’-end using polynucleotide kinase and passed through a Minispin column (Worthington). Total cellular RNA (10 pg) was hybridized to a 10fold molar excess of labeled oligonucleotide in 5 mM Na phosphate buffer, pH 6.75, 5 mM EDTA by heating to 90 “C for 10 min and subsequent slowcooling to room temperature. After 30 min the solution was then made 65 mM NaCl, 1mM dNTP, 50 mM Tris-HC1, pH 8.3, 10 mM MgC12,and 10 mM dithiothreitol in a final volume of 50 pl. One microliter of RNase inhibitor (RNasin, Promega Biotec), and 10 units of reverse transcriptase (IBI) were added. Incubation was for 90 min at 37 “C. The reaction was stopped by adding EDTA to 20mM. The samples were extracted with phenol/chloroform, precipitated with ethanol, washed, dried, dissolved in 90% formamide, and electrophoresed on a 6% denaturing polyacrylamide gel.
RESULTS
Transcription of Vector-derived HMG-14 and -1 7 mRNAsThe construction and the relevant features of the various vectors used in this study are diagrammatically presented in Fig. 1. In COS cells, the human HMG-14 and -17 cDNAs, cloned into thepolylinker of the eukaryotic vector pSVL, are expressed under the transcriptional control of the SV40 late promoter. The transcripts are accurately spliced and polyadenylated using the SV40 VP1 signals. The vector, which lacks the pBR322 “poison sequences” replicates efficiently in COS cells and gives rise to relatively high levels of translation products originating from the ATG codon of the cloned insert. In the present study these properties are of specific importance since the abundance of endogenous HMG-14 and -17 mRNA present in tissue culturecell lines2 may interfere with the transcription and detection of additional copies of HMGlike mRNAs. The slot blot analysis presented in Fig. 2 indicates that quantitative changes in the level of HMG RNA can be easily detected, in transformed cells, against the relative large background of the endogenous HMG transcripts present in COS cells. The stronger autoradiographic signals obtained in the transfected cells are not due to differences in hybridization efficiencies since the intensity of the signal obtained with the RNA derived from COS cells was similar to that obtained from HeLa cells, suggesting that their endogenous mRNA levels are similar and that thehuman cDNA hybridizes efficiently to the RNA extracted from the COS cells. Quantitative analysis of the radioactivity bound to each slot indicates that, compared to COS cells transfected with pSVL or pSVL14s, transfection with pSVL17s (i.e. HMG-17 in the
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