©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Stimulation of Transcription Accompanying Relaxation of Chromatin Structure in Cells Overexpressing High Mobility Group 1 Protein (*)

Yoshimasa Ogawa , Shigemi Aizawa , Hitoshi Shirakawa , Michiteru Yoshida (§)

From the (1) Department of Biological Science and Technology, Science University of Tokyo, Yamazaki, Noda-shi, Chiba 278, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We developed murine C-127 cell lines that stationarily overexpress high mobility group (HMG) proteins 1 and 2 by transfecting them with the bovine papilloma virus plasmid carrying their respective cDNA sequences. Using these cell lines, we examined the effects of these HMG proteins on the modulation of chromatin structure that accompanied transcription. The levels of HMG1 mRNA and protein in cells overexpressing HMG1 protein were enhanced about 7- and 3-fold, respectively, in comparison with control cells, whereas those in cells overexpressing HMG2 protein were enhanced about 17- and 9-fold. The expression of reporter genes transfected into the cells was enhanced approximately 2-fold in cells overexpressing HMG1, but not HMG2, in comparison with those in control cells, irrespective of the sources of the genes and promoters. The minichromosome derived from the reporter plasmid in cells overexpressing HMG1 protein was more susceptible to micrococcal nuclease digestion than those in cells overexpressing HMG2 protein and control cells. The enhanced accessibility to micrococcal nuclease was not restricted to the expressing gene and promoter but involved the entire minichromosome, suggesting that the enhancement of gene expression resulted from changes in the condensation of the entire minichromosomal region by HMG1 protein. Minichromosomes in cells overexpressing HMG contained enhanced amounts of the respective HMG proteins and simultaneously reduced amounts of histone H1s. These results suggest that HMG1 and -2 proteins have different functions in the modulation of chromatin structure, and that HMG1 protein may sustain the structure of the respective gene to ensure that its activity as a template is expressed fully. These observations on the modulation of chromatin structure accompanying gene transcription in cells overexpressing HMG protein may provide important information on the function of these proteins.


INTRODUCTION

High mobility group (HMG)() proteins 1 and 2, the ubiquitous non-histone components of chromatin, are considered to be implicated in DNA replication and cellular differentiation (for reviews, Bustin et al. (1990) and Einck and Bustin (1985)). In addition, HMG1 protein may be associated with the actively transcribed regions of chromatin (Beatriz and Dixon, 1978; Vidali et al., 1977). Microinjection of antibodies against HMG1 protein into living oocyte nuclei causes retraction of the transcription loops (Kleinschmidt et al., 1983). The HMG1 and -2 proteins stimulate in vitro transcription (Tremethick and Molloy, 1986) by facilitating the formation of effective initiation complexes (Tremethick and Molloy, 1988) or by stimulating the binding of a specific transcription factor to the promoter (Watt and Molloy, 1988). They also function as general class II transcription factors (Singh and Dixon, 1990; Ge and Roeder, 1994; Stelzer et al., 1994). These observations have suggested that the HMG proteins play a direct role in transcriptional events on the genes. On the other hand, they have roles in modulating DNA and chromatin structures in relation to their ability to bind preferentially to single-stranded DNA (Bidney and Reeck, 1978; Isackson et al., 1979; Yoshida and Shimura, 1984, Hamada and Bustin, 1985), unwind double-stranded DNA (Yoshida and Shimura, 1984; Makiguchi et al., 1984), and vice versa (Marekov et al. 1984), suppress (Waga et al., 1989) or mediate (Bonne-Andrea et al., 1984) nucleosome assembly, and remove the transcriptional blocks caused by left-handed Z-form DNA (Waga et al., 1988) and cruciform DNA (Waga et al. 1990), resulting in stimulation of in vitro transcription (Waga et al., 1988, 1990; Tremethick and Molloy, 1986, 1988; Singh and Dixon, 1990). These findings appear to support the idea that HMG proteins play an active role in gene regulation by functioning as quasitranscription factors (Landsman and Bustin, 1991). One of the most desirable methods for elucidating the roles of HMG proteins in transcriptional events is to construct a system for monitoring the transcription reaction together with the probable alterations in chromatin structure caused by the quasitranscription factors. In preceding studies, we assessed the transcriptional activation potential of HMG1 protein using cultured cell systems. The results showed that HMG1 protein can stimulate transcription to a certain extent. In addition, the acidic carboxyl terminus of the HMG1 protein molecule is essential for the enhancement of gene expression in addition to elimination of the repression caused by the DNA binding (Aizawa et al., 1994). Virtually all this work was carried out using vectors based on SV40 that transiently express a large amount of HMG1 protein or the variant. If it were possible to establish a permanent cell line which stationarily expresses a large amount of the HMG protein molecule, but is not destined to die as a consequence of such overexpression, such cells would provide a continuous source of this protein for use in studies of the biological mechanisms in which HMG proteins may participate, especially the alteration of chromatin structure accompanying gene expression.

In the present study, we used the bovine papilloma virus (BPV) as a vector to overexpress HMG1 or -2 protein in murine C-127 cells. The BPV DNA can propagate extrachromosomally as circular DNA of 20-100 copies per cell (Fukunaga et al., 1984; Sambrook et al., 1985). We have developed cell lines that overexpress HMG1 or -2 protein by transfecting them with the BPV plasmid carrying their respective cDNA sequences. Using these cell lines, we examined the effects of these HMG proteins on the modulation of chromatin structure accompanying transcription. The results strongly suggest that HMG1 protein may be a gene quasiactivator that modulates chromatin structure to orient the respective gene to ensure that its activity as a template is expressed fully.


MATERIALS AND METHODS

Preparation of Plasmids The plasmids used for this work were prepared as follows.

Plasmids pBPV-SR, pBPV-HMG1, and pBPV-HMG2

The plasmid pBPV (Pharmacia Biotech Inc.) was cleaved with XhoI at the multicloning site. In order to construct pBPV-SR as a control vector, a 2,970-bp DNA fragment containing the neogene followed by the SR promoter was ligated downstream from the SV40 origin/early promoter region in the XhoI-cleaved pBPV vector. For the constructions of pBPV-HMG1 and pBPV-HMG2, a 2200-bp HMG1 cDNA (Tsuda et al., 1988) and a 1412-bp HMG2 cDNA (Shirakawa et al., 1990) were ligated downstream from the SR promoter in pBPV-SR, respectively. The nucleotide sequences of the coding regions for these expression plasmids were verified by DNA sequencing.

Reporter Plasmids

The pCH110 plasmid carrying the lacZ gene downstream from the SV40 origin/early promoter was obtained from Pharmacia. Plasmids pmiwz carrying the lacZ gene downstream from the Rous sarcoma virus promoter (obtained from the Japan Cancer Research Resources Bank, Tokyo), pSV2CAT carrying the chloramphenicol acetyltransferase (CAT) gene downstream from the SV40 origin/early promoter (Gorman et al., 1982), pactCAT carrying the cat gene downstream from the -actin promoter (Schmidt et al., 1985), and ptkCAT carrying the cat gene downstream from the thymidine kinase promoter (McKnight, 1980) were also used as reporter plasmids. They were prepared by the alkaline-sodium dodecyl sulfate (SDS) method, followed by purification using equilibrium density gradient centrifugation in cesium chloride (Seyedin and Kistler, 1979). Transfection of C-127 Cells with pBPV Plasmids The murine cell line C-127 was maintained in Dulbecco's modified Eagle's medium (DMEM, Nissui) supplemented with 10% fetal calf serum (FCS; Life Technologies, Inc.) in a humidified atmosphere containing 5% COat 37 °C. The transfection of C-127 cells with pBPV plasmid DNA by the liposome method was carried out as described by Felgner et al. (1987) with a slight modification. Aliquots (2 µg) of each required plasmid and 15 µg of the transfection reagent, Lipofectin (Life Technologies Inc.), were mixed gently, according to the manufacturer's manual, in Hepes-buffered saline (20 m M Hepes, pH 7.4, 150 m M NaCl) and kept for 15 min, followed by transfection. After incubation for 3 h, the medium was exchanged for DMEM supplemented with 10% FCS, and the cells were incubated for an additional 48 h. Then, the cultures were subdivided (split ratio 1:4) into medium containing G418 (0.4 mg/ml, Life Technologies, Inc.). The cells usually started to die 3 days later, and surviving colonies were apparent after incubation for 10-14 days. Surviving colonies were picked at after 3 weeks using the filter method (Masuji et al., 1974) and incubated in DMEM supplemented with 10% FCS for about 1 week. The colonies that grew in the selective G418 medium were assumed to have arisen from single cells and were not subcloned. Northern Dot Hybridization Northern dot hybridization to examine the expression levels of the mRNAs for HMG proteins was carried out using the method of Butler et al. (1984). A 478-bp PstI- PstI HMG1 cDNA fragment (Tsuda et al., 1988) or a 1.2-kbp XhoI- XhoI HMG2 cDNA fragment (Shirakawa et al., 1990) labeled with [P]dCTP by a random primer labeling kit (Takara, Kyoto) or by amplification in a reaction mixture containing 5`-[P]dATP primer, respectively, were used as hybridization probes. Northern Blot Hybridization The total RNAs were prepared from the cells by the guanidium thiocyanate method (Sambrook et al., 1989), fractionated on 1.1% (w/v) agarose gel, transferred to a Zeta-Probe membrane (Bio-Rad), and hybridized with the [P]DNA probes described above and with a 1-kbp fragment from the lacZ gene in pCH110 excised by double digestion with EcoRI and ScaI, a 1.4-kbp DNA fragment from the cat gene in pSV2CAT excised by double digestion with HindIII and BamHI, or a 0.9-kbp DNA fragment from -tubulin cDNA (Lewis et al., 1985) used as an expression standard, and processed for autoradiography. Southern Blot Hybridization Plasmid DNAs were prepared by the method of Hirt (1967) from transformed cell lines, digested with HindIII, separated by electrophoresis on 0.7% (w/v) agarose gel, transferred to a GeneScreen Plus membrane (Dupont NEN), hybridized with the [P]SR promoter region upstream from the HMG genes labeled with a random primer DNA labeling kit (Takara, Kyoto), and processed for autoradiography. Immunoblotting Analysis The whole-cell proteins from C-127 cells transfected with the pBPV plasmid were fractionated on 5-20%(w/v) gradient polyacrylamide gel containing SDS and then transferred to a nitrocellulose membrane filter (Schleicher and Schuell). After blocking the filter with non-fat dried milk, an appropriate dilution of polyclonal rabbit antiserum against HMG1 or HMG2 protein was added, and the bound antibodies were detected with biotinylated anti-rabbit antibody and alkaline phosphatase streptavidin conjugate (Amersham). In some cases, the filters were scanned by a laser densitometer (Molecular Dynamics) to analyze the relative amounts of HMG1 and -2 proteins quantitatively. Transfection with the Reporter Plasmid The required reporter plasmid was transfected into C-127 cell lines by the cationic liposome method using transfection reagent (Boehringer Mannheim) as described by Felgner et al. (1987). The plasmid DNA-liposome complex was added to semiconfluent cells in Hepes-buffered saline and incubated for 4 h. After transfection, the medium was exchanged for DMEM supplemented with 10% FCS, and the cells were incubated further for various times. Determination of Enzyme Activities The harvested cells were suspended in 0.25 M Tris-HCl, pH 7.8, followed by 3 cycles of freezing and thawing. The supernatant was obtained by centrifugation at 12,000 g for 10 min. An aliquot of each extract was assayed for -galactosidase activity (Sambrook et al., 1989), which was expressed as units/mg of protein. The CAT protein was measured using CAT ELISA (Boehringer Mannheim). Nuclear Run-on Transcription Assay Nuclear run-on transcription assay was conducted as described by Ayusawa et al. (1986). Micrococcal Nuclease Digestion Cells overexpressing HMG1 protein (1-OE cells), cells overexpressing HMG2 protein (2-OE cells), cells not overexpressing HMG proteins (N-OE cells), and C-127 cells were transfected with the pSV2CAT plasmid. After 12 h of transfection, the cell nuclei were prepared by the method of Laitinen et al. (1990) and incubated for 0, 1, 2, 5, 10, 30, and 60 min with micrococcal nuclease. The DNA fractions were prepared from the nuclear digests and electrophoresed on 1.6% (w/v) agarose gel. The gel was stained with ethidium bromide, followed by photography. Thereafter, the DNA samples were transferred to a GeneScreen plus membrane, hybridized with probes of [P]DNA fragments from the SV40 promoter, the cat gene, the ampgene, shown in Fig. 5F, and a 0.7-kbp DNA fragment from the -actin cDNA (Tokunaga et al., 1986), and processed for autoradiography. Preparation of Minichromosomes for Analysis of their Protein Component The 1-OE, 2-OE, N-OE, and C-127 cells were transfected with the pSV2CAT plasmid. After 12 h of transfection, the cell lysates were prepared by the method of Oudet et al. (1989). The minichromosomes were purified by 5-30% (w/v) sucrose density gradient centrifugation, and the proteins were separated by 14% SDS-polyacrylamide gel electrophoresis and stained with SERVA Blue G (SERVA). The amounts of proteins were determined by tracing with a laser densitometer (Molecular Dynamics).


Figure 5: Micrococcal nuclease digestion of nuclei and minichromosomes from C-127, N-OE, 1-OE, and 2-OE cells. At 12 h after transfection with pSV2CAT, cell nuclei were incubated for 0 ( lane 2), 1 ( lane 3), 2 ( lane 4), 5 ( lane 5), 10 ( lane 6), 30 ( lane 7), and 60 ( lane 8) min with micrococcal nuclease. Lane 1 contained the sample with no enzyme and was not incubated. The DNA fractions prepared from the digests were electrophoresed on an agarose gel, stained with ethidium bromide ( A), transferred to a membrane, hybridized with [P]DNA fragments from the -actin gene ( B), SV40 promoter ( C), cat gene ( D), and ampgene ( E). A simplified map of the pSV2CAT DNA used for the probes is presented in F. The 340-, 250-, and 750-bp DNA fragments were used as probes for the SV40 promoter, cat gene, and ampgene, respectively.




RESULTS

Development of Cell Lines Expressing HMG Proteins from pBPV Vectors

The murine C-127 cell line was chosen for development of cell lines overexpressing HMG proteins because the cells can be transformed morphologically with high efficiency by BPV DNA (Sambrook et al., 1985). The pBPV-HMG1 and -2 vectors, containing the complete coding regions of HMG1 or -2 cDNAs, respectively, were constructed. In order to carry out comparative studies between HMG1 and -2 proteins, pig thymus HMG cDNAs, which were the only pair isolated so far from a single experimental source, were chosen for plasmid construction. Transcription of these genes was controlled by the SR promoter. A vector plasmid (pBPV-SR) containing the SR promoter, but no gene controlled by it, was also used as a control.

The C-127 cells were transfected with the pBPV-HMG1, pBPV-HMG2, and pBPV-SR plasmids, and foci of cells rendered resistant to the antibiotic G418 were isolated. Foci of cells which arose at frequencies of 98, 96, and 58 per 2 µg of plasmid vector were obtained by transfection with pBPV-HMG1, pBPV-HMG2, and pBPV-SR, respectively. Colonies that grew in the presence of G418 were expanded into cell lines and screened directly by Northern dot hybridization to select those that overexpressed the desired genes. As the level of mRNA expression for HMG protein varied considerably among the colonies, 11 or 10 cell colonies expressing 5 to 7 times more mRNA for HMG1 or -2 protein than those transfected with pBPV-SR were isolated. Southern, Northern, and Western blot analyses were conducted to determine accurately the levels of expression in these HMG1 and -2 protein overexpressing (1-OE and 2-OE, respectively) cells in comparison with those of control cells transfected with the pBPV-SR (N-OE cells) and nontransfected C-127 cells (C-127 cells).

In order to examine the state of the BPV-recombinant molecules in the transformed murine cells overexpressing HMG mRNA, DNA was prepared from each cell line. Southern hybridization using as a probe the SR promoter region of the undigested molecules which migrated to the positions of form I (supercoiled) DNA demonstrated the extrachromosomal presence of BPV-recombinant molecules in all the cell lines developed in this study. Cleavage of the DNA with SacI yielded single bands of 16.1 kbp for 1-OE cells, 15.3 kbp for 2-OE cells, and 14.1 kbp for N-OE cells. Southern hybridization of the DNA digests with HindIII yielded bands of the expected sizes from the respective constructs (Fig. 1 A). These results suggested that the DNA introduced into the murine cells retained the same nucleotide size as the original DNA introduced into the cells and replicated extrachromosomally.


Figure 1: Analysis of cell lines expressing HMG proteins. A, Southern blot hybridization analysis of DNA in the cell lines. An aliquot (10 µg) of DNA digest with HindIII each from the transfected cell line, 1-OE ( lane 3, clone 1-21; lane 4, clone 1-27; lane 5, clone 1-42; lane 6, clone 1-61; lane 7, clone 1-78; lane 8, clone 1-96), 2-OE ( lane 11, clone 2-8; lane 12, clone 2-14; lane 13, clone 2-47; lane 14, clone 2-53; lane 15, clone 2-79), and N-OE ( lanes 2 and 10, clone N-66), and from nontransfected C-127 cells ( lanes 1 and 9) was electrophoresed and subjected to Southern hybridization analysis using the [P]SR promoter region as a probe. The relative electrophoretic mobilities between lanes 1-8 and lanes 9-15 are not the same due to the use of separate experiments. B, Northern blot hybridization analysis of transfected cell lines. The total RNAs prepared from several transfected cell lines, 1-OE ( lane 3, clone 1-21; lane 4, clone 1-27; lane 5, clone 1-42; lane 6, clone 1-61; lane 7, clone 1-78; lane 8, clone 1-96), 2-OE ( lane 11, clone 2-8; lane 12, clone 2-14; lane 13, clone 2-47; lane 14, clone 2-53; lane 15, clone 2-79), and N-OE ( lanes 2 and 10, clone N-66), and from nontransfected C-127 cells ( lanes 1 and 9) were electrophoresed and subjected to Northern hybridization analysis using a P-labeled 478-bp PstI- PstI HMG1 cDNA fragment ( top), a 1.2-kbp XhoI- XhoI HMG2 cDNA fragment ( middle), and the -tubulin cDNA fragment ( bottom) as probes. The relative electrophoretic mobilities between lanes 1-8 and lanes 9-15 are not the same due to the use of separate experiments. C, Western blot analysis of proteins in the cell lines. The whole-cell proteins prepared from several transfected cell lines, 1-OE ( lane 6, clone 1-21; lane 7, clone 1-27; lane 8, clone 1-42; lane 9, clone 1-61; lane 10, clone 1-96), 2-OE ( lane 13, clone 2-8; lane 14, clone 2-14; lane 15, clone 2-35; lane 16, clone 2-47, lane 17, clone 2-53; lane 18, clone 2-79), and N-OE ( lanes 3 and 19, clone N-66; lanes 4 and 20, clone N-99; lane 5, clone N-80), and from nontransfected C-127 cells ( lanes 1, 2, 11, and 12) were electrophoresed and subjected to Western blot analysis with rabbit antisera against HMG1 ( lanes 1-10) and 2 ( lanes 11-20) proteins. The arrowheads indicate the HMG1 ( lanes 1-10) and -2 ( lanes 11-20) protein bands. The relative electrophoretic mobilities between lanes 1-10 and lanes 11-20 are not the same due to the use of separate experiments. Top, autoradiograms; bottom, amounts of proteins relative to those of C-127 cells.



In order to determine accurately the levels of expression of HMG mRNAs, the total RNAs from the cells were prepared and analyzed by Northern blot hybridization (Fig. 1 B). The autoradiograms, whose exposures were within the linear response range of the films, obtained in three independent experiments, were traced densitometrically. The relative mRNA levels were calculated by standardization with that of -tubulin. The level of HMG1 mRNA was enhanced about 7-fold in 1-OE cells in comparison with that in parental C-127 cells, but no significant changes in 2-OE and N-OE cells were observed. In contrast, the level of HMG2 mRNA was enhanced about 3-fold in 1-OE and N-OE cells in comparison with that in C-127 cells, which is consistent with our previous observation that the level of HMG2 mRNA is generally enhanced in transformed cells.() Furthermore, the level of HMG2 mRNA in 2-OE cells was enhanced approximately 6-fold in comparison with that in N-OE cells, showing that the expression of HMG2 mRNA was enhanced about 17-fold by transfecting C-127 cells with pBPV-HMG2.

In order to examine whether the enhanced expression of HMG mRNAs resulted in increased amounts of the proteins, the relative amounts of the HMG proteins in the cells were estimated. The whole-cell proteins were separated by electrophoresis on SDS-polyacrylamide gel followed by Western blot analysis using antibodies against HMG1 or HMG2 protein (Fig. 1 C). The amount of each protein relative to that in C-127 cells was estimated from the densitometric tracings of the immunostained filters. The amount of HMG1 protein in 1-OE cells was at least 3-fold that in C-127 cells, whereas the amounts in 2-OE, N-OE, and C-127 cells were similar. The amounts of HMG2 protein in 1-OE and N-OE cells were increased about 3-fold in comparison with C-127 cells, and the amount of HMG2 protein in 2-OE cells was increased about 3-fold in comparison with N-OE cells, showing that the overall amount of HMG2 protein was enhanced approximately 9-fold by transfection with the pBPV-HMG2 plasmid. The mRNA and protein levels of HMG1 and HMG2 expression in the cells overexpressing HMG proteins are summarized in .

Therefore, it was shown to be possible to develop murine C-127 cell lines that stationarily overexpress HMG proteins by transfection with BPV vectors carrying the HMG cDNAs. Although the 1-OE, 2-OE, and N-OE cells showed no morphological changes, the growth rate of 1-OE cells was approximately 10% faster than that of N-OE and C-127 cells. Two-dimensional polyacrylamide gel electrophoresis of whole-cell proteins prepared from the HMG-overexpressing cells showed patterns similar to those of N-OE and C-127 cells except for a few differences in several proteins (data not shown), although the natures of their protein components have not been characterized. The nuclear proteins of 1-OE and 2-OE cells showed two-dimensional gel electrophoretic patterns similar to those of N-OE and C-127 cells, except for the apparent differences in the amounts of HMG proteins.

Stimulation of Reporter Plasmid Gene Expression in 1-OE, but Not 2-OE Cells

In order to examine the effects of overexpression of HMG proteins on gene expression, reporter plasmids were transfected into the HMG protein overexpressing cells. Plasmids pCH110 and pSV2CAT, carrying no replication origin for propagation in the cells transfected, were used to exclude any effects of the copy numbers of the reporter plasmids. Cell extracts were prepared at various times after transfection, and the -galactosidase activity and CAT protein were determined. The -galactosidase activity of 1-OE cells was maximum at 72 h after transfection with pCH110 and approximately twice that of C-127 cells. However, no such increases in the activities of 2-OE and N-OE cells were observed (Fig. 2 A). Similar results were obtained for CAT protein in the cells transfected with pSV2CAT (Fig. 2 B).


Figure 2: Expression of -galactosidase activity ( A) and CAT protein ( B) by reporter plasmids in the transfected cells. The -galactosidase activity and CAT protein in extracts from C-127 ( open columns), N-OE ( hatched columns), 1-OE ( filled columns), and 2-OE ( shadowed columns) cells transfected with the respective reporter plasmids were determined at 12, 24, 48, 72, 96, and 120 h after transfection. The values are means of three independent determinations, and bars represent standard deviations.



Northern blot hybridization analysis of RNAs prepared from the cells at 24 and 48 h after transfection was carried out using fragments from the lacZ (Fig. 3 A) and cat genes (Fig. 3 B) as probes to examine whether these increases in enzyme activity and protein resulted from enhanced levels of gene expression from reporter plasmids. When the level of respective expression was standardized with that of -tubulin, the expression of -galactosidase mRNA in 1-OE cells was enhanced approximately 4- and 5-fold at 24 and 48 h, respectively, after transfection in comparison with those in C-127 and N-OE cells, but that in 2-OE cells was not enhanced (Fig. 3 A). The relative increase in the amount of CAT mRNA in 1-OE cells transfected with pSV2CAT was similar to the above results (Fig. 3 B). To determine whether the marked enhancement of these mRNA levels in 1-OE cells was due to an increase in either the transcription rate or post-transcriptional events, we determined the transcriptional complexes on the cat gene in nuclei isolated from the cells transfected with pSV2CAT by run-on transcription activity. The amount of transcriptional complex was 3 to 4 times higher in 1-OE cells than in 2-OE, N-OE, and C-127 cells (Fig. 4). These results suggest that the enhancements of these mRNA levels observed in the above experiments (Fig. 3) must have been due mainly to increases in transcriptional events, and that HMG1, but not HMG2, protein over-expressed in the cells is capable of stimulating expression during the process of transcription of the reporter genes.


Figure 3: Northern blot analysis of the expression of the lacZ ( A) and cat ( B) genes in cells transfected with the respective reporter plasmids. Lanes 1 and 5, the total RNAs prepared from C-127. N-OE ( lanes 2 and 6), 1-OE ( lanes 3 and 7), and 2-OE ( lanes 4 and 8) cells at 24 h ( lanes 1-4) and 48 h ( lanes 5-8) after transfection were electrophoresed on agarose gel, blotted onto a membrane, and hybridized with [P]DNA fragments from the lacZ ( top in A) and cat ( top in B) genes. A DNA fragment excised from the -tubulin cDNA was used as a standard expression probe ( bottoms in A and B).



Stimulation of Gene Expression in 1-OE Cells Is Independent of the Sources of the Promoter and Gene

The above results suggest that the increase in gene expression may be independent of the source of the gene. In order to explore this possibility, several reporter plasmids carrying various promoters and reporter genes were transfected into 1-OE and 2-OE cells, and the enzyme activity and amount of protein were determined. As summarized in , those in 1-OE cells transfected with reporter plasmids carrying the SV40, -actin, thymidine kinase, and Rous sarcoma virus promoters and the lacZ and cat genes were twice as high as those in C-127 and N-OE cells. In contrast, those in 2-OE cells were similar to the controls. These results suggest that stimulation of gene expression by HMG1 protein is independent of the sources of the promoter and gene.

Changes in Conformation of the Minichromosome in Cells Overexpressing HMG

The differential effect of overexpression of the HMG proteins on gene expression from reporter plasmids led us to hypothesize that the structure of the minichromosome derived from the reporter plasmids may be altered in the respective cells. In order to examine this possibility, nuclei from cells transfected with plasmid pSV2CAT were prepared at 12 h after transfection and digested with micrococcal nuclease for various times. The DNA samples prepared from the digests were separated on agarose gel and stained with ethidium bromide, and their relative susceptibilities to micrococcal nuclease were compared (Fig. 5 A). The chromatin in all the cell nuclei was digested progressively in proportion to the time of incubation with micrococcal nuclease. Substantial amounts of mononucleosome, which migrated fastest on the gel, were produced in 1-OE and 2-OE cells 1 min after digestion, but very little was produced in N-OE and C-127 cells. In contrast, the larger polynucleosomes in the 1-OE and 2-OE cells disappeared rapidly during digestion in comparison with those in N-OE and C-127 cells. If we assume that the degree of accessibility of chromatin to micrococcal nuclease was proportional to the rate of disappearance of the polynucleosome ladders, then the degree of accessibility of 1-OE and 2-OE cellular chromatin was approximately 2 and 1.5 times that of control cellular chromatin, respectively. These results suggest that the entire chromatin structure in the nuclei of cells overexpressing HMG protein is decondensed. This was investigated by carrying out Southern hybridization analysis of the DNA digests using the -actin cDNA fragment as a probe for the host cell chromosomal DNA (Fig. 5 B). A 0.34-kbp DNA fragment containing the SV40 promoter (Fig. 5 C), a 0.25-kbp 5`-terminal DNA fragment excised from the CAT gene (Fig. 5 D), and a 0.75-kbp DNA fragment containing the 5`-terminal region of the ampgene (Fig. 5 E) were also used as probes (Fig. 5 F) to analyze the structural alterations in the minichromosome derived from the reporter plasmid. The susceptibilities of the -actin gene in 1-OE and 2-OE cells to the nuclease were not markedly different from the control cell value (Fig. 5 B), suggesting that the conformation of the chromatin in the host cells is not uniform. However, the minichromosome in 1-OE cells probed with the SV40 promoter, CAT gene, and the ampgene was markedly more susceptible to nuclease digestion than those in N-OE and C-127 cells (Fig. 5, C-E). Their degree of accessibility to micrococcal nuclease was approximately 5 times that of the controls.

However, the minichromosome in the 2-OE cells was rather resistant to nuclease digestion in comparison with that in the 1-OE cells, and the maximal degree of accessibility was only twice that of the controls. These results indicate that HMG1 protein overexpressed in the cells may have decondensed the structures of the minichromosome and the host cell chromatin, and that the decondensation of the minichromosome was not restricted to the promoter region and the gene expressing mRNA, but involved the entire minichromosome. Thus, the ability of the overexpressed HMG2 protein to change the conformation of the minichromosome may be weaker than that of HMG1 protein.

Enrichment of Minichromosome with HMG Protein in HMG-overexpressing Cells

In order to analyze the protein composition of the minichromosome in cells overexpressing HMG, the minichromosome was purified from the lysate of cells transfected with the reporter plasmid. The minichromosomal proteins were separated by SDS-polyacrylamide gel electrophoresis, stained, and traced densitometrically. They were also transferred to a nitrocellulose membrane, and the mobility of the HMG proteins was confirmed by immunostaining using antisera against them. The compositions of the minichromosomal proteins obtained from the four cell lines were fairly uniform, except for the HMG proteins and histone H1s (Fig. 6, A and B). The relative amounts of these proteins to whole core histone standard are presented in Fig. 6 C and . The relative amount of HMG1 protein in 1-OE cells was approximately 1.7-fold that in C-127 cells. Similar enrichment of HMG2 protein in 2-OE cells was observed. The relative amounts of HMG2 protein in minichromosomes from 1-OE and N-OE cells were almost identical with that in control C-127 cells. In contrast, the relative amounts of histones H1A, H1B, and H1in minichromosomes were reduced in 1-OE cells and 2-OE cells. These results indicate that overexpression of the HMG1 and -2 proteins in these cells affects the protein composition of the minichromosomes, and that the relative amount of histone H1s correlates well with the amounts of these HMG proteins bound to the minichromosomes.


Figure 6: Electrophoretic separation of minichromosomal proteins on polyacrylamide gel. A, profile of the stained gel. Lane 4, minichromosomes prepared from C-127. N-OE ( lane 5), 1-OE ( lane 6), and 2-OE ( lane 7) cells were solubilized in sample buffer containing SDS and applied on a 14% SDS-polyacrylamide gel. Pig whole histones ( lane 1), HMG1 ( lane 2), and HMG2 ( lane 3) proteins were also electrophoresed. The mobilities of the core histones (H2A, H2B, H3, and H4), H1 histones, and HMG1 and -2 proteins are marked at the left-hand side of the panel. B, densitometric tracing of the stained gels. C, the relative amounts of HMG1 and -2 proteins, H1, and its modified components were standardized with reference to those of the whole core histones determined from the densitometric tracings of three gels. Bars represent the standard deviations.




DISCUSSION

One of the most desirable methods for elucidating the roles of HMG proteins in transcriptional events is to construct a system for monitoring the transcription reaction together with the probable alterations in chromatin structure caused by the quasitranscription factor, as suggested by our previous observations (Aizawa et al., 1994). We used the BPV plasmid as a vector, which may propagate extrachromosomally with a large copy number and stationarily overexpress HMG protein in murine C-127 cells. The levels of HMG1 mRNA and protein in 1-OE cells were enhanced about 7- and 3-fold, respectively, in comparison with control cells, whereas those in 2-OE cells were enhanced about 17- and 9-fold, respectively ( Fig. 1 and ). The limited amounts of HMG1 and -2 proteins in these cells, despite the high mRNA expression level, suggest that the amounts of these proteins that a cell can tolerate may be limited. Similarly, in COS cells transfected with plasmids expressing HMG14 and -17 proteins, up to 50-fold excesses of mRNA were observed, although the levels of both proteins increased only about 3-fold (Giri et al., 1987). Taken together, these results suggest that the expression levels of major HMG proteins are well controlled in the cells due to their importance in chromatin functions. The levels of HMG2 mRNA and protein were enhanced about 3-fold in 1-OE and N-OE cells in comparison with those in C-127 cells. This enhancement is consistent with our previous finding that the levels of HMG2, but not HMG1, mRNA in several derivative cells obtained by transfection with the viral genes of the parental rat fibroblast 3Y1 cells were enhanced markedly. Enhanced expression of HMG2 protein was also observed in exponentially growing cells, suggesting a positive relationship between the amount of this protein in cells and their proliferation.Such enhanced expression of HMG2 by transformation is not contrary to the observation that an increase in the level of one HMG protein does not affect the level of others, suggesting that regulation of neither the transcription nor translation of these proteins is coordinated (Bustin et al., 1990). The production of excess amounts of HMG1 and -2 proteins did not materially alter the cellular morphology, although a slight increase in the growth rate of 1-OE cells, apparent alterations in the relative composition of cellular proteins (Fig. 6) and differences in the susceptibility of chromatin to micrococcal nuclease digestion in 1-OE and 2-OE cells (Fig. 5) were observed. It is also probable that the presence of large numbers of copies of the transforming region of the BPV genome may affect the protein composition, but not sufficiently to cause the cells to display a fully transformed type (Law et al., 1983; Lusky and Botchan, 1984).

Previous data (Johns, 1982; Einck and Bustin, 1985) have indicated that the primary binding site of both HMG1 and -2 proteins to nucleosomes is the linker DNA between core particles. This finding is supported by recent cross-linking experiments with cisplatin (102) and by experiments involving mild micrococcal nuclease digestion (Scovell et al., 1987). On the other hand, a chemical cross-linking study with various types of cross-linking reagents has indicated that these proteins can bind to core particles lacking linker DNA (Stros and Kolibalova, 1987). Therefore, investigations attempting to determine whether the HMG proteins can replace histone H1 have yielded conflicting results (Bustin et al., 1990). The protein compositions of minichromosomes in 1-OE and 2-OE cells differed from those of control cells ( Fig. 6and ). The relative amounts of HMG1 protein in 1-OE cells and of HMG2 protein in 2-OE cells increased 1.7- to 1.8-fold in comparison with the control levels, but the relative increases in the amounts of HMG proteins were not proportional to their levels of expression. It should be noted that the relative amounts of HMG2 protein in minichromosomes from 1-OE and N-OE cells did not increase, despite the enhanced expression of this protein that resulted from the transformation of the cells. The reason for this has not been resolved. In contrast with the increased amounts of HMG proteins in minichromosomes, the relative amounts of histone H1A, H1B, and H1clearly declined by about 15 to 30% in comparison with those in C-127 cells. These opposite changes in protein compositions lend support to the previous data (Johns, 1982; Einck and Bustin, 1985), indicating that the primary binding site of HMG1 and -2 proteins to nucleosomes is the linker DNA between core particles, and suggest that HMG1 protein in 1-OE cells and HMG2 protein in 2-OE cells may accumulate in the internucleosomal regions to take the place of histone H1s. In order to demonstrate the HMG protein binding sites in the nucleosome, a new method for detecting their positions without digesting the minichromosome should be developed. The minichromosome obtained in the present study appears to be useful for demonstrating the structure of chromatin and changes within it, because the minichromosome is prepared without using micrococcal or other nucleases or shearing, which may distort their highly ordered structures. In any event, the changes in minichromosomal protein composition accompanying overexpression of the HMG proteins suggest that chromatin structure may be controlled, at least partially, by the level of expression of the proteins in the cells and/or the relative distribution of the proteins between the nuclei and cytosol, depending on the cellular functions (Bustin et al., 1990).

Minichromosomes prepared from cells overexpressing HMG showed a higher susceptibility to micrococcal nuclease digestion than those from control cells. Furthermore, the chromatin structure of the host cell appeared to be more sensitive to such digestion, as demonstrated by ethidium bromide staining (Fig. 5). There is now a considerable amount of evidence to indicate that some of the HMG proteins are involved in maintenance of the structure of transcriptionally active chromatin (Johns, 1982). The proteins solubilized together with transcribed sequences by brief DNase I digestion contained HMG1 and -2, but not HMG14 and -17, proteins (Goodwin and Johns, 1978, Vidali et al., 1977, Teng et al., 1979). The facts that HMG1 and -2 bind selectively to single-stranded DNA (Bidney and Reeck, 1978; Isackson et al., 1979; Yoshida and Shimura, 1984; Hamada and Bustin, 1985), reduce the linking number of circular DNA when it is ligated in their presence (Waga et al., 1989), and removes the blocks resulting from formation of cruciform (Waga et al., 1990) or B-Z junction (Waga et al., 1988) structures due to supercoiling of closed-circular DNA suggest that these proteins may play a role in unwinding the DNA structure (Waga et al., 1990). These characteristics led us to expect that these proteins might play a role in modulating the structure of chromatin. In vitro reconstitution of chromatin in the presence of the HMG proteins showed that they suppress nucleosome assembly at physiological ionic strength to give a destabilized chromatin structure (Waga et al., 1989), contrary to the earlier results obtained by Bonne-Andrea et al. (1984). Our present results demonstrated that the chromatin structure in the presence of large quantities of the HMG1 and -2 proteins was decondensed. The protein compositions of the minichromosomes suggested that the decondensation was due to the presence of large amounts of the HMG protein bound to the minichromosome or to a deficiency of histone H1s, which stabilize the chromatin structure (van Holde, 1989), as discussed above. Interestingly, the minichromosome in 1-OE cells appeared to be more decondensed than that in 2-OE cells (Fig. 5). As the extra amounts of the individual HMG proteins contained in the respective minichromosomes were similar, irrespective of the level of expression of the HMG proteins in the cells, HMG1 protein itself, or possibly some factor that functions with the protein, may have a higher capacity to change the chromatin structure than the HMG2 protein. The decondensation of minichromosomes induced by the HMG proteins was not restricted to the promoter and coding genes but involved the entire region. Even if the HMG protein binds to the chromatin of a transcriptionally active region, or assembly of the HMG protein activates the gene to be transcribed, changes in chromatin conformation may be an important process of gene transcription on chromatin. This first, to our knowledge, direct demonstration of changes in chromatin condensation due to binding of HMG proteins may provide valuable information that will further our understanding of the mechanisms responsible for changes in chromatin structure during the transcription reaction.

Previously, we carried out studies to assess whether HMG1 protein has transcriptional activation potential by employing two assay systems. Introduction of HMG1 protein into COS-1 cells as a complex with a reporter plasmid carrying the lacZ gene enhanced the level of the gene expression. Co-transfection of an expression plasmid carrying HMG1 cDNA into the cells with a reporter plasmid carrying the lacZ gene enhanced the activity of -galactosidase 2- to 3-fold in comparison with that of a control effector plasmid (Aizawa et al., 1994). In the present study, the enzyme activities originating from the reporter plasmids carrying the lacZ and cat genes were enhanced approximately 2-fold in 1-OE, but not in 2-OE cells, in comparison with those in N-OE and C-127 cells (Fig. 2). The maximum activity was expressed at 72 h after transfection of the reporter plasmids, although the amount of mRNA in the cells decreased quickly after 48 h of transfection (data not shown), similar to our previous results (Aizawa et al., 1994). Because the reporter plasmids used carried no replication origin for propagation in the cells transfected, these results suggest that these enzymes may be relatively stable proteins in C-127 and their derivative cells in spite of the instability of their mRNAs, and that the enzyme activity at various times after transfection of the reporter plasmids may represent the amount of enzyme protein synthesized and accumulated in the cells before preparation of the cell extract. Therefore, the enhanced activity did not result from differences in the copy number of the reporter plasmid, but was attributable to the level of gene expression ( Fig. 3and ). This enhancement, which is consistent with our previous observations, indicates that HMG1 protein does, in fact, have transcriptional activation potential and that HMG2 protein does not, at least in the three assay systems we employed. The gene expression enhancements by HMG1 protein were independent of the sources of the promoter and the gene, suggesting that HMG1 protein function may not be dependent on the DNA sequence. The minichromosome prepared from 1-OE cells was extremely susceptible to micrococcal nuclease digestion in comparison with that from 2-OE and control cells. Furthermore, the ampgene, as well as the promoter sequence and reporter gene, was also susceptible to such digestion. These results suggest that the enhancement of gene expression resulted from the decondensation of the entire minichromosomal region by HMG1 protein. Decondensation of chromatin structure may be necessary, but insufficient alone, for enhancement of gene expression, because the minichromosome in 2-OE cells was also decondensed. In vitro experiments showed that the carboxyl-terminal truncated HMG1 molecule, which has a stronger DNA binding activity than the intact HMG1 molecule, repressed the transcription of reporter plasmids, suggesting that the acidic carboxyl-terminal domain decreases DNA binding activity and is engaged in enhancement of transcription (Aizawa et al., 1994). The DNA binding ability of DNA binding regions in HMG2 protein appear similar to that of HMG1 protein, because their primary sequences are similar (Shirakawa et al., 1990). Therefore, the different lengths and sequences of the acidic residues in these proteins may confer differential effects on their DNA binding and gene expression enhancement activities. However, we cannot rule out the important possibility that the modulatory effect of HMG1 protein on chromatin structure is fundamentally different from that of HMG2 protein, because we also observed apparent changes in the conformation of the minichromosome by HMG2 protein. The function of HMG1 protein may be to modulate the chromatin structure so that it assumes a diffuse form suitable for effective transcription of the gene.

  
Table: Summary of the levels of HMG1 and HMG2 expression in the cells overexpressing HMG proteins and in the minichromosomes purified from lysate of those cells transfected with pSV2CAT

The values relative to those of C-127 cells and the standard deviations are presented.


  
Table: Summary of the levels of gene expression in 1-OE and 2-OE cells by reporter plasmids carrying the various promoters and genes

The cells were transfected with the expression plasmids carrying the various promoters and genes listed in the table, and the enzyme activities and proteins expressed in the cells were determined by assaying the cell extracts. The values relative to those of N-OE cells and the standard deviations are presented.



FOOTNOTES

*
This work was supported, in part, by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan and by a grant from the Science Research Promotion Fund from Japan Private School Promotion Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 81-271-24-1501 (Ext. 4403); Fax: 81-271-25-1841.

The abbreviations used are: HMG, high mobility group; bp, base pair(s); kbp, kilobase pair(s); BPV, bovine papilloma virus; CAT, chloramphenicol acetyltransferase; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; 1-OE cells, cells overexpressing HMG1 protein; 2-OE cells, cells overexpressing HMG2 protein.

F. Yamazaki, Y. Nagatsuka, H. Shirakawa, and M. Yoshida, submitted for publication.


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