From the
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.
High mobility group (HMG)
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.
Preparation of Plasmids The plasmids used for this work were prepared as follows.
The C-127 cells were transfected with the
pBPV-HMG1, pBPV-HMG2, and pBPV-SR
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
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.
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.
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.
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 H1
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
The values relative to those of C-127 cells and the standard
deviations are presented.
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.
(
)
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.
Plasmids pBPV-SR
The
plasmid pBPV (Pharmacia Biotech Inc.) was cleaved with XhoI at
the multicloning site. In order to construct pBPV-SR, pBPV-HMG1, and pBPV-HMG2
as a control
vector, a 2,970-bp DNA fragment containing the neo
gene 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% CO
at 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 amp
gene, 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 amp
gene ( 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 amp
gene, respectively.
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.
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).
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.
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
amp
gene (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
amp
gene 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.
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.
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).
clearly 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).
-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 amp
gene, 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
Table:
Summary of the
levels of gene expression in 1-OE and 2-OE cells by reporter plasmids
carrying the various promoters and genes
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.