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Molecular and Cellular Biology, June 2000, p. 4062-4074, Vol. 20, No. 11
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Interference of the Simian Virus 40 Origin of
Replication by the Cytomegalovirus Immediate Early Gene Enhancer:
Evidence for Competition of Active Regulatory Chromatin
Conformation in a Single Domain
Peng-Hui
Chen,1
Wen-Bin
Tseng,2
Yi
Chu,3,
and
Ming-Ta
Hsu3,*
Graduate Institute of Life Sciences, National
Defense Medical Center,1 Institute of
Biomedical Sciences, Academia Sinica,2 and
Institute of Biochemistry, School of Life Science, National
Yang Ming University,3 Taipei, Taiwan, Republic
of China
Received 26 October 1999/Returned for modification 4 December
1999/Accepted 14 March 2000
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ABSTRACT |
Replication origins are often found closely associated with
transcription regulatory elements in both prokaryotic and eukaryotic cells. To examine the relationship between these two elements, we
studied the effect of a strong promoter-enhancer on simian virus 40 (SV40) DNA replication. The human cytomegalovirus (CMV) immediate early
gene enhancer-promoter was found to exert a strong inhibitory effect on
SV40 origin-based plasmid replication in Cos-1 cells in a position- and
dose-dependent manner. Deletion analysis indicated that the effect was
exerted by sequences located in the enhancer portion of the CMV
sequence, thus excluding the mechanism of origin occlusion by
transcription. Insertion of extra copies of the SV40 origin only
partially alleviated the inhibition. Analysis of nuclease-sensitive
cleavage sites of chromatin containing the transfected plasmids
indicate that the chromatin was cleaved at one of the regulatory sites
in the plasmids containing more than one regulatory site, suggesting
that only one nuclease-hypersensitive site existed per chromatin. A
positive correlation was found between the degree of inhibition of DNA
replication and the decrease of P1 cleavage frequency at the SV40
origin. The CMV enhancer was also found to exhibit an inhibitory effect
on the CMV enhancer-promoter driving chloramphenicol acetyltransferase
expression in a dose-dependent manner. Together these results suggest
that inhibition of SV40 origin-based DNA replication by the CMV
enhancer is due to intramolecular competition for the formation of
active chromatin structure.
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INTRODUCTION |
A majority of DNA replication
origins in both prokaryotic (3, 11, 35, 42, 53, 56, 59) and
eukaryotic (1, 5, 12, 17-19, 24, 25, 36, 38, 41, 43, 48, 60, 62-66, 68, 71, 75, 78, 81, 83, 84, 93-95) cells are closely
associated with a transcription unit. Recent characterization of
origins of replication in mammalian cells has shown that the origins
are located very close to a transcription unit or even within a
transcription unit (see review in reference 20). The tight association between transcription and replication units has
raised the question of whether or not these two processes are coupled
(32, 36, 42, 64, 91).
Mutual influences between replication and transcription processes have
been demonstrated. Activation of DNA replication by transcription has
been found in both prokaryotic (3, 11, 35, 56, 70) and
eukaryotic (48, 60) cells. On the other hand, transcription
has been found to suppress replication in bacterial plasmid DNA
(47, 53) and in yeast (77, 82), tetrahymena
(61), and human (33) cells. Conversely, DNA
replication can enhance transcription (28, 32, 64, 90, 91).
Evidence for coupling or a correlation between transcription and
replication processes has also been observed in Bacillus
subtilis (42), in Physarum (64),
in temporal regulation of transcription in active genes in early S
phase (26, 37), and in several mammalian genes (5, 41,
71, 94). Transcription factors have also been implicated in
regulating replication origins (7, 13, 18, 19, 30, 31, 38,
52).
To understand the role of transcription factor regulatory elements and
the transcription process in the regulation of eukaryotic DNA
replication, we studied the effect of strong promoter-enhancer on the
simian virus 40 (SV40) replication origin. Our results indicate that
the human cytomegalovirus (HCMV) immediate early (IE) gene enhancer
inhibits SV40 origin-dependent replication in a position- and
dose-dependent manner. A similar inhibitory effect was observed in a
plasmid containing a Rous sarcoma virus (RSV)-mouse mammary tumor virus
(MMTV) enhancer-promoter. Our results based on probing chromatin
structure at regulatory sequences by P1 nuclease suggest that
inhibition of the SV40 origin by the CMV IE enhancer is likely to be
the result of competition between the SV40 origin and CMV enhancer for
the formation of active chromatin conformation. P1 nuclease analysis
provided strong evidence that only one of the regulatory elements in a
DNA containing multiple regulatory elements can form active chromatin structure.
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MATERIALS AND METHODS |
Plasmid constructions.
Table 1
describes the plasmids used in this
study. Plasmid pCMVIE, a gift from Felicia Wu, contains a CMV IE
enhancer-promoter in front of a chloramphenicol acetyltransferase (CAT)
reporter gene. For the construction of pSCM1 plasmids (Fig.
1a), the CMV sequence in pCMVIE,
containing the HCMV IE gene enhancer and promoter, was excised by
digestion with restriction endonucleases ClaI and HindIII, extended with HindIII linker,
and then inserted at the HindIII site (nucleotide [nt]
5001/1 in pSV2cat) of pSV2cat or pSV2neo to generate plasmid pSCM-1cat
or pSCM-1neo. Insertion of the CMV IE gene enhancer-promoter at other
sites of pSV2cat was achieved by adding appropriate restriction
endonuclease linkers to the CMV fragment or by blunt-end ligation using
T4 DNA polymerase or Klenow fragment. Plasmids were named
pSCM-x(y), where x is the nucleotide
number of pSV2cat at which the CMV enhancer-promoter was inserted and
y represents the inserted orientation relative to the
direction of transcription from the SV40 early promoter, either the
same [(+)] or opposite [(
)] direction.
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FIG. 1.
(a) Map and replication efficiency of pSCM plasmids. The
circular map represents the parental plasmid, pSV2cat. Positions of
restriction sites at which the HCMV IE gene enhancer-promoter is
inserted are indicated on the map, together with the replication
efficiency of that plasmid relative to the parental plasmid. + and represent the orientation of the CMV promoter relative to the SV40
early promoter. (b) Map of pCATBasic vector. AmpR, ampicillin
resistance.
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Insertion of restriction fragments of CMV enhancer-promoter sequence
into the
HindIII site of pSV2cat was performed
similarly.
BAL31 deletion mutations of the CMV enhancer or
promoter sequence
were constructed by deletion from a restriction site
as indicated
in the
text.
Plasmid pL, used as an internal control of replication efficiency, was
constructed by replacing the
HindIII-
SmaI
fragment
of pSV2neo with a 564-bp
HindIII fragment of
lambda phage
DNA.
Plasmid clones containing a human triplet CAG repeat, bent DNA sequence
from
Leishmania minicircle (
49), and monkey cell
alpha satellite repeat sequence of monomer, dimer, and trimer
sizes
(171, 342, and 513 bp, respectively) were constructed by
inserting the
appropriate DNA fragment into the
HindIII site of
plasmid pSV2cat or pSV2neo. Inserted sequences were checked by
DNA
sequencing.
Oligonucleotides corresponding to SV40 21-bp repeats, human telomere
repeat (5 to 15 repeats of TTAGGG), and NF
B sequence
(ATCAACGGGACTTTCCAA repeated twice) with
HindIII linker sequence
at the ends were synthesized
using an oligonucleotide synthesizer.
These oligonucleotides were
cloned into pSV2cat at the
HindIII
site. The presence of
the target insert was confirmed by DNA
sequencing.
pSC series plasmids were constructed from the parental plasmid,
pCATBasic (Promega), by the insertion of an SV40 origin fragment
or its
polymer (SV40 nt 5197 to 325) at the
HindIII site (nt
2266
in the parental plasmid) to obtain plasmids pSC-2266, pS2C-2266
(containing two copies of the SV40 origin), and pS3C-2266 (containing
three copies of the SV40 origin). The CMV enhancer/promoter was
then
inserted at the
HindIII site of these plasmids to obtain
the corresponding plasmids pSC-2266/CMV, pS2C-2266/CMV, and
pS3C-2266/CMV.
Plasmid pCM was constructed by inserting one copy of the
CMV promoter-enhancer
(
NcoI-
HindIII fragment)
at the
XbaI (nt 2266) site of pCATBasic
(Promega). Plasmids
pCM-en1, pCM-en2, and pCM-en4 were derived
from pCM by the insertion of
an additional copy of the CMV enhancer
fragment,
ClaI to
NcoI (
598 to
223), at the positions indicated
in Table
1.
Plasmid pMAMneo-CAT was obtained from Clontech. pMneo-CAT was
constructed from pMAMneo-CAT by removing the
MluI-to-
BspEI fragments,
deleting the RSV-MMTV
sequences.
Transient DNA replication assay.
Cos-1 cells at 50 to 60%
confluence were cotransfected by the DEAE-dextran method
(79) with 1 µg of each plasmid DNA to be tested and pL.
This cell line expresses SV40 T antigen constitutively, thus allowing
the replication of plasmids containing the SV40 DNA replication origin.
Transfection efficiency was around 1 to 2% as determined by
transfection with plasmid containing 
-galactosidase reporter gene.
At 48 h after transfection, plasmid DNA was recovered from the
transfected cells by the Hirt method (39), digested with
restriction nuclease DpnI to remove parental unreplicated DNA and then with appropriate restriction endonucleases as indicated in
the figure legends, and analyzed by agarose gel electrophoresis and
Southern blotting. Replication efficiency of a test plasmid relative to
the parental pSV2cat plasmid was determined using the cotransfecting pL
as the internal standard. The radioactivity of each DNA band was
measured with an Instantimager (Packard). The ratio of the replicated
test plasmid relative to pL was corrected for the input amount in the
same Southern blot.
Two-dimensional gel electrophoresis analysis of DNA replication
intermediates.
Plasmid DNA recovered from transfected cells was
cleaved with restriction endonucleases StuI and
FspI and analyzed by a two-dimensional electrophoresis
technique described previously (87).
CAT assay.
CAT activity was measured by the method of Gorman
et al. (27). Each set of measurements included a mock
control and a pSV2cat positive control.
P1 nuclease digestion of intracellular nucleoprotein
complexes.
Cos-1 cells transfected with plasmids were lysed at
48 h after transfection with lysis buffer (0.5% NP-40, 10 mM
Tris-HCl [pH 7.5], 0.2 mM ZnCl2), and the nuclei were
digested with P1 nuclease as described previously (15).
After P1 nuclease digestion, plasmid DNA was extracted by the Hirt
method and purified by phenol and chloroform extractions. The purified
plasmid DNA was digested with restriction endonucleases as described in
the text and figure legends and then analyzed by agarose gel
electrophoresis and Southern blot analysis using appropriate probes
that abut one end of the cutting sites (indirect end labeling).
Transformation of Cos-1 cells.
Cos-1 cells were transfected
with plasmid pSCM-1neo, and the transfected cells were selected with
G418 (300 µg/ml). Surviving cell colonies were picked using Pasteur
pipettes and grown in the presence of G418 (300 µg/ml).
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RESULTS |
Position-dependent inhibition of the SV40 origin activity by the
CMV IE gene enhancer-promoter.
To study the effect of a
neighboring strong transcription unit on the activity of the SV40
origin of replication, we inserted the CMV IE gene enhancer-promoter at
the HindIII site (nt 1/5001) located immediately on the
early promoter side of the SV40 replication origin in plasmid pSV2cat
(Fig. 1a). The resulting plasmid, pSCM-1(), with the CMV promoter in
the opposite direction of the SV40 early promoter, was found to
accumulate about 1 to 3% of the amount of DNA of the parental plasmid,
pSV2cat, using the cotransfected plasmid pL as a quantitative internal
standard (Fig.
2a;
compare the region of test plasmid DNA in lanes 2 and 8). Similar
results were obtained when the CMV sequence was inserted in the
opposite direction (data not shown). Control plasmids containing
insertions of various DNA fragments similar in size to the CMV
enhancer-promoter at the HindIII site were also analyzed
(Fig. 2b). Insertion of lambda phage DNA fragments, alpha satellite,
telomeric, and CAG repeats, and Leishmania bent DNA had no
significant effect on replication of plasmid DNA. Thus, the inhibition
of replication of plasmid pSCM-1 is specifically mediated by the CMV IE
sequence.
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FIG. 2.
Gel electrophoretic and Southern analysis of replication
of test plasmids transfected into Cos-1 cells. Lane sets A and B
represent input plasmid DNA samples used for transfection and plasmid
DNA extracted from transfected cells 48 h posttransfection and
treated with restriction endonuclease DpnI to remove
unreplicated and methylated input DNA, respectively. Plasmid pL
cotransfected with the test plasmids is the internal standard used to
standardize transfection efficiency. (a) Analysis of pSCM plasmids.
Lanes 1 and 7, pSV2cat parental plasmid; lanes 2 and 8, pSCM-1();
lanes 3 and 9, pSCM-4755(); lanes 4 and 10, pSCM-3369(); lanes 5 and 11, pSCM-3502(); lanes 6 and 12, pSCM-2618(). All DNA samples
had been digested with endonuclease EcoRI and probed with pSCM-2618() plasmid DNA. The amount of
test plasmid DNA before and after transfection relative to the internal
standard was determined by the radioactivity in the Southern blot as
described in Materials and Methods. (b) Analysis of replication of
control plasmids with insertion of a 513-bp monkey alpha satellite
repeat (lanes 1 and 3) and 564-bp lambda phage HindIII
fragment (lanes 2 and 4) at the HindIII site of pSV2cat.
(c) Analysis of transient DNA replication of pMAMneo-CAT and its
derivative pMAneo-CAT, with the RSV-MMTV transcription
enhancer-promoter deleted. Lanes 1 and 4, pSV2cat; lanes 2 and 5, pMAMneo-CAT; lanes 3 and 6, pMAneo-CAT. With pL as an internal control,
pMAMneo-CAT replication efficiency is about 25% of that of pSV2cat,
whereas that of the deletion mutation is about 75%. Sizes of markers
(M) are indicated in nucleotides.
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When the CMV sequence was moved away from the SV40 origin, the
suppression of SV40 origin-dependent DNA replication was reduced,
but
the effect depended on the position of the CMV IE enhancer-promoter
sequence relative to the SV40 origin (Fig.
2a, with one orientation
of
CMV; results were similar with the other orientation [Fig.
1]). There
is no simple relationship between the distance of the
two regulatory
elements and the degree of inhibition (see Discussion).
For example,
insertion of the CMV sequence at the
PvuII site (nt
323)
immediately on the late promoter side of origin reduced the
accumulated
DNA amount to about 1 to 5% of that for pSV2cat, but
CMV sequence
located at the
NdeI site at nt 554 reduced DNA replication
to only about 45% of the control. On the other hand, the CMV
enhancer-promoter
at the
EcoRI site about 2.6 kb away from
the SV40 origin reduced
the accumulated DNA amount to about 20% of the
control (Fig.
2a,
lanes 6 and 12). These results suggest a
position-dependent effect
of the CMV IE gene enhancer-promoter on SV40
origin-based DNA
replication. The position-dependent inhibitory effect
is highly
reproducible, since all experiments were performed at least
three
times and each set of data was carefully analyzed by counting
the
radioactivity in each DNA band with an Instantimager. Similar
results were obtained when plasmid pSV2neo was used as the starting
vector.
To examine whether the suppression effect is specific to the CMV
sequence, we carried out replication analysis of a commercially
available plasmid, pMAMneo-CAT, that contains an RSV-MMTV transcription
regulatory element and an SV40 origin. This plasmid replicated
poorly
compared to pSV2cat. Deletion of the RSV-MMTV transcription
regulatory
element induced replication activity about threefold,
indicating that
this transcription regulatory element also exerts
a negative effect on
the SV40 origin (Fig.
2c).
Dosage effect of the CMV enhancer-promoter on SV40 DNA
replication.
The results above indicate a position-dependent
effect of CMV sequence on SV40 origin activity. To further analyze the
mechanism of inhibition of the SV40 origin by the CMV sequence, we
inserted multiple copies of the CMV sequence, either in tandem or
separately, in pSV2cat. Insertion of extra copies of the CMV sequence
either in tandem or at different sites further inhibited SV40
origin-dependent replication (Fig. 3).
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FIG. 3.
Dosage effect of CMV IE enhancer-promoter on SV40 origin
activity. Replication efficiency relative to that of the parental
plasmid pSV2cat was determined for plasmids containing one copy (1×)
or two copies (2×) of the HCMV IE enhancer-promoter (P/E) inserted at
the positions indicated. The orientation of the CMV promoter relative
to the SV40 early promoter is indicated as (+) or (). Plasmids
containing two copies of the CMV enhancer-promoter replicate much less
efficiently than plasmids with only one.
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Inhibition of SV40 origin-dependent replication is located in the
enhancer portion of the CMV regulatory element; exclusion of an origin
occlusion mechanism.
To determine whether inhibition of the SV40
origin is due to interference of initiation at the origin by
transcription from the CMV promoter, we removed the CMV promoter by
BAL31 nuclease deletion from the 3' end of the CMV
enhancer-promoter. As shown in Fig. 4,
deletion up to nt 91 including the TATA and CAAAT promoter elements
did not diminish the ability of the CMV sequence to suppress SV40
origin-dependent replication when the CMV sequence was inserted at the
HindIII site in either orientation. Furthermore, the
SV40 origin was not suppressed by the insertion of the minimal CMV
promoter from nt 42 to +107 at either HindIII (early
side of origin) or NdeI (late side of origin) in either
orientation. These results indicate that transcription toward or away
from the origin from either the early or late side does not affect SV40
origin-dependent DNA replication and that the CMV promoter is
dispensable for the inhibitory effect.
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FIG. 4.
Effect of deletion of the HCMV IE enhancer-promoter on
SV40 origin activity. The sequence organization of various repeats of
the IE enhancer (filled bars) and promoter elements (open bars) is
shown at the top. The portion retained after deletion is shown as solid
lines. The deleted CMV sequence was inserted at the
HindIII site (nt 1/5001) of pSV2cat, and the replication
efficiency of these plasmids relative to that of the parental plasmid
was determined by using the cotransfected pL internal standard.
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The fact that promoter deletion is still fully capable of inhibiting
SV40 DNA replication indicates that the suppressing sequences
of CMV
are located within the enhancer sequence. To further locate
the
suppression elements, we inserted portions of the CMV enhancer
at the
HindIII site of pSV2cat and studied the transient
replication
of these plasmids in Cos-1 cells. The 5' portion of the
enhancer
represented by the restriction fragments
ClaI-
NdeI (nt
598 to
348 relative to CMV IE
transcription start site) and
ClaI-
NcoI
(nt
598
to
223) reduced the plasmid DNA amount to about 11 to
18% and 6%,
respectively, of the control plasmid (Fig.
4). Thus,
the 5' portion of
the enhancer still contained repressor elements,
but the effect was
less than that of the whole enhancer. Similarly,
the 3' portion of the
enhancer, represented by the restriction
fragments
NdeI-
HindIII and
NcoI-
HindIII, also suppressed plasmid
replication to about 6% of the control (Fig.
4). These data indicate
that SV40 origin-suppressing elements are located throughout the
enhancer.
To map the suppressing elements further, we studied the effect of
deletion in the 3' portions of the CMV enhancer. As summarized
in Fig.
4,
BAL31 deletion analysis suggests a suppressing element
between nt
135 and
76. This region includes an NF-
B and two
SP1
transcription factor binding sites. Similarly, deletion of
sequence in
the 5' enhancer portion from
598 to
507 raised the
replicated DNA
amount from 18 to 75% of the level for pSV2cat.
This result suggests a
suppressing element between nt
598 and
507.
Since the SV40 enhancer also contains NF-
B and SP1 sites, we sought
to determine if these transcription factor binding sequences
in the 3'
portion of the CMV enhancer could inhibit the SV40 origin
through some
kind of competition with the SV40 enhancer. To this
end, we constructed
plasmids containing NF-
B binding site oligonucleotides
corresponding
to the 18-bp repeats in the CMV IE enhancer or its
oligomers and
inserted them at the
HindIII site of pSV2cat. No
suppression of plasmid replication was observed (data not shown).
Similarly, insertion of 21-bp repeats which contained multiple
SP1
binding sites from SV40 DNA at the
HindIII site of
pSV2cat
also did not appreciably affect the replication of plasmid DNA
in Cos-1 cells (data not shown). Thus, neither the SP1 nor NF-
B
binding site alone seems capable of inhibiting SV40 DNA replication
when inserted at the
HindIII site immediate to the early
side
of the SV40
origin.
Suppression of replication of plasmids containing two or three
copies of origin of replication by the CMV enhancer-promoter.
The
severe inhibition of the SV40 origin by the CMV enhancer-promoter
inserted at the EcoRI site 2.6 kb from the SV40 origin suggests a long-range effect of the CMV enhancer-promoter. To examine
whether this effect is due to long-range interactions between SV40 and
CMV regulatory elements or due to global effects of DNA topology or
nucleoprotein organization by the presence of CMV regulatory elements,
we inserted one or more extra copies of the SV40 origin in pSCM-1().
We reasoned that if the inhibition is through direct one-to-one
protein-mediated interactions between CMV and SV40 regulatory elements,
then one of the SV40 origins should always escape the inhibitory effect
and replication proficiency should be restored by the insertion of
extra copies of the SV40 origin. As shown in Fig.
5a, insertion of extra origins in this plasmid did not result in recovery of full replication activity. A
plasmid with insertion of an extra copy of the origin at nt 2618 in
pSCM-1() was still severely inhibited by a single copy of CMV
[plasmid pS2CM-1()/ori-2618(+) in Fig. 5a]. Even for the plasmid
containing three copies of the SV40 origin, one at the normal position
and tandem copies at nt 4751, replication was still repressed by the
CMV sequence to about 36% of the level for the control plasmid
pSV2cat. Similar inhibition efficiency by the CMV promoter-enhancer was
observed for plasmid constructs containing one, two, or three copies of
the SV40 origin in tandem but with the CMV enhancer inserted on the
late (Fig. 5b) or early (data not shown) side of the origin. These
results suggest that the CMV enhancer-promoter exerts a global
inhibitory effect that is not due to a direct one-to-one interaction
between SV40 and CMV regulatory sequences.
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FIG. 5.
Inhibition of replication of plasmids containing
multiple copies of the SV40 origin by a single copy of the HCMV
enhancer-promoter. (a) Additional copies of the SV40 origin (ori) were
inserted in plasmid pSCM-1 at the positions indicated in the plasmid
names. Average replication efficiency of parental plasmid pSV2cat is
taken as 100%. (b) pSC constructs (see Materials and Methods)
containing one, two, or three tandem copies of the SV40 origin or their
derivatives with a single HCMV enhancer-promoter insertion were
analyzed for replication efficiency. The minus sign following the
number 2266 indicates that the SV40 late promoter was used to drive CAT
gene expression. Replication efficiency was determined using pL as
internal standard. Replication efficiency of the pSC plasmids (pSC,
pS2C, and pS3C) without CMV insertion is taken as 100%.
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Competition between SV40 origin and CMV IE enhancer for the
establishment of active chromatin conformation; evidence for the
presence of only one nuclease-hypersensitive site per chromatin
domain.
The results described above indicate that the inhibition
of SV40 origin-dependent replication by the CMV enhancer is not due to
one-to-one interaction between SV40 and CMV regulatory sequences. To
further elucidate the mechanisms of inhibition of SV40 origin activity
by the CMV enhancer, we examined the possibility that the CMV enhancer
alters the chromatin conformation at the SV40 origin.
Active regulatory sequences either as origin of replication or as
promoter-enhancer have been shown to exhibit nucleosome-free
conformation and are hypersensitive to nuclease digestion (
7,
29). The special nucleoprotein organization at the regulatory
sequences has also been shown to establish a nucleosome phase
in
chromatin (
52,
73,
76,
92). We first considered the
possibility that the organization of transcription factors in
the CMV
enhancer altered the nucleosome phase, causing occlusion
of the SV40
origin. Analysis of nucleosome phases at the SV40
origin using
micrococcal nuclease digestion mapping did not reveal
gross alteration
of nucleosome phases at the SV40 origin between
plasmids pSV2cat and
pSCM-1(
) in the transfected cells (data
not shown). We then examined
the possibility that competition
for the formation of active chromatin
conformation as defined
by nuclease hypersensitivity may cause the
reduced frequency of
forming active chromatin at the SV40 origin and
thus the reduction
of replication activity. Evidence for such
competition has been
inferred from a previous study of SV40 genomes
containing two
separate copies of origin. These variant SV40 genomes
replicate
using only one origin at a time, and only one origin is
nuclease
hypersensitive in the DNA (
74,
89). This origin
interference
mechanism has also been observed in yeast (
8,
9,
22,
45,
54). If this mutual exclusion also occurs between the SV40
origin
and CMV enhancer, then the frequency of initiation at the SV40
origin would be reduced relative to the parental pSV2cat and the
effect
would be manifested as inhibition of DNA replication as
described
above.
To test this hypothesis, we assayed active chromatin conformation at
the CMV enhancer and SV40 origin using nuclease hypersensitivity
analysis. It has been well established that nuclease hypersensitivity
is a hallmark of active chromatin at the regulatory region
(
29).
Since we are interested in the relative accessibility
between
different regulatory sites in a single DNA molecule, a
steady-state
digestion pattern would be needed. The traditional DNase I
digestion,
however, is unsuitable for such an analysis because all
chromatin
DNA is eventually digested into small fragments. A detailed
kinetic
analysis of different sites digested would be required to
obtain
relative accessibility that may be changed during DNase I
digestion.
In the present work, we used the unique property of P1
nuclease
to determine the relative nuclease accessibility in different
regions of chromatin. Our lab has shown that P1 nuclease does
not
attack nucleosomal DNA under low-salt conditions even in prolonged
digestion and with an excess amount of the enzyme, whereas naked
DNA or
DNA in the nucleosome-free region is readily cleaved. Furthermore,
P1
cleaved SV40 chromatin only once at the regulatory sites without
further degrading viral DNA even in prolonged digestion with an
excess
amount of enzyme (
15). This unique property allowed us
to
assay the accessibility of various regions of a chromatin to
P1
nuclease without worrying about conditions for limited digestion
as in
the case of DNase I
digestion.
P1 nuclease digestion of nuclei transfected by plasmids containing
different combinations of SV40 and CMV enhancer-promoter
elements
showed that plasmids were mainly cleaved once to generate
full-length
linear DNA (Fig.
6a and b, lanes 1 to 4).
These results
indicate that only one site in the DNA is accessible to
P1 nuclease
irrespective of how many regulatory elements are present;
no products
corresponding to simultaneous double or multiple cleavages
at
regulatory elements were observed even in plasmids containing
four
regulatory elements. For example, plasmid
pS3CM-1(
)/ori(2X)-4751(
)
contains three copies of the SV40
origin, one at nt 1/5001, tandem
copies at nt 4751, and a CMV element
at nt 1/5001. P1 digestion
generated only full-length linear DNA (lanes
4); no significant
amount of products corresponding to double, or
triple cleavages
at regulatory sequences were observed. Mapping of P1
cleaved sites
in this plasmid by indirect end labeling showed that P1
cleaved
only at one of the four sites containing the regulatory
sequences
in a single molecule but not two, three, or four sites
simultaneously
(lanes 8). The fact that four cleavage products
corresponding
to the sites of the regulatory elements were observed
using by
indirect end labeling indicates unambiguously that each site
is
independently cleaved. Had they been cleaved simultaneously, one
would see only the band closest to the probe. Similar results
were
obtained for plasmids containing two copies of the CMV
enhancer-promoter
in addition to the SV40 origin and for viral DNA
containing two
copies of the SV40 origin, or one SV40 origin and an
MMTV promoter
(data not shown). These results suggest that only one
regulatory
region could form a nuclease-hypersensitive, active
chromatin
conformation in a single chromatin domain containing more
than
one regulatory sequence.
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FIG. 6.
Mapping of P1 nuclease cleavage sites of transfected
plasmid DNA in transfected cell nuclei. Cells at 48 h after
transfection were treated with lysis buffer, and the nuclei were
digested with P1 nuclease as described in Materials and Methods. (a)
Mapping of P1 cleavage sites in plasmids containing multiple regulatory
sequences. Lanes 1 to 4, plasmid DNA extracted after P1 digestion. The
main product is full linear DNA (L). The positions of forms I, II, and
III of pSV2cat DNA are indicated. Lanes 5 to 8, FspI
restriction enzyme digestion products of DNA from lanes 1 to 4. The
blot was probed with the FspI-EcoRI (nt 1845 to
2618) fragment of pSV2cat (indirect end labeling). This mapped the P1
cleavage sites from the clockwise direction in the pSV2cat map in Fig.
1. Lanes 1 and 5, pSV2cat parental plasmid. A cluster of cleavage sites
at the SV40 regulatory sequence not well resolved in this gel
electrophoresis condition is labeled "S." Lanes 2 and 6, pSCM-554(), with insertion of the CMV sequence at nt 554. Two major
clusters of cleavage sites corresponding to SV40 origin (S) and CMV (H)
enhancer were observed. Lanes 3 and 7, plasmid pSCM2-3502(+)/2618(),
with one SV40 origin and two copies of CMV at nt 2618 and 3502, respectively. Three major cleavage clusters were seen, one at the SV40
origin (S) and one each at the two copies of the CMV enhancer (H).
Lanes 4 and 8, plasmid pS3CM-1()/ori(2x)-4751(), containing three
SV40 origins, one at nt 1 and tandem copies at 4751 in addition to the
CMV sequence at nt 1. Four major cleavage clusters corresponding to the
four regulatory sites were seen. (b) The same blot as in panel a but
with an FspI-HaeII (1845 to 976) fragment. This
maps the cleavage sites from the counterclockwise direction in the
pSV2cat map. Sizes of markers (M) are indicated in nucleotides.
|
|
The single cleavage of plasmid DNA in nuclei was not due to incomplete
P1 digestion since P1 nuclease was added in excess
with prolonged
digestion (3 h). Exogenous plasmid DNA added to
nuclei before P1
digestion was rapidly cleaved into tiny fragments
within 20 min.
Digestion of in vitro-assembled chromatin by P1
nuclease indicated that
partially assembled linear plasmid DNA
was cleaved into small fragments
whereas fully assembled chromatin
from linear plasmid DNA was fully
resistant to P1 cleavage (data
not shown). If chromatin DNA in nuclei
was first cleaved with
endonuclease
BspE1, which cleaved the
plasmid once, and then digested
with P1, we observed again P1 cleavage
at either the SV40 origin
or CMV enhancer (data not shown). These
results indicate that
(i) P1 could cut linear DNA containing partially
assembled nucleosomes
in vitro and (ii) linearization of plasmid in
vivo with a restriction
endonuclease did not prevent P1 from cutting at
either of the
regulatory sites in the plasmid. Therefore, we believe
that limited
cleavage of transfected plasmid DNA is indeed due to
existence
of only one nuclease-hypersensitive site per plasmid
DNA.
If competition between regulatory sites in a single molecule for the
formation of active chromatin conformation is the mechanism
for
suppressing SV40 replication, then one would expect a direct
correlation between the relative frequency of P1 cleavage at the
SV40
origin and the degree of inhibition of SV40 origin-based
DNA
replication. As shown in Fig.
7, this
expectation is indeed
borne out; a positive correlation between
relative replication
activity and percentage of P1 cleavage at the SV40
origin was
observed (average of three independent experiments),
indicating
that the higher the sensitivity of SV40 origin to P1
nuclease,
the better its replication activity. The large effect on the
accumulated
plasmid DNA by a relatively small change in P1 cleavage is
due
to the fact that DNA replication is an amplification process.
The
analysis shown in Fig.
7 is a linear regression plot and does
not imply
any specific mechanism for the correlation observed.
This result,
together with the observations described above, suggests
that reduction
of SV40 origin-based DNA replication in plasmids
containing a strong
enhancer element is the result of competition
for the formation of
active chromatin conformation at the origin
of SV40 DNA replication.
View larger version (21K):
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|
FIG. 7.
Correlation between percentage of P1 cleavage at SV40
origin and DNA replication efficiency in pSCM plasmids. The ration of
intensity of DNA bands representing cleavage at the SV40 origin to the
total intensity of DNA bands from the cleavage of SV40 and CMV
regulatory sequence is plotted against the replication efficiency of
the plasmid relative to the parental plasmid pSV2cat.
|
|
Using higher-resolving gels, we found that there were six P1 cleavage
sites in SV40 regulatory sequences that map to SV40
enhancers, 21-bp
repeat, and SV40 core origin (data not shown).
There were two major P1
cleavage sites in the CMV enhancer that
map to the 5' and 3' portions
of the enhancer. These sites were
unchanged in different plasmids, but
there were subtle changes
in the relative intensity in different
plasmids, suggesting a
potential modulating effect of local sequence
microenvironment
on the fine organization of chromatin structure at the
regulatory
sites.
Interference of CMV enhancer-promoter-dependent transcription by
the CMV enhancer.
To further test the competition model described
above, we analyzed the effect of inserting a CMV IE enhancer fragment
on CAT reporter gene activity driven by the CMV enhancer-promoter. We reasoned that competition between the two enhancers in the
establishment of active chromatin conformation would reduce the
transcription initiated from the CMV enhancer-promoter in a manner
dependent on the copy number of the competing enhancers. This was
indeed observed (Fig. 8). Relative to the
control without enhancer insertion, CAT activity decreased in a manner
relative to the number of the competing enhancer. This result supports
the competitive model described above.
View larger version (15K):
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|
FIG. 8.
Suppression of transcription of CAT gene by the HCMV
enhancer-promoter by the HCMV enhancer fragment. pCM is a plasmid with
a CAT gene driven by the HCMV enhancer-promoter. CAT activity of this
plasmid is taken as 100%. pCM-en1, pCM-en2, and pCM-en4 contain one,
two, and four copies of the HCMV 5' enhancer
(ClaI-NcoI fragment) inserted at the positions
indicated in Table 1. Extra copies of the enhancer inhibit CAT activity
driven by the HCMV enhancer-promoter.
|
|
Enhancement of CMV promoter-initiated CAT activity by the
combination of SV40 origin and either CMV or SV40 enhancer.
In
contrast to the severe inhibition of SV40 origin-dependent DNA
replication by the CMV enhancer-promoter in pSCM-1(+), CAT activity of
this plasmid was actually 6-fold higher than that of the parental
plasmid pSV2cat and 200-fold higher than that of plasmid pCM without
the SV40 origin when assayed at 48 h posttransfection in Cos-1
cells (Fig. 9). Since the pSCM-1(+) DNA
amount is about 1 to 2% of the pSV2cat amount, the CAT activity per
DNA template was actually 200- to 400-fold higher than that of pSV2cat
at 48 h posttransfection. These results showed that although DNA
replication was suppressed by the CMV sequence, transcription was
stimulated by the presence of the SV40 origin.
View larger version (17K):
[in this window]
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|
FIG. 9.
Enhancement of transcription activity by CMV. CAT
activity in cells transfected with pSV2cat, pSCM-1(+), pSCM-1(),
pSCM-1(+)-1, and pSCM-1(+)-2 was analyzed at 48 h posttransfection
as described in Materials and Methods. CAT activity was sixfold higher
in pSCM-1(+) than in pSV2cat. Only a background level was observed for
pSCM-1(). pSCM-1(+)-1, a derivative of pSCM-1(+), contains the
portion of the CMV enhancer-promoter from nt 135 to +107 including
the promoter element plus a small segment of the CMV enhancer.
pSCM-1(+)-2 contains nt 598 to 223 of the enhancer.
|
|
The increased transcription is the result of enhancement of the CMV
promoter by the SV40 regulatory sequence, since reversing
the direction
of the CMV promoter or deletion of the CMV promoter
resulted in only a
background level of CAT activity (Fig.
9).
Removal of the SV40 promoter
reduced the enhancement level only
about threefold. Removal of both
SV40 and CMV enhancer-promoters
(21- and 72-bp repeats) resulted in
only background CAT activity.
These results indicate that the high
level of transcription observed
is initiated by the CMV promoter and
that stimulation of CAT activity
from the CMV promoter does not depend
on the SV40 promoter. Enhanced
CAT activity depended on the presence of
either the SV40 or CMV
enhancer. High-level CAT activity was still
observed in plasmids
devoid of either the SV40 or CMV enhancer.
However, deletion of
both enhancers resulted in poor CAT activity from
the minimum
CMV promoter (positions
42 to +107). The enhancement,
however,
depended on the presence of the SV40 origin of replication.
These
results suggest an up-regulation of the CMV promoter by the
combined
effect of an enhancer and SV40 core
origin.
| |
DISCUSSION |
Mechanisms of inhibition of SV40 origin-dependent replication by
the CMV IE gene enhancer.
We considered several mechanisms for the
effect of the CMV IE gene enhancer on SV40 origin-dependent
replication. First, we can exclude the transcription-mediated origin
occlusion mechanism (33, 47, 53, 61, 82) since the promoter
is dispensable (Fig. 4). Second, the possibility that the inhibition is
due to protein-mediated one-to-one interaction between the SV40 origin and CMV enhancer using a looping mechanism (57, 67, 78) seems unlikely because insertion of extra copies of the SV40 origin did
not reverse the effect. Third, we found no alteration in the bulk
nucleosome phase relative to the SV40 origin after insertion of the CMV
enhancer. However, since we looked at only the overall bulk nucleosome
phase, we could not exclude the possibility that a subpopulation of
plasmid existed with an altered nucleosome phase. Until there is a
method to separate different chromatin populations, this possibility
remains to be tested.
It is also possible that the CMV sequence blocks replication fork
passage. However, we observed no significant accumulation
of
replication intermediates in the agarose gels even in plasmids
that
were severely inhibited by the CMV enhancer. Analysis of
replication
intermediates by two-dimensional gel electrophoresis
showed that there
is no significant delay or block of replication
fork movement in the
region containing the CMV sequence. Furthermore,
since SV40 DNA
replication is bidirectional, it is unlikely that
a block of one of the
replication forks would cause significant
inhibition of DNA
replication. Because we were comparing the replication
between two DNA
molecules differing in the insertion of a 700-bp
CMV sequence, a change
in the elongation rate must be due to the
slowdown of elongation in the
CMV sequence. Since elongation occupies
only about 10% of the time of
overall replication, a 20% reduction
in replication would mean a
1,400% (20% × 10 × 5/0.7) decrease
in elongation rate through
the CMV enhancer. This was not observed
in the two-dimensional gel
analysis of replication intermediates
containing the CMV sequence.
Thus, we believe that the inhibition
of DNA replication by the CMV
sequence is not due to retardation
of replication fork movement but
must be due to inhibition of
DNA replication
initiation.
Finally, based on previous examples of origin dominance and
interference in SV40 and in yeast, we examined the possibility
that
inhibition of SV40 origin-based DNA replication by the CMV
enhancer
could be due to competition for the formation of active
chromatin
conformation at the regulatory sites. Our P1 nuclease
mapping data
indeed are consistent with this interpretation. First,
the fact that
only one nuclease-sensitive site is present per
DNA molecule
irrespective of how many regulatory sites are present
supports a
mechanism of competition for the formation of active
chromatin at SV40
origin. Second, the correlation between percentage
of origin existing
as nuclease-sensitive conformation and DNA
replication efficiency is
consistent with this interpretation.
The relatively small change in P1
cleavage compared to the large
reduction in replication activity is
mainly due to the fact that
replication is an amplified process. Thus,
a reduction of 20%
in replication initiation efficiency would result
in a 10-fold
reduction of accumulated DNA after 10 rounds of
replication. However,
the specific relationship between P1 sensitivity
and initiation
efficiency is not known, and the quantitative
relationship between
chromatin structure and DNA replication initiation
remains to
be
examined.
An interesting observation in this report is the position-dependent
effect of the CMV enhancer on SV40 origin activity. Although
enhancer
action is generally considered to be position independent,
Fromm and
Berg showed that the SV40 enhancer at a certain position
of the plasmid
failed to activate promoters (
23). Activity of
the yeast
origin of replication has also been found to depend
on the position in
a plasmid (
8,
9,
16,
54) and on chromosomal
location
(
21,
86). In yeast, hierarchy of origins is influenced
by
the origin-specific replication enhancer (
22,
45). These
observations suggest that local sequence microenvironment may
influence
the activity of a
cis-regulatory element. How local
sequences affect the activity of regulatory elements remains to
be
determined.
A prediction from the competition model is that replacement of the SV40
enhancer by the CMV enhancer should not inhibit SV40
origin-dependent
replication since there would be no competition.
To test this
prediction, we replaced the SV40 enhancer with the
CMV 5' enhancer
(
ClaI to
NcoI site), with the direction of the
CMV enhancer in the orientation toward the early SV40 promoter.
This
construction not only did not cause inhibition of SV40 origin-dependent
replication but actually increased replication threefold relative
to
that of plasmid pSV2cat. Thus, the CMV 5' enhancer was actually
superior to the endogenous enhancer in stimulating SV40 origin
activity, and the prediction was borne out. However, the SV40
origin
was inhibited when the CMV 5' enhancer was inserted in
the opposite
orientation. In contrast, the 3' portion of the enhancer
had only a
slight effect on SV40 origin activity used to replace
the SV40
enhancer. These results suggest that specific protein-protein
interactions are required for stimulation of the SV40 origin by
the
enhancer as described previously (
20).
Single P1 nuclease cleavage site in plasmids containing more than
one regulatory sequence.
In this study we used P1 nuclease to
probe the chromatin conformation at the regulatory sequences in the
various plasmids in vivo. Previously we showed that P1 made only a
single cut at the SV40 regulatory sequence while leaving the rest of
DNA intact even after overdigestion with an excess amount of the enzyme
(15). In vitro nucleosome reconstitution experiments using
pBR322 plasmid DNA confirmed the resistance of nucleosomal DNA to P1
nuclease digestion under low-salt conditions, whereas naked DNA is
degraded into acid-soluble fragments (data not shown). This unique
property of P1 nuclease allows us to probe the relative accessibility
of different regulatory sequences in a single molecule to P1 digestion in limiting digestion conditions. This type of analysis would be much
more difficult with the traditional DNase I method because the
sensitivity to DNase I is only relative and eventually all DNA
sequences are degraded into small fragments.
Using P1 nuclease as a probe, we were able to clearly show that in the
plasmid containing more than one regulatory sequence,
only one is
accessible to P1 nuclease digestion. This was observed
in different
plasmid constructs containing either homologous or
heterologous
regulatory sequences in two to three copies in various
locations and
therefore is unlikely to be due to some specific
sequence effect. To
our knowledge, this is the first demonstration
of mutual exclusion of
regulatory sequences to form active conformation
in a single chromatin
domain. The mutual exclusion may result
from simple competition of the
binding of regulatory factors,
with the binding of the first site
setting up a nucleosome array
that precludes the binding of
transcription factors to the other
sites. Alternatively, competitive
binding of regulatory sequences
to nuclear matrix may determine the
biological activity of regulatory
elements and exclude the other
regulatory elements activated at
the same time. It is also possible
that the formation of nuclease-hypersensitive
sites require topological
tension and the tension can support
the formation of only one active
chromatin conformation. In fact,
Leonard and Patient (
51)
have provided evidence that P1 nuclease
cleaves the AT sequence
upstream of the
Xenopus beta-globin gene
under torsional
stress. Torsional stress has also been implicated
in the generation of
DNase I sensitivity of active genes (
85).
Several published observations are consistent with the mutual exclusion
model. Shenk showed that in viral DNA containing two
origins of
replication, only one is active at one time (
74).
Replicator
interference or dominance similar to that of SV40 has
also been found
in yeast (
8,
16,
22,
45,
54,
94).
In plasmids that contain
multiple copies of a given yeast origin,
only one origin is activated
per DNA molecule. Furthermore, the
activity of the origin depends on
the location of in the DNA,
indicating a local sequence effect. Our
result of dose-dependent
suppression of CMV enhancer-promoter-driven
CAT activity by the
CMV enhancer is also consistent with the model of
mutual exclusion
for the formation of active regulatory sites within a
given chromatin
domain. Suppression of DNase I-hypersensitive sites by
a distant
exogenous hypersensitive site has been documented (
10,
34,
73). These observations are consistent with the competition
model described above. However, origin interference could also
be
resulted from replication fork interference by a nearby dominant
origin
(
86). Such a possibility needs to be considered in further
study of origin interference in order to distinguish between initiation
and elongation as the source of
interference.
Modulation of SV40 replication origin by transcription regulatory
elements.
Modulation of SV40 origin activity by other
transcription binding sites has also been previously observed. Arizumi
et al. showed that SV40 origin activity could be modulated by
immunoglobulin heavy-chain enhancer in a manner that depended on the
SV40 enhancer-promoter and the relative orientation of the two
regulatory sequences (2). Their observation that replacement
of the SV40 enhancer-promoter with the immunoglobulin enhancer either
enhanced or inhibited SV40 origin activity is similar to our data for
the CMV IE gene enhancer. In the case of the immunoglobulin enhancer,
the 5' enhancer element activates SV40 origin activity when placed on
the early side but suppresses replication when placed on the late side
of the core origin without the SV40 enhancer-promoter. This result is
the reverse of what we observed for the CMV enhancer. Unfortunately, Arizumi et al. did not study the effect of inverting the immunoglobulin enhancer. In our case, the CMV enhancer activates the SV40 core origin
in one orientation but inhibits in the other orientation when placed on
the late side of the core origin (data not shown).
In contrast to the CMV IE gene enhancer, other transcription factor
binding sites such as the c-Myc enhancer (
46), BK virus
NFI
site, multimers of MMTV NFI or ATF transcription factor binding
site
(
30,
31) and a 69-bp monkey cell DNA containing AP1 sites
(
80), and multiple copies of promoter elements
(
40) have been
shown to activate SV40 minimum or
enhancerless origin-dependent
replication. In the case of monkey cell
DNA containing an AP1
site, SV40 origin activity is enhanced only when
the sequence
is inserted on the late side of the origin and the effect
is abolished
when it is inserted at a distance. Activation of the
minimal SV40
origin by the transcription regulatory elements is similar
to
our observation of activation of enhancerless SV40 origin by the
CMV
5' enhancer. On the other hand, SV40 origin activity is suppressed
by
the RSV long terminal repeat enhancer (
6) and by the bovine
papillomavirus origin in a composite plasmid (
69). These
results
could be understood from our competition model. However,
further
experimentation is required to confirm this
hypothesis.
Stable maintenance of SV40 origin-based plasmids as extrachromosomal
DNA in permissive cells is difficult, possibly due to
cell killing by
runaway DNA replication (
14,
67). The finding
that pSCM-1cat
has five- to six-times-higher CAT activity compared
to the parental
plasmid but at the same time much less replication
efficiency allows us
to obtain stable transfectants containing
plasmid pSCM-1(+)neo, using
G418 as the selection agent. We were
able to maintain eight such cell
clones for at least 3 months,
with each cell containing up to 500 copies of free plasmid DNA.
These G418-resistant cell lines could still
support the replication
of plasmids containing the SV40 origin. The
observation suggests
that plasmid pSCM-1neo may be a useful vector for
the expression
of eukaryotic genes in mammalian
cells.
| |
ACKNOWLEDGMENTS |
This work is supported by a grant from the National Science
Council of Republic of China.
We thank L. P. Ting for critical reading and comments on the
manuscript. We also thank S. F. Tsai, S. T. Lee, and Y. S. Wu for providing plasmids used for control experiments.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Biochemistry, School of Life Science, National Yang Ming University,
Taipei, Taiwan, Republic of China. Phone: 011 8862 2820 1854. Fax: 011 8862 2820 1886. E-mail: MTH{at}ym.edu.tw.
Present address: Division of Cardiology, Vanderbilt University
School of Medicine, Nashville, TN 37232-2170.
| |
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Molecular and Cellular Biology, June 2000, p. 4062-4074, Vol. 20, No. 11
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
