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(Received for publication, June 6,
1995; and in revised form, August 16, 1995) From the
The expression of multidrug resistance/P-glycoprotein genes mdr1b(mdr1) and mdr1a(mdr3) is elevated during
hepatocarcinogenesis. To investigate the regulation of mdr1b gene expression, we used transient transfection expression assays
of reporter constructs containing various 5`-mdr1b flanking
sequences in hepatoma and non-hepatoma cells. We found that nucleotides
-233 to -116 preferentially enhanced the expression of
reporter gene in mouse hepatoma cell lines in an orientation- and
promoter context-independent manner. DNase I footprinting using nuclear
extracts prepared from hepatoma and non-hepatoma cells identified four
protein binding sites at nucleotides -205 to -186 (site A),
-181 to -164 (site B), -153 to -135 (site C),
and -128 to -120 (site D). Further analyses revealed that,
while site B alone played a major part for the enhancer function, sites
A and B combined conferred full enhancer activity. Site-directed
mutagenesis results also supported these results. Gel retardation
experiments using oligonucleotide competitors revealed that the site B
contains a dominant binding protein. This is the first report
demonstrating a cell type-specific enhancer in the mdr locus.
The role of this enhancer in the activation of mdr1b gene
during hepatocarcinogenesis is discussed.
The role of the multidrug resistance transporter P-glycoprotein
(P-gp) ( Tissue-specific expression of different mdr genes has been observed in the rodents, i.e. mdr1a in
intestine and blood-brain barrier, mdr1b in kidney and adrenal
gland, and mdr2 in the liver. Homozygous disruption of the
murine mdr1a resulted in accumulation of cytotoxic drugs in
the brain and increased drug sensitivity to the animals(12) .
Likewise, disruption of the murine mdr2 led to an impairment
of hepatic phospholipid secretion in homozygous animals(13) ,
consistent with the idea that mdr2 product is a phospholipid
transporter(14) . Overexpression of MDR1 gene has
been detected in human malignant biopsies(15) , and in some
cases, correlation between elevated MDR1 expression and poor
response to chemotherapeutic agents has been reported(16) . In
animals, enhanced expression of mdr1a and mdr1b mRNA
has been seen during
hepatocarcinogenesis(17, 18, 19, 20, 21, 22, 23, 24) .
However, the underlying mechanisms for the activation remain to be
determined. To explore the mechanisms that control the expression of mdr gene during hepatocarcinogenesis, we have isolated a
genomic DNA containing the 5` portion of the mouse mdr1b.
Using a transient expression assay, we report here the identification
of an enhancer sequence proximal to the promoter of this gene that
functions preferentially in hepatoma-derived cell lines.
A
fragment containing nucleotides (nt) -1069 to +97 of mdr1b gene was synthesized by PCR DNA using the subcloned DNA
as template, SP6 promoter sequence (in the vector) as left side primer,
and oligonucleotide 3 as the right side primer. The PCR product was
cloned into the PstI-HincII sites of pGEM3Z, released
by XbaI/HindIII digestion, and recloned into the XbaI-SmaI sites of a vector containing the
chloramphenicol acetyltransferase (CAT) gene(26) , generating
-1069M1CAT. A KpnI-PstI fragment was excised
from -1069M1CAT and subcloned into the KpnI-PstI sites of the CAT vector to generate the
first 5`-deletion mutant, -586M1CAT. Three additional 5`-deletion
recombinant DNA were constructed by PCR using -1069Ml3Z as
template and oligonucleotide primers 4-6 (all contain a KpnI site), each paired with SP6 promoter sequence (in the
vector) as left side primer. The PCR products were digested with KpnI/PstI and inserted into the CAT
vector(26) , generating recombinants -233M1CAT,
-158M1CAT, and -116M1CAT, respectively. Construction of
3`-deletion mutants was performed by the same strategy using
oligonucleotides 7-9, each paired with oligonucleotide 4 and
-1069M1CAT as template. The PCR products were cut with KpnI and cloned into the KpnI site of
-116M1CAT, yielding -233(del-130/-116)M1CAT,
-233(del-155/-116)M1CAT, and -233(del-172/-116)M1CAT,
respectively.
Pairwised
oligonucleotides containing complementary DNA sequences, i.e. oligonucleotides 14 and 15, 16 and 17, 18 and 19, and 20 and 21,
were annealed, and each were cloned into the BamHI site of
pBLCAT2 vector, generating -210/-185TKCAT,
-185/-155TKCAT, -153/-135TKCAT, and
-127/-120TKCAT, as well as their reverse version of
recombinants, respectively.
The mobility shift assays were performed essentially
as described previously(32) . DNase I footprinting was carried
out using the protocol described by Ohisson and Ediund(33) .
Figure 1:
Transient transfection assay of
promoter function of the mouse mdr1b gene. A, various
lengths of the 5`-flanking sequences from -1069 to -116
plus 58 nucleotides from transcription start site (arrows)
were inserted into the CAT vector (constructs a-e). Constructs f-h represent the 3`-deletion mutants with
the deleted sequences indicated by broken lines. The putative
transcription factor binding sites (SPl and TATA) and (AT)
Recombinant -1069M1CAT (Fig. 1, construct a) was transfected into these cells.
2 days after transfection, the CAT activity in the transfected cells
was determined. Levels of CAT expression in BprC1 cells were comparable
to those in Hepalclc and Hepal-6 cells (not shown) but were about 2-
and 20-fold higher than those in NIH3T3 cells and HeLa cells (not
shown), respectively, using the cotransfected pH2B- The levels of CAT expression from
-1069M1CAT, -586M1CAT, and -233M1CAT recombinants (Fig. 1B, constructs a-c) in the
transfected BprC1 cells were not significantly different. However,
deleting nt -233 to -158 and further downstream (Fig. 1B, constructs d and e)
resulted in a reduction of more than 90% of CAT activity. These results
suggest that sequences downstream from nt -233 are important for
the expression of the mdr1b gene in mouse hepatoma cells.
Similar results were seen in Hepal-6 and Hepalclc hepatoma cells (data
not shown), in which high levels of mdr1b expression were also
seen(25) . When the same set of recombinant plasmids were
transfected into NIH3T3 cells and HeLa cells, different expression
patterns emerged. In the transfected NIH3T3 cells (Fig. 1B), the level of CAT activity progressively
decreased as the lengths of 5`-mdr1b region decreased.
Furthermore, only less than 20% reduction in the levels of CAT
expression was found between -233M1CAT (construct c) and
-158M1CAT (construct d). In the transfected HeLa cells,
there was no significant difference in the CAT activity between
-233M1CAT and -158M1CAT recombinants (not shown). These
results suggest that positive cis-regulatory element(s)
located downstream from -233 nt of the mdr1b gene
functions preferentially in hepatoma cell lines. To determine the 3`
boundary of the cis-regulatory element, we carried out similar
analysis using -233MlCAT constructs with progressive deletions
from -116 to -172 (Fig. 1A, recombinants f, g, and h). Removing sequences between
-116 and -130 or between -116 to -155 resulted
in reduction of no more than 30% CAT activity in BprC1 cells. Removal
of additional sequences, i.e. -155 to -172,
resulted in reduction of 45% of activity. These results, together with
those from the 5`-deletion assay, suggest that the sequence located
between -233 and -172 plays a major role for the expression
of the reporter constructs in BprC1 cells, whereas the sequence between
-172 and -155 may also be contributory but comparatively
less substantial.
Figure 2:
Transient transfection CAT expression
assay of the mdr1b enhancer in hepatoma BprC1 and NIH3T3 cells
in different promoter contexts. mdr1b sequences -233 to
-116 were inserted into mdrlCAT (a, b)
or pBLCAT2 (d, e) in both the forward (a, d) and reverse (g, e) orientations. The
resultant recombinants and the vector (c, f) were
transfected into BprC1 and NIH3T3 cells as indicated. The levels of CAT
expression of these recombinants were shown in A (average of
three experiments). B shows the structure of the recombinant
DNA.
To
investigate whether nt -233 to -116 can enhance reporter
gene expression from a heterologous promoter, we inserted this sequence
in both forward and reverse orientations into pBLCAT2 (Fig. 2B, constructs d-f). This vector
contains a CAT reporter gene driven by the basal promoter from the
thymidine kinase (TK) gene. As shown in Fig. 2A, the forward version of construct (construct d) displayed a 10-fold increase in the CAT activity
in BprC1 cells, whereas it displayed about a 2-fold increase for the
reverse version (construct e). Again, all of these constructs
exhibited a very low enhancement of CAT activity in NIH3T3 cells. These
results indicate that nt -233 to -116 can enhance the
reporter gene expression in an orientation- and promoter
context-independent manner. Furthermore, this sequence showed hepatoma
specificity in directing the expression of the reporter gene even in a
heterologous promoter. Therefore, this sequence can be considered as an
enhancer preferentially active in the hepatoma cells.
Figure 3:
DNase I
footprinting assay of enhancer binding proteins in nuclear extract
prepared from BprC1 cells. The DNA fragment containing the enhancer
sequence (nt -233 to -116) (panel A) was
end-labeled with
To
determine whether additional protein binding sites located downstream
from -164 that could not be adequately resolved under the gel
electrophoretic conditions were favorable for detection of sites A and
B, we carried out footprinting analyses using end-labeled fragments
spanning nt -50 to -185 for the coding strand and
-210 to -80 for the non-coding strand (Fig. 3B). At least two additional protein binding
sites, located approximately at -153 to -135 (site
C) and -128 to -119 (site D), were detected.
For the reason as mentioned above, site C could be better detected in
the non-coding strand (by the characterized flanking hypersensitive
sites) than in the coding strand. The reason for this is not clear but
could be due to the unfavorable cleavage bias of A and C residues by
the nuclease (see lanes 1 and 2 in Fig. 3B). These results demonstrated multiple protein
binding sites in the mdr1b enhancer region. Fig. 3B also shows one footprint (indicated by bars) downstream
from -120 nt. The functional aspect of this footprint was not
further characterized.
Figure 4:
CAT expression analysis of various
portions of the mdr1b enhancer. CAT expression from various
recombinant plasmid DNA (a-g) in BprC1 and NIH3T3 cells
are shown. In all cases, F and R refer to forward and
reverse versions, respectively, of the mdr constructs that were used.
The structures of the plasmid DNA are schematically shown. CAT
expression was normalized to the
To further substantiate this finding, we carried out site-specific
mutagenesis of nucleotides downstream from -233 in
-233M1CAT. A total of 11 mutants were prepared (Fig. 5A). These constructs were transfected into BprC1
cells. The CAT activities in the transfected cells were determined (Fig. 5B). Mutants b through e, which
contain specific mutations between nt -226 and -200, had
CAT activities comparable to that of the wild-type construct (construct a). Mutants f through i, which
contain mutations spanning from the 3`-half of site A to the 5`-half of
site B, however, exhibited a 90% reduction of CAT activity in BprC1
cells but no reduction in NIH3T3 cells. Mutations in sites C and D (mutants j-l) exhibited minor reduction in CAT
activities in BprC1 cells in relative to those found in mutant e, and no significant reduction in NIH3T3 cells. These results
collaborate the notion that sites A and B are important for the
enhancer function as mentioned above.
Figure 5:
Site-directed mutagenesis of mdr1b enhancer and CAT assay. A, DNA sequence of the mdr1b enhancer region. The nucleotides chosen for mutagenesis are
indicated by boldface, and the corresponding mutated
nucleotides are shown below the arrows and underscored. Locations of footprints, sites A through D, are
indicated by brackets. B, quantitative analysis of
the CAT activities of these mutants (b through l), in
reference to the activity of -233M1CAT (a) in BprC1
cells (hatched bars) and in NIH3T3 cells (open bars),
are shown. The results are from three independent transfection
experiments.
Figure 6:
Gel mobility retardation assay of
protein-DNA complexes. End-labeled probe nt -233 to -116
containing sites A through D (panels A and D),
-210 to -155 (sites A and B, panels B and E), and -162 to -116 (sites C and D, panels C and F) were mixed with nuclear extract prepared from
BprC1 cells (panels A-C) or from NIH3T3 cells (panels D-F) in the presence of poly dI
The precise reasons that oligonucleotides
containing site A and site D sequences failed to show competitions in
gel shift assays are not clear but could be due to the following
possibilities: (i) protein binding to these sites, as detected by DNase
footprinting assays (Fig. 3), may require neighboring sequences,
since the probes used in these two assay systems were not entirely the
same; (ii) protein bindings to site A and site D may require protein
occupancies to site B and site C for stabilization; (iii) site A and
site D may not be genuine protein binding sites; instead, they may be
created by protein-protein interactions, using sites B or C as
anchoring points, thereby masking nuclease accessibilities of the
neighboring sequences. Likewise, the failure of detecting enhancing
activities for site C and site D in CAT assay (Fig. 4) suggests
that proteins recognizing these sites may serve only a structural role.
Further studies are needed to clarify these possibilities. Recent studies from several laboratories have demonstrated
that several putative positive and negative cis-regulatory
elements are present in the promoter regions of the rodent and human mdr genes(35, 36, 37, 38, 39, 40, 41) .
In addition, several protein binding sites have been located at the
promoter region of the murine mdr1b gene(42, 43) . The study presented here
identified an enhancer located between nt -233 and -116 in
the mouse mdr1b gene in which four DNase I footprints are
located. Further analyses showed that site A and site B together
possess full enhancer function, but site B plays a predominant role.
Strikingly, these sequences can promote preferentially in
hepatoma-derived cell lines. This is the first cell type-specific
enhancer found in the mdr locus (a cell type-specific enhancer
upstream from the human MDR1 gene was reported(44) ,
but association to the MDR1 could be due to cloning
artifact(45) ). Site A contains the imperfect inverted
repeat sequence 5`-ACTTACCTGAACACGTAAAG (underscored). Mutations in the
first half repeat (Fig. 5, mutant e) failed to abolish
the function of the enhancer, whereas the second half did (Fig. 5, f and g), suggesting that the 5`
boundary of the enhancer may begin at the second half of site A. We
searched DNA sequences recognized by transcription factors in the
GenBank and found that the second half repeat of site A contains a
subsequence resembling the sequence motif CGT(A/C)A that is critical
for binding of a group of cellular transcription factors, i.e. ATP, CREB, E4F1, or E4TF3, and EivF. Although these transcription
factors are capable of activating E1a- and cyclic AMP (cAMP)-inducible
promoters(46, 47, 48, 49) ,
different promoters respond very differently to these inducers. Thus,
whether mdr1b enhancer can be modulated by E1a or by cAMP
remains to be determined. However, it may be relevant to note that
cAMP-dependent protein kinase regulated sensitivity of mammalian cells
to multiple drugs has been reported(50) . Site B contains two
tetranucleotide direct repeats 5`-GTATGTAAATGTCTGAGG (Fig. 5)
and a potential HNF-1 binding site
((G/A)TTAATN(A/T)T(T/C)AG)(51) . In addition, one copy of p53
binding site ( DNase I footprinting and gel mobility
retardation assays apparently suggested that protein factors
recognizing specific binding sites in the mdr1b enhancer are
present in both hepatoma and non-hepatoma cells. This does not preclude
the possibility that these proteins have a role in the regulation of mdr1b expression, specifically in hepatoma cells. For example,
ATBF1, a multiple homeodomain zinc finger protein that selectively
down-regulates hepatoma cell-specific enhancer of human
Levels of mdr1b mRNA are elevated
during hepatocarcinogenesis. Studies of mdr1b expression in
this system are hampered by the lack of culture cell systems that mimic
the in vivo situation. Culturing primary hepatocytes in
vitro for several hours shows spontaneous activation of mdr1b expression(61, 62, 63) . Whether the
enhancer described here is involved in the mdr1b expression in vivo remains to be determined. Investigations using
transgenic animals and/or targeting gene delivery to HCC (54, 64) may allow us to address these issues. These
experiments are currently under way in our laboratory. In summary,
we have characterized a hepatoma-specific enhancer in cultured cells.
The identification of this enhancer may serve as a molecular basis for
future studies of the regulation of mdr1b expression during
hepatocarcinogenesis. These studies may eventually increase our
understanding of how mdr gene expression in HCC is controlled
and hence facilitate the development of approaches to control intrinsic
drug resistance in this disease.
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBank(TM)/EMBL Data Bank with accession number(s)
M57524[GenBank].
Volume 270,
Number 43,
Issue of October 27, 1995 pp. 25468-25474
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
)in the development of resistance to a wide variety
of lipophilic compounds in animal cells has been well documented (for
reviews, see (1, 2, 3) ). P-gp is a membrane
protein containing multiple transmembrane domains and two
intracellularly localized nucleotide binding sites. It is generally
believed that P-gp functions as an efflux pump, through which cytotoxic
lipophilic compounds are expelled. P-gp is encoded by a small gene
family consisting of three members in rodents (4, 5, 6) but only two in
humans(7, 8) . However, only two rodent genes (mdr1a or mdr3 and mdr1b or mdr1) (9, 10) and one human gene (MDR1) (11) are related to the multidrug resistance function in
cultured cells.
Isolation of DNA Fragment Containing a 5`-mdr1b
Sequence and Construction of mdr1b-CAT Chimeric
Recombinants
A DNA fragment containing the 5` portion of mdr1b cDNA was synthesized by the reverse
transcriptase-polymerase chain reaction (PCR) using oligonucleotide 1
as the left side primer and oligonucleotide 2 as the right side primer
and total RNA prepared from mouse L1210/Dox 0.5 cells (25) as
template. Table 1shows the sequences of oligonucleotides 1 and 2
and those used in the subsequent experiments. The PCR product was
nick-translated with [P]dCTP and used as a probe
to screen a cosmid genomic DNA library prepared from L1210/Dox 0.5
cells(25) . A positive clone designated cosmuDR11 was isolated.
The restriction enzyme sites in the insert were mapped, and a
1.5-kilobase PstI-SmaI fragment containing the 5`-end
of the mdr1b sequence was subcloned in pGEM3Z (Promega).
Construction of mdr1 Enhancer Subsequences in the
Homologous and Heterologous Promoters
A DNA fragment
containing nt -233 to -116 was synthesized using
oligonucleotides 4 and 10 primers and -1069M13Z DNA as template.
The fragment was cloned into pCRII vector (Invitrogen),
released by HindIII digestion, and recloned into pBLCAT2
vector (27) in both forward and reverse orientations to
generate -233/116TKCAT and -116/-233TKCAT,
respectively. The same fragment with reverse orientation was recloned
into the KpnI site of -116M1CAT to generate
-116/-233M1CAT. Similarly, a DNA fragment containing nt
-210 and -155 was synthesized by PCR using primer pair
oligonucleotide 11 and oligonucleotide 12, inserted into pCRII
intermediate, excised by BamHI, and recloned into the BamHI site of pBLCAT2 vector in both forward and reverse
orientations, yielding recombinants -210/-155TKCAT and
-155/-210TKCAT, respectively. To generate recombinant
-162/116TKCAT, DNA fragment containing nt -162 to
-116 was synthesized by PCR using oligonucleotides 15 and 10
primers, cloned into pCRII
vector, excised by HindIII digestion, and recloned into the HindIII site
of pBLCAT2 in both forward and reverse orientations.
Site-directed Mutagenesis
A total of 11
site-specifically mutagenized constructs of -233M1CAT were
prepared by replacing the wild-type sequence with mutated sequences
generated by PCR. Eight left side primers (oligonucleotides
22-29) and three right side primers (oligonucleotides
30-32) were used. All of these primers contain a KpnI
site (underscored) and site-specifically altered nucleotides (boldface letters) (Table 1). PCR products were
synthesized using -133M1CAT as a template (or -1069ML3Z for
left side mutant primers), and these oligonucleotides were paired with
their opposite side primers. The PCR products were digested with KpnI/PstI and cloned into the CAT vector to generate
the first set of eight mutants or with KpnI and cloned into
-116 MlCAT to generate the second set of three mutants. All the
constructs described here were confirmed by DNA sequencing.Cell Culture, Transfection, and CAT
Assay
NIH3T3, HeLa cells, mouse hepatoma cell lines
Hepalclc and Hepalclc-BprC1 (hereafter referred to as
BprC1)(28) , and Hepal-6 (29) were maintained in
Dulbecco's modified Eagle's medium (Life Technologies,
Inc.) supplemented with 10% fetal bovine serum (Life Technologies,
Inc.). Cells were transfected with CsCl-purified (2
) plasmid DNA
(5 µg) by the polybrene method (34) using the cotransfected
pH2B-
gal plasmid DNA as an internal reference to normalize
transfection efficiency for CAT expression according to the procedure
described(25) . The pH2B-gal contains a lacZ gene
linked to a basal promoter from Xenopus laevis histone H2B
that promotes constitutive expression in all cells. In some
experiments, pCMV-gal plasmid DNA was used as internal control for
transfection calibration. No significant difference was found between
the use of these two control plasmids.Preparation of Nuclear Extracts, Gel Mobility Shift
Assays, and DNase I Footprinting Analyses
Nuclear extracts
were prepared cultured cells and mouse HCC according to the procedure
described by Dignam et al.(30) . Mouse HCC were
spontaneously developed in the SV40 T-antigen transgenic mice ((31) , gift of Dr. Janet Butel, Baylor College of Medicine,
Houston, TX).
Cell Type-specific Activity of cis-Acting
Regulatory Elements in the Mouse mdr1b Promoter
Various
lengths of the mdr1b 5`-flanking sequence were inserted into a
vector containing a promoterless CAT gene (Fig. 1A).
These constructs were transfected into mouse hepatoma cells (Hepalclc,
BprC1, and Hepa1-6) and nonhepatoma cells (NIH3T3 and HeLa). A
previous study has demonstrated that these hepatoma-derived cell lines
exhibited high levels of steady-state mdr1b mRNA(25) ,
whereas NIH3T3 cells contained very low levels of mdr1b mRNA(19) . The levels of mdr1b mRNA in these
hepatoma cells are comparable to those in mouse kidneys, one of the
organs that contain abundant mdr1b mRNA(25) . Levels
of mdr1b mRNA in NIH3T3 cells are comparable to those in the
normal livers(19) . The murine mdr1b homolog is
missing in HeLa cells, which display very low levels of MDR1 and MDR2 expression.
repeats (35, 36) are shown on
-1069M1CAT. These recombinant DNA were transfected into BprC1
cells and NIH3T3 cells (B), and the levels of expression were
assayed using cotransfected pH2B-
gal plasmid DNA as internal
control for calibration of transfection efficiency. The results are
average of three experiments.
gal as an
internal control (Fig. 1B). Similar results were
obtained with pCMV-gal, in which the expression of lacZ gene is controlled by cytomegalovirus enhancer/promoter (not
shown). These results suggest that a sequence within nt -1069 can
direct reporter gene expression preferentially in hepatoma-derived
cells. Cell type-specific activity of mdr1b upstream sequences
have also been demonstrated previously by transient transfection
assay(35, 36) .The cis-Regulatory Element Can Function in Both an
Orientation- and Promoter Context-independent Manner
To
further characterize the hepatoma-related cis-regulatory
element located downstream from nt -233 of the mdr1b promoter, we inserted nt -233 to -116 into
-116M1CAT vector in the reverse orientation (Fig. 2A, constructb). Transient
transfection assay showed that the resultant construct exhibited about
6-fold enhancement in CAT activity, in comparison with the 9-fold
increase seen in the same construct with forward orientation (Fig. 2A, construct a). This result indicated
that the cis-regulatory element located -233 to
-116 can function in both forward and reverse orientations. All
the constructs, regardless of either orientation, yielded very poor
enhancement activity in NIH3T3 cells (Fig. 2A),
supporting the cell type specificity of the regulatory element.
Multiple Protein Binding Sites in the
Enhancer
To determine whether there were specific protein
binding sites in the enhancer, we carried out DNase I footprinting
analysis. A DNA fragment containing nt -233 to -116 was
synthesized by PCR using labeled primers at either end. The labeled DNA
was incubated with nuclear extracts from BprC1 cells and digested with
nuclease. Remarkably reproducible footprints (four experiments) were
observed in the crude nuclear extracts, suggesting that the trans-acting factors are either in high abundance or have high
affinity for their target sequences. Two DNase I protection sites,
located approximately nt -205 to -186 (site A) and
-181 to -164 (site B), were found in both the
coding and noncoding strands (Fig. 3A). Some of these
footprints were characterized by the flanking DNase I hypersensitive
sites in the presence of nuclear extracts, e.g. site B in the
coding strand and site A in the non-coding strand. Due to technical
limitations, the borders of these sites could only be assigned with
approximation. Furthermore, binding sites located on one strand were
not always exactly superimposed on another strand, and the footprints
detected in one strand were not equally discernible on the other.
P on either the coding strand (lanes
1-3) or the noncoding strand (lanes 4-6). The
labeled DNA was mixed with (lanes 2, 3, 5,
and 6) or without (lanes 1 and 4) BprC1
nuclear extract in the presence of poly dI
dC competitors and
digested by DNase I. Lanes G and T contain molecular
size markers generated by dideoxynucleotide sequencing ladders. The two
footprints (sites A and B) in both coding and noncoding strands are
shown by brackets. For detecting footprints downstream from
-160, end-labeled oligonucleotides with sequence -185 to
-50 (coding strand, lanes 1-4)) and -210 to
-80 (noncoding strand, lanes 5-7) were used as
probes. Two footprints, C and D, are shown by brackets (panel B). Another footprint downstream from -120
is shown by bars.
Sites A and B Are Important for the Hepatoma-specific
and Orientation-independent Enhancer Functions
To
investigate whether these protein binding sites are important for the
enhancer function and to dissect the essential domain in the enhancer,
oligonucleotide sequences spanning these binding sites, either as an
individual or in combination, were inserted into pBLCAT2 vector in both
forward and reverse orientations. Fig. 4shows that BprC1 cells
transfected with recombinant -210/-155TKCAT (construct
b), which contains both sites A and B, exhibited about 9-fold
increase in CAT activity, as compared with those transfected with
vector alone. This level of increase is comparable to that of the same
cells transfected with -233/-116 recombinant (construct
a). Even the reverse version of -210/155TKCAT (construct
b) still showed 3-fold increase of CAT activity. NIH3T3 cells
transfected with these recombinants showed no more than 2-fold increase
in either orientations. This result suggests that sites A and B contain
most, if not the entire, enhancer function seen in
-233/-116TKCAT. Recombinant -210/-185TKCAT (construct c), which contains site A only, exhibited no
enhancing activity in both BprC1 and NIH3T3 cells, whereas recombinants
-185/-155TKCAT (construct d, containing site B) in
both orientations displayed 7-fold increased activity in BprC1 cells
lines. No enhancer activities were found with recombinants
-162/-116TKCAT, -153/-135TKCAT, and
-127/-120TKCAT (constructs e through g)
containing sites C and D, combined or in individual, respectively. We
conclude that the entire enhancer function is mostly located in sites A
and B, with site B as a major player. These results are in general
agreement with those using deletion mutants as shown in Fig. 1.
-galactosidase activity using the
cotransfected pH2B-gal plasmid DNA. mdr1b sequences in
these constructs are as follows: a, -233 to -116; b, -210 to -155, c, -210 to
-185; d, -185 to -155; e,
-162 to -116; f, -153 to -135; and g, -127 to -120.
Multiple Proteins Bind to the mdr1b
Enhancer
To investigate the protein(s) that recognize the
enhancer sequence, we used the DNA fragment containing nt -233 to
-116 (sites A through D) as a probe in gel mobility retardation
assays. Nuclear extracts from BprC1 cells were mixed with P-labeled probe and nonspecific competitor,
poly(dI)
poly(dC), followed by various amounts of specific
competitors containing sequences in sites A-D. DNA-protein
complexes were resolved by polyacrylamide gel electrophoresis under
neutral conditions and visualized by autoradiography. In the absence of
competitors, a major retarded band (Fig. 6A, arrow) and several minor bands were observed from the
autoradiograms. Under the conditions used, this major band could be
effectively competed by a sequence containing site B but not by those
containing A, C, and D. This result suggests that the major retarded
band must arise by protein binding to site B. This was also evident
when nt -210 to -155 (containing sites A and B) was used as
probe (Fig. 6B). These results suggest that either
protein recognizing site B sequence is much more abundant than those
recognizing other sites or that the affinity of the protein bound to
site B supersedes those to other sites. When individual sequences
containing sites A-D were used as competitors to probe protein
binding to nt -162/-116, which contains sites C and D, only
site C sequence could compete efficiently, suggesting that protein
binding to site C dominates site D (Fig. 6C). Similar
results were observed when nuclear extracts from NIH3T3 (Fig. 6, D-F), normal mouse livers, and mouse HCC (not shown)
were used. These results, taken together, suggest that multiple
proteins in hepatoma- and nonhepatoma-derived cell extracts recognize
the enhancer with different capacities. The finding that site B
contains a dominant binding protein is consistent with the functional
assay that distinguishes site B from other binding sites (Fig. 4).
dC and
various amounts of unlabeled, double-stranded enhancer DNA competitors,
whose nucleotide sequences span A (-210 to -185), B (-185 to -155), C (p153 to -135),
and D (-127 to -120) elements as
indicated.
AAGACAAGTCT
) (52) is located between sites A and B of the mdr1b enhancer. However, it has been reported that a single copy of the
recognition sequence was insufficient for transcriptional activation by
p53(52, 53) . In this context, mdr1b enhancer
may not be sensitive to the function of p53, although previous studies
have demonstrated that the promoters of human MDR1(38) and Chinese hamster Pgp1(55) (both
are homologs of murine mdr1a) are modulated differentially by
wild-type and mutant p53. In any event, the identities of interacting
protein factors that confer cell type-specific enhancer function remain
to be demonstrated.
-fetoprotein gene, is present in both hepatoma and nonhepatoma
cells(56, 57) . At this time, we cannot exclude the
possibility of differential posttranscriptional modification of these
proteins (e.g. phosphorylation, poly(ADP-ribosylation), etc.)
resulting in transcriptional activation of mdr1b gene in liver
tumors. Alternatively, these enhancer binding proteins may interact
with other non-DNA binding factors to confer tissue specificity in the
similar manner as the recently identified novel B cell-derived
coactivator (OCA-B), which potentiates the activation of immunoglobulin
promoters by octamer-binding transcription factors(58) .
Several such coactivators have also been implicated in the
transcriptional regulation of liver-specific gene
expression(59, 60) . Molecular cloning of genes
encoding these mdr1b enhancer binding proteins should
facilitate understanding of the complex control mechanism of mdr1b gene expression.
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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