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INTRODUCTION |
Transcriptional regulation of eukaryotic genes are extremely
diverse, strictly controlled and coordinated events and involve complex
mechanisms for up- and down-regulation of gene expression (1, 2). Such
complexity and diversity of gene expression are obviously needed for
maintaining intricate balances among biological reactions and systems
in response to various internal and external stimulations and stress.
For characterization of promoter functions of various genes,
heterologous reporter genes, such as CAT,
-galactosidase,
luciferase, and green fluorescent protein genes, have often been
utilized (3-5). Use of such heterologous reporter genes often gives
convenience in both qualitative and quantitative analyses of promoter
activities. At the same time, however, use of such heterologous
reporter genes may result in significant biases in assessing subtle
structure-function relationships of promoters and irrelevant
observations, which are primarily due to the introduction of foreign
elements or due to the elimination of intrinsic elements of test genes
in the assay system. Any regulatory machinery including those in the
promoter regions must have evolved in the context of the rest of gene
structures, neighboring genomic elements as well as of higher order
structures of chromatin and chromosome. Therefore, use of a
heterologous reporter gene in studying regulatory mechanisms of an
unrelated gene may have a risk of resulting in irrelevant observations
and conclusions. To date, a large number of mammalian genes have been
analyzed for their promoter functions with various heterologous
reporter genes. However, literally no systematic studies have been
conducted addressing the issue concerning the relevancy and limitation
of commonly used heterologous reporter genes.
Many functional analyses of promoters of eukaryotic genes to date by
using CAT1 reporter genes
have shown weak to strong negative regulatory activities or silencer
activities, which are associated with specific 5'-flanking regions
containing promoter sequences (6, 7). Human genes for FIX and PC,
important factors involved in blood coagulation and anti-coagulation
pathways, respectively (8, 9), are among them (10, 11). The 5'-upstream
region of the hFIX gene promoter beyond approximately nt
800 up to at least nt
1900 showed a very strong silencer activity
in the CAT vector context (10). The hPC gene promoter was
also shown to have a substantial silencer activity associated with the
5'-upstream region beyond nt
82 up to nt
802 (11). Literally
nothing was known about the mechanisms by which these silencer
activities are generated and what roles they play in the natural
regulation of hFIX and hPC gene expression.
In this report, using transient expression assay system with HepG2 cell
or HTC cell lines, we first demonstrate that CAT reporter gene
constructs reproducibly show silencer activities associated with
hFIX and hPC gene promoters. We then present
experimental evidence that such silencer activities are irrelevant
artifacts specifically associated with the CAT reporter gene, but not
with other reporter genes including autologous hFIX or hPC gene as well
as the
-galactosidase gene, another commonly used heterologous reporter gene.
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EXPERIMENTAL PROCEDURES |
Materials--
Restriction enzymes and DNA modification enzymes
were purchased from Invitrogen and New England Biolabs. Radioactive
nucleotides, [
-32P]dCTP, were obtained from Amersham
Biosciences. Mouse anti-hPC monoclonal antibody and rabbit polyclonal
anti-hPC antibodies were purchased from Celsus Laboratory. Horseradish
peroxidase-linked goat anti-rabbit IgG was purchased from Invitrogen.
Anti-hFIX polyclonal and monoclonal antibodies for enzyme-linked
immunosorbent assay (ELISA) were obtained from Hematologic Technologies
Inc and Enzyme Research Laboratory, respectively, as previously
described (12). Media, fetal calf serum, penicillin, and streptomycin for mammalian cell cultures were obtained from Invitrogen. FuGENE-6 transfection reagent and CAT ELISA kit were purchased from Roche Molecular Biochemicals. HepG2 cells were obtained from ATCC. Rat hepatoma cells, HTC cells, was kindly provided by Dr. Thomas Gelerhter in this department. Human PC vector pUC119-hPC was kindly provided by
Dr. Francis Castelino at the University of Notre Dame.
o-nitrophenyl
-D-galactoside for the
-gal
assay was purchased from Sigma. All other reagents were of the highest
quality commercially available.
Construction of CAT and
-Gal Expression Vectors--
Plasmids
pCH110 and pSV2CAT contain
-gal and CAT genes
under the control of the SV40 early promoter, respectively (10, 13).
Plasmid pUMSVOCAT, a promoter-less CAT vector with virtually no
background CAT expression activity, was used as a control CAT expression vector (14).
Construction of CAT expression vectors under controls of the
hFIX or hPC promoters are as follows: expression
vectors with hFIX 5'-flanking sequences,
1895FIX/CAT and
416FIX/CAT, were constructed by inserting hFIX gene
fragments spanning nt
1895 through nt +29 or nt
416 through nt +29,
which were PCR-amplified from minigene
2231FIXm1 (15), into pUMSVOCAT
at the SmaI site, respectively (Table
I). Expression vectors,
802PC/CAT and
82PC/CAT, were constructed by inserting PCR-amplified fragments with
SmaI linkers, which span the region nt
802 through nt +66
or nt
82 through nt +66 of the hPC gene, into pUMSVOCAT,
at the SmaI site, respectively (11, 14) (Table I). A
lacZ gene fragment with BamHI and
HindIII sticky ends (3736 bp in size) were released from
pCH110 (10, 16), followed by their insertion into pUC19 between
BamHI and HindIII sites (3), thus generating
pOGAL. A PCR-amplified fragment of 1924 bp (nt
1895 through nt +29 of the hFIX gene) or 445 bp (nt
416 through nt +29) with
HindIII sticky end was then inserted into pOGAL at
HindIII site, thus generating
1895FIX/
GAL or
416FIX/
GAL. All the PCR-amplified sequences and ligation site
sequences of expression vectors were subjected to automated dideoxy
sequencing to confirm their accuracy.
Construction of hFIX and hPC Expression Vectors--
Human FIX
minigene (hFIXm1) and cDNA (hFIXc) expression vectors,
1895FIXm1,
416FIXm1,
1895FIXc, and
416FIXc, were constructed with plasmid
pUC19. These vectors contained 5'-end regulatory regions of the
hFIX gene identical to those used in the CAT vectors. Constructs
416FIXm1 and
416FIXc were prepared as previously described (12). Constructs
416FIXm1 or
416FIXc were digested with
SphI and StuI, removing a 483-bp fragment
encompassing a region nt
416 through nt +67 of the hFIX
gene (the unique StuI site at nt +67 in exon I). A
PCR-amplified 1967-bp fragment (nt
1895 through nt +72 of the
hFIX gene) with SphI and StuI sticky ends at 5'- and 3'-ends, respectively (Table I), was then inserted at
the sites, thus producing
1895FIXm1 and
1895FIXc, respectively. Minigenes
1895FIXm1 and
416FIXm1 were digested by BamHI
and KpnI, removing the 3'-UTR and poly(A) signal sequence of
the hFIX gene, followed by treatment with Klenow enzyme. A
fragment containing the SV40 early region and poly(A) signal sequence
(135 bp in size) was released by HpaI and BamHI
double digestion of pUMSVOCAT, followed by treatment with Klenow
enzyme, and then inserted into the above fragment vectors, generating
1895FIX/SV40 and
416FIX/SV40, respectively. Minigene
416FIXm1 was
digested by SphI/NheI to remove its
hFIX promoter region, followed by Klenow enzyme treatment and self-ligation, thus generating pFIXm1, a promoter-less hFIX minigene control vector for hFIX expression assays. A promoter-less hFIX cDNA control vector, pFIXc, was similarly generated by
SphI/NheI double digestion of
416FIXc, removing
the promoter region.
Human PC minigene (hPCm1) expression vectors,
802PCm1 and
82PCm1,
were constructed as follows: a fragment spanning nt
802 through nt
+1560 of the hPC gene (2362 bp in size) with SphI
and MscI linkers at 5'- and 3'-ends, respectively, was
PCR-amplified by using human genomic DNA as a template (11, 17) (Table
I), and inserted into pUC119-hPC between SphI and
MscI sites to replace its 5'-end portion. The 3'-end portion
of the resultant construct containing the internal Sse8387I
site in the 3'-UTR through an EcoRI site present at the
3'-end immediately outside of the poly(A) attachment site, was released
by Sse8387I/EcoRI double digestion. A
PCR-amplified fragment (615 bp in size, spanning nt +10494 through nt
+11108 of the hPC gene) with Sse8387I and
EcoRI sticky ends (17), was then inserted to fill the gap,
thus generating
802PCm1. Construct
82PCm1 was similarly generated
by replacing the 5' portion of
802PCm1 (nt
802 to nt +1560) with a
fragment spanning nt
82 to nt +1560. The 5'-end region, non-coding
exon 1 and partial intron 1 of the hPC gene were removed
from
802PCm1 by complete and partial digestion with SphI
and MscI, respectively. The remaining fragment from
802PCm1 was treated by Klenow fragment, and then self-ligated, generating promoter-less control vector, pPCm1. Expression vectors,
802PC/FIXc and
82PC/FIXc, were constructed as
follows: a fragment spanning nt
802 through nt +66 of the hPC gene with SphI and NheI linkers at
the 5'- and 3'-ends, respectively, was PCR-amplified from
802PCm1
(Table I). After digestion with SphI/NheI, this
fragment was inserted into
416FIXc, which was digested in advance
with SphI/NheI, removing the hFIX
promoter sequence, thus generating
802PC/FIXc. Construct
82PC/FIXc
was similarly generated by replacing the 5'-end fragment (nt
802 through nt +66) of
802PC/FIXc with a 148-bp hPC fragment
spanning nt
82 through nt +66 of the hPC gene. All the
PCR-amplified sequences and ligation site sequences of newly
constructed expression vectors were confirmed by automated dideoxy
sequencing for their accuracy.
Cell Culture and Transfection--
HepG2 cells were cultured in
Dulbecco's modified Eagle's medium (DMEM) supplemented with
L-glutamine, 25 mM HEPES buffer, 110 mg/liter
sodium pyruvate, antibiotics (penicillin, streptomycin, and neomycin),
and 10% fetal bovine serum in a 5% CO2 atmosphere at
37 °C. HTC cells were cultured under similar conditions except 5%
fetal bovine serum supplemented. Cell transfection was carried out with
the FuGENE 6 transfection reagent as previously described (18). Plasmid
pCH110 was used as a transfection internal control for CAT, hFIX and
hPC expression vectors, while pSV2CAT was similarly used for
-gal
expression vectors. 4-5 independent assays were carried out, and
average activities were shown with S.D.
CAT and
-Gal Assays--
The CAT protein assay was carried
out by using a CAT ELISA kit according to the manufacturer's
instruction.
-gal activity was assayed as previously reported (19).
Cell extracts obtained from cells co-transfected with pCH110 or pSV2CAT
were first assayed for
-gal or CAT activities, respectively. These
activities were used for normalizing transfection efficiencies among
culture dishes. Amounts of cell extracts taken for activity assays were
adjusted for the optimal assay range of CAT or
-gal activity.
Human FIX and hPC ELISA Assay--
Human FIX produced into the
conditioned culture medium was quantified by hFIX-specific ELISA as
previously described (15). This ELISA system reproducibly detected hFIX
antigen as low as 1 ng/ml. Human PC produced was assayed by ELISA as
previously described (20). Horseradish peroxidase-conjugated goat
anti-rabbit IgG was used as the detection antibody. For each expression
vector, minimally three independent ELISA were carried out with
duplicated assays for each diluted culture medium sample, and average
values were calculated to determine the amounts of protein produced.
Northern Blot Analysis of hFIX, hPC, and CAT mRNA Levels in
the Transfected Cells--
Northern blot analysis of HepG2 cells
transfected with hFIX, hPC, or CAT constructs was carried out as
previously described (15, 20). Total RNA samples prepared from the
transfected HepG2 cells were subjected to agarose gel electrophoresis
(15 µg per lane). For CAT mRNA detection, a coding region
fragment of CAT gene (624 bp in size) was PCR-amplified with
5' and 3' primers (5'-ACCACCGTTGATATATCC-3' and
5'-CTGCCACTCATCGCAGTA-3', respectively) by using pUMSVOCAT as a
template, and was used as a hybridization probe. A fragment (588 bp in
size) prepared by SspI/BamHI digestion of
416FIXm1 was used for hFIX hybridization. Human PC hybridization
probe (365 bp in size, from nt +8385 to +8749 in genomic nucleotide
numbering) was prepared as previously described (20). These probes were
labeled with [
-32P]dCTP by random priming (Amersham
Biosciences) to a specific activity of ~1 × 109
cpm/µg. To confirm the presence of equal amount of RNAs, blotted filters were washed twice at 75 °C, each for 30 min, in 10% sodium dodecyl sulfate, and hybridized with labeled RNR 18 probe.
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RESULTS |
Transient Expression Activities of CAT, hFIX Minigenes, and
cDNA Expression Vectors--
Transient expression activities of
newly generated CAT reporter gene constructs with different
hFIX 5' promoter sequences,
1895FIX/CAT and
416FIX/CAT,
in HepG2 cells are shown in Fig. 1A. Construct pUMSVOCAT, a
promoter-less control, gave no detectable CAT activities. Construct
1895FIX/CAT containing the hFIX promoter region nt
1895
through nt +29 showed only 15.9% CAT activity of that of the
416FIX/CAT containing the region nt
416 through nt +29. In
agreement, Northern blot analyses showed a similarly lowered mRNA
level in HepG2 cells transfected with
1895FIX/CAT, ~20% of that of
cells transfected with
416FIX/CAT (Fig. 1D). These results
indicated the existence of a strong silencer activity associated with
the 5'-upstream region of the hFIX gene, confirming our
previous observation (10).

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Fig. 1.
Transient expression assay of CAT, hFIX
minigenes, and cDNA expression vectors in HepG2 cells.
A, transient expression activities of CAT reporter gene
constructs with hFIX promoter sequences. Only essential
structures of CAT expression vectors with hFIX gene promoter
sequences are shown. Promoter sequences are depicted by thick
horizontal lines at the left with 5'-end nt numbering.
CAT reporter gene sequences are shown by gray rectangles
with thin vertical lines depicting stop codon positions, and
SV40 early gene-derived 3'-UTR with poly(A) signal sequence are shown
as open rectangles. Right-angled arrows depict
transcription start sites provided by the promoter sequences. Flanking
sequences at the 3'-end downstream of the poly(A) site are depicted by
thick horizontal lines (continuous parts of SV40 sequence).
Expression activities relative to that of 416FIX/CAT (average 40 ng/106 cells/24 h) are presented. B, transient
expression activities of hFIX minigene (FIXm1) expression
constructs. Structures of hFIX promoters are depicted by
thick horizontal lines at left with the 5'-end nt
numbering. Autologous hFIX gene sequences are shown by
shaded gray rectangles with thin horizontal lines
connecting rectangles representing introns. Sequences of
3'-UTR and contiguous flanking regions (thick horizontal
lines on the right) are all hFIX gene
sequences. Thin vertical lines inside of the
rectangles represent stop codon positions.
Right-angled arrows indicate transcription start sites.
Expression activities relative to that of 416FIXm1 are presented.
C, expression activities of hFIX cDNA constructs.
Various definitions are as described in B. Expression
activities relative to that of 416FIXc are presented.
D, Northern blot analysis of CAT mRNA in HepG2
cells. Lane 1, non-transfected HepG2 cells; lane
2, HepG2 cells transfected with pUMSVOCAT, negative control;
lane 3, HepG2 cells transfected with 1895FIX/CAT;
lane 4, HepG2 cells transfected with 416FIX/CAT.
Equivalent loading of RNA samples was confirmed by rehybridization of
the filter with RNR 18 probe (lower panel). The ratio of CAT
mRNA levels of the cells transfected with 1895FIX/CAT to that of
416FIX/CAT was ~1:5. Positions of CAT mRNA and 18 S rRNA are
marked with CAT and RNR 18 on the right of the upper and
lower panels, respectively. E, Northern blot
analysis of hFIX mRNA levels in HepG2 cells. Lane 1,
non-transfected HepG2 cells; lane 2, HepG2 cells transfected
with pFIXm1 (control); lane 3, HepG2 cells transfected with
1895FIXm1; lane 4, HepG2 cells transfected with
416FIXm1. Human FIX mRNA levels produced from HepG2 cells
transfected with 1895FIXm1 and that of 416FIXm1 are very similar
with a ratio of 1:1 after normalizing for RNA loading. Positions of
hFIX mRNA and 18 S rRNA are marked with hFIX and RNR 18, respectively.
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Relative expression activities of hFIXm1 constructs containing the
hFIX promoter sequences identical to those used in CAT constructs,
1895FIXm1,
416FIXm1,
1895FIXc, and
416FIXc, are shown in Fig. 1, B and C. Construct
416FIXm1
expressed hFIX at a level of ~40 ng/106 cells per 24 h and was defined to be at 100% activity. HepG2 cells expressed
non-detectable levels of hFIX as previously reported (15). As expected,
the promoter-less control vector, pFIXm1, showed only minimal hFIX
expression activity. Both
1895FIXm1 and
416FIXm1 showed equivalent
transient expression activities, which were correlated well with
similar hFIX mRNA levels in HepG2 cells transfected either with
1895FIXm1 or with
416FIXm1 (Fig. 1E). These results
indicated no appreciable silencer activities associated with the region
nt
1895 through nt
416 of the hFIX gene. Similar to the
hFIX minigene constructs, expression vectors with hFIX
cDNA sequence,
416FIXc and
1895FIXc, showed equivalent hFIX
expression activities in HepG2 cells, confirming no silencer activities
associated with the region nt
1895 through nt
416 (Fig. 1C). This
result also suggested that there is no specific contribution of the
functional hFIX intron 1 sequence to the silencer activity
shown by CAT reporter analysis.
Transient Expression Activities of CAT and hPC Minigenes Expression
Vectors--
Transient expression activities of newly constructed CAT
reporter gene vectors with hPC promoter sequences,
802PC/CAT and
82PC/CAT, are shown in Fig.
2A. The average CAT transient
expression activity of
802PC/CAT was only 21.6% of that of
82PC/CAT, and in agreement, the CAT mRNA level of HepG2 cells
transfected with
802PC/CAT was also lowered to ~25% of
82PC/CAT
(Fig. 2C). These results indicated the presence of
substantial silencer activity associated with the 5'-upstream region
(nt
82 to nt
802) of the hPC gene, which is consistent
with the previous report by Miao et al. (11).

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Fig. 2.
Transient expression assay of CAT and hPC
minigene expression vectors in HepG2 and HTC cells.
A, transient expression activities of CAT reporter gene
constructs with hPC promoters. Promoter sequences are
depicted by thick open bars at left with the
5'-end nt numbering. All others are as described in the legend to Fig.
1. Expression activities relative to that of 82PC/CAT are presented.
B, expression activities of hPC minigene
(PCm1) constructs in HepG2 cells and HTC cells. Human
PC promoter sequences are depicted by thick open
bars at left with the 5'-end nt numbering. Homogeneous
hPC structures are shown with checkerboard rectangles with
thin horizontal lines representing the first intron.
Thin vertical lines in the box indicate stop
codon positions. All the constructs contain homogeneous hPC 3'-UTR
sequences and immediate 3'-flanking sequences shown by thick
horizontal lines. Right-angle arrows indicate
transcription start sites. Expression activities relative to that of
82PCm1 are presented. C, Northern blot analysis of
CAT mRNA levels in HepG2 cells. Lane 1, non-transfected
HepG2 cells; lane 2, HepG2 cells transfected with pUMSVOCAT
(control); lane 3, HepG2 cells transfected with 802PC/CAT;
lane 4, HepG2 cells transfected with 82PC/CAT. The
ratio of the CAT mRNA level in the cells transfected with 802/CAT
to that of 82FIX/CAT was ~1:4.5 after normalizing for RNA loading.
CAT mRNA and 18 S rRNA are labeled with CAT and RNR 18 on the
right side of the upper and lower
panels, respectively. D, Northern blot analysis of
hPC mRNA levels in HepG2 cells. Lane 1, non-transfected
HepG2 cells; lane 2, HepG2 cells transfected with pPCm1
(control); lane 3, HepG2 cells transfected with 802PCm1;
lane 4, HepG2 cells transfected with 82PCm1. Ratio of hPC
mRNA level of HepG2 cells transfected with 802PCm1 to that of
cells transfected with 82PCm1 was ~1:1. Positions of hPC mRNA
and 18 S rRNA are shown by hPC and RNR 18, respectively.
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Transient expression activities of hPC minigenes expression
vectors were assayed with both HepG2 cells and HTC cells, respectively. Relative expression activities of hPC miningene constructs
with that of
82PCm1 defined as 100% are shown in Fig. 2B.
HepG2 cells showed a low, but significant level of endogenous hPC
expression (~12 ng/106 cells per 24 h, data not
shown), which correlated with the significant background expression
activity of pPCm1, a promoter-less construct (average 12.8 ng/106 cells per 24 h). Construct
82hPCm1 produced
101 ng/106 cells per 24 h into the culture medium.
Constructs
82PCm1 and
802PCm1 gave equivalent hPC expression
activities in HepG2 cells, correlating well with the similar levels of
hPC mRNA in the transfected HepG2 cells (Fig. 2D).
Relative expression activities of hPC expression vectors assayed with
HTC cells, a rat hepatoma cell line with no endogenous hPC expression,
agreed well with those obtained in HepG2 cells (Fig. 2B).
These results obtained with native gene constructs contradict the
observation with CAT reporter gene (Fig. 2A).
Possible Effects of the SV40-derived 3'-UTR on the Expression
Activities of hFIX Expression Vectors--
Relative expression
activities of chimeric pFIXm1/SV40,
1895FIXm1/SV40 and
416FIXm1/SV40 vectors, in which SV40 3'-UTR including poly(A) signal
sequence replaced the counterpart of the hFIXm1 gene, are
shown in Fig. 3A. Both
constructs showed similar hFIX expression activities, indicating no
contribution by the SV40 sequences in the CAT constructs to the
silencer activity observed with
1895FIX/CAT.

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Fig. 3.
Transient expression assay of chimeric hFIX
or -gal expression vectors.
A, transient expression activities of hFIXm1 expression
vectors with SV40 3'-UTR sequences. The structure is depicted with the
hFIX promoter sequences by thick horizontal lines
at left with the 5'-end nt numbering, followed by hFIX
minigene sequences as shown by shaded gray rectangles with
thin horizontal lines representing an intron, and SV40
derived 3'-UTR including poly(A) signal depicted by open
rectangles. Expression activities relative to that of
416FIXm1/SV40 are presented. B, transient expression
assays of -gal reporter gene expression vectors with hFIX
promoter sequences. -gal reporter gene sequences are shown by
dotted rectangles. The rest is identical to the descriptions
in the legend to Fig. 1. Expression activities relative to that of
416FIX/ GAL are presented. C, transient expression
activities of hFIX reporter gene expression vectors with hPC
promoter sequences. Human PC promoter sequences are depicted by
thick open bars at left with the 5'-end nt
numbering. Human FIX reporter sequences (cDNA) are shown with
shaded gray rectangles. Thin vertical lines in
the box indicate stop codon positions. All the constructs contain the
hFIX 3'-UTR sequence and immediate 3'-flanking sequence shown by
thick horizontal lines. Expression activities relative to
that of 82PC/FIXc are presented
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Transient Expression Activities of
-Gal Expression Vectors with
hFIX Promoters and hFIX cDNA Expression Vectors with hPC
Promoters--
Transient expression of
-gal reporter gene
constructs under the control of the hFIX gene promoter
sequences,
1895FIX/
GAL and
416FIX/
GAL, were assayed with
HepG2 cells (Fig. 3B). Construct pO
Gal, a promoter-less
control, gave no appreciable
-gal expression in HepG2 cells. No
silencer activity was found to be associated with the region nt
1895
through nt
419 of the hFIX gene in the
-gal reporter
gene context. Constructs composed of hPC promoter sequences
and hFIX cDNA reporter,
802PC/FIXc or
82PC/FIXc, also showed no
silencer activity associated with the 5'-flanking region nt
802
through nt
82 of the hPC gene (Fig. 3C).
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DISCUSSION |
Heterologous reporter genes have been widely used for functional
characterization of many eukaryotic gene promoters. Use of such
reporter genes, however, may have a risk of generating irrelevant results and conclusions on gene regulation. This is the issue that we
systematically addressed in the present study. Such irrelevant results
may be generated due to the absence of structural elements in the
heterologous reporter gene, which are present in the native test gene
and required for its regulation in the context of its own promoter.
Alternatively, structural elements in the heterologous genes, which are
absent in the test genes, may critically affect the transcriptional
regulation of test genes.
In the present studies, we first demonstrated that CAT reporter gene
expression vectors reproducibly showed strong silencer activities
associated with the 5'-flanking regions of the hFIX gene
(Fig. 1A), confirming the previous findings (10). Such silencer activities, apparently due to transcriptional suppression as
demonstrated by the similarly reduced mRNA levels (Fig.
1D), however, were not found when hFIX minigene
or cDNA were used as a reporter in place of the CAT gene
(Fig. 1, B and C). These results indicated that
the silencer activities found to be associated with the 5'-specific
upstream region of the hFIX gene may be irrelevant to the
hFIX gene, and were generated through a specific combination of the hFIX promoter and the CAT reporter gene. This
hypothesis was further supported by the similar findings obtained with
hPC gene expression vectors (Fig. 2). Use of the CAT
reporter gene in combination with the hPC promoter region
gave substantial silencer activity associated with the 5'-flanking
region nt
802 through nt
82 (Fig. 2A), whereas no such
silencer activity was observed when the hPC minigenes were
used as a reporter (Fig. 2B).
It is possible that some elements present in the hFIX
minigene, such as the functional intron sequence which grossly elevates mRNA levels (12, 21), might eliminate the silencer activity observed with CAT reporter. However, this possibility was not supported, since hFIX cDNA constructs without the intron sequence did not show any silencer activities (Fig. 1C). The SV40
3'-UTR sequence used in the CAT reporter gene was also shown to be not responsible for generating the silencer activity (Fig. 3A).
Furthermore, the observed correlation between levels of produced CAT,
hFIX, or hPC protein and their mRNA levels eliminates the
possibility of reporter gene-dependent differences in
translational efficiency, intracellular protein trafficking or
secretion process as a possible cause for generating CAT reporter
gene-specific silencer activities (Figs. 1, D and
E and 2, C and D). Examinations with
another series of chimera constructs composed of promoter sequences of
the hPC gene connected with the hFIX cDNA sequence also
showed no silencer activity associated with the hPC promoter
region nt
802 through nt
82 (Fig. 3C). It is important
to emphasize that hFIX and hPC genes share a
significant similarity in their coding regions (22). However, their
promoter regions are grossly different from each other, suggesting that
these promoter regions took different evolutional pathways (20,
23).
Expression vectors with the
-gal gene, another common
reporter gene, also do not show any silencer activity with
hFIX promoter sequences, further supporting our conclusion
that the silencer activities observed with the 5'-flanking
upstream-specific regions of hFIX as well as the
hPC gene are unique artifacts generated by use of CAT
reporter genes (Fig. 3B). The detailed mechanisms responsible for generating such silencer activities associated with the
CAT reporter genes in combination with the promoters of genes of
interest remain to be determined. Particularly, identification of any
specific parts of the CAT gene structure involved in
cross-talk with the specific regions of test promoter sequences would
be critical.
Other heterologous reporter genes, which are not included in the
present study, may also exert unusual effects including silencer activities on various gene promoters. Furthermore, specific
combinations of a tested gene promoter and a heterologous reporter gene
may even generate pseudo-high activity for the promoter although to our
knowledge no systematic studies are reported to date. These possibilities are yet to be tested.
Our findings indicate that observations on the promoter
structure-function relationship analyzed with the commonly used
CAT reporter gene may not necessarily represent the true
promoter regulatory mechanisms of many genes of interest, and strongly suggest a need for their systematic re-examination. Analysis of a
specific promoter may be best done, if possible, with its autologous coding and subsequent downstream structure sequences as a reporter gene.