(Received for publication, October 22, 1996, and in revised form, November 19, 1996)
From the Palo Alto Medical Foundation Research
Institute, Palo Alto, California 94301 and the § Department
of Medicine, Division of Gastroenterology, Stanford University School
of Medicine, Stanford, California 94304
Cholesterol 7-hydroxylase is the rate-limiting
enzyme in the degradation of cholesterol to bile salts and plays a
central role in regulating cholesterol homeostasis. The mechanisms
involved in the transcriptional control of the human gene are largely
unknown. HepG2 cells represent an appropriate model system for the
study of the regulation of the gene. To identify liver-specific DNA sequences in the promoter of the human CYP7 gene, we first
examined the DNase I hypersensitivity in the 5
-region of the gene. An area of hypersensitivity was observed in the region from
50 to
200
of the human gene in nuclei from transcriptionally active HepG2 cells,
but was absent in transcriptionally inactive HeLa cell nuclei or in
free DNA. Various 5
-promoter deletion constructs were made and
transfected into HepG2 cells. About 300 base pairs of upstream sequence
are required for high level promoter activity of the human
CYP7 gene in HepG2 cells. DNase I footprinting of the
hypersensitive region revealed nine protected sequences. Gel retardation experiments demonstrated binding of HNF-3 to the segment from
80 to
70 and of hepatocyte nuclear factor HNF-4 (and ARP-1) to
the segment from
148 to
127 of the human CYP7 promoter.
Deletion of either of these sites depressed promoter activity in HepG2 cells. A third region from
313 to
285 is bound by members of the
HNF-3 family and acts as an enhancer. Additionally, the segment from
197 to
173 binds a negative regulatory protein that is present in
Chinese hamster ovary cell extracts and in HepG2 cell extracts. These
experiments define the key control elements responsible for basal
transcription of the human CYP7 gene in HepG2 cells.
Cholesterol 7-hydroxylase catalyzes the rate-limiting step in
the pathway that leads to the catabolism of cholesterol to bile acids
(for review, see Ref. 1). Cholesterol 7
-hydroxylase is a microsomal
enzyme member of the cytochrome P-450 family. In human and rat, the
major products of this metabolic pathway are cholic acid and
chenodeoxycholic acid. Bile acids have an important role in cholesterol
homeostasis; their synthesis and excretion cause a decrease in hepatic
cholesterol levels, while their presence in the intestine facilitates
the solubilization of dietary fats and is required for the absorption
of cholesterol and fat-soluble vitamins. Because of the importance of
these functions, bile acid synthesis in the liver is carefully
regulated to maintain cholesterol homeostasis (1). To date, little is
known about the molecular mechanisms that control cholesterol
catabolism and bile acid synthesis.
The cDNAs and genes for cholesterol 7-hydroxylase have been
isolated from rat (2-4), human (5, 6), hamster (7), and mouse (8).
CYP7 mRNA is found exclusively in the liver (9), making
this gene a target for the study of the molecular mechanisms implicated
in hepatic-specific gene expression. Work by several groups has
demonstrated that CYP7 mRNA levels are modulated in
cultured cells by a number of effectors. For example, in cultured rat
hepatocytes (10) and human hepatoma (HepG2) cells, the addition of bile
acids to the culture media suppresses CYP7 mRNA levels (11), and dietary cholesterol and dexamethasone increase
CYP7 mRNA levels (12). In vivo, however,
cholesterol feeding increases but dexamethasone reduces CYP7
mRNA levels in rats (12, 13). The elegant work by Lavery and
Schibler (14) demonstrated that in rats, CYP7 gene
expression follows a strict diurnal rhythm, with mRNA levels
peaking in the evening; this phenomenon is controlled at the level of
transcription by a specific transcription factor, DBP.
Our long-term goal is to elucidate the molecular mechanisms that operate to regulate transcription of the human CYP7 gene in the liver as well as those that promote modulation by diet and hormones. As a first step toward our goal, we have pursued the identification of liver-specific elements that regulate basal transcription of the human CYP7 gene in hepatic cells. HepG2 cells have been used successfully as a model system to study CYP7 gene expression in a number of laboratories (11, 15, 16). Up-regulation of CYP7 mRNA by cholesterol as well as down-regulation by bile acids have been demonstrated (11).
Our rationale has been to use DNase I hypersensitivity as a tool to map
liver-specific elements that are relevant in vivo, followed
by a thorough analysis of the underlying hepatic control elements in
HepG2 cells prior to testing them in in vivo animal models.
Using this approach, we established that liver-specific promoter
elements of the CYP7 gene lie between 213 and +1. Within this region are functional binding sites for the liver-enriched transcription factors HNF-3 (
80 to
70), HNF-4, and ARP-1 (
144 to
127). Furthermore, a ubiquitous transcription factor that binds to
the region from
197 to
173 reduces promoter activity of the human
CYP7 gene, and an enhancer resides within an HNF-3-like binding site at
300 to
293.
All constructs were derived from
plasmid D230 20.5-1 (kindly provided by the late Dr. Mike Komaromy).
This plasmid contains the segment from 780 to +133 of the human
cholesterol 7
-hydroxylase gene and was constructed by
PCR1 using oligonucleotide primers derived
from the GeneBankTM data base sequence. The genomic
sequence is flanked by BamHI sites, allowing it to be
excised by digestion with BamHI. Construct
764CAT was made
by insertion of the
764/+46 BamHI fragment into a pOCAT
vector (17) that had been digested with BamHI and treated with calf intestinal phosphatase. Plasmid
764CAT was used as a
template for the remaining 5
-promoter deletion plasmids. The 5
-primers used for amplification were as follows:
341 to
317 for
the
341CAT construct,
313 to
289 for the
313CAT construct,
285 to
261 for the
285CAT construct,
268 to
244 for the
268CAT construct,
227 to
203 for the
227CAT construct,
213 to
189 for the
213CAT construct,
91 to
67 for the
91CAT
construct, and
65 to
41 for the
65CAT construct. In every case,
the sequence of the primer started with CGCGGATTC (the
BamHI site). The 3
-primer for all these constructs extended
from +46 to +22 and also contained a BamHI site at its
5
-end. In each case, the amplified product was digested with
BamHI, followed by ligation to the pOCAT vector that had
been digested with BamHI. To prepare plasmid
313CAT
(
80/
70), the following PCR primers were used: primer 1, from
313 to
292 (with the BamHI site at the 5
-end); primer 2, from
55 to
95, but without the sequence from
70 to
80; primer 3, from
95 to
55 without
80 to
70; and primer 4, from +46 to +22 with the BamHI site at the 5
-end. The first PCR utilized primers 3 and 4 and generated an intermediate product spanning from
95 to +46 without
80 to
70. The two intermediate PCR products were mixed, denatured, reannealed, and used as primers on
a third PCR to generate a product spanning from
313 to +46 without
80 to
70. This BamHI fragment was then cloned into
pOCAT. The orientation of the insert was determined by PCR. Constructs
227CAT
(
197/
173),
213CAT
(
144/
127), and
313CAT
(
300/
293) were made in a similar manner as the
construct described above. Construct
313mut-1 was made by introducing
a primer in which five point mutations (CGTAC instead of AAACA at
298
to
294) were made to disrupt HNF-3 binding. The DNA sequence of every promoter deletion construct was verified by sequencing.
Human
hepatoma cells (HepG2), Chinese hamster ovary (CHO) cells, and HeLa
cells were grown as described previously (18). Transient transfections
with the various plasmid constructs were performed by the calcium
phosphate coprecipitation method as described previously (19) with 7 µg of the CAT gene expression plasmid and 6 µg of an internal
reference plasmid (pRSV-gal). In some experiments, 6 µg of an
expression plasmid for HNF-3
(20) or control vector was included.
The CAT assays were performed according to the protocol of Gorman
et al. (21), and CAT activities were normalized according to
the results of the
-galactosidase activity in order to correct for
differences in transfection efficiency.
Nuclear extracts from HepG2 and CHO cells were prepared by the method of Dignam et al. (22). Extracts from mouse liver nuclei were made by the procedure of Gorski et al. (23). Whole cell extracts from COS cells were prepared as described previously (20). In some cases, cells were transfected with 20 µg of an expression plasmid for HNF-3 (24) using the calcium phosphate coprecipitation technique as described above. For gel mobility shift assays, 1-4 µg of nuclear extract was incubated for 30 min at room temperature with 0.5-1 ng of 32P-labeled double-stranded oligonucleotide and 4 µg of poly(dI-dC) in buffer containing 15 mM Hepes (pH 7.9), 15% glycerol, 0.6 mM EDTA, 60 mM KCl, 5 mM MgCl2, and 0.6 mM dithiothreitol. Incubation mixtures were then fractionated on 5% native polyacrylamide gels in 0.5 × Tris borate/EDTA. Gels were soaked in 10% glycerol for 10 min, dried, and exposed to x-ray films.
DNase I HypersensitivityDNase I hypersensitivity studies were performed as described by Levy-Wilson et al. (18).
DNase I FootprintingDNase I footprinting was performed as described before (19).
OligonucleotidesOligonucleotides were purchased from the Stanford University Digestive Disease Center. Complementary sets of single-stranded oligonucleotides were annealed to form double-stranded oligonucleotides and then purified on nondenaturing 15% acrylamide gels.
To determine whether there are any DNase I-hypersensitive
sites in the 5-end of the human CYP7 gene, several sets of
experiments were performed. A representative example is shown in Fig.
1. Nuclei from HepG2 cells or HeLa cells or free DNA was
treated with increasing amounts of DNase I, followed by digestion of
the purified DNA with EcoRV. The Southern blots were
hybridized with the probe indicated at the bottom of Fig. 1. The
original 2917-bp EcoRV fragment was progressively digested
by incubation of HepG2 nuclei with increasing amounts of DNase I. Concomitantly, a broad band appeared and was designated DH1
(
Nase I-
ypersensitive). From the mobility of
the DH1 band relative to known restriction fragment markers run in
parallel with the samples, DH1 was localized to between
50 and
200
of the CYP7 gene, as shown above the map in the bottom part
of Fig. 1. When nuclei from HeLa cells (in which the CYP7
gene is transcriptionally inactive) were treated in an identical manner
with DNase I, the EcoRV fragment was resistant to
degradation, and no DNase I-hypersensitive sites were detected (data
not shown). The hypersensitive region was not observed when free DNA
from HepG2 cells was subjected to the same procedure (Fig. 1,
right panel), suggesting that chromatin structural features in the vicinity of the transcriptional start site of the human CYP7 gene in HepG2 nuclei are selectively open and available
for DNase I cutting.
DNase I Footprinting of the 5
The
DNase I hypersensitivity studies suggest that hepatic-specific
transcription factors bind to the region from 50 to
200, thus
causing its hypersensitivity. Binding of hepatic-specific nuclear
proteins to the 5
-proximal region of the human CYP7 gene was examined by DNase I footprinting. Nine protected regions were observed in the DNA segment from
341 to +46 using nuclear extracts from HepG2 cells (Fig. 2, A and
B). Footprint 1 extends from
35 to
48, footprint 2 from
54 to
62, footprint 3 from
67 to
81, footprint 4 from
91 to
104, footprint 5 from
129 to
144, footprint 6 from
174 to
191, footprint 7 from
213 to
227, footprint 8 from
268 to
285, and footprint 9 from
313 to
341. The protected regions have
been evolutionarily conserved, as evidenced by a correspondence among
the footprints in human, mouse, and rat liver cells as shown in Fig. 2
(C and D), suggesting that the nuclear proteins
that bind to these sequences have an important functional role in
CYP7 gene transcription.
Functional Assays to Test the Role of the Footprints in Promoter Activity
The combined results from the hypersensitivity studies
and DNase I footprinting strongly suggest that key liver-specific
promoter elements reside in the 300 bp immediately upstream of the
transcriptional start site of the CYP7 gene. This hypothesis
was tested as follows. Several constructs were made in which
5-segments of the human CYP7 gene of varying lengths were
cloned upstream of the reporter CAT gene, and their promoter activity
was tested in transient transfection assays in HepG2 cells. A schematic
illustration of the constructs and their promoter activities are shown
in Fig. 3.
The construct with the largest 5-extension was
764CAT, followed by
341CAT. To elucidate the potential functional role of the footprinted
regions, subsequent constructs were designed to exclude either one
footprint at a time or the region between two footprints. Transient
transfections into HepG2 cells revealed a small but reproducible
reduction of CAT activity (~20%) upon deletion of sequences between
764 and
341, suggesting that weak positive control elements reside
in this region. On the other hand, a weak negative element may reside
between
341 and
313 (footprint 9), as judged by the 30% increase
in CAT activity of the
313CAT construct as compared with the
341CAT
construct. Deletion of the segment from
313 to
285 caused a 40%
decrease in CAT activity, suggesting that this segment may contain
binding sites for a positive regulatory element. Further deletions from
285 to
227 and from
227 to
213 increased CAT activity by 30% each, respectively, suggesting that footprints 7 and 8 may harbor binding sites for negative regulatory elements. The
213CAT construct exhibited the highest promoter activity, suggesting that most of the
important elements are localized within 213 bp upstream of the start
site, in agreement with the DNase I data of Fig. 1. Deletion of the
segment from
213 to
91 that includes footprints 4-6 caused a 40%
reduction in promoter activity, suggesting the removal of a positive
element. The activity of the
65CAT deletion was 2-fold lower than
that of the
91CAT construct, suggesting that a positive element
resides between
91 and
65. Therefore, from the data in Fig. 3, we
conclude that the segment from
213 to +1 contains the minimal
regulatory elements required for transcription from the human
CYP7 promoter in transient assays in HepG2 cells.
The decline in transcriptional activity
observed when sequences between 213 and
91 were deleted (Fig. 3)
suggests that DNA sequences in this region play a key role in basal
promoter activity. Three nuclear protein-binding sites, namely
footprints 4-6, are located in this region.
To identify the protein factors responsible for footprints 4-6, gel
retardation experiments were conducted. A double-stranded oligonucleotide corresponding to the segment from 197 to
173 (representing footprint 6) was incubated with nuclear extracts from
HepG2 and CHO cells (Fig. 4). Two specific complexes
were formed with HepG2 extracts (lane 1) that were competed
for by an excess of unlabeled oligonucleotide (lane 2). The
same specific complexes were seen when using CHO extracts (lanes
3 and 4), suggesting that the transcription factor
binding to this region may be ubiquitous. The specific complex of lower
molecular weight may represent a proteolytic fragment of the major
binding protein since the intensity of this band varied from extract to
extract. However, we cannot rule out the possibility that two different
proteins bind to this sequence. An excess of unlabeled oligonucleotides
representing in each case the binding site for a known transcription
factor such as HNF-1, C/EBP, HNF-3, and HNF-4 failed to abolish complex formation (data not shown). Comparison of the DNA sequence from
197
to
173 to a data base of transcription factor-binding sites revealed
only some similarity to the binding site for the GATA factor that plays
a role in erythroid expressed genes (25, 26).
The segment from 148 to
127 (representing footprint 5) exhibited
sequence similarity to the binding sites for the liver-enriched transcription factors HNF-4 (27) and ARP-1 (28). These two proteins
bind to the same DNA sequence with different affinities. When the
labeled
148/
127 oligonucleotide was incubated with an extract from
COS cells that had been transfected with an HNF-4 expression vector
(Fig. 5, lane 2), we observed a complex that was not detected with control extract. This complex was competed for by
an excess of unlabeled oligonucleotide (lane 3), confirming its identity as the HNF-4 complex. A weaker but specific HNF-4·DNA complex was also detected with HepG2 nuclear extracts (lanes
6 and 7). When the
148/
127 probe was incubated with
an extract from COS cells that had been transfected with an ARP-1
expression plasmid, we observed formation of a retarded complex
(lane 4), not evident with control extract, that was also
specific (lane 5). However, the affinity of ARP-1 for the
footprint 5 sequence appears to be severalfold lower than the affinity
of HNF-4 for that same sequence.
Incubation of an oligonucleotide representing footprint 4 with nuclear extracts yielded one weak specific complex of high molecular weight (data not shown). Computer analysis of footprint 4 revealed no similarities to the binding sites of known transcription factors.
Analysis of the Function of the Footprinted SequencesTo
ascertain the functional importance of the proteins binding to
footprints 5 and 6, the effect of deleting these regions upon promoter
activity was determined. The 144/
127 deletion (footprint 5) was
made in the context of the
213CAT construct, and the
197/
173
deletion (footprint 6) was made in the context of the
227CAT
construct (Fig. 6). Deletion of the sequence from
197
to
173 (footprint 6) increased promoter activity by 2-fold, suggesting that it binds a negative regulatory element. On the other
hand, deletion of the
144/
127 segment (footprint 5) decreased CAT
activity by ~2.5-fold, indicating that the protein(s) binding to
footprint 5 also play a role in the transcriptional activation of the
CYP7 gene. Having demonstrated that both HNF-4 and ARP-1 can
bind to footprint 5 (Fig. 5), we examined the effect of an excess of
each of these transcription factors upon the CAT activities of the
wild-type and deletion constructs. To this end, the reporter constructs
were cotransfected with expression plasmids for either HNF-4 or ARP-1.
Cotransfection of the wild-type
213CAT construct with HNF-4 failed to
alter promoter activity, suggesting that HNF-4 may not be limiting in
our HepG2 cells. As expected, the additional HNF-4 protein did not
affect the CAT activity of the
213CAT construct in which the HNF-4
site had been deleted (Fig. 6). An excess of ARP-1 protein reduced the
transcriptional activity of the wild-type
213CAT construct by 2-fold,
a result consistent with the fact that ARP-1, when binding to its
cognate sequence, usually exerts a repressor effect (28). As expected,
an excess of ARP-1 protein had no effect in the absence of the
ARP-1-binding site.
These data suggest that both HNF-4 and ARP-1 can bind to the
144/
127 region. When HNF-4 binds, it promotes activation of transcription; when ARP-1 binds, transcription is decreased. This is
analogous to the situation in the proximal promoter of the human apoB
gene, where an HNF-4 (ARP-1) site resides (20).
Computer analysis of
the sequence of the proximal promoter region of the human
CYP7 gene revealed a perfect match to the HNF-3-binding site
(TGTTTGCT) (20) in the segment from 80 to
70, a sequence that is
100% conserved among the human, rat, and mouse genes. Binding of
nuclear proteins from HepG2 cells and mouse liver to an oligonucleotide
encompassing this sequence (
88 to
65) was examined. In Fig.
7, we observe that the labeled
88/
65 oligonucleotide forms two major retarded complexes with HepG2 extracts, designated as A
and B (lane 3). The mobility of these complexes is identical to that of complexes A and B formed between a labeled oligonucleotide representing a consensus HNF-3-binding site and HepG2 nuclear proteins
(lane 1). Specificity of binding is established in
lane 2. Furthermore, the
88/
65 probe also formed two
specific complexes with mouse liver nuclear extracts (lane
4) that were competed for by the homologous oligonucleotide
(lane 5) as well as by the HNF-3 consensus oligonucleotide
(lane 6). The HNF-3 consensus oligonucleotide formed
retarded complexes with mouse liver proteins that were identical in
mobility to those formed by the
88/
65 oligonucleotide (lane
7) and were specific (lane 8) and also competed for by
the
88/
65 oligonucleotide (lane 9). These observations confirm that HNF-3 does bind to the
88/
65 segment of the
CYP7 promoter.
The functional significance of this binding was studied by making a
promoter construct in which the HNF-3 site at 80 to
70 was deleted
in the context of the
313CAT construct (Fig. 8). The
313CAT construct has an activity of 1.10 as compared with the
764CAT construct. Deletion of the
80/
70 segment reduced promoter
activity by 3-fold, demonstrating that binding of HNF-3 to this
sequence is important for promoter activity in HepG2 cells. When an
HNF-3
expression vector was cotransfected with our constructs, the
activity of the wild-type
313CAT construct was 5-fold higher, implying that HNF-3
is limiting in HepG2 cells. When the region from
80 to
70 was deleted from the construct, transcriptional activity
in the presence of an excess of HNF-3
was again reduced, thus
underscoring the importance of this region in hepatic transcription of
the CYP7 promoter.
Based on computer analysis, there are three other potential
HNF-3-binding sites within the region from 320 to
240 of the human
CYP7 promoter, namely
316 to
306,
288 to
278, and
235 to
243 (29). The footprinting data of Fig. 2 revealed a
protected region from
285 to
268. However, deletion of this segment
did not affect promoter activity (data not shown), suggesting that the
putative HNF-3 site at
285 to
268 is not functional in our assays.
Similarly, the segment from
255 to
245 was not protected by nuclear
proteins from liver or HepG2 cells (Fig. 2), suggesting that HNF-3 may
not bind to that sequence, and deletion of the region from
268 to
227 did not significantly affect the activity of the human
CYP7 promoter. To evaluate the possible role of HNF-3 in
these regions, we cotransfected our promoter deletion constructs together with an HNF-3
expression vector (Fig. 9).
The transcriptional activity of the
764CAT,
341CAT, and
313CAT
constructs was 5-7-fold higher in the presence of excess HNF-3
. On
the other hand, the activity of the
285CAT construct dropped 2-fold
as compared with that of the
313CAT construct in the presence of
cotransfected HNF-3
, suggesting that a functional HNF-3-binding site
may reside in the segment from
313 to
285 and that the putative
site at
243 to
235 may not be functional. Examination of the DNA
sequence from
313 to
285 revealed two (5 out of 8 bp) matches to
the HNF-3 recognition sequence (
297 to
293 and
290 to
286). The sequence of this segment of the human CYP7 gene is shown in
Fig. 10A, with the two 5-bp matches to the
HNF-3 recognition sequences indicated by brackets.
Binding of the 313/
285 sequence to nuclear proteins from HepG2
cells was examined. As shown in Fig. 11A,
the
313/
285 oligonucleotide probe formed five specific retarded
complexes with HepG2 nuclear proteins, namely complexes A, B, C, and E
and a lower molecular weight complex (lanes 1 and
2). The HNF-3 consensus oligonucleotide from the
transthyretin promoter competes for formation of complexes A, B, and C,
but not complex E or the smaller complex (lane 3). On the
other hand, the HNF-3 consensus probe forms two major specific retarded
complexes with HepG2 proteins, namely complexes C and D, and a lower
molecular weight complex migrating between the nonspecific complex and
complex E (lanes 4 and 5) that are partially competed for by the
313/
285 oligonucleotide (lane
6).
These data suggest that the 313/
285 segment does bind members of
the HNF-3 family. The question arises as to whether these DNA/protein
interactions are functionally significant. To elucidate the functional
role of these sequences, we first mutagenized the putative
HNF-3-binding site centered at
297 to
293 by altering those 5 bp as
shown for the
313mut-1 construct in Fig. 10A. The CAT
activity of the
313mut-1 construct was 3-fold lower than that of the
wild-type construct, thus validating the importance of those 5 bp in
the transcriptional activity of the human CYP7 promoter
(Fig. 10B). The involvement of HNF-3
in binding to this segment was demonstrated by cotransfection of the wild-type and mutant
313CAT constructs with an HNF-3
expression plasmid. The transcriptional activity of the
313mut-1 construct in the presence of
an excess of HNF-3
was reduced by >2-fold as compared with the
wild-type construct (Fig. 10B). In Fig. 11B, the
DNA binding properties of the
313mut-1 and wild-type sequences are
compared. As shown above in Fig. 11A, the wild-type
oligonucleotide forms four major specific retarded complexes with HepG2
nuclear proteins, namely complexes A, B, C, and E, with complex B being
the strongest (lanes 1 and 2). The mutant
oligonucleotide as well as the HNF-3 consensus oligonucleotide compete
mainly for the formation of complex C (lanes 3 and
4). The
313mut-1 oligonucleotide forms two major specific
complexes, complex C and a lower molecular weight complex (lanes
5 and 6). Interestingly, both the HNF-3 oligonucleotide
and the wild-type
313/
285 sequence compete for formation of complex
C (lanes 7 and 8). The HNF-3 oligonucleotide forms complexes C and D (lanes 9 and 10). Thus, a
5-bp mutation in the segment from
297 to
293 disrupts binding of
the proteins responsible for the formation of complexes A and B, but
not complex C, suggesting that the second HNF-3 site at
290 to
286
may be responsible for complex C formation and for the residual CAT
activity of the
313mut-1 construct (Fig. 10). In parallel
experiments, we deleted the segment from
300 to
293 in the context
of the
313CAT construct. Gel retardation experiments with the
313CAT
(
300/
293) oligonucleotide revealed no retarded complexes
(data not shown), suggesting that the 8-bp deletion can disrupt binding
of nuclear proteins to the adjacent
290/
286 sequence. Transfections
of the wild-type and mutant
313CAT
(
300/
293) constructs into
HepG2 cells revealed a 5.5-fold decrease in CAT activity of the
deletion construct as compared with the wild-type construct, thus
confirming that the HNF-3 site at
300 to
293 is important for
CYP7 promoter activity (Fig. 10). Cotransfection of the
HNF-3
expression plasmid with the wild-type and
313CAT deletion
constructs yielded similar results, i.e. a 4-fold reduction
in promoter activity of the deletion construct.
In summary, mutagenesis or deletion of the segment from 300 to
293
in the context of a
313CAT promoter construct severely impairs
promoter activity, demonstrating that protein binding to this segment
is functionally significant. Results of the gel retardation experiments
show that members of the HNF-3 family of transcription factors are
involved in binding to the
313/
285 region and thus play an
important functional role in CYP7 gene regulation in HepG2
cells. Our overall data can be reconciled by postulating that, in
transient assays, ~213 bp of upstream sequence are required for basal
promoter activity of the human CYP7 gene. However, in the
context of the entire gene, the sequence from
313 to
285 binds
HNF-3 and related proteins, and it functions as a transcriptional
enhancer because deletion or mutagenesis of the HNF-3 sequence at
300
to
293 severely reduces transcription.
The mechanisms by which expression of the CYP7 gene is
regulated are worthy of study because of the important role that
cholesterol 7-hydroxylase plays in regulating overall cholesterol
homeostasis. However, information available to date is fragmentary.
Using transgenic mice, Ramirez et al. (30), using large
constructs encompassing
1633 bp of the rat CYP7 gene
5
-upstream region, ligated to a mouse albumin enhancer and linked to
the reporter LacZ gene, demonstrated that the regulation of
the reporter gene by bile salts that is observed in vivo can
be reproduced. However, in the absence of the mouse albumin enhancer,
expression of the reporter gene in the liver was not detectable. Using
a line of transformed cultured mouse hepatocytes and stable
transfections, a liver-specific enhancer was localized some 7 kilobase
pairs upstream of the transcriptional start site of the rat
CYP7 gene and was required for high level transcription from
the CYP7 promoter. But, this enhancer may not function
in vivo in transgenic mice. Using HepG2 cells and transient transfection assays, Molowa et al. (31) implicated the
transcription factor HNF-3 as playing a role in transcription of the
human CYP7 gene by binding to a site located between
432
and
220. Chiang and Stroup (15), studying the proximal promoter
region of the rat CYP7 gene, described two regions protected
from DNase I digestion that they designated footprints A and B. Rat
footprint A (
81 to
35) corresponds to human footprints 1-3 of Fig.
2, and footprint B is equivalent to our footprint 5. These
investigators have suggested (based on computer analysis of the DNA
sequence) that HNF-3, C/EBP, HNF-4, retinoic acid receptor, COUP, and
glucocorticoid receptor may bind to the rat promoter region and
influence transcription. They have also proposed that recognition sites
for binding proteins that respond to corticosteroids as well as insulin
and bile acids may also reside in this region. Lavery and Schibler (14)
have shown that the liver-enriched basic leucine zipper protein DBP binds to an element centered at
225 of the rat CYP7 gene
and plays an important role in the circadian transcription of the gene.
A similar sequence is present in footprint 7 (
227 to
213) of the
human gene (Fig. 2). Thus, the apparent complexity and multiplicity of
interactions among various transcription factors that may regulate this
gene warrant a comprehensive and systematic approach.
Our long-term goal with the human CYP7 gene is to identify
all of the control elements that regulate this gene and that modulate its in vivo expression. Encouraged by our earlier results
with the apoB gene (30) and by the suitability of HepG2 cells as a
model system for hepatic-specific expression of the CYP7
gene (11, 32, 33), we began our studies by identifying a key DNase
I-hypersensitive region in the segment from 50 to
200 of the human
CYP7 gene. This DNase I-hypersensitive region is absent in
cells in which the CYP7 gene is transcriptionally inactive, such as HeLa cells, and in free DNA, suggesting that it is the chromatin structural features of the CYP7 gene that make
this region open and available for interaction with transcription
factors.
Transfections of 5-CAT deletion constructs illustrated in Fig. 3
yielded results that were consistent with those from the DNase I
hypersensitivity studies. Together, both approaches point to the
location of key hepatic-specific elements between
213 and +1. Within
this region, we found functional binding sites for HNF-3 (
80 to
70), HNF-4 (and/or ARP-1) (
144 to
127), and a ubiquitous
transcription factor (
197 to
173) present also at high
concentrations in CHO cell extracts. Deletion of either the HNF-3 or
HNF-4 sites reduced promoter activity, while deletion of the
197/
173 sequence increased promoter activity. The HNF-3 and HNF-4
sites are also present in the rat gene, but their functional role has
not been determined. HNF-4 and HNF-3 may act synergistically, as has
been shown to occur in the promoter of another liver-specific gene, the
apoA-I gene (34). The negative influence of the sequence from
197 to
173 upon transcription is of interest. It is not uncommon to find
that a ubiquitous factor, working in conjunction with other
cell-specific factors, plays a key role in transcriptional activation
or repression. For example, in the human apoC-III gene, an interaction
between SP1, bound to distal regulatory sites, and HNF-4, bound to a
more proximal promoter site, is required for transcriptional activation
(35).
Another interesting observation relates to the role of the sequence
from 300 to
285 in CYP7 promoter activity. This sequence binds one or more proteins related to the HNF-3 family of transcription factors. Deletion of the 8-bp sequence (
300 to
293) in the context of the
313CAT construct decreased promoter activity by 70%.
Mutagenesis of the HNF-3-binding site within this sequence also
decreased promoter activity, suggesting that the segment from
313 to
285 functions as a hepatic-specific enhancer for the CYP7
promoter. This observation is in good agreement with the hypothesis
formulated by Molowa et al. (31), suggesting that a
liver-specific enhancer harboring HNF-3-binding sites is present in the
segment from
432 to
220 of the human CYP7 gene.
It has recently been proposed (29) that a number of regulatory elements
responsive to many effectors such as phorbol ester, insulin,
glucocorticoids, and thyroid hormone may reside within the segment of
the human CYP7 promoter from 764 to +46 studied in this
work. However, the precise location of these control sequences has not
yet been determined. Our studies provide detailed information regarding
the hepatic-specific elements required for basal transcription of the
human gene in HepG2 cells. It would be interesting to elucidate whether
the DNA sequence elements responsive to thyroid hormone, for example,
interact with one or more of the hepatic elements described here or
whether different mechanisms operate to modulate the expression of this
gene.
Because our ultimate goal is to identify all of the regulatory elements of the human CYP7 gene that are functional in vivo, we want to ascertain the role that the hepatic-specific sequences characterized in HepG2 cells play in liver expression of these constructs in transgenic mice. Future experiments will address these questions.
We thank Drs. Rob Costa, Sotirios Karathanasis, and Frances Sladek for gifts of expression plasmids for HNF-3, ARP-1, and HNF-4, respectively. We also thank Rick Cuevas for assistance in the preparation of this manuscript and to Drs. Alan R. Brooks and Brian J. McCarthy for comments on the manuscript.