Hypertension. 1996;27:980-1008
(Hypertension. 1996;27:980-1008.)
© 1996 American Heart Association, Inc.
Transcriptional Regulation of the Genes Involved in Lipoprotein Transport
The Role of Proximal Promoters and Long-range Regulatory Elements and Factors in Apolipoprotein Gene Regulation
Dimitris Kardassis;
Maria Laccotripe;
Iannis Talianidis;
Vassilis Zannis
From the Section of Molecular Genetics, Boston (Mass) University Medical
Center (D.K., M.L., V.Z.), and the University of Crete Medical School and
Institute of Molecular Biology and Biotechnology of Crete (Greece) (D.K.,
I.T., V.Z.).
Correspondence to D. Kardassis, Section of Molecular Genetics, Boston University Medical Center, 700 Albany St, Boston, MA 02118-2394.
Key Words: apolipoproteins • genes, regulator • gene expression regulation • transcription factors • eukaryotic genes
 |
Introduction
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The transcription of
eukaryotic genes is a complex biological
event involving
numerous proteins—including RNA polymerase
II, the proteins of
the basal transcription initiation complex,
and a variety of
promoter- and enhancer-specific transcription
factors—and
requiring an ATP-dependent activation step.
1 2 3 4 5 6 7 8 9 10 11 12 The
regulation of transcription is
responsible for the tissue-specific
gene expression as well
as gene expression during differentiation and
development and
in response to intracellular and extracellular stimuli
such
as hormones and metabolites.
Numerous studies have established that a precise array of regulatory
elements exists in each promoter/enhancer and these elements are
occupied by transcription factors. It has been proposed that this
promoter/enhancer-specific arrangement of factors permits the
formation of stereospecific DNA-protein complexes. These complexes may
directly or indirectly interact with the basal transcription system,
thus leading to the transcriptional activation of the target
gene.8 13
|
Methodologies Used for Study of Transcriptional Regulation of
Genes
|
|---|
Several experimental advances have facilitated the study of
eukaryotic
promoters and have led to the identification and
characterization
of several eukaryotic transcription
factors. These include the
following: (1) Definition of the
long-range regulatory elements
that confer tissue specificity or
developmentally regulated
expression. This analysis utilizes
transgenic mouse technologies.
14 15 (2) Definition of the
promoter region a few kilobases upstream
of the transcription
initiation site necessary for gene transcription.
This analysis
monitors the expression of a reporter gene under
the control of normal
and mutated promoters after transfection
of cell cultures. (3)
Identification of the different factors
that bind to a specific
promoter region and definition of their
binding sites on the DNA. For
this purpose, several techniques
are used, including DNase I
footprinting, in vivo footprinting,
16 17 18 gel
electrophoretic mobility shift assays,
19 supershift
assays,
and DNA binding interference assays that involve modification
of
T residues by KMnO
4 and G residues by dimethyl
sulfate.
20 The
relationship of a factor that binds to a
specific regulatory
element to previously described factors can be
assessed by competition
assays, by direct comparison with the purified
factor, and by
use of antifactor antibodies in DNA binding assays.
Finally,
in vitro mutagenesis of the promoter region can be used for
assessment
of the importance of specific elements for transcription in
cell
cultures usually with CAT assays and in vitro transcription
assays.
This information can then be correlated with the ability of
a
mutated sequence to bind to the factor. The above methodologies
also
allow the purification of transcription factors and cloning
of cDNAs
encoding them. A key step in the protein purification
is a DNA
sequence–specific affinity chromatographic method
using
concatamers of the DNA binding site of the factor as
ligand.
21 Two main approaches are used for the isolation
of cDNAs encoding
mammalian transcription factors. The first involves
screening
of cDNA libraries with oligonucleotide probes
corresponding
to a partial protein sequence of the factor. The second
approach
involves screening of expression cDNA libraries with
32P-labeled
synthetic double-stranded
oligonucleotides corresponding to
the DNA
binding site of the corresponding factor or with appropriate
antibodies.
22 All known transcription factors are modular
in nature and contain
a DNA binding domain and transcriptional
activation domain.
23 In addition, several factors contain
a dimerization or multimerization
domain that permits them to
form homodimers and heterodimers
or multiprotein complexes. Finally, a
variety of receptors for
steroids, thyroids, retinoids, etc, contain a
ligand binding
site.
24 Isolation, expression, and
functional analysis of the
cloned factors by in vitro
mutagenesis provide the biological
material required for study of
specific mechanisms responsible
for transcriptional activation of
eukaryotic genes.
In this review, we summarize our current knowledge on the regulatory
elements and factors that control the transcription of several
apolipoprotein genes. We emphasize recent advances in the regulation of
transcription of the human apoA-I/C-III/A-IV gene cluster and the human
apoE/C-I/C-IV/C-II gene cluster.
|
Proximal cis-Acting Regulatory Elements and Factors
Involved in the Regulation of Transcription of Human
Apolipoprotein Genes
|
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Plasma levels of apolipoproteins could in principle be increased
by
increasing the level of gene transcription. Thus, the genetic
information
pertinent to regulatory mechanisms governing apolipoprotein
gene
transcription is important. Existing biochemical and genetic
data
suggest that increased plasma apoA-I and decreased plasma
apoB levels
could decrease the ratio of low-density lipoprotein
to
high-density lipoprotein and thus protect humans against
atherosclerosis.
25 Similarly, reductions
in plasma apoA-II levels could have some
protective role against
atherosclerosis,
26 and reductions in
plasma
apoC-I and apoC-III levels could have beneficial effects in
reducing
plasma triglyceride levels.
27
Finally, increases in plasma
apoE levels could accelerate the removal
of lipoprotein remnants
and thus protect against the development of
atherosclerosis.
28 Use of the techniques
outlined above resulted in the mapping
of the proximal regulatory
elements of most of the apolipoprotein
promoters and the factors that
bind to them. Fig 1
shows the
information obtained for apoA-I, apoC-III, apoA-IV, apoB, and
apoA-II.
29 The information on the apoE/C-I/C-IV/C-II gene
complex is presented
below at the end of the article. To
facilitate the description
of the nuclear activities that recognize the
different regulatory
elements of the apolipoprotein genes, we have
adopted a uniform
nomenclature system that identifies each activity by
three characteristics:
(1) the name of the target gene, (2) the element
to which the
factors bind, and (3) the mobility of the DNA/protein
complexes.
This mobility is indicated by the numbers 1, 2, and 3, going
from
the slowest to the most rapidly migrating complexes. With this
nomenclature,
the activities that bind to the regulatory element C of
the
apoA-I promoter are designated A-IC1, A-IC2, A-IC3, etc (Fig
2
). Previously described factors, ie, C/EBP, HNF-1,
HNF-3, HNF-4,
etc, maintain their names.
30 Our systematic
analysis of five
apolipoprotein promoters resulted in the
identification of 37
regulatory elements. Other investigators have also
identified
4 elements in the proximal apoE promoter, 6 in the HCR of
the
apoE/C-I/C-IV/C-II gene locus, 6 in the second intron enhancer,
3
in the third intron enhancer of apoB, and 1 in the 5' silencer
of the
apoB gene. A careful examination of the identified activities
indicates
that several previously described factors participate
in the
transcriptional regulation of the apolipoprotein genes
(Fig 1A
through
1H). This includes the liver-enriched factors
C/EBP, HNF-1, HNF-3,
and HNF-4
31 32 33 34 as well as ubiquitous
factors such as
NF-1, NFY, SP1, and GA binding protein/E-twenty-six
specific
(GABP/Ets-1) (Table 1
).
35 36 37 38
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Figure 2. DNA binding gel electrophoresis assay with the
apoA-I regulatory element C as probe. The figure explains the
nomenclature used to describe the factors that bind to the different
regulatory elements of the apolipoprotein promoters (see text and Fig 1 ).
|
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Although Fig 1A through 1H and Table 1 indicate that several previously
described transcription factors may recognize different apolipoprotein
promoters, the arrangement of the factors within each promoter is
unique. This unique arrangement of the regulatory elements and factors
bound to them (referred to as promoter context) may allow the formation
of a unique and stereospecific DNA-protein complex that results in the
transcriptional activation of the corresponding gene. Analysis
of the promoter strength by transient transfection assays with the use
of wild-type and mutated promoter CAT constructs showed that
despite the apparent complexity of the apolipoprotein promoters, only a
few regulatory elements and the corresponding factors may be essential
for optimal transcription in cell culture. The most important
regulatory regions are indicated by one or two asterisks. One asterisk
is used when mutations that eliminate the binding to an indicated
element of the corresponding factor reduced transcription from 1% to
14%, and two asterisks when mutations reduced transcription 15% to
30% (Fig 1A through 1H).
|
Involvement of Enhancers, Silencers, and Tissue-Specific Elements
in Apolipoprotein Gene Regulation
|
|---|
The apoC-III gene is closely linked to the human apoA-I and
apoA-IV
genes
39 and is localized 2.5 kb downstream of the
apoA-I gene
and 5 kb upstream of the apoA-IV gene. The direction of
transcription
of the apoC-III gene is opposite to that of the apoA-I
and apoA-IV
genes (Fig 1A
). A series of in vitro and in vivo studies
have
pointed out that the distal apoC-III regulatory elements may
act
as homologous enhancers for apoC-III
40 41 as well as for
the
other two genes of the cluster.
42 43 44 45 46 47 The in vitro
experiments
showed that deletion of the distal apoC-III promoter region
reduced
the strength of the proximal promoter to 10% to 20% of its
original
value, implying that these elements are required to
enhance
the transcription of the apoC-III gene (Fig 3A
).
40 41 Further
studies presented
in detail below indicated that constructs
which contain the upstream
apoC-III regulatory elements F through
J increased the strength of the
other two promoters of the cluster,
the apoA-I
44 45 46 47 (Fig 3B
) and apoA-IV
43 promoters (Fig
3C
), as well as the
strength of the heterologous apoB promoter
44 (Fig 3D
).
Similarly, expression of segments of the apoA-I/C-III/A-IV
gene cluster
in transgenic mice indicated that hepatic expression
requires only 5'
regulatory elements in the apoA-I and apoC-III
genes, whereas the
intestinal expression of the apoA-I and apoA-IV
genes requires elements
localized in the intergenic sequence
between the apoC-III and apoA-IV
genes.
42 45 48 A different
type of tissue-specific
enhancer is also found in the apoA-II
gene. Without the apoA-II
enhancer, the transcription driven
by the proximal and middle apoA-II
regulatory elements is approximately
1% of control (Fig 4A
).
49 50 This enhancer is functional and
increases
10-fold the promoter strength of the heterologous
liver-specific
promoter of the hepatic lipase
51 (Fig 4B
). Tissue-specific
transcriptional enhancers have been found in
the second and
third introns of the human apoB gene
52 53 54
(Fig 1G
and 1I
)
as well as in the intergenic regions between the apoC-I
gene
and apoC-I' pseudogene
55 and the apoC-I' pseudogene
and apoC-IV
gene
56 and are discussed below.
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Figure 3. Schematic representations show
effect of the apoC-III enhancer on transcriptional activity of the
apoC-III (A), apoA-I (B), apoA-IV (C), and apoB (D) promoters. Distal
apoC-III regulatory elements enhance the strength of homologous and
heterologous promoters and may increase HNF-4–dependent
transactivation. The letters a through g indicate reference values
(100%) for each set of experiments.
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Figure 4. Schematic representations show
effects of the apoA-II enhancer on transcriptional activity of
homologous (A) and heterologous (B) promoters as indicated on the
figure. Distal apoA-II regulatory elements I through N act as a
liver-specific enhancer in homologous and heterologous promoters.
The letters a and b indicate reference values (100%) for each panel.
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|
|
Elements and Factors Involved in Transcriptional Regulation of
the Human ApoA-II Gene
|
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Footprinting analysis identified a set of 4 proximal (A
through
D), 4 middle (E through H), and 7 distal (I through N)
regulatory
elements between nucleotides -903 and
-33 of the apoA-II promoter.
49 50 57 The identity of
the factors that bind to the distal A-II
enhancer element as well as
the middle and proximal apoA-II
elements was verified by DNA binding
and competition assays.
This analysis showed that elements AB,
K, and L bind a heat-stable
factor of 41 kD that also recognizes
the regulatory element
C-IIIB of apoC-III and was designated
C-IIIB1.
58 59 Simultaneous
nucleotide
substitutions that prevented the binding of
C-IIIB1 activity
in elements AB, K, and L reduced the strength of the
apoA-II
promoter in HepG2 and CaCo-2 cells to 6% to 7% of
control.
59 Elements AB and K bind, in addition to C-IIIB1,
a heat labile
activity designated A-IIAB1. Mutations in the A-IIAB1
binding
site reduce the promoter activity to background
levels.
60 The
nature and importance of the A-IIAB1
activity have not been
clarified. A new activity designated A-IIN3
binds to the regulatory
element N. Deletion of element N reduced
hepatic and intestinal
transcription of the apoA-II gene to 7% and
18% of control,
respectively, indicating that A-IIN3 is
important.
49 Finally,
two new activities designated A-IIM1
and A-IIM2 bind to the
regulatory element M, and one activity
designated A-IID1 binds
to element D. Element D is also recognized by
GABP, an Ets-related
protein, as well as C/EBP family members. It
appears that A-IID1
acts as a negative and GABP as a positive
regulator.
61 A-IIM1
is present in the liver and in
CaCo-2 cells, whereas A-IIM2
is present only in the
liver.
60 The contribution of the A-IIM1
and A-IIM2
activities in hepatic and intestinal transcription
is unclear because
deletion of element M did not significantly
affect the promoter
strength in HepG2 or CaCo-2 cells. C/EBP
and other proteins that
recognize the CCAAT motif bind with
high affinity to the regulatory
elements L, C, and D. Low-affinity
binding sites for C/EBP are also
found in elements F, G, and
AB (Table 1
). The most important C/EBP site
is on element L.
Mutations in this site that prevented the binding of
C/EBP and
other CCAAT box binding proteins reduced both hepatic and
intestinal
transcription to 30% of control.
60 Elements H
and I bind the
previously described homeodomain factors HNF-1 and NF-1,
respectively.
Deletion of these elements reduced the promoter activity
in
HepG2 and CaCo-2 cells to 60% to 80% of control.
60
The regulatory
element J contains on the noncoding strand two direct
repeated
sequences, AGGGTA(A)AGGTTG, with one spacer
nucleotide between
them (included in parentheses). This
sequence has homology to
a consensus half-site motif, AGG/TTCA,
which is the binding
site of hormone nuclear
receptors.
62 63 64 As shown in Fig
5A
, element
A-IIJ binds HNF-4 and other orphan and ligand-dependent
nuclear
receptors.
31 65 66 67 68 69 Deletion of this element
reduces the
apoA-II promoter strength 70% and 32% of control
in HepG2 and CaCo-2
cells, respectively.
60 Cotransfection experiments
showed
that HNF-4 activates 2.2-fold the hepatic transcription
driven
by the -911 to +29 construct of the apoA-II promoter,
whereas
ARP-1, EAR-2, and EAR-3 repressed transcription to 35%
to 40% of
control.
68 Interestingly, when element J was deleted,
HNF-4
as well as EAR-2 and EAR-3 repressed the transcription of the
reporter
gene.
68 This repression most likely results from
the weak binding
of these factors to the regulatory elements K and L of
apoA-II,
which, as we showed previously, play an essential role in
apoA-II
gene transcription.
60 Recent studies in our
laboratory have
shown that the regulatory element AB and other sites of
the
apoA-II promoter are recognized by sterol response element binding
protein-1
(SREBP-1).
70 Cotransfection experiments have
shown that SREBP-1
represses the transcription of the apoA-II
gene.
71 This potential
participation of sterol response
factors in apoA-II gene regulation
is exciting and the subject of
ongoing research.
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Figure 5. DNA binding gel electrophoresis assays show binding
of orphan nuclear receptors HNF-4, ARP-1, EAR-2, and EAR-3 and of
hepatic nuclear extracts (NE) to the HRE A-IIJ of the human apoA-II
(A), B-A1 of human apoB (B), A-ID of human apoA-I (C), C-IIIB of human
apoC-III (D), and A-IVC of human apoA-IV (E) genes. The regulatory
elements involved are A-ID, A-IIJ, B-AI, C-IIIB, and A-IVC,
respectively (see Fig 1B, 1C, 1D, and 1F). NHR indicates nuclear
hormone receptor.
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The first intron of the human apoA-II gene between
nucleotides +38 and +206 acts as silencer and reduces the
strength of the apoA-II promoter (-911 to +38) to 15% to 18% of
its original value in HepG2 and CaCo-2 cells. This region also reduces
the strength of the heterologous thymidine kinase
promoter.72
|
Elements and Factors Involved in Transcriptional Regulation of
the Human ApoB Gene
|
|---|
The human apoB gene is localized in a 47.5-kb region flanked
by
matrix association regions (MARs).
73 The proximal apoB
promoter
region between nucleotides -150 and +124 can
direct the expression
of a reporter gene in hepatic and intestinal
cells but not in
HeLa cells. Longer promoter fragments extending to
nucleotide
-1800 have lower promoter
activity.
74 DNase I footprinting
analysis
identified three regulatory elements designated A,
CB, and E (Fig 1E
and 1F
). The regulatory element CB binds two
types of activities in
overlapping sites.
75 Site I (-118 to
-98)
binds heat-stable activities related to C/EBP, and site
II
(-112 to -88) binds three chromatographically
separable activities
initially designated B-CB1, 2, and
3
75 (Fig 1F
). Subsequently
it was shown that site II binds
to members of HNF-3 (HNF-3
,ß,
).
76 The regulatory
element A binds heat-stable activities related
to C/EBP in two
locations, site IV (-72 to -53) and site V (-53
to
-33).
75 77 The regulatory element A also contains
the direct
repeated sequence AGGTCC(AAA)AGGGCG on the noncoding strand
(with
three spacer nucleotides included in parentheses).
This sequence
has homology to the consensus AGG/TTCA half-site
motif that
is recognized by hormone nuclear
receptors.
63 64 65 Element
A binds members of the nuclear
receptor family HNF-4, ARP-1,
EAR-2, and EAR-3
68 (Fig 5B
).
Using the element as a ligand,
we have purified by affinity
chromatography from rat hepatic
nuclear extracts a
protein with an approximate
Mr of 60 kD that
was
designated NF-BA1.
78 Mutagenesis of the binding site of
NF-BA1
as well as in vitro transcription assays indicated that NF-BA1
is
a positive regulator. Bandshift clipping experiments with different
proteases
showed that the degradation products of NF-BA1 are
similar to
those obtained by HNF-4 but different from those obtained
with
the other nuclear receptors ARP-1, EAR-2, and EAR-3, which repress
the
activity of the apoB promoter (C. Cladaras, unpublished
observations,
1994). Heat-stable activities related to C/EBP also
bind to
element E (+33 to +52), which is located in the first exon of
the
apoB gene
75 (Fig 1F
). Element E and element CB are
weak C/EBP
binding sites, whereas site IV (-72 to -53) on
element A is
a strong C/EBP binding site. In vitro mutagenesis of the
promoter
region showed that the mutations at the HNF-3 binding site II
(-112
to -94), the nuclear hormone receptor binding site
III (-86
to -62), or the strong C/EBP binding site IV
(-72 to -53) reduced
hepatic and intestinal transcription
to 9%, 2%, and 10% to 13%
of control, respectively, indicating the
potential importance
of the factors that recognize these elements for
apoB gene transcription.
75 The proximal apoB promoter
elements extending to nucleotide
-898 are not
sufficient for tissue-specific expression of the
apoB gene in
vivo.
54 Tissue culture experiments have shown
that the
second intron of the apoB gene between nucleotides
+621 and
+1064 enhances threefold and fivefold the strength
of the apoB promoter
in HepG2 and CaCo-2 cells, respectively,
but not in HeLa
cells.
52 This region contains six regulatory
elements
designated A through F. The element E (+806 to +940)
is essential for
the enhancer activity and is recognized by
HNF-1, C/EBP, and several
other unidentified activities designated
a, b, c, d, e, f, protein I,
and protein II.
52 79 The organization
of the different
activities in the second intron enhancer is
shown in Fig 1G
. Similarly,
the apoB sequence between nucleotides
1065 and 2977
enhances the strength of the apoB promoter approximately
twofold in
HepG2 and CaCo-2 cells, respectively.
53 Deletion
analysis
localized the enhancer to a 155-bp fragment, which is
flanked
by DNase I–hypersensitive sites. This region contains three
footprints
designated A, B, and C. The activities that bind to these
elements
have not been identified rigorously. Finally, the region
between
nucleotides -3067 and -2734 represses
the strength of the apoB
promoter in CaCo-2 but not in HepG2
cells.
76 This region contains
a binding site for the
transcription factor ARP-1 between nucleotides
-2801
and -2728, and it has been suggested that ARP-1 reduces
transcription
by interfering with the function of HNF-3, which binds to
the
proximal promoter site BC
76 (Fig 1F
). The factors that
recognize
these positive and negative regulatory elements are shown in
Fig
1G
. Transgenic mouse experiments have shown that the second
intron
enhancer region is sufficient to direct expression of
apoB promoter
constructs in the liver but not in the intestine.
Incorporation of both
the second and third intron enhancers
and sequences containing the 5'
and 3' MARs in the apoB constructs
increased their expression but did
not eliminate the integration-related
position effects on the
expression levels of the transgene.
The inclusion of the 5' upstream
negative regulatory region
in this construct did not affect its hepatic
expression in vivo
(Fig 1I
).
54
|
Role of ApoC-III Enhancer and Proximal HREs on Transcriptional
Regulation of the Human ApoA-I/C-III/A-IV Gene Cluster
|
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Transcriptional Regulation of the Human ApoA-I Gene
As indicated above, the distal regulatory elements of apoC-III
increase
the strength of homologous as well as heterologous promoters
(Fig
3
). To understand the mechanism of this transcriptional
activation,
it is important to identify the factors that bind to the
proximal
promoters of the target genes as well as the factors that bind
the
apoC-III enhancer. This knowledge would provide information
on the
interactions that lead to the transcriptional activation
of the target
genes. The three proximal regulatory elements
A-IB (-128 to
-77), A-IC (-175 to -148), and A-ID (-220 to
-190)
of the apoA-I gene are necessary and sufficient for its
hepatic
expression in vivo and in vitro.
30 46 48 Sequence
comparisons
showed that the regulatory elements A-ID and A-IB contain
sequences
with high similarity to an AGG/TTCA half-site motif found
on
the promoter sites of genes responsive to members of the
steroid/thyroid
receptor superfamily.
63 64 65 The HRE
present on element A-ID
and A-IB is composed of two direct repeats
with sequences GGGTCA(GA)GGTTCA
and AGTTCA(A)GGATCA, respectively,
on the noncoding strands.
The spacing between the half repeat sites of
A-ID and A-IB are
two and one nucleotides, respectively.
DNA binding and competition
assays showed that elements A-IB and A-ID
support the binding
of HNF-4; other nuclear orphan receptors (Fig 5C
);
and ligand-dependent
nuclear receptors RXR
, RXR
/RAR
, and
RXR
/T
3Rß
68 80 81 82 83 84 (Fig 6A
and 6B
). Potassium permanganate and dimethyl
sulfate
interference experiments showed that RXR
homodimers
and
RXR
/RAR
and RXR
/T
3Rß heterodimers participate in
protein-DNA
interactions with 12, 13, and 11 out of the 14
nucleotides,
respectively, that span repeats 1 and 2 of
element A-ID and
the spacer region separating them (Fig 6C
).
Cotransfection experiments
in HepG2 cells with normal and mutated
promoter constructs and
plasmids expressing nuclear hormone receptors
showed that RXR
homodimers transactivated the
wild-type promoter 150% of control
in the presence of
9-
cis-retinoic acid, whereas RXR
/T
3Rß
heterodimers
repressed transcription to 60% of control in the presence
of
triiodothyronine. RXR
/RAR
and HNF-4 did not affect the
transcription,
which was driven by the proximal apoA-I
promoter.
82 83 Drastic
mutagenesis that altered either
part of both repeats in the
HRE of element A-IB or repeat 2 and the
adjacent spacer region
in the HRE of element A-ID eliminated the
binding of hepatic
activities present in rat liver nuclei and
reduced the promoter
strength to approximately 5% to 7% of control.
These findings
suggest that both HREs are essential for optimal hepatic
expression
of the apoA-I gene and that the factors which occupy them
may
act alone or in synergy with other factors to increase
transcription.
Another interesting feature of the proximal apoA-I
promoter
is that the regulatory region C is recognized by both positive
and
negative regulators that bind to overlapping domains. The region
-148
to -168 is recognized by two activities designated
A-IC1 and
A-IC3. Mutations that affected the binding of A-IC1 increased
transcription
4.6-fold, indicating that this protein acts as a negative
regulator.
Element C is also recognized by the heat-stable
activities that
bind in several elements of the apoB and apoC-III
promoters
as well as by C/EBP. Mutations that affected the binding of
these
activities reduced transcription to 8% to 14% of
control.
30 Cotransfection experiments with C/EBP
transactivated the apoA-I
promoter 1.5- to
2-fold,
83 85 whereas cotransfection with the
early growth
response factor-1 (Erg-1) transactivated the apoA-I
promoter
eightfold.
85 Erg-1 binds to the -220 to
-211 and -189 to -180
promoter regions, and it was
suggested that under conditions
of liver regeneration, it may play some
role in apoA-I gene
transcription.
86 Cotransfection
experiments of HepG2 cells
with HNF-3 did not increase the apoA-I
promoter strength in
HepG2 cells.
87 A weak HNF-3 binding
site exists within the
apoA-I regulatory element C. Gene inactivation
experiments in
mice suggest that HNF-3 may not play a significant role
in apoA-I
gene regulation (K. Kastner, G. Schutz, personal
communication).
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Figure 6. DNA binding gel electrophoresis assays of
ligand-dependent nuclear hormone receptors to the HRE of apoA-I. A
and B, Binding of homodimers of RXR and heterodimers of RXR with
RAR and T3Rß to the regulatory elements A-ID and A-IB,
respectively. The combinations of nuclear hormone receptors used are
indicated at the top of the blots. C, Summary of KMnO4 and
dimethyl sulfate modification pattern of RXR homodimers, RXR,
RAR, and T3Rß heterodimers using the A-ID element as
probe. Nucleotides of the repeats are numbered 1 through 6
on the noncoding strand. Nucleotides in the spacer region
or nucleotides 5' of the first nucleotide are
numbered -1 and -2. NE indicates nuclear extract. Large
squares, diamonds, and ovals indicate strong DNA-protein interactions
of the A-ID probe with RXR, RXR-RAR, and
RXR-T3Rß, respectively. Small squares, diamonds, and
ovals indicate weak DNA-protein interactions of the A-ID probe with
RXR, RXR-RAR, and RXR-T3Rß, respectively.
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The distal apoC-III promoter region containing the regulatory elements
F through J acts as an enhancer to increase the strength of the
proximal apoA-I promoter in HepG2 cells (Fig 3B). The enhancement in
HepG2 cells is approximately 13-fold when the apoC-III promoter is
cloned 5' of the apoA-I promoter and fivefold when it is cloned 3' of
the CAT gene in either orientation. DNase I footprinting identified
five regulatory elements within the enhancer designated F through J.
DNA binding and competition experiments showed that the regulatory
element H of the enhancer forms three DNA-protein complexes (Fig 7A). Competition experiments with
oligonucleotides corresponding to other distal
regulatory elements of apoC-III as well as
oligonucleotides containing the binding site of the
transcription factor SP1 showed that all three complexes that bound to
the oligonucleotide C-IIIH were competed completely by
oligonucleotides C-IIIH, C-IIII, and SP1.
Oligonucleotide C-IIIF competed out the formation of
complex 3 and partially that of complexes 1 and 2, whereas
oligonucleotide C-IIIJ did not compete out any of the
complexes (Fig 7A), suggesting that the factors which bind to the
regulatory elements H, I, and F of apoC-III are common.
Analysis of nuclear extracts from different tissues and cells
showed that the activity which binds to the regulatory element H is a
ubiquitous factor (Fig 7B). Additional DNA binding, competition, and
supershift assays with the other upstream apoC-III elements as
probes established that the apoC-III promoter contains multiple binding
sites for the ubiquitous transcription factor SP1, which recognizes the
regulatory elements F, H, and I. Similar analysis showed that
the regulatory element G represents a specialized HRE that is
recognized by the orphan receptors ARP-1 and EAR-3 but not by
HNF-4.41 A single activity designated C-IIIJ1 binds to the
regulatory element J. This or a similar activity also binds as a minor
component to the regulatory elements F and I where SP1 is the
predominant binding activity. Finally, a minor activity designated
C-IIII5 binds to the regulatory element I. The factors that bind to the
apoC-III enhancer are shown in Fig 1C.
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Figure 7. A, DNA binding gel electrophoresis and competition
assay with use of the upstream apoC-III regulatory element H
(-705 to -690) as probe and rat liver nuclear extracts
(N.E.). Competitor oligonucleotides were added in all
except the first lane at 100-fold molar excess relative to
32P-labeled oligonucleotides.
Oligonucleotides used are indicated by abbreviations at
the top of the figure. B, Binding assays with the regulatory element
C-IIIH as probe and nuclear extracts prepared from rat liver, kidney,
and spleen and HeLa and CaCo-2 cells. Note that the binding activities
that bind are ubiquitous and related to SP1.
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Contribution of Distal ApoC-III Regulatory Elements and Proximal
ApoA-I Promoter Elements to the Strength of the ApoA-I Promoter
in HepG2 Cells
The contribution of apoC-III regulatory elements to the
strength of the proximal apoA-I promoter in HepG2 cells was evaluated
by transient transfection experiments with promoter constructs
containing 5' deletions.44 This analysis showed
that deletion of the 5' apoC-III promoter region extending to
nucleotide -890 increased by 30% the activity of the
apoA-I promoter/apoC-III enhancer cluster. The promoter/enhancer
activity was nearly abolished by deletion of the regulatory elements J,
I, and H (Fig 8A, left column). The contribution of the
distal apoC-III regulatory elements to the enhancer activity was also
evaluated by point mutations that abolish the binding of specific
factors to their cognate sites (Table 2). This
analysis showed that the promoter/enhancer activity was reduced
to 40% to 45% of its value by mutations in elements H and G and to
55% to 70% of its value by mutations in elements I, J, or F. As shown
in Fig 1C, element G binds activities related to orphan receptors ARP-1
and EAR-3, and element F binds SP1 as a major and C-IIIJ1 as a minor
activity. The findings indicate that all the factors that bind to the
upstream apoC-III promoter region are required to enable it to
activate optimally the closely linked apoA-I promoter. Similar
mutagenesis analysis showed that the ability of the apoC-III
enhancer to activate transcription driven by the proximal
apoA-I promoter is affected greatly by mutations in the regulatory
element A-ID of apoA-I (Fig 8B, left column). This mutation reduced the
strength of the promoter/enhancer complex to 6% of its original value.
As discussed, the regulatory element A-ID contains an HRE and binds a
variety of orphan and ligand-dependent nuclear hormone
receptors.29 80 81 82 83 84 In contrast, mutations in the
regulatory element C of apoA-I that abolished the binding to this
region of heat-stable activities related to C/EBP30
reduced the strength of the promoter/enhancer complex only to 65% of
its original value.
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Table 2. Sequences of Normal and Mutated Elements in the
ApoC-III Enhancer and Proximal ApoA-I, ApoC-III, ApoA-IV, and ApoB
Promoters
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Contribution of Distal ApoC-III Regulatory Elements and
Proximal ApoA-I Promoter Elements to HNF-4–Mediated
Transactivation of the Promoter/Enhancer Complex in CaCo-2
Cells
The proximal -255 to -5 and the -1500 to
-5 nucleotides of the apoA-I promoter had low levels
of activity in CaCo-2 cells in both the presence and absence of the
enhancer. This activity could be increased to levels comparable to
those of HepG2 cells in the presence of HNF-4. For this reason, the
-1500 apoA-I promoter/apoC-III enhancer CAT constructs were used
in cotransfection experiments with plasmids expressing HNF-4 to assess
the effects of mutations in the proximal HREs, as well as the distal
apoC-III promoter elements (Table 2) on the HNF-4–mediated
transactivation in CaCo-2 cells. In general, the mutations affected the
HNF-4–mediated transactivation of the -1500 apoA-I
promoter/apoC-III enhancer cluster in CaCo-2 cells to
the same extent as they affected the strength of
the -255 to -5 apoA-I promoter/apoC-III enhancer cluster in
HepG2 cells. The wild-type -1500 apoA-I promoter/apoC-III
enhancer cluster was transactivated 7- to 10-fold by HNF-4.
The transactivation was reduced to 40% and 45% of its original value
by deletion of the regulatory element J and by point mutations in
element H, respectively, and was nearly abolished by deletions of
elements J, I, and H. Point mutations in elements J or F and I (Table 2) reduced the transactivation of the promoter/enhancer cluster to 65%
and 90% of its original value, respectively (Fig 8A, right column).
Mutations in the regulatory element A-ID (HRE) of the proximal apoA-I
promoter reduced the transactivation of the promoter/enhancer cluster
to 7% of its original value, whereas mutations in the regulatory
element A-IC of the proximal apoA-I promoter did not affect the
HNF-4–mediated transactivation of the promoter/enhancer cluster in
CaCo-2 cells (Fig 8B, right column). The findings suggest that the
HNF-4–mediated transactivation of the apoA-I promoter/apoC-III
enhancer cluster in CaCo-2 cells is promoted by interactions between
HNF-4, which binds to the HREs of the proximal apoA-I promoter, and
several of the factors that bind to the regulatory elements F through J
of the apoC-III enhancer.
Transcriptional Regulation of the Human ApoC-III Gene
DNA binding and footprinting analysis of the apoC-III
promoter region identified a set of four proximal (A through D) and six
distal (E through J) regulatory elements between
nucleotides -792 and -254 (Fig 1C). DNA binding and competition assays established the different
activities that recognize the proximal regulatory region. Element
C-IIIB binds two types of factors in overlapping binding motifs. One of
these motifs is an octameric CAGGTGAC sequence between
nucleotides -86 and -79 of the coding strand
that is recognized by a heat-stable activity. This activity,
designated C-IIIB1,58 has a molecular mass of 41 kD and
recognizes, in addition to C-IIIB, multiple sites on the apoA-II
promoter (Fig 1H).59 60 The other motif is an HRE between
nucleotides -82 and -70. This HRE consists of
two direct repeat sequences, GGGCAA AGGTCA, on the noncoding strand
with no spacing between them. Similar to the HREs found in other
apolipoprotein promoters, element C-IIIB binds HNF-4; the orphan
receptors ARP-1, EAR-2, and EAR-3; and heterodimers of RXR with RAR,
T3Rß, and peroxisome proliferator activated
receptor (PPAR).68 88 89 90 Mutations in element B that
eliminated the binding of both HNF-4 and C-IIIB1 abolished
transcription, whereas mutations that eliminated the binding of HNF-4
but allowed the binding of factor C-IIIB1 reduced hepatic transcription
to 36% of control.40 Finally, mutations that eliminated
the binding of C-IIIB1 but did not affect the binding of HNF-4
increased slightly the hepatic transcription. These data indicate that
both factors HNF-4 and C-IIIB1 are positive regulators; however, the
former has greater activation potential than the latter. The regulatory
elements C and D bind heat-stable activities as well as members of
the C/EBP family, which were also shown to bind to several locations in
the human apoB promoter. The CD region also contains two binding sites
for a new activity designated C-IIIC1.91 Mutations in
element C that prevented the binding of C-IIIC1 and of heat-stable
activities in this region did not affect significantly the hepatic and
intestinal transcription. Element D also binds NF-
B, suggesting a
potential role of this region in acute phase
response.91 92 The activities that bind to the regulatory
element E have not been identified. The sequence -460 to
-451 of apoC-III contains motif TCCAAACATC, which has high
homology to an insulin response element (IRE) found in the
phosphoenolpyruvate carboxyl kinase (PEPCK) promoter.93
ApoC-III steady-state mRNA levels and transcription rates were
elevated in diabetic rats and could be decreased significantly by
treatment with insulin in transient transfection assays. Insulin also
repressed apoC-III promoter activity, and it has been proposed that the
IRE of the apoC-III promoter is responsible for this
transcriptional repression.94 The organization of
the different activities on the apoC-III promoter is shown in Fig 1C.
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Contribution of Distal ApoC-III Regulatory Factors to the Strength
of the ApoC-III Promoter
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The contribution to intestinal and hepatic transcriptions by
factors
that occupy distal apoC-III regulatory elements was assessed
by
mutations that eliminate the binding of these factors to
their cognate
site or by deletions of certain elements. Similar
to the experiments
described for apoA-I, transfection of the
mutated promoter constructs
in HepG2 and CaCo-2 cells provided
information on the effect of
specific mutations on the promoter
strength. This analysis
yielded the following interesting data:
(1) The hepatic transcription
of apoC-III is affected mostly
by mutations in elements B, H, and G. As
shown in Fig 1C
, elements
B and G bind nuclear hormone receptors, and
element H binds
activities related to SP1. (2) The hepatic and
intestinal transcriptions
are affected differently by mutations in
element G. Thus, point
mutations in element G (enclosed in a box) or
deletion of elements
G and F reduced hepatic transcription to 26% of
control. In
contrast, the intestinal transcription was either
unaffected
by point mutation to element G or increased 1.6-fold by
deletion
of elements G and F. These findings indicate that different
combinations
of factors that occupy the distal regulatory elements of
the
apoC-III promoter are required for optimal transcription in
hepatic
and intestinal cells. Similar to the hepatic transcription,
mutations
in elements B and H affected severely the intestinal
transcription. (3)
Both hepatic and intestinal transcriptions
were also reduced
significantly by point mutations in the regulatory
elements F, I, and J
or by deletion of the 5' elements H, I,
and J (Fig 9
, left
column). Dramatic reduction in the apoC-III
promoter
strength in HepG2 cells was also obtained by mutagenesis
of an HRE
located in the 3' end of element I described below
(S. Lavrentiadou and
V.Z., unpublished observations, 1995).
As shown in Fig 1C
, elements I,
H, and F bind mainly activities
related to SP1. Elements B and G bind
nuclear hormone receptors,
and element J binds a new activity
designated C-IIIJ1. These
findings indicate that optimal activation of
the apoC-III gene
in hepatic and intestinal cells requires positive
regulatory
factors that bind to the HRE as well as all positive
regulatory
factors that bind to the distal regulatory elements F
through
J.
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Figure 9. Effect of mutations in proximal and distal apoC-III
promoter on the strength and transactivation by HNF-4. The figure shows
the effect of mutations of Table 2, shown on the left of the figure,
that affected binding of the corresponding factors to their cognate
sites, on the promoter strength, and the HNF-4–mediated
transactivation of the apoC-III promoter in different cell types. Note
that elements B and G are essential for transactivation in hepatic,
intestinal, and HeLa cells. Some mutations in other elements affect
differently hepatic and intestinal transactivation. Mutations that
caused the most drastic changes to the promoter strength or the
HNF-4–mediated transactivation are enclosed in boxes.
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Contribution of Factors Bound to Distal ApoC-III Regulatory
Elements to HNF-4–Mediated Transactivation of the ApoC-III
Promoter
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Cotransfection experiments of HepG2 and CaCo-2 cells with the
reporter
apoC-III promoter plasmids (Fig 9
) and an HNF-4–expressing
plasmid
were used for assessment of the role of this specific factor
on
the activation of the apoC-III promoter in different cell
types. This
analysis showed that HNF-4 transactivated the
apoC-III
promoter in HepG2, CaCo-2, and HeLa cells 5-, 6-, and 17-fold,
respectively.
Deletion of either the 5' elements J, I, and H or the 3'
elements
G and F of the distal promoter reduced transactivation to 10%
and
13%, respectively, of its original value in HepG2 cells (Fig
9
,
right column). The 5' region contains two SP1 binding sites
on elements
I and H, and the 3' region contains one weak SP1
binding site on
element F and an orphan receptor binding site
on element G. Reduction
in the magnitude of transactivation
by deletion of 5' or 3' elements is
also observed in CaCo-2
and HeLa cells although the deletion of
elements F and G had
less severe effects on the HNF-4–mediated
transactivation
of CaCo-2 cells compared with either HepG2 or HeLa
cells. Reduction
in the magnitude of HNF-4–mediated transactivation is
also
observed by several point mutations that eliminate the binding
of
the corresponding factor to its cognate site. The overall
mutagenesis
analysis of the distal promoter suggests that neither
the 3'
half of the distal promoter, which contains the regulatory
elements F
and G, nor the 5' half of the enhancer, which contains
elements H, I,
and J, is sufficient to provide optimal HNF-4–mediated
transactivation.
Rather, the optimal HNF-4–mediated transactivation
requires
complex interactions between HNF-4 and several factors that
bind
to the distal regulatory elements F through J. The extent of
transactivation
of the constructs carrying mutations in the apoC-III
enhancer
elements ranged from 7- to 12-fold, whereas the range of
transactivation
of the construct carrying a mutation in the HRE was
less than
2.2- to 1.8-fold in CaCo-2 and HepG2 cells, respectively.
The 3' end of the regulatory element I contains two direct repeats,
AGTGGG(TCCAG)AGGGCA, on the coding strand separated by five spacer
nucleotides (shown in parentheses). This sequence has
homology to the consensus half-site AGG/TTCA motif that is
recognized by hormone nuclear receptors.63 64 65 Element I is
recognized by the HNF-446 and other members of the
steroid/thyroid receptor superfamily (S. Lavrentiadou, unpublished
observations, 1995). Mutagenesis that abrogated the binding of HNF-4
and ligand-dependent nuclear receptors to this site reduced the
promoter activity to 5% of control and abolished the HNF-4–mediated
transactivation of the apoC-III promoter (S. Lavrentiadou, unpublished
observations, 1995). The reduction in transactivation observed by this
mutation is similar to that obtained by the mutation in the regulatory
element B that abolished the binding of hormone nuclear receptors to
this site (Fig 9, right). These findings suggest that the communication
of HNF-4 molecules bound to the proximal and distal sites is important
for the transcriptional activation of the apoC-III enhancer/proximal
promoter complexes. Other proteins of the promoter/enhancer cluster may
increase DNA binding and promote HNF-4–HNF-4 interactions.
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Transcriptional Regulation of the ApoA-IV Gene
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The apoA-IV gene displays tissue-specific expression in
primates,
with the intestine being the major and liver the minor sites
of
apoA-IV mRNA synthesis.
95 Previous studies showed that
the
transcription driven by the proximal apoA-IV promoter was increased
in
HepG2 and CaCo-2 compared with HeLa cells.
96 Tenfold
enhancement
of transcription in HepG2 and CaCo-2 cells was achieved by
larger
apoA-IV promoter constructs extending to nucleotide
-3500.
97 However, only a large DNA segment extending
up to the -7700
nucleotide position from the
transcriptional start site was
able to drive apoA-IV transcription in
transgenic mice.
42 This
construct included the apoC-III
promoter region that is located
at a 6.65-kb
distance.
40 98 DNase I footprinting analysis of
the
proximal apoA-IV promoter with the use of rat liver nuclear
extract
showed the presence of four protected regions: A-IVA
(-32 to
-22), A-IVB (-84 to -42), A-IVC (-148 to
-120), and
A-IVD (-274 to -250).
43 DNA
binding and competition assays
showed that element A-IVC binds the
orphan receptors HNF-4,
ARP-1, and EAR-3 with similar affinity
(
Kd, 4 to 7 nmol/L) (Fig
5E
). The
participation of these orphan nuclear receptors in
the formation of the
DNA-protein complex with oligonucleotide
A-IVC and
crude rat liver nuclear extracts was supported further
by competition
and antibody supershift assays. Antibodies raised
against HNF-4, which
recognize only HNF-4, and chicken ovalbumin
upstream
promoter transcription factor (COUP-TF), which recognizes
ARP-1 and
EAR-3 but not HNF-4, supershifted part of the complex
formed on the
A-IVC site. A substantial amount of unaltered
activity remained when
both antibodies were included in the
binding reaction, indicating that
besides these hormone receptors,
other nuclear factors can also
recognize this element. Methylation
interference of nuclear proteins
binding to the A-IVC oligonucleotide
probe indicated
that HNF-4, ARP-1, and EAR-3 recognize the direct
repeat
GGGTCA(CAAA)AGTCCA of the coding strand and have similar
but not
identical DNA protein contact points.
43 This sequence
has
substantial homology to the consensus half-site motif AGG/TTCA
found
in other hormone-responsive genes
63 64 65 and
contains a four-nucleotide
spacer region (shown in
parentheses). The factors that bind
to the regulatory elements A, B,
and D of the apoA-IV promoter
have not been identified. Transient
transfection assays showed
that the proximal apoA-IV promoter region
-700 to +10 had very
low activity in cells of hepatic (HepG2) or
intestinal (CaCo-2)
origin, thus the contribution of the proximal
regulatory elements
to the strength of the proximal apoA-IV promoter
could not be
evaluated using the proximal promoter
alone.
43
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Introduction
Methodologies Used for Study...
