(Hypertension. 1996;27:980-1008.)
© 1996 American Heart Association, Inc.
Articles |
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
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
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Introduction |
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 Top Introduction Methodologies Used for Study...Proximal cis-Acting...Involvement of Enhancers,...Elements and Factors Involved...Elements and Factors Involved...Role of ApoC-III Enhancer...Contribution of Distal ApoC-III...Contribution of Factors Bound...Transcriptional Regulation of...Contribution of Distal ApoC-III...Contribution of Distal ApoC-III...Distal ApoC-III Regulatory...Interactions Between Nuclear...Contribution of Apolipoprotein...Conclusions and Future...References |
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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
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Methodologies Used for Study of Transcriptional Regulation of Genes |
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TopIntroductionMethodologies Used for Study...Proximal cis-Acting...Involvement of Enhancers,...Elements and Factors Involved...Elements and Factors Involved...Role of ApoC-III Enhancer...Contribution of Distal ApoC-III...Contribution of Factors Bound...Transcriptional Regulation of...Contribution of Distal ApoC-III...Contribution of Distal ApoC-III...Distal ApoC-III Regulatory...Interactions Between Nuclear...Contribution of Apolipoprotein...Conclusions and Future...References |
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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 KMnO4 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.
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Proximal cis-Acting Regulatory Elements and Factors Involved in the Regulation of Transcription of Human Apolipoprotein Genes |
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TopIntroductionMethodologies Used for Study...Proximal cis-Acting...Involvement of Enhancers,...Elements and Factors Involved...Elements and Factors Involved...Role of ApoC-III Enhancer...Contribution of Distal ApoC-III...Contribution of Factors Bound...Transcriptional Regulation of...Contribution of Distal ApoC-III...Contribution of Distal ApoC-III...Distal ApoC-III Regulatory...Interactions Between Nuclear...Contribution of Apolipoprotein...Conclusions and Future...References |
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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-431 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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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).
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Involvement of Enhancers, Silencers, and Tissue-Specific Elements in Apolipoprotein Gene Regulation |
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TopIntroductionMethodologies Used for Study...Proximal cis-Acting...Involvement of Enhancers,...Elements and Factors Involved...Elements and Factors Involved...Role of ApoC-III Enhancer...Contribution of Distal ApoC-III...Contribution of Factors Bound...Transcriptional Regulation of...Contribution of Distal ApoC-III...Contribution of Distal ApoC-III...Distal ApoC-III Regulatory...Interactions Between Nuclear...Contribution of Apolipoprotein...Conclusions and Future...References |
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The apoC-III gene is closely linked to the human apoA-I and apoA-IV genes39 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-III40 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-I44 45 46 47 (Fig 3B) and apoA-IV43 promoters (Fig 3C), as well as the
strength of the heterologous apoB promoter44 (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 lipase51 (Fig 4B). Tissue-specific transcriptional enhancers have been found in
the second and third introns of the human apoB gene52 53 54
(Fig 1G and 1I) as well as in the intergenic regions between the apoC-I
gene and apoC-I' pseudogene55 and the apoC-I' pseudogene
and apoC-IV gene56 and are discussed below.
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Elements and Factors Involved in Transcriptional Regulation of the Human ApoA-II Gene |
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TopIntroductionMethodologies Used for Study...Proximal cis-Acting...Involvement of Enhancers,...Elements and Factors Involved...Elements and Factors Involved...Role of ApoC-III Enhancer...Contribution of Distal ApoC-III...Contribution of Factors Bound...Transcriptional Regulation of...Contribution of Distal ApoC-III...Contribution of Distal ApoC-III...Distal ApoC-III Regulatory...Interactions Between Nuclear...Contribution of Apolipoprotein...Conclusions and Future...References |
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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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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
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Elements and Factors Involved in Transcriptional Regulation of the Human ApoB Gene |
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TopIntroductionMethodologies Used for Study...Proximal cis-Acting...Involvement of Enhancers,...Elements and Factors Involved...Elements and Factors Involved...Role of ApoC-III Enhancer...Contribution of Distal ApoC-III...Contribution of Factors Bound...Transcriptional Regulation of...Contribution of Distal ApoC-III...Contribution of Distal ApoC-III...Distal ApoC-III Regulatory...Interactions Between Nuclear...Contribution of Apolipoprotein...Conclusions and Future...References |
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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
375 (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-368 (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 gene75 (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 BC76 (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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TopIntroductionMethodologies Used for Study...Proximal cis-Acting...Involvement of Enhancers,...Elements and Factors Involved...Elements and Factors Involved...Role of ApoC-III Enhancer...Contribution of Distal ApoC-III...Contribution of Factors Bound...Transcriptional Regulation of...Contribution of Distal ApoC-III...Contribution of Distal ApoC-III...Distal ApoC-III Regulatory...Interactions Between Nuclear...Contribution of Apolipoprotein...Conclusions and Future...References |
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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/T3Rß68 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/T3Rß 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/T3Rß
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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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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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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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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TopIntroductionMethodologies Used for Study...Proximal cis-Acting...Involvement of Enhancers,...Elements and Factors Involved...Elements and Factors Involved...Role of ApoC-III Enhancer...Contribution of Distal ApoC-III...Contribution of Factors Bound...Transcriptional Regulation of...Contribution of Distal ApoC-III...Contribution of Distal ApoC-III...Distal ApoC-III Regulatory...Interactions Between Nuclear...Contribution of Apolipoprotein...Conclusions and Future...References |
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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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Contribution of Factors Bound to Distal ApoC-III Regulatory Elements to HNF-4–Mediated Transactivation of the ApoC-III Promoter |
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TopIntroductionMethodologies Used for Study...Proximal cis-Acting...Involvement of Enhancers,...Elements and Factors Involved...Elements and Factors Involved...Role of ApoC-III Enhancer...Contribution of Distal ApoC-III...Contribution of Factors Bound...Transcriptional Regulation of...Contribution of Distal ApoC-III...Contribution of Distal ApoC-III...Distal ApoC-III Regulatory...Interactions Between Nuclear...Contribution of Apolipoprotein...Conclusions and Future...References |
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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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TopIntroductionMethodologies Used for Study...Proximal cis-Acting...Involvement of Enhancers,...Elements and Factors Involved...Elements and Factors Involved...Role of ApoC-III Enhancer...Contribution of Distal ApoC-III...Contribution of Factors Bound...Transcriptional Regulation of...Contribution of Distal ApoC-III...Contribution of Distal ApoC-III...Distal ApoC-III Regulatory...Interactions Between Nuclear...Contribution of Apolipoprotein...Conclusions and Future...References |
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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 genes63 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 |
Contribution of Distal ApoC-III Regulatory Elements to the Strength of the ApoA-IV Promoter |
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TopIntroductionMethodologies Used for Study...Proximal cis-Acting...Involvement of Enhancers,...Elements and Factors Involved...Elements and Factors Involved...Role of ApoC-III Enhancer...Contribution of Distal ApoC-III...Contribution of Factors Bound...Transcriptional Regulation of...Contribution of Distal ApoC-III...Contribution of Distal ApoC-III...Distal ApoC-III Regulatory...Interactions Between Nuclear...Contribution of Apolipoprotein...Conclusions and Future...References |
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As shown in Fig 3D
, the strength of the proximal apoA-IV promoter
increased ninefold and eightfold in HepG2 and CaCo-2 cells,
respectively, when the apoC-III promoter (-100 to -890) was
linked to it in tandem or in the reverse orientation. The contribution
of distal apoC-III regulatory elements to the strength of the apoA-IV
promoter in HepG2 and CaCo-2 cells was evaluated by transient
transfection experiments with constructs containing 5' and 3' deletions
of the enhancer. This analysis localized the optimal enhancer
activity within the -890 to -500 apoC-III promoter sequence
(Fig 10, lines 3 and 4) that contains the distal
regulatory elements F through J of the apoC-III gene.40 43
The enhancer activity was practically abolished by terminal 5' deletion
of elements J and I and was reduced to 12% and 18% of its original
value in HepG2 and CaCo-2, respectively, by 3' deletions of elements F
and G (Fig 10; compare line 4 with lines 6 and 7). Combinations of one
or two elements of the apoC-III enhancer with the proximal apoA-IV
promoter had no effect on apoA-IV promoter strength (Fig 10; compare
line 4 with lines 8 and 9). This finding indicates that one or more
proteins of the proximal apoA-IV promoter and the entire protein
complex that assembles on the apoC-III enhancer are required for
optimal enhancement of transcription. Mutagenesis of the HRE of the
proximal apoA-IV promoter that abolished the binding of hormone nuclear
receptor reduced the enhancer activity to 17% and 22% of its original
value in HepG2 and CaCo-2 cells, respectively (Fig 10; compare line 4
with line 5).
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Contribution of Distal ApoC-III Regulatory Elements to HNF-4–Mediated Transactivation of the ApoA-IV Promoter/ApoC-III Enhancer Cluster |
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TopIntroductionMethodologies Used for Study...Proximal cis-Acting...Involvement of Enhancers,...Elements and Factors Involved...Elements and Factors Involved...Role of ApoC-III Enhancer...Contribution of Distal ApoC-III...Contribution of Factors Bound...Transcriptional Regulation of...Contribution of Distal ApoC-III...Contribution of Distal ApoC-III...Distal ApoC-III Regulatory...Interactions Between Nuclear...Contribution of Apolipoprotein...Conclusions and Future...References |
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Cotransfection experiments with apoA-IV promoter/apoC-III enhancer constructs and plasmids expressing HNF-4 showed that HNF-4 transactivated the apoC-III enhancer/apoA-IV promoter cluster approximately 8.5- and 4-fold in HepG2 and CaCo-2 cells, respectively (Fig 10
, lines 3 and 4) in the presence of HNF-4. The
activity of the truncated apoC-III enhancer/apoA-IV promoter lacking
the 5' elements J and I or 3' elements G and F of the enhancer was
dramatically reduced; however, the extent of transactivation by HNF-4
was reduced to 8% and 60% of its original value in HepG2 and CaCo-2
cells, respectively (Fig 10, lines 6 and 7). Constructs containing one
or two elements of the apoC-III enhancer and proximal promoter
exhibited diminished enhancer activity and transactivation by HNF-4
(Fig 10, lines 8 and 9). The HNF-4–dependent transactivation of the
apoC-III enhancer/apoA-IV promoter CAT construct was abolished by
mutations that eliminated the binding of HNF-4 to its cognate site on
element A-IVC (Fig 10, line 5). In general, there was a correlation
between the effect of the apoC-III mutations on the strength of the
proximal apoA-IV promoter and the extent of the HNF-4–dependent
transactivation of the promoter (Fig 10, lines 1 through 9).
Consistent with the findings shown in Figs 8 and 9, the data
with the apoA-IV promoter suggest that the enhancement of transcription
is mediated by complex interactions between SP1 and other activities
that bind to the apoC-III enhancer and that hormone nuclear receptors
bind to the HRE of element A-IVC. |
Distal ApoC-III Regulatory Elements Act as a General Modular Enhancer and Potentiate the Strength of Heterologous Promoters That Contain HREs |
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TopIntroductionMethodologies Used for Study...Proximal cis-Acting...Involvement of Enhancers,...Elements and Factors Involved...Elements and Factors Involved...Role of ApoC-III Enhancer...Contribution of Distal ApoC-III...Contribution of Factors Bound...Transcriptional Regulation of...Contribution of Distal ApoC-III...Contribution of Distal ApoC-III...Distal ApoC-III Regulatory...Interactions Between Nuclear...Contribution of Apolipoprotein...Conclusions and Future...References |
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As discussed above, a common feature of several apolipoprotein promoters is that they contain one or two HREs that bind the orphan receptors HNF-4, ARP-1, EAR-2, and EAR-3 (Fig 5
). These HREs are
recognized by different combinations of homodimers of
cis-RXR and/or heterodimers of RXR with
trans-RAR and T3Rß (Fig 6A and 6B).39 43 66 67 68 69 76 80 81 82 83 The concept that the apoC-III
enhancer operates through interactions between upstream
activators bound to the enhancer and hormone nuclear
receptors bound to proximal sites was further reinforced by
cotransfection experiments involving heterologous promoters. The apoB
promoter was shown previously to contain both an HRE and a C/EBP
binding site on element A.75 77 Cotransfection experiments
with apoB promoter/apoC-III enhancer CAT constructs showed a 4- to
8-fold activation in HepG2 cells and a 7- to 10-fold activation in
CaCo-2 cells, depending on the length of the apoB promoter construct
(Fig 3D and Fig 11). HNF-4 transactivated
further the apoB promoter/apoC-III enhancer cluster in CaCo-2 cells but
not in HepG2 cells (data not shown). The enhancement of transcription
of the apoB promoter/enhancer cluster achieved in CaCo-2 cells in the
presence of HNF-4 was 13- to 19-fold (Fig 3D and Fig 11). The
-1800 to +124 apoB promoter constructs provided stronger
activation than the -267 to +8 apoB promoter constructs in both
HepG2 and CaCo-2 cells. More importantly, mutation in the HRE of
element A abolished the activity of the promoter/enhancer cluster,
whereas mutation in the adjacent C/EBP binding site reduced the
activity of the promoter/enhancer cluster to approximately 50% of its
original value in HepG2 and 33% of its original value in CaCo-2
cells.44 The activity of the mutated promoter/enhancer
cluster increased to 52% of the control value in the presence of
HNF-4 (Fig 11).
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The preceding analysis of the proximal apoA-I and apoB promoter
mutations indicates that the apoC-III enhancer has the ability to
bypass partially or totally the effect of mutations in C/EBP sites that
inactivate the proximal promoters, but its function is
abrogated by mutations in the proximal HREs. This is illustrated
clearly in the experiments of Figs 8B and 11. The data show that
mutations in the C/EBP binding site of the apoA-I and apoB promoters
reduced the proximal promoter strength to 8% and 13%, respectively.
Fusion of the mutated constructs with the apoC-III enhancer increased
the strength of the mutated apoA-I promoter/enhancer cluster to 40-fold
in HepG2 cells (Fig 8) and the strength of the mutated apoB
promoter/apoC-III enhancer 16- and 26-fold in HepG2 and CaCo-2 cells,
respectively (Fig 11). The level of transcription achieved by the
mutated promoter/enhancer cluster is 2.1- and 3-fold higher than that
achieved with the wild-type apoB and apoA-I promoters,
respectively, which lack the apoC-III enhancer. These findings show
that despite the inability of C/EBP to bind to its cognate site in
either of the two mutated promoters, there is a persistent synergism
between the remainder of the factors that bind to the proximal
promoters and the distal enhancer.
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Interactions Between Nuclear Hormone Receptors Bound to Proximal and Distal Sites and Factors Bound to ApoC-III Enhancer Can Be Either Synergistic or Antagonistic |
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TopIntroductionMethodologies Used for Study...Proximal cis-Acting...Involvement of Enhancers,...Elements and Factors Involved...Elements and Factors Involved...Role of ApoC-III Enhancer...Contribution of Distal ApoC-III...Contribution of Factors Bound...Transcriptional Regulation of...Contribution of Distal ApoC-III...Contribution of Distal ApoC-III...Distal ApoC-III Regulatory...Interactions Between Nuclear...Contribution of Apolipoprotein...Conclusions and Future...References |
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The synergistic or antagonistic interactions between nuclear hormone receptors bound to the HRE of element A-ID of apoA-I and SP1 or other activities bound to the apoC-III enhancer can also be visualized with the minimal AdML promoter linked to two regulatory elements A-ID (HRE) of apoA-I, the distal apoC-III regulatory elements F through J, or both (Fig 12
).
Cotransfection experiments with these constructs and plasmids
expressing HNF-4 showed that the strength of the minimal AdML promoter
linked either to two copies of the regulatory elements A-ID (HRE) of
apoA-I or to the distal apoC-III elements F through J increased
moderately up to threefold by HNF-4, depending on the cell line.
However, fusion of both sets of the regulatory elements to the 5'
region of the minimal AdML promoter increased 18.5- and 14.5-fold the
HNF-4–mediated transactivation of the minimal promoter in HepG2 and
CaCo-2 cells, respectively (Fig 12A). The change in transactivation
achieved by both elements is approximately 600% in HepG2 and CaCo-2
cells compared with the sum of the transactivation achieved by the HREs
and apoC-III enhancer alone (Fig 12A). The findings suggest that HNF-4,
which can bind to the proximal and distal HREs, and other factors that
bind to the enhancer can transactivate synergistically the
minimal AdML promoter. Interestingly, in the absence of HNF-4, the two
apoA-I HREs were unable to activate the AdML promoter in HepG2
or CaCo-2 cells. The apoC-III enhancer alone containing the regulatory
elements F through J activated sixfold and eightfold the AdML
promoter HRE cluster in HepG2 and CaCo-2 cells, respectively. However,
the combination of both the apoA-I HREs and apoC-III enhancer decreased
the strength of the AdML promoter/apoC-III enhancer cluster. Thus, the
activity of the AdML promoter linked to the apoA-I HRE and apoC-III
enhancer is 50% to 70% compared with the sum of the activities of the
AdML linked to the individual elements. This finding suggests that in
both cell types, the proteins that bind to the enhancer and the mixture
of nuclear receptors that occupy the HRE in the two cell types act
antagonistically, leading to a small reduction in
transcription. The finding also indicates that the types of nuclear
hormone receptors that can occupy the apoA-I HRE may determine the
synergistic or antagonistic interactions between the SP1 or
other factors that bind to the enhancer and hormone nuclear receptors
that occupy the proximal and distal sites. Synergistic interactions are
also observed with heterologous AdML promoter constructs containing the
HREs of element C-IIIB of apoC-III and A-IVC of
apoA-IV.41 43 The change in transcription achieved by the
combination of the C-IIIB HRE and apoC-III enhancer in HepG2 and CaCo-2
cells was approximately 400% to 500% in the absence of HNF-4 and
1800% in the presence of HNF-4 (Fig 12B). The change in transcription
achieved in HepG2 and CaCo-2 cells by the combination of the A-IVC HRE
and apoC-III enhancer was approximately 250% to 350% in the absence
and 1400% to 1500% in the presence of HNF-4 (Fig 12C). Similar
synergistic interactions can be visualized with the apoA-IV
promoter/apoC-III enhancer cluster. Thus, as shown in Fig 12D, the
relative activity of the promoter that contains an intact HRE and a
construct that contains an intact apoC-III enhancer but a mutated HRE
was 1.2 and 4.5, respectively, in the presence of HNF-4 (the sum of the
activities of the two constructs is 5.7). In contrast, the activity of
the construct that contains both an intact apoC-III enhancer and an
intact HRE is 157. This provides an increase in transactivation by a
factor of 27.5. Thus, the change in transcription achieved in HepG2 and
CaCo-2 cells by the combination of the proximal apoA-IV promoter
containing an intact HRE and the apoC-III enhancer is 2750% and
1000%, respectively, in the presence of HNF-4. Fig 12D also suggests
similar synergistic interactions between the factors bound to the HRE
and the factors bound to the apoC-III enhancer in HepG2 and CaCo-2
cells in the absence of HNF-4. In this case, however, the change in the
transactivation in HepG2 and CaCo-2 cells is 360% and 270%,
respectively, compared with that of 2750% and 1000% observed in the
presence of HNF-4. This can be explained by the fact that the element
A-IVC in hepatic cells, in addition to binding HNF-4, may also bind
other nuclear receptors that may have lower activation potential
compared with HNF-4. Cotransfection of these cells with HNF-4 results
in a very large increase in the HNF-4 concentration in the nuclei of
the transfected cells. The excess of HNF-4 may displace the other
factors from the HRE site, and this in turn may lead to increased
synergistic transactivation of the apoA-IV gene. Fig 12E summarizes the
findings of Fig 12A through 12D and indicates that the concentration in
the cell, the affinity of a specific type of nuclear receptor, and the
availability of ligands may determine the synergistic or
antagonistic interactions between the factors that bind to
the HRE and those that bind to the apoC-III enhancer.
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The ability of the apoC-III enhancer to potentiate the apoA-I promoter
in HepG2 and CaCo-2 cells was also confirmed in two other recent
studies.46 47 The promoter strength increase was 4- to
5-fold in CaCo-2 cells and 10-fold in HepG2 cells, depending on the
construct. These studies did not evaluate the contribution of the
different elements of the intact enhancer on the strength of the apoA-I
promoter/apoC-III enhancer cluster. One study showed that the
-595 to -192 apoA-I promoter region is essential for
expression of the apoA-I gene in CaCo-2 cells.47 This
region contained four additional regulatory elements, I (-408 to
393), II (-440 to -411), III (-488 to -467),
and IV (-523 to 492) (Fig 1B). Elements I through IV were
occupied by nuclear activities present in CaCo-2 cells, but only
elements I and II were occupied by nuclear activities present in
HepG2 cells. The nature of these factors was not determined. ApoA-I
promoter constructs containing the -192 to -595 regulatory
region in the presence and absence of the enhancer could be
transactivated 5- to 25-fold by HNF-4 in CaCo-2 but not
HepG2 cells.46 47 One of the studies also explored the
ability of the apoC-III enhancer to direct intestinal expression in
transgenic mice.46 Multilabel immunocytochemical
analysis in the intestine of transgenic mice showed that
apoC-III enhancer directs the expression of the apoA-I gene to
proliferating and nonproliferating jejunal crypt epithelial cells as
well as in villus-associated enterocytes. The expression in
enterocytes is along the duodenal-to-ileal axis, which
resembles that of mouse and human apoA-I. Confocal microscopy studies
also showed that the apoC-III enhancer causes anomalous expression of
apoA-I in enteroendocrine cells.46
The overall picture that emerges from the various studies is one in
which the distal apoC-III regulatory elements act as general modular
enhancers that can potentiate the strength of proximal promoters that
contain one or more HREs.40 41 42 43 44 45 46 47 This potentiation involves
synergistic interactions between hormone nuclear receptors and the
factors that bind to the apoC-III enhancer. Similar to other systems,
it is assumed that the hormone nuclear receptors that bind to the
proximal HREs and SP1 and the other factors that bind to the apoC-III
enhancer form a stereospecific DNA-protein complex.3 These
complexes may interact directly or indirectly via TATA box binding
protein–associated factors (TAFs) with the basal transcription
complex, thus leading to the transcriptional activation of the target
gene3 5 (Fig 13). Mutations in the
enhancer or proximal promoter that prevent the binding of one or more
of the participating factors may affect the configuration of this
complex and thus explain the reduction in the strength of the
promoter/enhancer complex. The data with the AdML promoter also
indicate that the types of nuclear hormone receptors that can occupy
the HREs of the different apolipoprotein promoters may determine the
synergistic or antagonistic interactions between SP1 and
the other factors that bind to the enhancer and the hormone nuclear
receptors that occupy the proximal site.
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Although SP1 is the major activity that binds to the apoC-III enhancer,
the mutagenesis analysis showed that SP1 molecules alone are
not sufficient for optimal enhancer activity. This implies that SP1
molecules must act in concert with the factors that bind to the other
regulatory elements of the enhancer. It is interesting that HNF-4
and/or other orphan receptors41 46 as well as
ligand-dependent nuclear receptors (S. Lavrentiadou and V.Z.,
unpublished data, 1995) bind to the regulatory elements G and I of the
enhancer. Mutations in these elements significantly reduce the enhancer
activity. Thus, it is possible that SP1, which binds to elements F, H,
and I, may act as an architectural component and facilitate
interactions among molecules of nuclear hormone receptors that bind to
proximal and distal sites (Fig 13). These interactions may be favorable
or unfavorable, thus resulting in transcriptional synergism or
transcriptional repression. Combinatorial interactions among factors
have been described in other enhancers, including the T-cell receptor
gene enhancer99 100 101 and the virus-induced human
interferon-ß enhancer.3 102 103 In the case of the
T-cell receptor enhancer, binding of lymphoid enhancer factor-1
promotes interactions among the other factors that bind to the
enhancer.99 100 101 In the case of the interferon-ß
enhancer, binding of high mobility group-1Y [HMG-1(Y)] protein
increases the binding affinity as well as the interactions among the
factors NF-B, activating transcription factor-2 (ATF-2), and
interferon regulatory factor-1 (IRF-1), which also bind to the
enhancer.3 102 103
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Contribution of Apolipoprotein Enhancers to Tissue-Specific Expression of Human Apolipoprotein Genes In Vivo |
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TopIntroductionMethodologies Used for Study...Proximal cis-Acting...Involvement of Enhancers,...Elements and Factors Involved...Elements and Factors Involved...Role of ApoC-III Enhancer...Contribution of Distal ApoC-III...Contribution of Factors Bound...Transcriptional Regulation of...Contribution of Distal ApoC-III...Contribution of Distal ApoC-III...Distal ApoC-III Regulatory...Interactions Between Nuclear...Contribution of Apolipoprotein...Conclusions and Future...References |
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Role of Distal ApoC-III Regulatory Elements as Intestinal Enhancer
An interesting question that requires further clarification is whether the distal apoC-III regulatory elements F through J, in addition to increasing the strength of the promoters of the apoA-I/C-III/A-IV gene cluster as well as other heterologous promoters, suffice to confer tissue-specific expression of one or more of these genes in vivo. To date, transgenic experiments suggest that intestinal expression of the apoA-IV gene requires the apoC-III promoter region extending 7.7 kb 5' of the apoA-IV promoter42 (Fig 14A
). This region contains
the apoC-III enhancer.40 41 43 Similarly, the proximal
apoC-III promoter extending to nucleotide -200 is
sufficient to direct hepatic expression of the apoC-III gene in vivo;
however, intestinal expression requires a 5.9-kb intergenic sequence
between the apoC-III and apoA-IV genes104 (Fig 14A). A
recent study has shown that the proximal apoA-I promoter extending to
nucleotide -300 linked to the regulatory elements F
through J of apoC-III could direct intestinal expression in
villus-associated enterocytes along the duodenal-to-ileal
axis but also produced inappropriate expression in
cryptoendothelial cells as well as in
subpopulations of enteroendocrine cells46 (Fig 14A). It
has been pointed out that other sequences are required for the correct
expression of the human apoA-I gene in the intestine.46 It
is possible that the sequences required for correct intestinal
expression may be located 5' or 3' of nucleotide
-255. As discussed, the -523 to -492 and -488
to -467 apoA-I promoter regions are occupied by nuclear
activities that are present in CaCo-2 but not HepG2
cells47 (Fig 1B).
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Potential Mechanisms of Transcriptional Activation of the
ApoA-I/C-III/A-IV Gene Cluster
Currently, tissue culture and transgenic mouse experiments from
different laboratories have provided the following information
pertinent to the expression of the apoA-I/C-III/A-IV gene cluster (Fig 14B).40 41 42 43 44 45 46 47 48
The hepatic transcription of the apoA-I gene is controlled by synergistic interactions between nuclear hormone receptors bound to elements D and B and factors bound to the apoC-III enhancer.30 45 48 82 The intestinal transcription of the apoA-I gene is controlled by synergistic interactions between nuclear hormone receptors bound to apoA-I elements D and B and factors bound to the apoC-III enhancer.30 45 82 Other factors bound to distal apoA-I regulatory elements may contribute to correct intestinal expression and may increase further the promoter strength. Availability of HNF-4, which can bind to the regulatory elements A-IB, A-ID, and C-III-I, may activate the transcription of the apoA-I gene in the intestine.46 47 82 The hepatic transcription of the apoC-III gene is controlled by synergistic interactions between nuclear hormone receptors bound to apoC-III element B, orphan and ligand-dependent receptors, or related activities bound to elements G and I and SP1 bound to elements F, H, and I of the apoC-III enhancer.40 41 104 The intestinal expression is controlled by orphan and ligand-dependent nuclear hormone receptors bound to elements B, G, and I and SP1 or other factors bound to the enhancer and possibly additional factors bound to distal regulatory sites.40 41 104
Finally, the intestinal and hepatic expressions of the apoA-IV gene are
controlled by synergistic interactions between nuclear hormone
receptors bound to the apoA-IV element C, factors bound to the apoC-III
enhancer,42 43 and possibly other factors bound to distal
regulatory sites. The regulatory elements that contribute to the
hepatic and intestinal expressions of the apoA-I/C-III/A-IV gene
complex in vivo are shown in Fig 14B.
Transcriptional Regulation of the ApoE/C-I Gene
Cluster
The human apoE, apoC-I, apoC-IV, and apoC-II genes are closely
linked. The cluster of the four genes maps on the long arm of
chromosome 19 and spans a 45-kb region.105 The apoC-I gene
is located 5.5 kb downstream of the apoE gene, and the apoC-I'
pseudogene is located 7.5 kb 3' to the apoC-I gene. The human apoC-II
gene is found 20 kb downstream of the apoC-I' pseudogene, and the human
apoC-IV gene is 0.55 kb upstream of the apoC-II
gene.105 106 The low-density lipoprotein receptor gene
is closely linked with the apoE/C-I/C-II/C-IV cluster105
(Fig 15A).
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Promoter deletions and footprinting analysis of the apoE
promoter identified six proximal regulatory elements (I through VI) in
the -336 to -44 region and two elements (DI and DII) within
the +69 to +191 region (Fig 15B).107 DNA binding and
competition assays with HeLa nuclear extracts showed that elements I,
III, and IV are recognized by SP1. Element II is also recognized by a
70-kD protein (Fig 15B), and element III is recognized by a 55-kD
protein.107 108 Deletion analysis of the
-246 to -81 region placed in front of the herpes thymidine
kinase promoter indicated that element III is important for
transcription of the reporter CAT gene in Chinese hamster ovary (CHO)
cells. Elements II and III are also essential for transcription of the
apoE gene in hepatic cells.107 Similar information was
obtained by in vitro transcription with the -383 to +73 region as
a template and HeLa nuclear extracts (Fig 15B). The in vitro
transcription analysis also showed that element I contributes
to the optimal transcription in HeLa cells.108 The distal
regulatory elements that control the transcription of the apoE gene in
different tissues have been determined with transgenic mice. High
expression levels of apoE in the kidney can be accomplished with a
construct containing -650 nucleotides of the 5' and
72 nucleotides of the 3' regions of the apoE
gene.109 Various regions in the intergenic sequence
between apoE and apoC-I are required for the expression of the apoE and
apoC-I genes in a variety of tissues tested, including liver, testis,
spleen, skin, submaxillary gland, kidney, brain, small intestine,
heart, stomach, and pancreas.110 111 This region also
contains a silencer that inhibits the expression of the apoE gene in
the kidney in a construct containing 0.65 kb 5' and 4-kb 3' apoE
sequences. Several elements contribute to the expression of apoE in
kidney. A positive element is localized in the 5' region between
-2 and -0.65 kb, and a silencer is localized in the second
intron of the apoE gene. Deletion of the second intron of the apoE gene
allows expression in kidney in constructs containing 0.65-kb 5' and
1.7-kb 5' regulatory sequences. Expression of apoE in kidney is also
abolished in constructs containing the region -2 kb upstream to
+1.7 kb downstream by deletion of the regulatory element III that binds
SP1 and the 55-kD regulatory protein (Fig 15B and 15D).108 111 Finally, the region in the intergenic
sequence between the apoC-I gene and apoC-I' pseudogene was originally
shown to contain a 2-kb element originating 4.4 kb 3' of the apoC-I
gene that is required for the hepatic expression of the human apoE and
apoC-I genes and was designated HCR-1109 110 111 (Fig 15A).
HCR-1 can also promote the hepatic expression of the apoA-IV gene in a
construct that contains heterologous 2.5-kb 5' and 1.7-kb 3'
sequences.111 The HCR-1–mediated expression of the apoE
gene containing 5-kb 5' and 1.7-kb 3' regions is significantly reduced
by deletion of the regulatory element III (Fig 15B). Deletion of
elements I and II did not affect hepatic expression but permitted
expression in other tissues.111 Thus, the deletion of
element I permitted high levels of expression in kidney and low levels
of expression in lung, whereas deletion of element II permitted
moderate levels of expression in kidney and lung and low levels of
expression in spleen, heart, stomach, testis, and
brain.111 Initial experiments suggested that HCR-1 was
localized within 219 bp.112 Subsequent experiments mapped
the HCR-1 required for the hepatic expression of the apoE gene within
319 bp, approximately 15 kb downstream of the apoE gene. HCR-1 contains
several DNase I–hypersensitive sites and has limited binding affinity
for nuclear scaffold55 (Fig 15A). Footprinting
analysis combined with DNA binding and competition assays
identified four regulatory elements within or 3' of the HCR-1
designated H-1 through H-4 (Fig 15C). Element H-1 and the surrounding
region contain four tandemly repeated motifs, TGTTTGC, in the antisense
strand designated c, d, e, and f (Fig 15C). In addition, two related
sequences designated a and b are found 5' upstream of footprint H-1.
The TGTTTGC motif is found in the promoters of several genes expressed
in the liver and binds a transcription factor that recognizes
single-stranded DNA.113 The TTTG core of this motif is
found in the core of the DNA binding site of high-mobility group
proteins114 that upon binding introduce pronounced bending
to the DNA.99 Thus, it is possible that binding of factors
to these sites helps in the formation of stereospecific DNA complexes
involving the proximal and distal activators as well as the
proteins of the basal transcription system (see Fig 13). Element H-2
binds a factor designated TF-LF2 that recognizes the -480 region
of the rat transferrin promoter.115 Element H-3 contains
two direct repeat sequences, AGGTCA(G)AGACCT, that can be classified as
an HRE, with one spacer nucleotide (shown in parentheses).
This sequence binds HNF-4 weakly; nevertheless, it is possible that it
binds with higher affinity other members of the orphan or
ligand-dependent nuclear receptors.62 63 64 The factor
that binds to the H-4 element is unknown and has been designated X.
Element H-5 contains a GATA motif found in the hemoglobin locus control
region, which binds GATA-binding factors.116 Element H-6
is within an Alu family sequence that contains several DNase
I–hypersensitive sites.55 This sequence binds activities
related to the C/EBP family members. The sequence of the HCR-1 between
nucleotides 1 and 461 that contains elements H-1 through
H-5 is a functional HCR. However, a smaller region between
nucleotides 6 and 325 that contains footprints H-1 through
H-3 as well as a longer region between nucleotides 6 and
587 that contains all the elements H-1 through H-6 can direct
liver-specific expression of apoE in a copy-independent manner
(Fig 15D). Recently, a second HCR designated HCR-2 was identified 27 kb
3' of the apoE gene in the middle of the intergenic sequence between
the apoC-I' pseudogene and the newly discovered apoC-IV gene (Fig 15A).
HCR-2 has 85% sequence identity to HCR-1 and is believed to have
arisen from the duplication of HCR-1.56 A 632-bp sequence
containing HCR-2 can by itself direct hepatic transcription of a DNA
segment containing the apoE gene, including 5-kb 5' and 1.7-kb 3'
sequences that lack the HCR-1 region (Fig 15D).
A variety of regulatory elements extending from 5 kb 5' of the apoE
gene to 1 kb downstream of the apoC-I' pseudogene also control
positively or negatively the expression of the apoC-I gene in different
tissues. Hepatic expression requires sequences extending 3.1 kb 5' of
apoC-I and the intergenic sequences between the apoC-I gene and apoC-I'
pseudogene that contains the HCR-1.109 110 Deletions or
additions in the 5' and 3' sequences cause different expression
patterns in various tissues studied (Fig 15E).
The contribution of the factors that bind to the HCR-1 of apoE to the
hepatic expression of apoE and apoC-I has not been assessed. Similar to
the apoA-I/C-III/A-IV enhancer of Fig 13, it is possible to envision
stereospecific DNA-protein interactions between SP1 and the other
proteins that bind to the proximal promoter with the factors bound to
HCR-1. Proteins that introduce DNA bending may orient properly the
proximal and upstream activators and promote their
interaction via TATA box binding protein–associated factors with
the proteins of the basal transcription machinery.
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Conclusions and Future Directions |
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TopIntroductionMethodologies Used for Study...Proximal cis-Acting...Involvement of Enhancers,...Elements and Factors Involved...Elements and Factors Involved...Role of ApoC-III Enhancer...Contribution of Distal ApoC-III...Contribution of Factors Bound...Transcriptional Regulation of...Contribution of Distal ApoC-III...Contribution of Distal ApoC-III...Distal ApoC-III Regulatory...Interactions Between Nuclear...Contribution of Apolipoprotein...Conclusions and Future...References |
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Transcription factors participate in the final step(s) of signal transduction pathways, leading to transcriptional activation or repression of specific genes. The transcription of eukaryotic genes is a complex biological process; understanding this process will provide insights into how genes can be switched on and off in normal and pathological states of an organism. Both in vitro and in vivo experiments are essential in identifying the physiologically relevant regulatory elements and factors. Both in vitro and in vivo approaches have advantages and limitations, and it is the combination of both approaches that can provide the optimal information. The identification of promoter and enhancer elements that are required for correct tissue-specific expression in vivo is essential and can be verified by both transgenic experiments and in vivo footprinting techniques. The identification of the factors that occupy these elements requires in vitro experiments, including purification, cloning, and immunochemical identification. The importance of specific factors and their potential ligands can be assessed by promoter transactivation assays and inhibition of the synthesis of these factors in cell cultures by antisense methodologies.
Rapid scientific advances have provided new insights into the domains
of the transcription factors required for transcriptional activation,
DNA binding, dimerization, and ligand interaction. In several cases,
gene inactivation experiments have provided information on the
importance of specific transcription factors. Rapid progress has also
been made in understanding the proteins of the basal transcription
machinery and how the transcription factors communicate with the basal
transcription factors through complex protein-protein and
DNA-protein interactions. Structural biology approaches have provided
the three-dimensional structure of the functional domains of the
transcription factors and their mode of interaction with DNA or
ligands. Although Figs 1 and 15 indicate that the apolipoprotein
promoters are recognized by a multitude of factors, the in vivo and in
vitro approaches described above make it possible to determine which of
these factors are physiologically relevant.
Subsequent studies can focus on numerous aspects of gene regulation,
including (1) the potential functional activation or inhibition of a
transcription factor by dimerization and/or interaction with ligands,
as well as the ligands involved in this process; (2) the direct or
indirect interaction of the transcription factors via their activation
domains with the components of the basal transcription systems; (3) the
three-dimensional protein-protein and DNA-protein interactions
within the specific promoter/enhancer cluster; and (4) the
intracellular or extracellular stimuli that initiate the signal
transduction pathway leading to transcriptional activation or
repression of a specific gene.
Understanding the mechanism of transcriptional regulation may allow us in the long run to selectively switch on and off the apolipoprotein genes. As indicated, upregulation of apoA-I and possibly apoA-IV genes and/or downregulation of apoB, apoA-II, and apoC-III genes may have beneficial effects in protecting humans from hyperlipidemia and atherosclerosis.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health (HL-33952), from the European Economic Community (BIOT-CT93-0473), and from KOS Pharmaceuticals, Inc, Miami, Fla. We would like to thank Anne Plunkett for excellent technical assistance and Dr Savvas Makrides for carefully reading the manuscript. This article is dedicated to the memory of our close associate and friend Christos Cladaras, who died on August 10, 1994.
Received May 16, 1995; first decision July 14, 1995; accepted February 5, 1996.
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References |
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TopIntroductionMethodologies Used for Study...Proximal cis-Acting...Involvement of Enhancers,...Elements and Factors Involved...Elements and Factors Involved...Role of ApoC-III Enhancer...Contribution of Distal ApoC-III...Contribution of Factors Bound...Transcriptional Regulation of...Contribution of Distal ApoC-III...Contribution of Distal ApoC-III...Distal ApoC-III Regulatory...Interactions Between Nuclear...Contribution of Apolipoprotein...Conclusions and Future...References |
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