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Hypertension. 1996;27:980-1008

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(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

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
up arrowTop
*Introduction
down arrowMethodologies Used for Study...
down arrowProximal cis-Acting...
down arrowInvolvement of Enhancers,...
down arrowElements and Factors Involved...
down arrowElements and Factors Involved...
down arrowRole of ApoC-III Enhancer...
down arrowContribution of Distal ApoC-III...
down arrowContribution of Factors Bound...
down arrowTranscriptional Regulation of...
down arrowContribution of Distal ApoC-III...
down arrowContribution of Distal ApoC-III...
down arrowDistal ApoC-III Regulatory...
down arrowInteractions Between Nuclear...
down arrowContribution of Apolipoprotein...
down arrowConclusions and Future...
down arrowReferences
 
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
up arrowTop
up arrowIntroduction
*Methodologies Used for Study...
down arrowProximal cis-Acting...
down arrowInvolvement of Enhancers,...
down arrowElements and Factors Involved...
down arrowElements and Factors Involved...
down arrowRole of ApoC-III Enhancer...
down arrowContribution of Distal ApoC-III...
down arrowContribution of Factors Bound...
down arrowTranscriptional Regulation of...
down arrowContribution of Distal ApoC-III...
down arrowContribution of Distal ApoC-III...
down arrowDistal ApoC-III Regulatory...
down arrowInteractions Between Nuclear...
down arrowContribution of Apolipoprotein...
down arrowConclusions and Future...
down arrowReferences
 
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.


*    Proximal cis-Acting Regulatory Elements and Factors Involved in the Regulation of Transcription of Human Apolipoprotein Genes
up arrowTop
up arrowIntroduction
up arrowMethodologies Used for Study...
*Proximal cis-Acting...
down arrowInvolvement of Enhancers,...
down arrowElements and Factors Involved...
down arrowElements and Factors Involved...
down arrowRole of ApoC-III Enhancer...
down arrowContribution of Distal ApoC-III...
down arrowContribution of Factors Bound...
down arrowTranscriptional Regulation of...
down arrowContribution of Distal ApoC-III...
down arrowContribution of Distal ApoC-III...
down arrowDistal ApoC-III Regulatory...
down arrowInteractions Between Nuclear...
down arrowContribution of Apolipoprotein...
down arrowConclusions and Future...
down arrowReferences
 
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 1Down 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 2Down). 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 1ADown 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 1Down).35 36 37 38



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Figure 1. A through 1D. See legend with 1H and 1I.



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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 1Up).


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Table 1. Binding Sites for C/EBP, Nuclear Receptors, C-IIIB1, HNF-1, and HNF-3 in Apolipoprotein Promoters

Although Fig 1AUp through 1H and Table 1Up 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 1AUp through 1H).


*    Involvement of Enhancers, Silencers, and Tissue-Specific Elements in Apolipoprotein Gene Regulation
up arrowTop
up arrowIntroduction
up arrowMethodologies Used for Study...
up arrowProximal cis-Acting...
*Involvement of Enhancers,...
down arrowElements and Factors Involved...
down arrowElements and Factors Involved...
down arrowRole of ApoC-III Enhancer...
down arrowContribution of Distal ApoC-III...
down arrowContribution of Factors Bound...
down arrowTranscriptional Regulation of...
down arrowContribution of Distal ApoC-III...
down arrowContribution of Distal ApoC-III...
down arrowDistal ApoC-III Regulatory...
down arrowInteractions Between Nuclear...
down arrowContribution of Apolipoprotein...
down arrowConclusions and Future...
down arrowReferences
 
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 1AUp). 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 3ADown).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 3BDown) and apoA-IV43 promoters (Fig 3CDown), as well as the strength of the heterologous apoB promoter44 (Fig 3DDown). 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 4ADown).49 50 This enhancer is functional and increases 10-fold the promoter strength of the heterologous liver-specific promoter of the hepatic lipase51 (Fig 4BDown). Tissue-specific transcriptional enhancers have been found in the second and third introns of the human apoB gene52 53 54 (Fig 1GUp and 1IUp) 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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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.


*    Elements and Factors Involved in Transcriptional Regulation of the Human ApoA-II Gene
up arrowTop
up arrowIntroduction
up arrowMethodologies Used for Study...
up arrowProximal cis-Acting...
up arrowInvolvement of Enhancers,...
*Elements and Factors Involved...
down arrowElements and Factors Involved...
down arrowRole of ApoC-III Enhancer...
down arrowContribution of Distal ApoC-III...
down arrowContribution of Factors Bound...
down arrowTranscriptional Regulation of...
down arrowContribution of Distal ApoC-III...
down arrowContribution of Distal ApoC-III...
down arrowDistal ApoC-III Regulatory...
down arrowInteractions Between Nuclear...
down arrowContribution of Apolipoprotein...
down arrowConclusions and Future...
down arrowReferences
 
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 1Up). 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 5ADown, 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 1BUp, 1CUp, 1DUp, and 1FUp). NHR indicates nuclear hormone receptor.

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
up arrowTop
up arrowIntroduction
up arrowMethodologies Used for Study...
up arrowProximal cis-Acting...
up arrowInvolvement of Enhancers,...
up arrowElements and Factors Involved...
*Elements and Factors Involved...
down arrowRole of ApoC-III Enhancer...
down arrowContribution of Distal ApoC-III...
down arrowContribution of Factors Bound...
down arrowTranscriptional Regulation of...
down arrowContribution of Distal ApoC-III...
down arrowContribution of Distal ApoC-III...
down arrowDistal ApoC-III Regulatory...
down arrowInteractions Between Nuclear...
down arrowContribution of Apolipoprotein...
down arrowConclusions and Future...
down arrowReferences
 
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 1EUp and 1FUp). 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 1FUp). Subsequently it was shown that site II binds to members of HNF-3 (HNF-3{alpha},ß,{gamma}).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 5BUp). 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 1FUp). 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 1GUp. 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 1FUp). The factors that recognize these positive and negative regulatory elements are shown in Fig 1GUp. 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 1IUp).54


*    Role of ApoC-III Enhancer and Proximal HREs on Transcriptional Regulation of the Human ApoA-I/C-III/A-IV Gene Cluster
up arrowTop
up arrowIntroduction
up arrowMethodologies Used for Study...
up arrowProximal cis-Acting...
up arrowInvolvement of Enhancers,...
up arrowElements and Factors Involved...
up arrowElements and Factors Involved...
*Role of ApoC-III Enhancer...
down arrowContribution of Distal ApoC-III...
down arrowContribution of Factors Bound...
down arrowTranscriptional Regulation of...
down arrowContribution of Distal ApoC-III...
down arrowContribution of Distal ApoC-III...
down arrowDistal ApoC-III Regulatory...
down arrowInteractions Between Nuclear...
down arrowContribution of Apolipoprotein...
down arrowConclusions and Future...
down arrowReferences
 
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 3Up). 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 5CUp); and ligand-dependent nuclear receptors RXR{alpha}, RXR{alpha}/RAR{alpha}, and RXR{alpha}/T368 80 81 82 83 84 (Fig 6ADown and 6BDown). Potassium permanganate and dimethyl sulfate interference experiments showed that RXR{alpha} homodimers and RXR{alpha}/RAR{alpha} and RXR{alpha}/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 6CDown). Cotransfection experiments in HepG2 cells with normal and mutated promoter constructs and plasmids expressing nuclear hormone receptors showed that RXR{alpha} homodimers transactivated the wild-type promoter 150% of control in the presence of 9-cis-retinoic acid, whereas RXR{alpha}/T3Rß heterodimers repressed transcription to 60% of control in the presence of triiodothyronine. RXR{alpha}/RAR{alpha} 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{alpha} and heterodimers of RXR{alpha} with RAR{alpha} 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{alpha} homodimers, RXR{alpha}, RAR{alpha}, 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{alpha}, RXR{alpha}-RAR{alpha}, and RXR{alpha}-T3Rß, respectively. Small squares, diamonds, and ovals indicate weak DNA-protein interactions of the A-ID probe with RXR{alpha}, RXR{alpha}-RAR{alpha}, and RXR{alpha}-T3Rß, respectively.

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 3BUp). 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 7ADown). 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 7ADown), 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 7BDown). 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 1CUp.



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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.

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 8ADown, 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 2Down). 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 1CUp, 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 8BDown, 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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Figure 8. (facing page). Effect of mutations on the strength of the -255 to -5 apoA-I promoter/apoC-III enhancer cluster in HepG2 cells and transactivation of the -1500 to -5 apoA-I promoter/apoC-III enhancer cluster by HNF-4 in CaCo-2 cells. A, Effects of mutations in the distal regulatory elements F through J of the apoC-III promoter (Table 2Up), and B, effects of mutations in the proximal regulatory elements C and D of the apoA-I promoter (Table 2Up) on the strength of the apoA-I promoter/apoC-III enhancer cluster in HepG2 cells (left) or its transactivation by HNF-4 in CaCo-2 cells (right). The relevant regulatory elements of the apoA-I promoter and apoC-III enhancer have been described30 40 41 and are symbolized by capital letters. Mutated sites are indicated by X. Note that the apoC-III enhancer can bypass mutations in the C/EBP binding site that inactivate the proximal promoter.


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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

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 2Up) 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 2Up) reduced the transactivation of the promoter/enhancer cluster to 65% and 90% of its original value, respectively (Fig 8AUp, 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 8BUp, 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 1CUp). 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 1HUp).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{alpha} 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-{kappa}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 1CUp.


*    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 1CUp, 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 9Down, 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 1CUp, 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 2Up, 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.


*    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 9Up) 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 9Up, 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 9Up, 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.


*    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 5EUp). 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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As shown in Fig 3DUp, 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 10Down, 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 10Down; 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 10Down; 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 10Down; compare line 4 with line 5).



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Figure 10. Localization of elements of the distal apoC-III enhancer and proximal apoA-IV promoter required for optimal strength of the apoA-IV promoter/apoC-III enhancer cluster in HepG2 and CaCo-2 cells and transactivation by HNF-4. W.T. indicates wild-type.


*    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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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 10Up, 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 10Up, 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 10Up, 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 10Up, 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 10Up, lines 1 through 9). Consistent with the findings shown in Figs 8Up and 9Up, 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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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 5Up). These HREs are recognized by different combinations of homodimers of cis-RXR{alpha} and/or heterodimers of RXR with trans-RAR{alpha} and T3Rß (Fig 6AUp and 6BUp).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 3DUp and Fig 11Down). 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 3DUp and Fig 11Down). 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 11Down).



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Figure 11. Activation of the -267 to +8 apoB promoter by the apoC-III enhancer in HepG2 and CaCo-2 cells. The relevant regulatory elements of the apoB promoter and apoC-III enhancer are symbolized by capital letters as described.40 41 74 Mutated sites are indicated by X. The lower portion of the figure shows the effect of mutations in the HRE and adjacent C/EBP site of the proximal promoter (Table 2Up) and on the strength of the apoB promoter/apoC-III enhancer cluster in HepG2 and CaCo-2 cells in the presence or absence of HNF-4. The distal apoC-III promoter enhances the strength of the mutated apoB promoter by 16- and 26-fold in HepG2 and CaCo-2 cells, respectively, and thus overcompensates for the mutation in the proximal C/EBP site.

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 8BUp and 11Up. 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 8Up) and the strength of the mutated apoB promoter/apoC-III enhancer 16- and 26-fold in HepG2 and CaCo-2 cells, respectively (Fig 11Up). 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.


*    Interactions Between Nuclear Hormone Receptors Bound to Proximal and Distal Sites and Factors Bound to ApoC-III Enhancer Can Be Either Synergistic or Antagonistic
up arrowTop
up arrowIntroduction
up arrowMethodologies Used for Study...
up arrowProximal cis-Acting...
up arrowInvolvement of Enhancers,...
up arrowElements and Factors Involved...
up arrowElements and Factors Involved...
up arrowRole of ApoC-III Enhancer...
up arrowContribution of Distal ApoC-III...
up arrowContribution of Factors Bound...
up arrowTranscriptional Regulation of...
up arrowContribution of Distal ApoC-III...
up arrowContribution of Distal ApoC-III...
up arrowDistal ApoC-III Regulatory...
*Interactions Between Nuclear...
down arrowContribution of Apolipoprotein...
down arrowConclusions and Future...
down arrowReferences
 
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 12Down). 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 12ADown). 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 12ADown). 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 12BDown). 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 12CDown). Similar synergistic interactions can be visualized with the apoA-IV promoter/apoC-III enhancer cluster. Thus, as shown in Fig 12DDown, 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 12DDown 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 12EDown summarizes the findings of Fig 12ADown 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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Figure 12. A through 12D. See legend with 12E.

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 1BUp). 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 13Down). 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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Figure 13. Schematic representation shows putative protein-protein interactions among the factors bound to the promoter/enhancer region and the proteins of the basal transcription machinery. The figure is based on the data presented here and in previous studies.39 40 41 42 43 44 45 46 47 The model speculates that SP1, which binds to three sites on the apoC-III enhancer, and other factors help orient properly an activator (assumed tentatively to be a nuclear hormone receptor) relative to another molecule of a nuclear hormone receptor that binds to a proximal promoter site. These activators in turn interact with the proteins of the basal transcription complex via TATA box binding protein–associated factors (TAFs), thus leading to the initiation of a transcriptional event. RNA Pol II indicates RNA polymerase II.

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 13Up). 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 {alpha} gene enhancer99 100 101 and the virus-induced human interferon-ß enhancer.3 102 103 In the case of the T-cell receptor {alpha} 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-{kappa}B, activating transcription factor-2 (ATF-2), and interferon regulatory factor-1 (IRF-1), which also bind to the enhancer.3 102 103


*    Contribution of Apolipoprotein Enhancers to Tissue-Specific Expression of Human Apolipoprotein Genes In Vivo
up arrowTop
up arrowIntroduction
up arrowMethodologies Used for Study...
up arrowProximal cis-Acting...
up arrowInvolvement of Enhancers,...
up arrowElements and Factors Involved...
up arrowElements and Factors Involved...
up arrowRole of ApoC-III Enhancer...
up arrowContribution of Distal ApoC-III...
up arrowContribution of Factors Bound...
up arrowTranscriptional Regulation of...
up arrowContribution of Distal ApoC-III...
up arrowContribution of Distal ApoC-III...
up arrowDistal ApoC-III Regulatory...
up arrowInteractions Between Nuclear...
*Contribution of Apolipoprotein...
down arrowConclusions and Future...
down arrowReferences
 
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 14ADown). 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 14ADown). 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 14ADown). 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 1BUp).



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Figure 14. A, Schematic representation of the constructs containing regulatory elements of the apoA-I, apoC-III, and apoA-IV promoter region required for intestinal and hepatic expression of the corresponding genes. The figure is based on References 42, 46, 48, and 104. B, Schematic presentation of the apoA-I/C-III/A-IV gene cluster and proposed role of the apoC-III enhancer in the transcriptional regulation of the apoA-I gene and other genes of the cluster. Numbers 1 through 9 indicate the following: ApoA-I gene: (1) The proximal promoter suffices for hepatic expression; (2) the enhancer increases hepatic expression; (3) the enhancer is required for intestinal expression; (4) other upstream elements may contribute to tissue specificity and optimal intestinal expression. ApoC-III gene: (5) the proximal promoter suffices for hepatic expression; (6) the enhancer increases hepatic expression; (7) the enhancer and possibly other upstream elements are required for intestinal expression. ApoA-IV gene: (8) the proximal promoter is inactive; (9) the enhancer and possibly other elements are required for hepatic and intestinal expression. HS indicates heat-stable.

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 14BUp).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 14BUp.

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 15ADown).



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Figure 15. A through C, Schematic representations of regulatory elements and transcription factors involved in regulation of the human apoE and apoC-I genes. A, Long-distance regulatory elements of the apoA-E/C-I/C-IV/C-II gene cluster. B, Proximal regulatory elements and factors controlling the transcription of the human apoE gene. Regulatory elements are designated by I, II, etc, starting from the initiation of transcription. Their boundaries are designated by brackets, with numbers specifying their location relative to the initiation of transcription. The factors are designated by ellipsoids carrying their names. Asterisks indicate mutations that reduce transcription 1% to 14% (*) and 15% to 30% (**) of control, respectively. C, Regulatory elements and factors of the HCR-1 of the apoE/C-I/C-IV/C-II cluster. D and E, Schematic representations of the constructs containing the regulatory elements of the apoE/C-I/C-IV/C-II cluster required for tissue-specific expression of the human apoE (D) and apoC-I (E) genes. LDL indicates low-density lipoprotein. Based on References 55, 56, and 106 through 111.

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 15BUp).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 15BUp), 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 15BUp). 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 15BUp and 15DUp).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 15AUp). 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 15BUp). 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 15AUp). 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 15CUp). Element H-1 and the surrounding region contain four tandemly repeated motifs, TGTTTGC, in the antisense strand designated c, d, e, and f (Fig 15CUp). 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 13Up). 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 15DUp). 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 15AUp). 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 15DUp).

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 15EUp).

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 13Up, 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.


*    Conclusions and Future Directions
up arrowTop
up arrowIntroduction
up arrowMethodologies Used for Study...
up arrowProximal cis-Acting...
up arrowInvolvement of Enhancers,...
up arrowElements and Factors Involved...
up arrowElements and Factors Involved...
up arrowRole of ApoC-III Enhancer...
up arrowContribution of Distal ApoC-III...
up arrowContribution of Factors Bound...
up arrowTranscriptional Regulation of...
up arrowContribution of Distal ApoC-III...
up arrowContribution of Distal ApoC-III...
up arrowDistal ApoC-III Regulatory...
up arrowInteractions Between Nuclear...
up arrowContribution of Apolipoprotein...
*Conclusions and Future...
down arrowReferences
 
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 1Up and 15Up 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.


*    Selected Abbreviations and Acronyms
 
AdML = adenovirus major late promoter
apo = apolipoprotein
ARP = apolipoprotein regulatory protein
C/EBP = CAAT/enhancer binding protein
CAT = chloramphenicol acetyltransferase
EAR = V-ERB–related receptor
HCR = hepatic control region
HNF = hepatic nuclear factor
HRE = hormone response element
NF = nuclear factor
RAR = retinoic acid receptor
RXR = retinoic acid X receptor
SP1 = stimulatory protein-1
T3R = triiodothyronine (thyroid hormone) receptor



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Figure 1EG. E through 1G. See legend with 1H and 1I.



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Figure 1HI. Schematic representation of regulatory elements and transcription factors that participate in regulation of human apolipoprotein genes. A and E, Long-distance regulatory elements of the apoA-I/C-III/A-IV gene cluster and apoB gene, respectively. B, C, D, F, and H, Regulatory elements and factors controlling the transcription of the human apoA-I, apoC-III, apoA-IV, apoB, and apoA-II genes, respectively. In most cases, regulatory elements are designated by letters (A, B, etc) starting from the point of transcription initiation. Their boundaries are designated by brackets, with numbers specifying their location relative to the point of transcription initiation. The factors are designated by ellipsoids carrying their names. Asterisks indicate mutations that reduce transcription 1% to 14% (*) and 15% to 30% (**) of control. The symbol + in B indicates a mutation that increased transcription 4.6-fold. The letters V, W, X, Y, and Z indicate nuclear activities of unknown identity. The diagrams of A through H are based on References 29 through 98. I, Regulatory elements of the apoB gene that permit its hepatic expression in transgenic mice. The diagram is based on Reference 54. Erg-1 indicates early growth response-1; HS, heat-stable; IRE, insulin response element; SREBP-1, sterol response element binding protein-1; GABP, GA binding protein; Ets-1, E-twenty-six specific; MAR, matrix association region; and ß-gal, ß-galactosidase.



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Figure 12E. A through C, Activation of the minimal AdML promoter by synergism between nuclear hormone receptors bound to the HRE and factors bound to the apoC-III enhancer. Shown are effects of the regulatory elements A-ID of apoA-I (A), C-IIIB of apoC-III (B), and A-IVC of apoA-IV (C) on the strength of the AdML promoter in HepG2 and CaCo-2 cells. D, Effect of the apoC-III enhancer and proximal apoA-IV promoter mutated in the HRE on the strength of the apoC-III enhancer/apoA-IV promoter cluster. Shown is percent change in transcription by interaction between nuclear hormone receptors and SP1 and other factors bound to the enhancer. Percent change in transcription is calculated by dividing the activity of the promoter construct with the sum of the activities obtained by the HRE construct or the apoC-III enhancer construct. The extent of transactivation is calculated for the promoter/enhancer construct only and represents the promoter activity obtained in the presence and absence of HNF-4. Asterisks indicate that in transfection experiments with HNF-4, the HRE is occupied by HNF-4. Otherwise, the HRE is occupied by a mixture of nuclear hormone receptors that are present in the cell nucleus. W.T. indicates wild-type. E, Magnitude of the synergistic interactions between different types of hormone nuclear receptors and the factors bound to the apoC-III enhancer. The concentration in the cell and affinity of factors that can bind the HRE may determine the extent of synergism between nuclear receptors and the factors bound to the apoC-III enhancer. The data summarize the experiments presented in A through D.



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Figure 15D. D. See legend with 15A-C.



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Figure 15E. E. See legend with 15A-C.


*    Acknowledgments
 
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.


*    References
up arrowTop
up arrowIntroduction
up arrowMethodologies Used for Study...
up arrowProximal cis-Acting...
up arrowInvolvement of Enhancers,...
up arrowElements and Factors Involved...
up arrowElements and Factors Involved...
up arrowRole of ApoC-III Enhancer...
up arrowContribution of Distal ApoC-III...
up arrowContribution of Factors Bound...
up arrowTranscriptional Regulation of...
up arrowContribution of Distal ApoC-III...
up arrowContribution of Distal ApoC-III...
up arrowDistal ApoC-III Regulatory...
up arrowInteractions Between Nuclear...
up arrowContribution of Apolipoprotein...
up arrowConclusions and Future...
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