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Transcriptional Regulation of the Genes Involved in Lipoprotein Transport

1996; Lippincott Williams & Wilkins; Volume: 27; Issue: 4 Linguagem: Inglês

10.1161/01.hyp.27.4.980

1524-4563

Dimitris Kardassis, Maria Laccotripe, Iannis Talianidis, Vassilis I. Zannis,

RNA Research and Splicing

HomeHypertensionVol. 27, No. 4Transcriptional Regulation of the Genes Involved in Lipoprotein Transport Free AccessResearch ArticleDownload EPUBAboutView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticleDownload EPUBTranscriptional 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 and Vassilis Zannis Dimitris KardassisDimitris Kardassis 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.). , Maria LaccotripeMaria Laccotripe 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.). , Iannis TalianidisIannis Talianidis 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.). and Vassilis ZannisVassilis 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.). Originally published1 Apr 1996https://doi.org/10.1161/01.HYP.27.4.980Hypertension. 1996;27:980–1008The 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.123456789101112 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.813Methodologies Used for Study of Transcriptional Regulation of GenesSeveral 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.1415 (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,161718 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 GenesPlasma levels of apolipoproteins could in principle be increased by increasing the level of gene transcription. Thus, the genetic information pertinent to regulatory mechanisms governing apolipoprotein gene transcription is important. Existing biochemical and genetic data suggest that increased plasma apoA-I and decreased plasma apoB levels could decrease the ratio of low-density lipoprotein to high-density lipoprotein and thus protect humans against atherosclerosis.25 Similarly, reductions in plasma apoA-II levels could have some protective role against atherosclerosis,26 and reductions in plasma apoC-I and apoC-III levels could have beneficial effects in reducing plasma triglyceride levels.27 Finally, increases in plasma apoE levels could accelerate the removal of lipoprotein remnants and thus protect against the development of atherosclerosis.28 Use of the techniques outlined above resulted in the mapping of the proximal regulatory elements of most of the apolipoprotein promoters and the factors that bind to them. Fig 1 shows the information obtained for apoA-I, apoC-III, apoA-IV, apoB, and apoA-II.29 The information on the apoE/C-I/C-IV/C-II gene complex is presented below at the end of the article. To facilitate the description of the nuclear activities that recognize the different regulatory elements of the apolipoprotein genes, we have adopted a uniform nomenclature system that identifies each activity by three characteristics: (1) the name of the target gene, (2) the element to which the factors bind, and (3) the mobility of the DNA/protein complexes. This mobility is indicated by the numbers 1, 2, and 3, going from the slowest to the most rapidly migrating complexes. With this nomenclature, the activities that bind to the regulatory element C of the apoA-I promoter are designated A-IC1, A-IC2, A-IC3, etc (Fig 2). Previously described factors, ie, C/EBP, HNF-1, HNF-3, HNF-4, etc, maintain their names.30 Our systematic analysis of five apolipoprotein promoters resulted in the identification of 37 regulatory elements. Other investigators have also identified 4 elements in the proximal apoE promoter, 6 in the HCR of the apoE/C-I/C-IV/C-II gene locus, 6 in the second intron enhancer, 3 in the third intron enhancer of apoB, and 1 in the 5′ silencer of the apoB gene. A careful examination of the identified activities indicates that several previously described factors participate in the transcriptional regulation of the apolipoprotein genes (Fig 1A through 1H). This includes the liver-enriched factors C/EBP, HNF-1, HNF-3, and HNF-431323334 as well as ubiquitous factors such as NF-1, NFY, SP1, and GA binding protein/E-twenty-six specific (GABP/Ets-1) (Table 1).35363738Although Fig 1A through 1H and Table 1 indicate that several previously described transcription factors may recognize different apolipoprotein promoters, the arrangement of the factors within each promoter is unique. This unique arrangement of the regulatory elements and factors bound to them (referred to as promoter context) may allow the formation of a unique and stereospecific DNA-protein complex that results in the transcriptional activation of the corresponding gene. Analysis of the promoter strength by transient transfection assays with the use of wild-type and mutated promoter CAT constructs showed that despite the apparent complexity of the apolipoprotein promoters, only a few regulatory elements and the corresponding factors may be essential for optimal transcription in cell culture. The most important regulatory regions are indicated by one or two asterisks. One asterisk is used when mutations that eliminate the binding to an indicated element of the corresponding factor reduced transcription from 1% to 14%, and two asterisks when mutations reduced transcription 15% to 30% (Fig 1A through 1H).Involvement of Enhancers, Silencers, and Tissue-Specific Elements in Apolipoprotein Gene RegulationThe apoC-III gene is closely linked to the human apoA-I and apoA-IV genes39 and is localized 2.5 kb downstream of the apoA-I gene and 5 kb upstream of the apoA-IV gene. The direction of transcription of the apoC-III gene is opposite to that of the apoA-I and apoA-IV genes (Fig 1A). A series of in vitro and in vivo studies have pointed out that the distal apoC-III regulatory elements may act as homologous enhancers for apoC-III4041 as well as for the other two genes of the cluster.424344454647 The in vitro experiments showed that deletion of the distal apoC-III promoter region reduced the strength of the proximal promoter to 10% to 20% of its original value, implying that these elements are required to enhance the transcription of the apoC-III gene (Fig 3A).4041 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-I44454647 (Fig 3B) and apoA-IV43 promoters (Fig 3C), as well as the strength of the heterologous apoB promoter44 (Fig 3D). Similarly, expression of segments of the apoA-I/C-III/A-IV gene cluster in transgenic mice indicated that hepatic expression requires only 5′ regulatory elements in the apoA-I and apoC-III genes, whereas the intestinal expression of the apoA-I and apoA-IV genes requires elements localized in the intergenic sequence between the apoC-III and apoA-IV genes.424548 A different type of tissue-specific enhancer is also found in the apoA-II gene. Without the apoA-II enhancer, the transcription driven by the proximal and middle apoA-II regulatory elements is approximately 1% of control (Fig 4A).4950 This enhancer is functional and increases 10-fold the promoter strength of the heterologous liver-specific promoter of the hepatic lipase51 (Fig 4B). Tissue-specific transcriptional enhancers have been found in the second and third introns of the human apoB gene525354 (Fig 1G and 1I) as well as in the intergenic regions between the apoC-I gene and apoC-I′ pseudogene55 and the apoC-I′ pseudogene and apoC-IV gene56 and are discussed below.Elements and Factors Involved in Transcriptional Regulation of the Human ApoA-II GeneFootprinting 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.495057 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-IIIB1.5859 Simultaneous nucleotide substitutions that prevented the binding of C-IIIB1 activity in elements AB, K, and L reduced the strength of the apoA-II promoter in HepG2 and CaCo-2 cells to 6% to 7% of control.59 Elements AB and K bind, in addition to C-IIIB1, a heat labile activity designated A-IIAB1. Mutations in the A-IIAB1 binding site reduce the promoter activity to background levels.60 The nature and importance of the A-IIAB1 activity have not been clarified. A new activity designated A-IIN3 binds to the regulatory element N. Deletion of element N reduced hepatic and intestinal transcription of the apoA-II gene to 7% and 18% of control, respectively, indicating that A-IIN3 is important.49 Finally, two new activities designated A-IIM1 and A-IIM2 bind to the regulatory element M, and one activity designated A-IID1 binds to element D. Element D is also recognized by GABP, an Ets-related protein, as well as C/EBP family members. It appears that A-IID1 acts as a negative and GABP as a positive regulator.61 A-IIM1 is present in the liver and in CaCo-2 cells, whereas A-IIM2 is present only in the liver.60 The contribution of the A-IIM1 and A-IIM2 activities in hepatic and intestinal transcription is unclear because deletion of element M did not significantly affect the promoter strength in HepG2 or CaCo-2 cells. C/EBP and other proteins that recognize the CCAAT motif bind with high affinity to the regulatory elements L, C, and D. Low-affinity binding sites for C/EBP are also found in elements F, G, and AB (Table 1). The most important C/EBP site is on element L. Mutations in this site that prevented the binding of C/EBP and other CCAAT box binding proteins reduced both hepatic and intestinal transcription to 30% of control.60 Elements H and I bind the previously described homeodomain factors HNF-1 and NF-1, respectively. Deletion of these elements reduced the promoter activity in HepG2 and CaCo-2 cells to 60% to 80% of control.60 The regulatory element J contains on the noncoding strand two direct repeated sequences, AGGGTA(A)AGGTTG, with one spacer nucleotide between them (included in parentheses). This sequence has homology to a consensus half-site motif, AGG/TTCA, which is the binding site of hormone nuclear receptors.626364 As shown in Fig 5A, element A-IIJ binds HNF-4 and other orphan and ligand-dependent nuclear receptors.316566676869 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.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.72Elements and Factors Involved in Transcriptional Regulation of the Human ApoB GeneThe human apoB gene is localized in a 47.5-kb region flanked by matrix association regions (MARs).73 The proximal apoB promoter region between nucleotides −150 and +124 can direct the expression of a reporter gene in hepatic and intestinal cells but not in HeLa cells. Longer promoter fragments extending to nucleotide −1800 have lower promoter activity.74 DNase I footprinting analysis identified three regulatory elements designated A, CB, and E (Fig 1E and 1F). The regulatory element CB binds two types of activities in overlapping sites.75 Site I (−118 to −98) binds heat-stable activities related to C/EBP, and site II (−112 to −88) binds three chromatographically separable activities initially designated B-CB1, 2, and 375 (Fig 1F). Subsequently it was shown that site II binds to members of HNF-3 (HNF-3α,β,γ).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).7577 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.636465 Element A binds members of the nuclear receptor family HNF-4, ARP-1, EAR-2, and EAR-368 (Fig 5B). Using the element as a ligand, we have purified by affinity chromatography from rat hepatic nuclear extracts a protein with an approximate Mr of 60 kD that was designated NF-BA1.78 Mutagenesis of the binding site of NF-BA1 as well as in vitro transcription assays indicated that NF-BA1 is a positive regulator. Bandshift clipping experiments with different proteases showed that the degradation products of NF-BA1 are similar to those obtained by HNF-4 but different from those obtained with the other nuclear receptors ARP-1, EAR-2, and EAR-3, which repress the activity of the apoB promoter (C. Cladaras, unpublished observations, 1994). Heat-stable activities related to C/EBP also bind to element E (+33 to +52), which is located in the first exon of the apoB gene75 (Fig 1F). Element E and element CB are weak C/EBP binding sites, whereas site IV (−72 to −53) on element A is a strong C/EBP binding site. In vitro mutagenesis of the promoter region showed that the mutations at the HNF-3 binding site II (−112 to −94), the nuclear hormone receptor binding site III (−86 to −62), or the strong C/EBP binding site IV (−72 to −53) reduced hepatic and intestinal transcription to 9%, 2%, and 10% to 13% of control, respectively, indicating the potential importance of the factors that recognize these elements for apoB gene transcription.75 The proximal apoB promoter elements extending to nucleotide −898 are not sufficient for tissue-specific expression of the apoB gene in vivo.54 Tissue culture experiments have shown that the second intron of the apoB gene between nucleotides +621 and +1064 enhances threefold and fivefold the strength of the apoB promoter in HepG2 and CaCo-2 cells, respectively, but not in HeLa cells.52 This region contains six regulatory elements designated A through F. The element E (+806 to +940) is essential for the enhancer activity and is recognized by HNF-1, C/EBP, and several other unidentified activities designated a, b, c, d, e, f, protein I, and protein II.5279 The organization of the different activities in the second intron enhancer is shown in Fig 1G. Similarly, the apoB sequence between nucleotides 1065 and 2977 enhances the strength of the apoB promoter approximately twofold in HepG2 and CaCo-2 cells, respectively.53 Deletion analysis localized the enhancer to a 155-bp fragment, which is flanked by DNase I–hypersensitive sites. This region contains three footprints designated A, B, and C. The activities that bind to these elements have not been identified rigorously. Finally, the region between nucleotides −3067 and −2734 represses the strength of the apoB promoter in CaCo-2 but not in HepG2 cells.76 This region contains a binding site for the transcription factor ARP-1 between nucleotides −2801 and −2728, and it has been suggested that ARP-1 reduces transcription by interfering with the function of HNF-3, which binds to the proximal promoter site BC76 (Fig 1F). The factors that recognize these positive and negative regulatory elements are shown in Fig 1G. Transgenic mouse experiments have shown that the second intron enhancer region is sufficient to direct expression of apoB promoter constructs in the liver but not in the intestine. Incorporation of both the second and third intron enhancers and sequences containing the 5′ and 3′ MARs in the apoB constructs increased their expression but did not eliminate the integration-related position effects on the expression levels of the transgene. The inclusion of the 5′ upstream negative regulatory region in this construct did not affect its hepatic expression in vivo (Fig 1I).54Role of ApoC-III Enhancer and Proximal HREs on Transcriptional Regulation of the Human ApoA-I/C-III/A-IV Gene ClusterTranscriptional Regulation of the Human ApoA-I GeneAs indicated above, the distal regulatory elements of apoC-III increase the strength of homologous as well as heterologous promoters (Fig 3). To understand the mechanism of this transcriptional activation, it is important to identify the factors that bind to the proximal promoters of the target genes as well as the factors that bind the apoC-III enhancer. This knowledge would provide information on the interactions that lead to the transcriptional activation of the target genes. The three proximal regulatory elements A-IB (−128 to −77), A-IC (−175 to −148), and A-ID (−220 to −190) of the apoA-I gene are necessary and sufficient for its hepatic expression in vivo and in vitro.304648 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.636465 The HRE present on element A-ID and A-IB is composed of two direct repeats with sequences GGGTCA(GA)GGTTCA and AGTTCA(A)GGATCA, respectively, on the noncoding strands. The spacing between the half repeat sites of A-ID and A-IB are two and one nucleotides, respectively. DNA binding and competition assays showed that elements A-IB and A-ID support the binding of HNF-4; other nuclear orphan receptors (Fig 5C); and ligand-dependent nuclear receptors RXRα, RXRα/RARα, and RXRα/T3Rβ688081828384 (Fig 6A and 6B). Potassium permanganate and dimethyl sulfate interference experiments showed that RXRα homodimers and RXRα/RARα and RXRα/T3Rβ heterodimers participate in protein-DNA interactions with 12, 13, and 11 out of the 14 nucleotides, respectively, that span repeats 1 and 2 of element A-ID and the spacer region separating them (Fig 6C). Cotransfection experiments in HepG2 cells with normal and mutated promoter constructs and plasmids expressing nuclear hormone receptors showed that RXRα homodimers transactivated the wild-type promoter 150% of control in the presence of 9-cis-retinoic acid, whereas RXRα/T3Rβ heterodimers repressed transcription to 60% of control in the presence of triiodothyronine. RXRα/RARα and HNF-4 did not affect the transcription, which was driven by the proximal apoA-I promoter.8283 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,8385 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).The distal apoC-III promoter region containing the regulatory elements F through J acts as an enhancer to increase the strength of the proximal apoA-I promoter in HepG2 cells (Fig 3B). The enhancement in HepG2 cells is approximately 13-fold when the apoC-III promoter is cloned 5′ of the apoA-I promoter and fivefold when it is cloned 3′ of the CAT gene in either orientation. DNase I footprinting identified five regulatory elements within the enhancer designated F through J. DNA binding and competition experiments showed that the regulatory element H of the enhancer forms three DNA-protein complexes (Fig 7A). Competition experiments with oligonucleotides corresponding to other distal regulatory elements of apoC-III as well as oligonucleotides containing the binding site of the transcription factor SP1 showed that all three complexes that bound to the oligonucleotide C-IIIH were competed completely by oligonucleotides C-IIIH, C-IIII, and SP1. Oligonucleotide C-IIIF competed out the formation of complex 3 and partially that of complexes 1 and 2, whereas oligonucleotide C-IIIJ did not compete out any of the complexes (Fig 7A), suggesting that the factors which bind to the regulatory elements H, I, and F of apoC-III are common. Analysis of nuclear extracts from different tissues and cells showed that the activity which binds to the regulatory element H is a ubiquitous factor (Fig 7B). Additional DNA binding, competition, and supershift assays with the other upstream apoC-III elements as probes established that the apoC-III promoter contains multiple binding sites for the ubiquitous transcription factor SP1, which recognizes the regulatory elements F, H, and I. Similar analysis showed that the regulatory element G represents a specialized HRE that is recognized by the orphan receptors ARP-1 and EAR-3 but not by HNF-4.41 A single activity designated C-IIIJ1 binds to the regulatory element J. This or a similar activity also binds as a minor component to the regulatory elements F and I where SP1 is the predominant binding activity. Finally, a minor activity designated C-IIII5 binds to the regulatory element I. The factors that bind to the apoC-III enhancer are shown in Fig 1C.Contribution of Distal ApoC-III Regulatory Elements and Proximal ApoA-I Promoter Elements to the Strength of the ApoA-I Promoter in HepG2 CellsThe contribution of apoC-III regulatory elements to the strength of the proximal apoA-I promoter in HepG2 cells was evaluated by transient transfection experiments with promoter constructs containing 5′ deletions.44 This analysis showed that deletion of the 5′ apoC-III promoter region extending to nucleotide −890 increased by 30% the activity of the apoA-I promoter/apoC-III enhancer cluster. The promoter/enhancer activity was nearly abolished by deletion of the regulatory elements J, I, and H (Fig 8A, left column). The contribution of the distal apoC-III regulatory elements to the enhancer activity was also evaluated by point mutations that abolish the binding of specific factors to their cognate sites (Table 2). This analysis showed that the promoter/enhancer activity was reduced to 40% to 45% of its value by mutations in elements H and G and to 55% to 70% of its value by mutations in elements I, J, or F. As shown in Fig 1C, element G binds activities related to orphan receptors ARP-1 and EAR-3, and element F binds SP1 as a major and C-IIIJ1 as a minor activity. The findings indicate that all the factors that bind to the upstream apoC-III promoter region are required to enable it to activate optimally the closely linked apoA-I promoter. Similar mutagenesis analysis showed that the ability