The regulatory elements CIIC (−159/−116) and CIIB (−102/−81) of the apolipoprotein CII (apoCII) promoter have distinct specificities for orphan nuclear receptors (Vorgia, P., Zannis, V. I., and Kardassis, D. (1998) J. Biol. Chem.273, 4188–4199). In this communication we investigated the contribution of ligand-dependent and orphan nuclear receptors on the transcriptional regulation of the humanapoCIIgene. It was found that element CIIC in addition to ARP-1 and EAR-2 binds RXRα/T3Rβ heterodimers strongly, whereas element CIIB binds hepatic nuclear factor 4 (HNF-4) exclusively. Binding is abolished by mutations that alter the HRE binding motifs.Transient cotransfection experiments showed that in the presence of T3, RXRα/T3Rβ heterodimers transactivated the −205/+18 apoCII promoter 1.6- and 11-fold in HepG2 and COS-1 respectively. No transactivation was observed in the presence of 9-cis-retinoic acid. Transactivation requires the regulatory element CIIC, suggesting that this element contains a thyroid hormone response element. HNF-4 did not affect the apoCII promoter activity in HepG2 cells. However, mutations in the HNF-4 binding site on element CIIB and inhibition of HNF-4 synthesis in HepG2 cells by antisense HNF-4 constructs decreased the apoCII promoter activity to 25–40% of the control, indicating that HNF-4 is a positive regulator of the apoCII gene. ARP-1 repressed the −205/+18 but not the −104/+18 apoCII promoter activity in HepG2 cells, indicating that the repression depends on the regulatory element CIIC. In contrast, combination of ARP-1 and HNF-4 transactivated different apoCII promoter segments as well as a minimal adenovirus major late promoter driven by the regulatory element CIIB. Mutagenesis or deletion of elements CIIB or CIIC established that the observed transactivation requires DNA binding of one of the two factors and may result from HNF-4-ARP-1 interactions that elicit the transactivation functions of HNF-4.The combined data indicate that RXRα/T3Rβ in the presence of T3 and HNF-4 can upregulate the apoCII promoter activity by binding to the regulatory elements CIIC and CIIB, respectively. In addition, ARP-1 can either have inhibitory or stimulatory effects on the apoCII promoter activity via different mechanisms. The regulatory elements CIIC (−159/−116) and CIIB (−102/−81) of the apolipoprotein CII (apoCII) promoter have distinct specificities for orphan nuclear receptors (Vorgia, P., Zannis, V. I., and Kardassis, D. (1998) J. Biol. Chem.273, 4188–4199). In this communication we investigated the contribution of ligand-dependent and orphan nuclear receptors on the transcriptional regulation of the humanapoCIIgene. It was found that element CIIC in addition to ARP-1 and EAR-2 binds RXRα/T3Rβ heterodimers strongly, whereas element CIIB binds hepatic nuclear factor 4 (HNF-4) exclusively. Binding is abolished by mutations that alter the HRE binding motifs. Transient cotransfection experiments showed that in the presence of T3, RXRα/T3Rβ heterodimers transactivated the −205/+18 apoCII promoter 1.6- and 11-fold in HepG2 and COS-1 respectively. No transactivation was observed in the presence of 9-cis-retinoic acid. Transactivation requires the regulatory element CIIC, suggesting that this element contains a thyroid hormone response element. HNF-4 did not affect the apoCII promoter activity in HepG2 cells. However, mutations in the HNF-4 binding site on element CIIB and inhibition of HNF-4 synthesis in HepG2 cells by antisense HNF-4 constructs decreased the apoCII promoter activity to 25–40% of the control, indicating that HNF-4 is a positive regulator of the apoCII gene. ARP-1 repressed the −205/+18 but not the −104/+18 apoCII promoter activity in HepG2 cells, indicating that the repression depends on the regulatory element CIIC. In contrast, combination of ARP-1 and HNF-4 transactivated different apoCII promoter segments as well as a minimal adenovirus major late promoter driven by the regulatory element CIIB. Mutagenesis or deletion of elements CIIB or CIIC established that the observed transactivation requires DNA binding of one of the two factors and may result from HNF-4-ARP-1 interactions that elicit the transactivation functions of HNF-4. The combined data indicate that RXRα/T3Rβ in the presence of T3 and HNF-4 can upregulate the apoCII promoter activity by binding to the regulatory elements CIIC and CIIB, respectively. In addition, ARP-1 can either have inhibitory or stimulatory effects on the apoCII promoter activity via different mechanisms. Plasma apolipoprotein CII (apoCII) 1The abbreviations used are: apoCII, apolipoprotein CII; apoCI′, apoCI pseudogene; HRE, hormone response element; TRE, thyroid hormone response element; hHNF-4, human hepatic nuclear factor 4; hEAR-2, human v-erbA-related factor 2; hARP-1, human apoA-I regulatory protein 1; CAT, chloramphenicol acetyltransferase; AdML, adenovirus major late promoter; hRXRα, human retinoid X receptor α; hT3Rβ, human thyroid hormone receptor β; hRARα, human retinoic acid receptor α; hPPARα, human peroxisome proliferator-activated receptor α; T3, triiodothyronine; CREB, cAMP response element binding protein. 1The abbreviations used are: apoCII, apolipoprotein CII; apoCI′, apoCI pseudogene; HRE, hormone response element; TRE, thyroid hormone response element; hHNF-4, human hepatic nuclear factor 4; hEAR-2, human v-erbA-related factor 2; hARP-1, human apoA-I regulatory protein 1; CAT, chloramphenicol acetyltransferase; AdML, adenovirus major late promoter; hRXRα, human retinoid X receptor α; hT3Rβ, human thyroid hormone receptor β; hRARα, human retinoic acid receptor α; hPPARα, human peroxisome proliferator-activated receptor α; T3, triiodothyronine; CREB, cAMP response element binding protein. is a potent activator of the lipoprotein lipase, has known protein and gene sequence, and plays an important role in the catabolism of triglyceride-rich lipoproteins (1Jackson R.I. Baker H.N. Gilliam E.B. Gotto Jr., A.M. Proc. Natl. Acad. Sci U. S. A. 1977; 74: 1942-1945Crossref PubMed Scopus (120) Google Scholar, 2Miller A.L. Smith L.C. J. Biol. Chem. 1973; 248: 3359-3362Abstract Full Text PDF PubMed Google Scholar, 3Zannis V.I. Kardassis D. Zanni E.E. Harris H. Hirschorn K. Advances in Human Genetics. 21. Plenum Publishing Corp., New York1993: 145-319Google Scholar, 4Nilsson-Ehle P. Garfinkel A.S. Schotz M.C. Annu. Rev. Biochem. 1980; 49: 667-693Crossref PubMed Scopus (570) Google Scholar, 5Breckenridge W.C. Little J.A. Steiner G. Chow A. Poapst M. New Engl. J. Med. 1978; 298: 1265-1273Crossref PubMed Scopus (409) Google Scholar, 6Brunzell J.D. Scriver C. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill Inc., New York1989: 1165-1180Google Scholar, 7Lusis A.J. Heinzmann C. Sparkes R.S. Scott J. Knott T.J. Geller R. Sparkes M.C. Mohand T. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3929-3933Crossref PubMed Scopus (86) Google Scholar, 8Wei C.F. Tsao Y.K. Robberson D.L. Gotto Jr., A.M. Brown K. Chan L. J. Biol. Chem. 1985; 260: 15211-15221Abstract Full Text PDF PubMed Google Scholar).We have shown recently that the 0.55-kilobase intergenic region between the apoCII and apoCIV genes is a strong cell type-specific promoter, and its activity is enhanced by hepatic control region 1 (9Allan C.M. Walker D. Segrest J.P. Taylor J.M. Genomics. 1995; 28: 291-300Crossref PubMed Scopus (69) Google Scholar, 10Vorgia P. Zannis V.I. Kardassis D. J. Biol. Chem. 1998; 273: 4188-4199Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The apoCII promoter contains five footprints defined by hepatic nuclear extracts and designated CII-A (−74/−44), CII-B (−102/−81), CII-C (−159/−116), CII-D (−288/−265), and CII-E (−497/−462). An important role in apoCII gene regulation and transcriptional enhancement is mediated by two hormone response elements, which map within the footprinted regions CIIB (−102/−81) and CIIC (−159/−116) and have different specificities for orphan nuclear receptors. CIIC is recognized by ARP-1, EAR-2, but not HNF-4, whereas CIIB is recognized exclusively by HNF-4 (10Vorgia P. Zannis V.I. Kardassis D. J. Biol. Chem. 1998; 273: 4188-4199Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar).Orphan nuclear receptors as well as receptors for retinoids and thyroids are members of a nuclear receptor superfamily that controls diverse biological functions including growth, development, and homeostasis (11Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6292) Google Scholar, 12Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6027) Google Scholar, 13Kastner P. Mark M. Chambon P. Cell. 1995; 83: 859-869Abstract Full Text PDF PubMed Scopus (933) Google Scholar, 14Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2818) Google Scholar, 15LeBlanc B.P Stunnenberg H.G. Genes Dev. 1995; 9: 1811-1816Crossref PubMed Scopus (132) Google Scholar). They recognize specific hexameric AG(G/T)TCA motifs with variations in sequence, spacing, and orientation, designated hormone response elements (HREs) (16Umesono K. Murakami K.K. Thompson C.C. Evans R.M. Cell. 1991; 65: 1255-1266Abstract Full Text PDF PubMed Scopus (1488) Google Scholar, 17Giguere V. Endocr. Rev. 1994; 15: 61-79Crossref PubMed Google Scholar, 18Glass C.K. Endocr. Rev. 1994; 15: 391-407PubMed Google Scholar, 19Mangelsdorf D.J. Umesono K. Evans R.M. Sporn M.B. Roberts A.B. Goodman The Retinoids: Biology, Chemistry and Medicine. 2nd Ed. Raven Press, Ltd., New York1994: 319-349Google Scholar, 20Glass C.K. Holloway J.M. Biochem. Biophys. Acta. 1990; 1032: 157-176PubMed Google Scholar). In the current study, we demonstrate that in the presence of T3, RXRα/T3Rβ heterodimers bind to a thyroid hormone response element (TRE) present in element CIIC and transactivate the human apoCII promoter. Binding of ARP-1 to the same site repressed the promoter activity. Furthermore, antisense methodologies and promoter mutagenesis established that HNF-4 is a positive activator required for optimal activity of the apoCII promoter in HepG2 cells. Finally, combination of ARP-1 and HNF-4 superactivate the apoCII promoter via novel mechanisms that may require interaction of the two factors on the apoCII promoter. Plasma apolipoprotein CII (apoCII) 1The abbreviations used are: apoCII, apolipoprotein CII; apoCI′, apoCI pseudogene; HRE, hormone response element; TRE, thyroid hormone response element; hHNF-4, human hepatic nuclear factor 4; hEAR-2, human v-erbA-related factor 2; hARP-1, human apoA-I regulatory protein 1; CAT, chloramphenicol acetyltransferase; AdML, adenovirus major late promoter; hRXRα, human retinoid X receptor α; hT3Rβ, human thyroid hormone receptor β; hRARα, human retinoic acid receptor α; hPPARα, human peroxisome proliferator-activated receptor α; T3, triiodothyronine; CREB, cAMP response element binding protein. 1The abbreviations used are: apoCII, apolipoprotein CII; apoCI′, apoCI pseudogene; HRE, hormone response element; TRE, thyroid hormone response element; hHNF-4, human hepatic nuclear factor 4; hEAR-2, human v-erbA-related factor 2; hARP-1, human apoA-I regulatory protein 1; CAT, chloramphenicol acetyltransferase; AdML, adenovirus major late promoter; hRXRα, human retinoid X receptor α; hT3Rβ, human thyroid hormone receptor β; hRARα, human retinoic acid receptor α; hPPARα, human peroxisome proliferator-activated receptor α; T3, triiodothyronine; CREB, cAMP response element binding protein. is a potent activator of the lipoprotein lipase, has known protein and gene sequence, and plays an important role in the catabolism of triglyceride-rich lipoproteins (1Jackson R.I. Baker H.N. Gilliam E.B. Gotto Jr., A.M. Proc. Natl. Acad. Sci U. S. A. 1977; 74: 1942-1945Crossref PubMed Scopus (120) Google Scholar, 2Miller A.L. Smith L.C. J. Biol. Chem. 1973; 248: 3359-3362Abstract Full Text PDF PubMed Google Scholar, 3Zannis V.I. Kardassis D. Zanni E.E. Harris H. Hirschorn K. Advances in Human Genetics. 21. Plenum Publishing Corp., New York1993: 145-319Google Scholar, 4Nilsson-Ehle P. Garfinkel A.S. Schotz M.C. Annu. Rev. Biochem. 1980; 49: 667-693Crossref PubMed Scopus (570) Google Scholar, 5Breckenridge W.C. Little J.A. Steiner G. Chow A. Poapst M. New Engl. J. Med. 1978; 298: 1265-1273Crossref PubMed Scopus (409) Google Scholar, 6Brunzell J.D. Scriver C. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Disease. McGraw-Hill Inc., New York1989: 1165-1180Google Scholar, 7Lusis A.J. Heinzmann C. Sparkes R.S. Scott J. Knott T.J. Geller R. Sparkes M.C. Mohand T. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3929-3933Crossref PubMed Scopus (86) Google Scholar, 8Wei C.F. Tsao Y.K. Robberson D.L. Gotto Jr., A.M. Brown K. Chan L. J. Biol. Chem. 1985; 260: 15211-15221Abstract Full Text PDF PubMed Google Scholar). We have shown recently that the 0.55-kilobase intergenic region between the apoCII and apoCIV genes is a strong cell type-specific promoter, and its activity is enhanced by hepatic control region 1 (9Allan C.M. Walker D. Segrest J.P. Taylor J.M. Genomics. 1995; 28: 291-300Crossref PubMed Scopus (69) Google Scholar, 10Vorgia P. Zannis V.I. Kardassis D. J. Biol. Chem. 1998; 273: 4188-4199Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The apoCII promoter contains five footprints defined by hepatic nuclear extracts and designated CII-A (−74/−44), CII-B (−102/−81), CII-C (−159/−116), CII-D (−288/−265), and CII-E (−497/−462). An important role in apoCII gene regulation and transcriptional enhancement is mediated by two hormone response elements, which map within the footprinted regions CIIB (−102/−81) and CIIC (−159/−116) and have different specificities for orphan nuclear receptors. CIIC is recognized by ARP-1, EAR-2, but not HNF-4, whereas CIIB is recognized exclusively by HNF-4 (10Vorgia P. Zannis V.I. Kardassis D. J. Biol. Chem. 1998; 273: 4188-4199Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Orphan nuclear receptors as well as receptors for retinoids and thyroids are members of a nuclear receptor superfamily that controls diverse biological functions including growth, development, and homeostasis (11Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6292) Google Scholar, 12Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6027) Google Scholar, 13Kastner P. Mark M. Chambon P. Cell. 1995; 83: 859-869Abstract Full Text PDF PubMed Scopus (933) Google Scholar, 14Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2818) Google Scholar, 15LeBlanc B.P Stunnenberg H.G. Genes Dev. 1995; 9: 1811-1816Crossref PubMed Scopus (132) Google Scholar). They recognize specific hexameric AG(G/T)TCA motifs with variations in sequence, spacing, and orientation, designated hormone response elements (HREs) (16Umesono K. Murakami K.K. Thompson C.C. Evans R.M. Cell. 1991; 65: 1255-1266Abstract Full Text PDF PubMed Scopus (1488) Google Scholar, 17Giguere V. Endocr. Rev. 1994; 15: 61-79Crossref PubMed Google Scholar, 18Glass C.K. Endocr. Rev. 1994; 15: 391-407PubMed Google Scholar, 19Mangelsdorf D.J. Umesono K. Evans R.M. Sporn M.B. Roberts A.B. Goodman The Retinoids: Biology, Chemistry and Medicine. 2nd Ed. Raven Press, Ltd., New York1994: 319-349Google Scholar, 20Glass C.K. Holloway J.M. Biochem. Biophys. Acta. 1990; 1032: 157-176PubMed Google Scholar). In the current study, we demonstrate that in the presence of T3, RXRα/T3Rβ heterodimers bind to a thyroid hormone response element (TRE) present in element CIIC and transactivate the human apoCII promoter. Binding of ARP-1 to the same site repressed the promoter activity. Furthermore, antisense methodologies and promoter mutagenesis established that HNF-4 is a positive activator required for optimal activity of the apoCII promoter in HepG2 cells. Finally, combination of ARP-1 and HNF-4 superactivate the apoCII promoter via novel mechanisms that may require interaction of the two factors on the apoCII promoter. We thank Dr. Hinrich Gronemeyer for providing us with 9-cis-retinoic acid and Dr. Fulvio Mavilio for providing the phosphoglycerol kinase β-galactosidase plasmid. We also thank Dr. Helen Dell, Margarita Hadzopoulou-Cladaras, Horng-Yuan Kan, and Aris Moustakas for helpful comments and Anne Plunkett for typing the manuscript.
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