Title: Expression and Regulation of Multiple Murine ATP-binding Cassette Transporter G1 mRNAs/Isoforms That Stimulate Cellular Cholesterol Efflux to High Density Lipoprotein
Abstract: The murine Abcg1 gene is reported to consist of 15 exons that encode a single mRNA (herein referred to as Abcg1-a) and protein. We now demonstrate that (i) the murine gene contains two additional coding exons downstream of exon 1, (ii) transcription involves the use of multiple promoters, and (iii) the RNA undergoes alternative splicing reactions. As a result, three mRNAs are expressed that encode three putative protein isoforms that differ at their amino terminus. ABCG1 transcripts are induced in vivo in multiple tissues in response to the liver X receptor ligand T0901317. Identification and characterization of four liver X receptor response elements in the intron downstream of exon 2 provides a mechanism by which this induction occurs. Importantly, cholesterol efflux to high density lipoprotein was stimulated following transfection of Hek293 cells with plasmids encoding individual ABCG1 isoforms. In situ hybridization studies in embryonic day 11.5–15.5 mouse embryos revealed strong expression of ABCG1 transcripts in the olfactory epithelium, hind brain, eye, and dorsal root ganglia. The relatively high levels of expression in neuronal tissues and the eye suggest that ABCG1-dependent cholesterol efflux may be critical for normal neuronal function in addition to its role in macrophages. The murine Abcg1 gene is reported to consist of 15 exons that encode a single mRNA (herein referred to as Abcg1-a) and protein. We now demonstrate that (i) the murine gene contains two additional coding exons downstream of exon 1, (ii) transcription involves the use of multiple promoters, and (iii) the RNA undergoes alternative splicing reactions. As a result, three mRNAs are expressed that encode three putative protein isoforms that differ at their amino terminus. ABCG1 transcripts are induced in vivo in multiple tissues in response to the liver X receptor ligand T0901317. Identification and characterization of four liver X receptor response elements in the intron downstream of exon 2 provides a mechanism by which this induction occurs. Importantly, cholesterol efflux to high density lipoprotein was stimulated following transfection of Hek293 cells with plasmids encoding individual ABCG1 isoforms. In situ hybridization studies in embryonic day 11.5–15.5 mouse embryos revealed strong expression of ABCG1 transcripts in the olfactory epithelium, hind brain, eye, and dorsal root ganglia. The relatively high levels of expression in neuronal tissues and the eye suggest that ABCG1-dependent cholesterol efflux may be critical for normal neuronal function in addition to its role in macrophages. There are more than 373 members in the ATP-binding cassette (ABC) 1The abbreviations used are: ABC, ATP-binding cassette; LXR, liver X receptor; LXRE, LXR response element; RXR, retinoid X receptor; HDL, high density lipoprotein; UTR, untranslated region; EMSA, electrophoretic mobility shift assay; TK, thymidine kinase; RACE, rapid amplification of cDNA ends. superfamily of transmembrane transporters that are expressed in species as diverse as plants, yeast, Drosophila, and mammals (1Sanchez-Fernandez R. Davies T.G. Coleman J.O. Rea P.A. J. Biol. Chem. 2001; 276: 30231-30244Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar). To date, 52 members of this superfamily have been identified in mammals (2Dean M. Hamon Y. Chimini G. J. Lipid Res. 2001; 42: 1007-1017Abstract Full Text Full Text PDF PubMed Google Scholar, 3Dean M. Rzhetsky A. Allikmets R. Genome Res. 2001; 11: 1156-1166Crossref PubMed Scopus (1499) Google Scholar). ABC transporters have been subdivided into full and half transporters; full transporters contain two ATP-binding domains and two multitransmembrane domains, each typically containing six transmembrane α-helices. In contrast, half transporters contain one ATP-binding cassette and one transmembrane domain and must dimerize to form a functional pump (4Ambudkar S.V. Gottesman M.M. Abelson J.N. Simon M.I. ABC Transporters: Biochemical, Cellular, and Molecular Aspects. 292. Academic Press, San Diego, CA1998: 101-115Google Scholar, 5Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3375) Google Scholar, 6Luciani M.F. Chimini G. EMBO J. 1996; 15: 226-235Crossref PubMed Scopus (250) Google Scholar). Binding and hydrolysis of ATP by these membrane-bound pumps provides the energy to transport small hydrophobic substrates from the cytoplasm either to the outside of the cell or into intracellular compartments such as the endoplasmic reticulum, peroxisomes, or mitochondria (5Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3375) Google Scholar, 7Childs S. Ling V. Important Adv. Oncol. 1994; : 21-36PubMed Google Scholar). The importance of these proteins is illustrated by the fact that numerous human diseases are caused by mutations in specific members of the ABC transporter superfamily (2Dean M. Hamon Y. Chimini G. J. Lipid Res. 2001; 42: 1007-1017Abstract Full Text Full Text PDF PubMed Google Scholar, 3Dean M. Rzhetsky A. Allikmets R. Genome Res. 2001; 11: 1156-1166Crossref PubMed Scopus (1499) Google Scholar, 8Klein I. Sarkadi B. Varadi A. Biochim. Biophys. Acta. 1999; 1461: 237-262Crossref PubMed Scopus (511) Google Scholar, 9Allikmets R. Gerrard B. Hutchinson A. Dean M. Hum. Mol. Genet. 1996; 5: 1649-1655Crossref PubMed Scopus (283) Google Scholar). ABC transporters are further subdivided into seven groups, ABCA through ABCG. Members of the ABCG subtype (ABCG1, ABCG2, ABCG3, ABCG4, ABCG5, ABCG8, and Drosophila white, brown, and scarlet) are unique in that the ATP-binding cassette of these half transporters is at the amino terminus and the transmembrane domains are at the carboxyl terminus of the protein. In the original studies the cDNAs corresponding to ABCG1 were referred to as either ABC8 and/or white (10Savary S. Denizot F. Luciani M. Mattei M. Chimini G. Mamm. Genome. 1996; 7: 673-676Crossref PubMed Scopus (52) Google Scholar, 11Croop J.M. Tiller G.E. Fletcher J.A. Lux M.L. Raab E. Goldenson D. Son D. Arciniegas S. Wu R.L. Gene (Amst.). 1997; 185: 77-85Crossref PubMed Scopus (78) Google Scholar). Both the murine and human cDNAs encode proteins of ∼67 kDa that have 34% amino acid identity with the Drosophila white protein (10Savary S. Denizot F. Luciani M. Mattei M. Chimini G. Mamm. Genome. 1996; 7: 673-676Crossref PubMed Scopus (52) Google Scholar, 11Croop J.M. Tiller G.E. Fletcher J.A. Lux M.L. Raab E. Goldenson D. Son D. Arciniegas S. Wu R.L. Gene (Amst.). 1997; 185: 77-85Crossref PubMed Scopus (78) Google Scholar). Drosophila white dimerizes with either scarlet or brown to form heterodimers (white/scarlet or white/brown, respectively) that transport precursors of the ommochromes and pteridines into the pigment granule of the fly eye (12Mackenzie S.M. Howells A.J. Cox G.B. Ewart G.D. Genetica (Dordrecht). 2000; 108: 239-252Crossref PubMed Scopus (92) Google Scholar, 13Dreesen T.D. Johnson D.H. Henikoff S. Mol. Cell. Biol. 1988; 8: 5206-5215Crossref PubMed Scopus (174) Google Scholar, 14Tearle R.G. Belote J.M. McKeown M. Baker B.S. Howells A.J. Genetics. 1989; 122: 595-606Crossref PubMed Google Scholar, 15Pepling M. Mount S.M. Nucleic Acids Res. 1990; 18: 1633Crossref PubMed Scopus (44) Google Scholar). However, no mammalian homologues of Drosophila brown or scarlet that might function as the heterodimeric partner of ABCG1 have been identified. The mammalian ABCG4 protein has 94% amino acid identity with ABCG1, consistent with gene duplication (16Annilo T. Tammur J. Hutchinson A. Rzhetsky A. Dean M. Allikmets R. Cytogenet. Cell Genet. 2001; 94: 196-201Crossref PubMed Scopus (44) Google Scholar, 17Oldfield S. Lowry C. Ruddick J. Lightman S. Biochim. Biophys. Acta. 2002; 1591: 175-179Crossref PubMed Scopus (51) Google Scholar). However, ABCG4 expression is reported to be limited to the brain and eye (17Oldfield S. Lowry C. Ruddick J. Lightman S. Biochim. Biophys. Acta. 2002; 1591: 175-179Crossref PubMed Scopus (51) Google Scholar) and this would preclude the formation of obligate ABCG1/ABCG4 functional heterodimers in many tissues. In an attempt to determine the function for mammalian ABCG1, Klucken et al. (18Klucken J. Buchler C. Orso E. Kaminski W.E. Porsch-Ozcurumez M. Liebisch G. Kapinsky M. Diederich W. Drobnik W. Dean M. Allikmets R. Schmitz G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 817-822Crossref PubMed Scopus (474) Google Scholar) treated human macrophages with antisense oligonucleotides to ABCG1. This treatment resulted in decreased expression of a protein of 110 kDa and decreased efflux of cholesterol and phospholipids to HDL3 (18Klucken J. Buchler C. Orso E. Kaminski W.E. Porsch-Ozcurumez M. Liebisch G. Kapinsky M. Diederich W. Drobnik W. Dean M. Allikmets R. Schmitz G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 817-822Crossref PubMed Scopus (474) Google Scholar). Surprisingly, the antisense oligonucleotides also decreased apoE secretion from macrophages (19Schmitz G. Langmann T. Curr. Opin. Lipidol. 2001; 12: 129-140Crossref PubMed Scopus (184) Google Scholar). The mammalian ABCG1 cDNA was originally identified from studies using degenerate PCR and RNA from either a murine macrophage cell line (10Savary S. Denizot F. Luciani M. Mattei M. Chimini G. Mamm. Genome. 1996; 7: 673-676Crossref PubMed Scopus (52) Google Scholar), a murine pre-B cell library (11Croop J.M. Tiller G.E. Fletcher J.A. Lux M.L. Raab E. Goldenson D. Son D. Arciniegas S. Wu R.L. Gene (Amst.). 1997; 185: 77-85Crossref PubMed Scopus (78) Google Scholar), or a human Jurkat T-cell line (11Croop J.M. Tiller G.E. Fletcher J.A. Lux M.L. Raab E. Goldenson D. Son D. Arciniegas S. Wu R.L. Gene (Amst.). 1997; 185: 77-85Crossref PubMed Scopus (78) Google Scholar) or after using exon-trapping and a human chromosome 21 cDNA library (20Chen H. Rossier C. Lalioti M.D. Lynn A. Chakravarti A. Perrin G. Antonarakis S.E. Am. J. Hum. Genet. 1996; 59: 66-75PubMed Google Scholar). These murine and human cDNAs had 97 and 87% identity at the amino acid and nucleic acid levels, respectively. In general, high expression of ABCG1 mRNA was noted in spleen, lung, thymus, and brain, whereas expression in kidney, liver, and heart was low or undetectable (10Savary S. Denizot F. Luciani M. Mattei M. Chimini G. Mamm. Genome. 1996; 7: 673-676Crossref PubMed Scopus (52) Google Scholar, 11Croop J.M. Tiller G.E. Fletcher J.A. Lux M.L. Raab E. Goldenson D. Son D. Arciniegas S. Wu R.L. Gene (Amst.). 1997; 185: 77-85Crossref PubMed Scopus (78) Google Scholar). Subsequent studies demonstrated that murine ABCG1 mRNA levels were highly induced when macrophages were either incubated with LXR agonists or were converted to cholesterol ester-loaded foam cells (18Klucken J. Buchler C. Orso E. Kaminski W.E. Porsch-Ozcurumez M. Liebisch G. Kapinsky M. Diederich W. Drobnik W. Dean M. Allikmets R. Schmitz G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 817-822Crossref PubMed Scopus (474) Google Scholar, 21Venkateswaran A. Repa J.J. Lobaccaro J.M. Bronson A. Mangelsdorf D.J. Edwards P.A. J. Biol. Chem. 2000; 275: 14700-14707Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, 22Tangirala R.K. Bischoff E.D. Joseph S.B. Wagner B.L. Walczak R. Laffitte B.A. Daige C.L. Thomas D. Heyman R.A. Mangelsdorf D.J. Wang X. Lusis A.J. Tontonoz P. Schulman I.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11896-11901Crossref PubMed Scopus (371) Google Scholar). Recently, we and others identified multiple human ABCG1 transcripts that are produced as a result of transcriptional initiation at different exons and alternative mRNA splicing (23Langmann T. Porsch-Ozcurumez M. Unkelbach U. Klucken J. Schmitz G. Biochim. Biophys. Acta. 2000; 1494: 175-180Crossref PubMed Scopus (43) Google Scholar, 24Lorkowski S. Rust S. Engel T. Jung E. Tegelkamp K. Galinski E.A. Assmann G. Cullen P. Biochem. Biophys. Res. Commun. 2001; 280: 121-131Crossref PubMed Scopus (63) Google Scholar, 25Kennedy M.A. Venkateswaran A. Tarr P.T. Xenarios I. Kudoh J. Shimizu N. Edwards P.A. J. Biol. Chem. 2001; 276: 39438-39447Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). Each of the resulting mRNAs has a unique 5′ sequence upstream of exons 11–23 (20Chen H. Rossier C. Lalioti M.D. Lynn A. Chakravarti A. Perrin G. Antonarakis S.E. Am. J. Hum. Genet. 1996; 59: 66-75PubMed Google Scholar, 25Kennedy M.A. Venkateswaran A. Tarr P.T. Xenarios I. Kudoh J. Shimizu N. Edwards P.A. J. Biol. Chem. 2001; 276: 39438-39447Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 26Langmann T. Klucken J. Reil M. Liebisch G. Luciani M.F. Chimini G. Kaminski W.E. Schmitz G. Biochem. Biophys. Res. Commun. 1999; 257: 29-33Crossref PubMed Scopus (429) Google Scholar). Each unique upstream sequence contains an in-frame ATG, resulting in the synthesis of multiple putative protein isoforms that contain variable amino termini of 56–203 amino acids and a conserved 583 amino acids at their carboxyl termini (24Lorkowski S. Rust S. Engel T. Jung E. Tegelkamp K. Galinski E.A. Assmann G. Cullen P. Biochem. Biophys. Res. Commun. 2001; 280: 121-131Crossref PubMed Scopus (63) Google Scholar, 25Kennedy M.A. Venkateswaran A. Tarr P.T. Xenarios I. Kudoh J. Shimizu N. Edwards P.A. J. Biol. Chem. 2001; 276: 39438-39447Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). 2M. A. Kennedy and P. A. Edwards, unpublished data. It is not known whether such variations at the amino terminus affect protein localization, dimerization, or function. In contrast to the multiple human isoforms, only a single murine ABCG1 cDNA has been identified to date. The LXR response element (LXRE) (27Willy P.J. Umesono K. Ong E.S. Evans R.M. Heyman R.A. Mangelsdorf D.J. Genes Dev. 1995; 9: 1033-1045Crossref PubMed Scopus (920) Google Scholar) that is presumed to be necessary for the activation of the murine gene by LXR/RXR has yet to be identified. In the current study we report on the identification of new exons and novel mRNAs that encode three putative ABCG1 protein isoforms. In addition, we identify multiple LXREs within an intron of the Abcg1 gene. Promoter-reporter studies suggest that these LXREs are necessary and sufficient to activate the gene in numerous tissues in response to LXR ligands. In situ hybridization studies show that ABCG1 is expressed at relatively high levels in the olfactory bulb epithelium, hind brain, hippocampus, choroid plexus, eye, and dorsal root ganglia of developing embryos and/or adults. Finally, we show that each ABCG1 isoform stimulates the efflux of cholesterol to HDL. These data suggest that ABCG1 may play a key role in regulating lipid efflux from neurons and epithelial cells in addition to macrophages. Reagents—Mammalian expression vectors for LXRα and RXRα (pCMX-LXRα, pCMX-RXRα) were gifts from Dr. Ron Evans (Salk Institute, La Jolla, CA). RXRα polyclonal antibody (D20, sc-553X) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Synthetic LXR ligand T0901317 (27Willy P.J. Umesono K. Ong E.S. Evans R.M. Heyman R.A. Mangelsdorf D.J. Genes Dev. 1995; 9: 1033-1045Crossref PubMed Scopus (920) Google Scholar) was purchased from Cayman Chemical (Ann Arbor, MI), and synthetic RXR ligand LG100153 (28Boehm M.F. Zhang L. Badea B.A. White S.K. Mais D.E. Berger E. Suto C.M. Goldman M.E. Heyman R.A. J. Med. Chem. 1994; 37: 2930-2941Crossref PubMed Scopus (384) Google Scholar) was a gift from Dr. Richard Heyman (Ligand Pharmaceuticals, La Jolla, CA). Human HDL3 was obtained from Intracell. Total HDL (density 1.063–1.21 g/ml) was obtained by ultracentrifugation of human plasma. Animals—C57BL/6 mice were purchased from Jackson Laboratories (Bar Harbor, ME) and maintained on standard rodent chow or chow supplemented with 0.05% T0901317. This corresponds to ∼50 mg/kilogram body weight/day. Oligonucleotides—Single-stranded oligonucleotides (MWG, Highpoint, NC) were annealed with their complimentary strands to generate double-stranded DNAs that were then used in electrophoretic mobility shift assays (EMSAs) or to produce promoter-reporter genes. Sequences of the oligonucleotides and primers used for PCR are given in Table I. The nucleotide sequences of all reporter genes was verified.Table IPrimers used in this studyPrimerSequenceLXRE-15′-AGGCTTGCCCAGAGCTAACTCTCCAG-3′EMSALXRE-25′-GAGAATGGACTGTCCTAACCACTCTG-3′EMSALXRE-35′-CTGTTTGCCCTGTAGTAACCTCTGTA-3EMSALXRE-45′-CTTCTGGGTTATAAAAGGCCATTGAA-3′EMSALXRE-55′-GGGCTTGCCCTAGTCTGAACTCCCAC-3′EMSALXRE-65′-CGCCGGGGTTACTATCGGTCAATGCT-3′EMSALXRE-75′-GAGAGAGGTTAATGTAGGTCATCTTG-3′EMSAmutLXRE-65′-CGCCGGGAATACTATCGAACAATGCT-3′EMSA2xLXRE-35′-GATCTGGCTGTTTGCCCTGTAGTAACCTCTGTAGGTTGGCTGTTTGCCCTGTAGTAACCTCTGTAGGT-3′Reporter2xLXRE-65′-GATCCCGCGCCGGGGTTACTATCGGTCAATGCTCGCCCGCGCCGGGGTTACTATCGGTCAATGCTCGC-3′ReporterGSP15′-ATGCCAGTCTCCCTGTATCCTG-3′5′-RACEGSP25′-CCAGCTCTCCACTGTTGAATTTC-3′5′-RACEExon 15′-ATGGCCGCTTTCTCGGTC-3′ (forward primer)Real time PCR (Exon 1-2)Exon 25′-TCCGTCATTGCGGCAGA-3′ (reverse primer)Probe 1-25′-CCGCCATGAATGCCAGCAGCTA-3′ (probe)Exon 1A5′-CGGCCACCTTGCTGCTT-3′ (forward primer)Real time PCR (Exon 1A-1B)Exon 1B5′-GGCTTGGTGTCAACCCTAACA-3′ (reverse primer)Probe A-B5′-CACGGTTCCCAGAACTTCCAGTCCG-3′ (probe)3′-UTR5′-CGCCTCCAAGCCAGCA-3′ (forward primer)Real time PCR (3′-UTR)3′-UTR5′-ACCTCCCAGCAGTGCCC-3′ (reverse primer)Probe 3′-UTR5′-CGAGGCAAAGCAGACATTGTGACCA-3′ (probe) Open table in a new tab Cell Culture, Construction of Luciferase Reporter Genes, and Transient Transfections—HepG2 cells were maintained as described (29Laffitte B.A. Kast H.R. Nguyen C.M. Zavacki A.M. Moore D.D. Edwards P.A. J. Biol. Chem. 2000; 275: 10638-10647Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Thymidine kinase (TK)-Luc reporter constructs under the control of two copies of either LXRE-3 or LXRE-6 were generated by annealing double-stranded oligonucleotides and then ligating the DNA into BamH1-digested pTK-Luc (30Selden R.F. Howie K.B. Rowe M.E. Goodman H.M. Moore D.D. Mol. Cell. Biol. 1986; 6: 3173-3179Crossref PubMed Scopus (471) Google Scholar). Transient transfection of HepG2 cells was performed in triplicate in 48-well plates as described (29Laffitte B.A. Kast H.R. Nguyen C.M. Zavacki A.M. Moore D.D. Edwards P.A. J. Biol. Chem. 2000; 275: 10638-10647Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 31Anisfeld A.M. Kast-Woelbern H.R. Meyer M.E. Jones S.A. Zhang Y. Williams K.J. Willson T. Edwards P.A. J. Biol. Chem. 2003; 278: 20420-20428Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Briefly, reporter constructs (100 ng) and pCMV-β-galactosidase (50 ng) were transiently transfected with either pCMX-LXRα (50 ng), pCMX-RXRα (5 ng) expression vectors, and/or control plasmid (pTKCIII) (total of 205 ng/well). After transfection, cells were treated with 1 μm T0901317 and 100 nm LG100153 or Me2SO for 24 h prior to cell lysis and assay of luciferase activity (29Laffitte B.A. Kast H.R. Nguyen C.M. Zavacki A.M. Moore D.D. Edwards P.A. J. Biol. Chem. 2000; 275: 10638-10647Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Luciferase activities were normalized for small variations in transfection efficiencies following the determination of β-galactosidase activity (29Laffitte B.A. Kast H.R. Nguyen C.M. Zavacki A.M. Moore D.D. Edwards P.A. J. Biol. Chem. 2000; 275: 10638-10647Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). Hek293 cells were grown in Dulbecco's modified Eagle's medium + 10% fetal bovine serum and plated in 24-well plates (0.3 × 106 cells/ml) and allowed to adhere overnight. We used LipofectAMINE 2000 (Invitrogen) to transiently transfect triplicate wells of Hek293 cells with the empty pCMX vector (1 μg/well) or the pCMX vector containing a cDNA encoding a specific ABCG1 isoform. Analysis of cells transfected with plasmids encoding green fluorescent protein and pCMX indicated that the transfection efficiency was >70%. Cholesterol Efflux—Following transfection of Hek293 cells with the indicated plasmids, the cells were washed twice with phosphate-buffered saline and incubated for 24 h in media A (Dulbecco's modified Eagle's medium + 0.2% bovine serum albumin) supplemented with an acyl-CoA:cholesterol O-acyltransferase inhibitor (58-035, 2 μg/ml) (32Kritharides L. Jessup W. Mander E.L. Dean R.T. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 276-289Crossref PubMed Scopus (89) Google Scholar), [3H]cholesterol (1μCi/ml), and where indicated, T0901317 (1 μm) and LG100153 (100 nm). The cells were washed twice with phosphate-buffered saline and incubated for an additional 4 h in media A and where indicated, T0901317 and LG100153. After this equilibration period, the cells were washed twice more with phosphate-buffered saline and then incubated for an additional 4 h in media A or in media A supplemented with either HDL3 (50 μg/ml) or total HDL (50 μg/ml). Media were removed and centrifuged at 14,000 × g for 2 min, and the radioactivity in the supernatant determined as described (33Venkateswaran A. Laffitte B.A. Joseph S.B. Mak P.A. Wilpitz D.C. Edwards P.A. Tontonoz P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12097-12102Crossref PubMed Scopus (844) Google Scholar). Isopropanol was added to each well to solubilize cell-associated radioactivity. After 24 h, aliquots were removed and the cell-associated radioactivity determined (33Venkateswaran A. Laffitte B.A. Joseph S.B. Mak P.A. Wilpitz D.C. Edwards P.A. Tontonoz P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12097-12102Crossref PubMed Scopus (844) Google Scholar). Cholesterol efflux was determined by dividing the radioactive content of the media by the sum of the radioactivity in the cells and media. Efflux was linear for >8 h. 5′-RACE—5′-RACE was performed using cDNA from Sure-RACE™ Multi-Tissue RACE panels (OriGene Technologies, Rockville, MD) according to the manufacturer's protocol. An adaptor containing sequences complimentary to two primers, ADP1 and ADP2, was attached to the 5′ end of the cDNAs. First round PCR was performed with a gene specific primer (GSP1) corresponding to exon 4 of the mouse gene and ADP1 under following conditions: 94 °C for 3 min, 10 cycles of 94 °C for 30 s, 68 °C for 30 s, 72 °C for 2 min, and 15 cycles of 94 °C for 30 s, 62 °C for 30 s, 72 °C for 2 min, then extension at 72 °C for 6 min. The first round PCR products were diluted 10-fold, and an aliquot was used as template for second round PCR with nested primers GSP2 and ADP2 under the following conditions: 94 °C for 3 min, 5 cycles of 94 °C for 30 s, 65 °C for 30 s, 72 °C for 2 min, 5 cycles of 94 °C for 30 s, 62 °C for 30 s, 72 °C for 2 min, 25 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 2 min, extension at 72 °C for 6 min. The PCR products were separated on agarose gels and recovered fragments were cloned into pCR2.1-TOPO vector (Invitrogen) for sequencing. Isolation of Murine Peritoneal Macrophages—Murine peritoneal macrophages were isolated as described (34Mak P.A. Laffitte B.A. Desrumaux C. Joseph S.B. Curtiss L.K. Mangelsdorf D.J. Tontonoz P. Edwards P.A. J. Biol. Chem. 2002; 277: 31900-31908Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Briefly, 1 ml of 4% thioglycolate was injected intraperitoneally into 12- to 16-week-old male C57BL/6 mice. Four days after the injection, macrophages were isolated and plated on 100-mm dishes at 1.2 million cells/ml. Isolated macrophages were allowed to adhere for 12 h in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The medium was then replaced with Dulbecco's modified Eagle's medium supplemented with 10% lipoprotein-deficient serum and 100 μm mevalonic acid. Cells were treated with either 1 μm T0901317 and 100 nm LG100153 or Me2SO for 24 h followed by RNA isolation. RNA Isolation and Real Time Quantitative PCR—Total RNA was isolated using TRIzol Reagent (Invitrogen). Real time PCR was performed as described (35Zhang Y. Kast-Woelbern H.R. Edwards P.A. J. Biol. Chem. 2003; 278: 104-110Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). The nucleotide sequences of the primers and probes are given in Table I. Probes were modified at the 5′ end with 6-carboxyfluorescein and at the 3′ end with 6-carboxytetramethylrhodamine (Integrated DNA Technology, Coralville, IA). EMSAs—EMSAs were performed as described (29Laffitte B.A. Kast H.R. Nguyen C.M. Zavacki A.M. Moore D.D. Edwards P.A. J. Biol. Chem. 2000; 275: 10638-10647Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). LXRα and RXRα were synthesized in vitro using the TNT T7-coupled reticulocyte system (Promega, Madison, WI) (36Kast H.R. Goodwin B. Tarr P.T. Jones S.A. Anisfeld A.M. Stoltz C.M. Tontonoz P. Kliewer S. Willson T.M. Edwards P.A. J. Biol. Chem. 2002; 277: 2908-2915Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar). In Situ Hybridization of Embryos—In situ hybridization was performed as described (37Hogan B.L.M. Beddington R. Costantini F. Lacy E. Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Plainview, NY1994Google Scholar). Briefly, a 905-bp fragment, corresponding to 725 bp of the 3′-UTR and 180 bp of the open reading frame of the murine ABCG1 cDNA, was subcloned into pCI-Neo (Promega) to generate pCI-Neo-mABCG1. The linearized plasmid was transcribed with either T7 or T3 RNA polymerase using the (digoxigenin)-UTP labeling kit (Roche Applied Science), to generate sense or antisense digoxigenin-labeled cRNA probes. For whole mount in situ hybridization, embryos were dissected at embryonic day 11.5 and fixed overnight in 4% paraformaldehyde in phosphate-buffered saline at 4 °C. For in situ hybridization of embryo sections, embryos were dissected at embryonic day 12.5 or embryonic day 15.5, embedded in optimal cutting temperature (OCT) compound (10.24% polyvinyl alcohol, 4.26% w/w polyethylene glycol) (VWR, West Chester, PA), and stored at –20 °C. Twenty-μm cryosections (or the whole mount embryos) were hybridized with the indicated probe for 18 h at 68 °C prior to addition of alkaline phosphatase-conjugated anti-digoxigenin. The sections then incubated in BM-Purple (Roche Applied Science) for 48 h to allow for color development. Identification of ABCG1 Transcripts That Encode Novel Isoforms—A single murine ABCG1 cDNA was originally reported by Savary et al. and Croop et al. (10Savary S. Denizot F. Luciani M. Mattei M. Chimini G. Mamm. Genome. 1996; 7: 673-676Crossref PubMed Scopus (52) Google Scholar, 11Croop J.M. Tiller G.E. Fletcher J.A. Lux M.L. Raab E. Goldenson D. Son D. Arciniegas S. Wu R.L. Gene (Amst.). 1997; 185: 77-85Crossref PubMed Scopus (78) Google Scholar). For comparative purposes, we refer to this transcript, corresponding to exons 1 and 2–15, as ABCG1-a (Fig. 1). To determine whether additional murine ABCG1 transcripts are produced, we used 5′-RACE to analyze cDNAs derived from multiple tissues. The approach, described in detail under "Materials and Methods," utilized cDNAs that contained a unique adapter at the 5′ end. These cDNAs were used as templates for PCR reactions, utilizing primers corresponding to the 5′ linker and to exon 4. Nested PCR reactions were subsequently performed utilizing internal primers corresponding to the linker and exon 4, and the resulting DNA products were cloned and sequenced. Many clones contained sequences with 100% identity to exons 1, 2, 3, and 4 and thus presumably corresponded to transcript ABCG1-a (data not shown). However, one cDNA, derived from the spleen library, contained a novel 220-bp sequence upstream of exon 2. Comparison of the novel sequence with genomic DNA databases indicated that all 220 bps were present in two new exons (exons 1A and 1B in Fig. 1A) that were localized 3′ of exon 1. The novel transcript, containing sequences corresponding to exons 1A + 1B + 2–15, is referred to as ABCG1-b (Fig. 1B). Subsequently, we utilized spleen RNA as a template to generate full-length cDNA using primers to exon 15 and either exon 1 or exon 1A. As expected, this approach resulted in the generation of cDNAs corresponding to ABCG1-a and ABCG1-b (Fig. 1B). Surprisingly, a third cDNA was isolated that corresponded to a transcript containing exon 1A fused to exons 2–15 (ABCG1-c in Fig. 1B). The 5′ sequences of these three transcripts are shown in Fig. 1C. Previous studies (10Savary S. Denizot F. Luciani M. Mattei M. Chimini G. Mamm. Genome. 1996; 7: 673-676Crossref PubMed Scopus (52) Google Scholar, 11Croop J.M. Tiller G.E. Fletcher J.A. Lux M.L. Raab E. Goldenson D. Son D. Arciniegas S. Wu R.L. Gene (Amst.). 1997; 185: 77-85Crossref PubMed Scopus (78) Google Scholar) identified an in-frame methionine in exon 1 of the ABCG1-a transcript (Fig. 1, B and C). Translation initiated at this methionine would produce a protein containing 666 amino acids (Fig. 1B). In contrast, ABCG1-b and ABCG1-c lack exon 1, but contain an in-frame methionine in exon 1A; translation initiated at this methionine would generate proteins containing either 714 or 682 amino acids, respectively (Fig. 1B). Thus, proteins derived from translation of the three mRNAs are predicted to contain an identical carboxyl terminus of 652 amino acids (encoded by exons 2–15) but to differ at their amino termini (Fig. 1, B and C). Comparison of Murine and Human ABCG1 Genomic Organization—We performed comparative studies of murine and human genomic DNA to identify conserved exonic sequences. These studies utilized VISTA and various exon predicting programs (including MZEF, GENIE, GRAIL 1, GRAIL2, GRAIL 1A, FGENE, and GENESCAN). The results indicate a very high degree of conservation between the nucleotide and amino acid sequences of exons 1 and 2–15 of the murine gene and exons 5, 7, and 11–23 of the human gene (Fig. 2). However, nucleotide sequences corresponding to the novel murine exons 1A and 1B w