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Alternative Usages of Multiple Promoters of the Acetyl-CoA Carboxylase β Gene Are Related to Differential Transcriptional Regulation in Human and Rodent Tissues

2004; Elsevier BV; Volume: 280; Issue: 7 Linguagem: Inglês

10.1074/jbc.m409037200

ISSN

1083-351X

Autores

So-Young Oh, Min Young Lee, Jong Min Kim, Sarah Yoon, Soonah Shin, Young Nyun Park, Yong‐Ho Ahn, Kyung‐Sup Kim,

Tópico(s)

Peroxisome Proliferator-Activated Receptors

Resumo

Acetyl-CoA carboxylase β (ACCβ) is a critical enzyme in the regulation of fatty acid oxidation and is dominantly expressed in the skeletal muscle, heart, and liver. It has been established that two promoters, P-I and P-II, control the transcription of the ACCβ gene. However, the precise mechanism involved in controlling tissue-specific gene expression of ACCβ is largely unknown yet. In this study we revealed that promoter P-I, active in the skeletal muscle and heart but not in the liver, could be activated by myogenic regulatory factors and retinoid X receptors in a synergistic manner. Moreover, P-I was also activated markedly by the cardiac-specific transcription factors, Csx/Nkx2.5 and GATA4. These results suggest that the proper stimulation of P-I by these tissue-specific transcription factors is important for the expression of ACCβ according to the tissue types. In addition, CpG sites around human exon 1a transcribed by P-I are half-methylated in muscle but completely methylated in the liver, where P-I is absolutely inactive. In humans, the skeletal muscle uses P-II as well as P-I, whereas only P-I is active in rat skeletal muscle. The proximal myogenic regulatory factor-binding sites in human P-II, which are not conserved in rat P-II, might contribute to this difference in P-II usage between human and rat skeletal muscle. Hepatoma-derived cell lines primarily use another novel promoter located about 3 kilobases upstream of P-I, designated as P-O. This study is the first to explain the mechanisms underlying the differential regulation of ACCβ gene expression between tissues in living organisms. Acetyl-CoA carboxylase β (ACCβ) is a critical enzyme in the regulation of fatty acid oxidation and is dominantly expressed in the skeletal muscle, heart, and liver. It has been established that two promoters, P-I and P-II, control the transcription of the ACCβ gene. However, the precise mechanism involved in controlling tissue-specific gene expression of ACCβ is largely unknown yet. In this study we revealed that promoter P-I, active in the skeletal muscle and heart but not in the liver, could be activated by myogenic regulatory factors and retinoid X receptors in a synergistic manner. Moreover, P-I was also activated markedly by the cardiac-specific transcription factors, Csx/Nkx2.5 and GATA4. These results suggest that the proper stimulation of P-I by these tissue-specific transcription factors is important for the expression of ACCβ according to the tissue types. In addition, CpG sites around human exon 1a transcribed by P-I are half-methylated in muscle but completely methylated in the liver, where P-I is absolutely inactive. In humans, the skeletal muscle uses P-II as well as P-I, whereas only P-I is active in rat skeletal muscle. The proximal myogenic regulatory factor-binding sites in human P-II, which are not conserved in rat P-II, might contribute to this difference in P-II usage between human and rat skeletal muscle. Hepatoma-derived cell lines primarily use another novel promoter located about 3 kilobases upstream of P-I, designated as P-O. This study is the first to explain the mechanisms underlying the differential regulation of ACCβ gene expression between tissues in living organisms. In mammals, acetyl-CoA carboxylase (ACC) 1The abbreviations used are: ACC, acetyl-CoA carboxylase; MRF, myogenic regulatory factor; 5′-UTR, 5′-untranslated region; RXR, retinoid X receptors; RAR, retinoic acid receptors; PIPES, 1,4-piperazinediethanesulfonic acid. is a critical enzyme in fatty acid metabolism. ACC exists as two isoforms, α and β, that are encoded by the separate genes and show different tissue distribution (1Lopez-Casillas F. Bai D.H. Luo X.C. Kong I.S. Hermodson M.A. Kim K.H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5784-5788Crossref PubMed Scopus (138) Google Scholar, 2Abu-Elheiga L. Almarza-Ortega D.B. Baldini A. Wakil S.J. J. Biol. Chem. 1997; 272: 10669-10677Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 3Ha J. Lee J.K. Kim K.S. Witters L.A. Kim K.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11466-11470Crossref PubMed Scopus (132) Google Scholar, 4Widmer J. Fassihi K.S. Schlichter S.C. Wheeler K.S. Crute B.E. King N. Nutile-McMenemy N. Noll W.W. Daniel S. Ha J. Kim K.H. Witters L.A. Biochem. J. 1996; 316: 915-922Crossref PubMed Scopus (67) Google Scholar, 5Abu-Elheiga L. Jayakumar A. Baldini A. Chirala S.S. Wakil S.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4011-4015Crossref PubMed Scopus (157) Google Scholar). Because ACCβ is associated with the mitochondrial outer membranes, the changes in its activity affect the concentration of malonyl-CoA around the mitochondria (3Ha J. Lee J.K. Kim K.S. Witters L.A. Kim K.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11466-11470Crossref PubMed Scopus (132) Google Scholar, 6Abu-Elheiga L. Brinkley W.R. Zhong L. Chirala S.S. Woldegiorgis G. Wakil S.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1444-1449Crossref PubMed Scopus (327) Google Scholar). Malonyl-CoA is a negative modulator of carnitine palmitoyltransferase-I (CPT-I), which is the rate-limiting enzyme in the fatty acyl-CoA transport system for fatty acid β-oxidation. Therefore, ACCβ plays a critical role for regulating mitochondrial fatty acid oxidation. ACCβ is expressed abundantly in heart, skeletal muscle, and liver, all places in which fatty acid oxidation actively occurs (2Abu-Elheiga L. Almarza-Ortega D.B. Baldini A. Wakil S.J. J. Biol. Chem. 1997; 272: 10669-10677Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 7Kim K.H. Annu. Rev. Nutr. 1997; 17: 77-99Crossref PubMed Scopus (313) Google Scholar, 8Zhang S. Kim K.H. Biochem. Biophys. Res. Commun. 1996; 229: 701-705Crossref PubMed Scopus (15) Google Scholar). ACCβ transcripts contain two species of 5′-UTRs, which contain either the sequence of exon 1a or of exon 1b via the alternative usage of two promoters, i.e. P-I and P-II. Exon 1a and exon 1b are located ∼15 kilobases apart in human genome but are both connected to the common exon 2 in mRNA after splicing. However, the two transcripts encode for the same protein because they both use the same ATG start codon for translation, which resides in exon 2 (3Ha J. Lee J.K. Kim K.S. Witters L.A. Kim K.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11466-11470Crossref PubMed Scopus (132) Google Scholar, 9Lee J.J. Moon Y.A. Ha J.H. Yoon D.J. Ahn Y.H. Kim K.S. J. Biol. Chem. 2001; 276: 2576-2585Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). In skeletal and cardiac muscles, ACCβ activities are reported to be rapidly regulated via phosphorylation by AMP-activated protein kinase in response to exercise, resulting in increases in fatty acid β-oxidation (4Widmer J. Fassihi K.S. Schlichter S.C. Wheeler K.S. Crute B.E. King N. Nutile-McMenemy N. Noll W.W. Daniel S. Ha J. Kim K.H. Witters L.A. Biochem. J. 1996; 316: 915-922Crossref PubMed Scopus (67) Google Scholar, 10Rasmussen B.B. Winder W.W. J. Appl. Physiol. 1997; 83: 1104-1109Crossref PubMed Scopus (129) Google Scholar, 11Dean D. Daugaard J.R. Young M.E. Saha A. Vavvas D. Asp S. Kiens B. Kim K.H. Witters L. Richter E.A. Ruderman N. Diabetes. 2000; 49: 1295-1300Crossref PubMed Scopus (102) Google Scholar, 12Boone A.N. Rodrigues B. Brownsey R.W. Biochem. J. 1999; 341: 347-354Crossref PubMed Scopus (44) Google Scholar, 13Dyck J.R. Kudo N. Barr A.J. Davies S.P. Hardie D.G. Lopaschuk G.D. Eur. J. Biochem. 1999; 262: 184-190Crossref PubMed Scopus (137) Google Scholar, 14Tomas E. Tsao T.S. Saha A.K. Murrey H.E. Zhang C.C. Itani S.I. Lodish H.F. Ruderman N.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16309-16313Crossref PubMed Scopus (844) Google Scholar). The liver is another organ that actively oxidizes fatty acids, although the purpose of fatty acid oxidation in the liver differs from its functions in skeletal and cardiac muscles. Hepatic fatty acid oxidation provides acetyl-CoA for the production of ketone bodies during periods of fasting. Recently, we reported that hepatic ACCβ is regulated by sterol regulatory element-binding protein-1 in response to feeding status, through the P-II (15Oh S.Y. Park S.K. Kim J.W. Ahn Y.H. Park S.W. Kim K.S. J. Biol. Chem. 2003; 278: 28410-28417Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The metabolic changes in the liver in response to environmental stimuli are not as rapid as those in skeletal and cardiac muscles. This implies that the change in ACCβ amounts by transcriptional regulation is important in the liver, although the rapid regulation of enzyme activity by phosphorylation/dephosphorylation is the major control in skeletal and cardiac muscles. P-II is also active in human skeletal muscle and is regulated by myogenic regulatory factors (MRFs) (9Lee J.J. Moon Y.A. Ha J.H. Yoon D.J. Ahn Y.H. Kim K.S. J. Biol. Chem. 2001; 276: 2576-2585Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). MRFs, including Myf5, MyoD, myogenin, and MRF4, are basic helix-loop-helix transcription factors involved in myogenic differentiation. Although these factors all recognize the common consensus sequence, E-box (CANNTG), four MRFs are expressed in a temporally distinct pattern during myocyte differentiation. Myf5 and MyoD have been shown to establish the myogenic lineage during embryogenesis, whereas myogenin and MRF4 play a major role in the expression of muscle genes in fully differentiated myotubes (16Smith T.H. Block N.E. Rhodes S.J. Konieczny S.F. Miller J.B. Development. 1993; 117: 1125-1133PubMed Google Scholar, 17Rudnicki M.A. Jaenisch R. BioEssays. 1995; 17: 203-209Crossref PubMed Scopus (375) Google Scholar, 18Sassoon D.A. Dev. Biol. 1993; 156: 11-23Crossref PubMed Scopus (145) Google Scholar, 19Sabourin L.A. Rudnicki M.A. Clin. Genet. 2000; 57: 16-25Crossref PubMed Scopus (558) Google Scholar). These factors physically interact with retinoic acid receptors and act as transcriptional activators during differentiation (20Froeschle A. Alric S. Kitzmann M. Carnac G. Aurade F. Rochette-Egly C. Bonnieu A. Oncogene. 1998; 16: 3369-3378Crossref PubMed Scopus (32) Google Scholar, 21Downes M. Mynett-Johnson L. Muscat G.E. Endocrinology. 1994; 134: 2658-2661Crossref PubMed Scopus (28) Google Scholar, 22Alric S. Froeschle A. Piquemal D. Carnac G. Bonnieu A. Oncogene. 1998; 16: 273-282Crossref PubMed Scopus (23) Google Scholar). The synergistic action between MFR4 and RXR, which are the abundant members of their families in fully differentiated myocytes, is most effective in the activation of ACCβ P-II activity in humans (23Kim J.Y. Lee J.J. Kim K.S. Exp. Mol. Med. 2003; 35: 23-29Crossref PubMed Scopus (9) Google Scholar). The level of ACCβ is higher in the heart than in skeletal muscle. However, it is currently not clear as to which promoter directs ACCβ expression in the heart. Cardiomyocyte-specific transcription factors, such as Csx/Nkx2.5, GATA4, MEF2, and eHand but not MRFs, have been implicated in cardiac development and cardiac gene expression. The cardiac-specific homeobox protein, Csx/Nkx2.5, and the zinc finger protein, GATA4, function as critical transcription factors in cardiac development (24Olson E.N. Srivastava D. Science. 1996; 272: 671-676Crossref PubMed Scopus (392) Google Scholar, 25Kasahara H. Usheva A. Ueyama T. Aoki H. Horikoshi N. Izumo S. J. Biol. Chem. 2001; 276: 4570-4580Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 26Komuro I. Izumo S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8145-8149Crossref PubMed Scopus (462) Google Scholar, 27Molkentin J.D. Lin Q. Duncan S.A. Olson E.N. Genes Dev. 1997; 11: 1061-1072Crossref PubMed Scopus (958) Google Scholar) and synergistically activate a number of cardiac genes, such as the atrial natriuretic factor gene, the iodothyronine deiodinase gene, and the α-actin gene (28Durocher D. Charron F. Warren R. Schwartz R.J. Nemer M. EMBO J. 1997; 16: 5687-5696Crossref PubMed Scopus (550) Google Scholar, 29Lee Y. Shioi T. Kasahara H. Jobe S.M. Wiese R.J. Markham B.E. Izumo S. Mol. Cell. Biol. 1998; 18: 3120-3129Crossref PubMed Scopus (245) Google Scholar, 30Dentice M. Morisco C. Vitale M. Rossi G. Fenzi G. Salvatore D. Mol. Endocrinol. 2003; 17: 1508-1521Crossref PubMed Scopus (45) Google Scholar, 31Sepulveda J.L. Belaguli N. Nigam V. Chen C.Y. Nemer M. Schwartz R.J. Mol. Cell. Biol. 1998; 18: 3405-3415Crossref PubMed Scopus (277) Google Scholar). In the present study our intention was to identify the promoter that directs ACCβ expression in the heart and to prove that the ACCβ promoter is indeed activated by the cardiac-specific transcription factors, Csx/Nkx2.5 and GATA4. ACCβ is actively expressed in the skeletal muscle, the heart, and the liver, and its gene expression is differentially regulated in the respective organs. The mechanisms underlying this phenomenon remain an enigma. In the present study we proved that ACCβ levels change drastically in liver as a response to feeding status, whereas they are maintained at a constant level both in skeletal muscle and in the heart. This differential regulation of ACCβ gene expression originates in the alternative usage of promoters, such as P-I and P-II. P-I is the sole promoter found in the heart and skeletal muscle of rats, although both P-I and P-II are active in human skeletal muscle. We demonstrated the activation of P-I via synergistic action between MRF4 and the retinoid X-receptor as well as Csx/Nkx2.5 and GATA4, which explains the tissue-specific activation of P-I in both the skeletal muscle and the heart. We also elucidate that the CpG sites around exon 1a are half-methylated in skeletal muscle, in contrast to their complete methylation in the liver, resulting in the silencing of P-I. This study is the first to explain the mechanisms underlying the differential regulation of ACCβ gene expression in human and rodent tissues. Animals and Diets—Male Sprague-Dawley rats, weighing 150–200 g, were used for all experiments. For the fasting and refeeding study, rats were put on a fast for 48 h and then refed with a fat-free high carbohydrate diet for 0, 24, or 48 h. All experiments were performed at least three times. The fat-free high carbohydrate diet contained 82% (w/w) carbohydrates (74% starch, 8% sucrose), 18% (w/w) casein, 1% (w/w) vitamin mix, and 4% (w/w) mineral mix. All the materials for the diet were purchased from Harlan Teklad Co. (Madison, WI). Western Blot Analysis—Rat tissues were homogenized in 50 mm sodium phosphate buffer, pH 7.4, containing 10% (v/v) glycerol, 10 mm β-mercaptoethanol, 0.1 mm phenylmethylsulfonyl fluoride, 1× protease inhibitor mixture with glass pestles and then centrifuged at 5000 rpm at 4 °C for 10 min. Supernatants were precipitated in 12.5% polyethylene glycol. Precipitated proteins were dissolved in ⅕ initial volume of homogenization buffer, and the concentration of soluble protein was determined by the Bradford assay (Bio-Rad). Extracts were separated in 5% SDS-polyacrylamide gel and transferred onto Protran nitrocellulose membranes (Schleicher & Schuell). Immunoblot analysis was carried out with horseradish peroxidase-conjugated streptavidin (Vector Laboratories, Burlingame, CA) and polyclonal anti-ACCβ antibody, and specific bands were visualized using a SuperSignal West Pico Trial Kit (Pierce). RNase Protection Assays—Rat cRNA probes were synthesized from the sequences of either exon 1a (90 bp) or exon 1b (52 bp) extending to exon 2 (69 bp) by in vitro transcription. Human cRNA probes I, II, and O were established from pCRII plasmids containing sequences of exon 1a (58 bp), 1b (60 bp), and 1o (56 bp) extending to 100 bp of exon 2. After linearization of each plasmid (1 μg) by HindIII digestion, 32P-labeled cRNA was synthesized by T7 RNA polymerase (Ambion, Austin, TX). Probes were purified by gel elution after electrophoresis with 6% polyacrylamide, 6 M urea gel. RNase protection assays with purified probes were performed with the RPAIII kit (Ambion, Austin, TX). The total RNA (20 μg) isolated from rat livers was hybridized with a probe (1.6 × 105 cpm) in 30 μl of hybridization buffer at 42 °C for 12–16 h. The unhybridized RNA was digested by adding 150 μl of the diluted solution (1:100) of RNase A/T1 at 37 °C for 30 min. Probes protected from RNase were precipitated by the addition of 225 μl of RNase inactivation/precipitation III solution followed by 15 min of centrifugation at 12,000 rpm. Precipitates were washed with 70% ethanol and then denatured with 4 μl of sequencing gel-loading buffer at 95 °C for 3 min, then resolved on 6% polyacrylamide, 6 M urea gel. Gels were dried and exposed to Kodak BioMax film at -70 °C with intensifying screens. A sequencing ladder was loaded in the adjacent lane to determine the size of the products. Primer Extension Analysis—Primer extension was performed as described by Kim et al. (32Kim K.S. Park S.W. Moon Y.A. Kim Y.S. Biochem. J. 1994; 302: 759-764Crossref PubMed Scopus (13) Google Scholar). Antisense oligonucleotides of rat exon 1a and human exon 1o, 1a_AS, and 1o_AS were labeled with [γ-32P]ATP (PerkinElmer Life Sciences) by T4 polynucleotide kinase. The labeled oligonucleotides (2 × 105 cpm) were mixed with 50 μg of rat skeletal muscle, heart, and HepG2 RNAs in 100 μl of hybridization buffer (40 mm PIPES, pH 6.8, 1 mm EDTA, 0.4 m NaCl, 80% deionized formamide). The mixtures were incubated at 90 °C for 3 min and hybridized overnight at 42 °C. Annealed mixtures were precipitated by ethanol and used for the extension reaction. These mixtures were extended with SuperScript™II (Invitrogen) at 42 °C for 1 h under buffer conditions specified by the manufacturer's instructions. After phenol:chloroform: isoamyl alcohol (25:24:1) extraction and ethanol precipitation, the sizes of the products were determined by 6% denaturing polyacrylamide gel electrophoresis. The lengths of rat exon 1a and human exon 1o were determined by comparing to sequencing products of the cloned promoter region in both rats and humans. Construction of Plasmids—The luciferase constructs of human ACCβ P-II, phP-IIβ(-569/+65), and phP-IIβ-(-93/+65), were described by Lee et al. (9Lee J.J. Moon Y.A. Ha J.H. Yoon D.J. Ahn Y.H. Kim K.S. J. Biol. Chem. 2001; 276: 2576-2585Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The oligonucleotides used in promoter construction are shown in Table I. phP-Iβ-(-1735/+100) was constructed by amplifying the human ACCβ promoter region and introducing it into the SmaI site of the pGL3-Basic vector. Constructs of prP-Iβ-(-1864/+14), prP-IIβ-(-485/+65), and prP-IIβ-(-90/+65) were generated from the rat ACCβ promoter region and cloned in the SmaI sites of pGL3basic. phP-Oβ-(-1143/+191) was constructed by amplifying the upstream region containing human ACCβ exon 1o and introducing it into the SacI/SmaI site of the pGL3-Basic vector. Rat GATA4 cDNA was amplified by PCR and inserted into the HindIII/XhoI site of pcDNA3. The plasmid of pcDNA3-mycCSX was a generous gift from Dr. Issei Komuro (Chiba University, Chiba, Japan).Table ISequences of oligonucleotides used in the experimentsNameSequencesprP-IIβ-(-1864/+14)-1864_S5′-CTC CTC CCT ACG CGT GGT TCT CTC TCA-3′+14_AS5′-TGA GTG GCA GCA GTG ACC TA-3′phP-Iβ-(-1735/+100)-1735_S5′-TGA GGC AGG AGG TAC CTT TGA GCC CA-3′+100_AS5′-TAA CCC TGA ATG CAC GGT GG 3′GATA4_S5′-AGA GGG ATC CGC GAT GTA CCA AAG-3′GATA4_AS5′-GAA GGC TCG AGT GAT TAC GCG GT-3′1a_AS5′-TTT CAA GCT CCT CTG TGG CT-3′1o_AS5′-ACA GGA ATC ATT AGG CCA GGT-3′phP-Oβ-(-1143/+191)-1143_S5′-AGC AGT GAG CTC CAA GTT TCC A-3′+191_AS5′-ACA GGA ATC ATT AGG CCA GGT-3′CpG_S5′-GGT AGA GTA AGT AGT TAG TAG G-3′CpG_AS5′-GGG TCT TCT AAT TAA CTT CCT TCA-3′hexon1o_S5′-AGC CTG CCT CTG CAA AGG CAG GAC C-3′hexon1b_S5′-TGT GGG CGC CTG TCA GCC TCA CTC A-3′hexon2_S5′-GCA AAC CTC ATC CCG AGC CAG GAG C-3′hexon2_AS5′-CCA GCA ACA GAG TCC TCG TCG GAG G-3′β-Actin_S5′-ATG GAA TCC TGT GGC ATC CA-3′β-Actin_AS5′-ACC AGA CAG CAC TGT GTT GG-3′ Open table in a new tab Cell Culture and Transient Transfection—All reagents for cell cultures and Lipofectamine PLUS reagents were purchased from Invitrogen. NIH3T3 (Dulbecco's modified essential medium (DMEM)), C2C12 (DMEM), Alexander (minimal essential medium (MEM)), HepG2 (MEM), Hep3B (RPMI1640), and PLC/PRF5 (RPMI1640) cells were cultured in medium supplemented with 10% (v/v) fetal bovine serum and 100 μg/ml antibiotics/antimycotics at 37 °C in an 80 ∼ 90% humidified CO2 incubator. Rat primary hepatocyte culture and transfection were performed as describe by Ahn et al. (33Ahn Y.H. Kim J.W. Han G.S. Lee B.G. Kim Y.S. Arch. Biochem. Biophys. 1995; 323: 387-396Crossref PubMed Scopus (19) Google Scholar). Cells were prepared for experiments on 6-well plates at 2.5 × 105 ∼ 1 × 106 cells per well and then incubated for about 20 h. When cells were 80% confluent, cells were transfected with the indicated plasmids using Lipofectamine PLUS according to the manufacturer's protocols. The plasmid DNA and 3 μl of PLUS reagent were mixed in 100 μl of serum-free media and then added to 100 μl of serum-free media containing 2 μl of Lipofectamine reagent. The total amounts of DNA per well were adjusted to the same amounts by the addition of mock vector plasmid. The cells were washed with PBS, and supplied with 800 μl of serum-free media during incubation. After 15 min, Lipofectamine-DNA mixture was added to the wells. The cells which had been transfected for 3 h were washed twice with phosphate-buffered saline then grown for 48 h in media supplemented with 10% fetal bovine serum and 100 μg/ml antibiotics/antimycotics. RXR and RAR ligands, 1 μm 9-cis-retinoic acid and all-trans-retinoic acid, were treated after 20 h since cells were transfected and cultured further for additional 24 h. Cells were harvested and lysed with 200 μl of reporter lysis buffer (Promega, Madison, WI), and cell debris was removed by centrifugation. Luciferase activities were measured using 10 μl of cell extract and 50 μl of luciferase assay reagent (Promega). For the β-galactosidase assay, the color changes of extracts by hydrolysis of o-nitrophenol-β-d-galactopyranoside (Sigma-Aldrich) were detected as kinetics at 420 nm at 37 °C for 5 min. Methylation Analysis of CpG Islands—These genomic DNA were prepared from human muscle, liver, and HepG2 cell lines. Each tissue was ground using liquid nitrogen and lysed in lysis buffer (10 mm Tris, pH 8.0, 100 mm EDTA, 0.5% SDS, 20 μg/ml RNase A, 1 mg/ml proteinase K) at 50 °C for 5 h. After phenol:chloroform:isoamyl alcohol extraction, DNA precipitated by ethanol was picked up and was dissolved in TE buffer (10 mm Tris, pH 8.0, 1 mm EDTA). After 10 μg of genomic DNA was digested by 10 units of EcoRI at 37 °C for 5 h, unmethylated C residues were converted into U residues via the bisulfite reaction. In brief, 2 μg of linearized DNA were denatured in 0.3 m NaOH at 37 °C for 20 min and treated with 550 μl of converting solution (10 mm hydroxyquinone, 2.8 m sodium bisulfite, pH 5.0) then incubated in 55 °C for 16 h in darkness. Sulfonated single-strand DNA fragments were purified using the Wizard DNA Clean-Up system (Promega). Sulfonated C residues were desulfonated and deaminated with 0.3 m NaOH at 37 °C for 15 min and neutralized with 3 m ammonium acetate, pH 7.0. The converted DNA in which C residues had been converted to U residues was precipitated with ethanol and dissolved in 50 μl of TE buffer. The primers were designed according to C-to-T converted sequence of the region surrounding exon 1a as denoted in Table I as CpG_S and CpG_AS. The PCR reaction mixture contained 10 μl of converted DNA, 0.2 pmol of primers, Gold Taq reaction buffer, 1.5 mm MgCl2, 1.25 mm dNTPs, and 1 unit of Gold Taq polymerase (Roche Applied Science) amplified as follows: denaturation for 5 min at 94 °C, 40 cycles of denaturation for 30 s at 94 °C, annealing for 30 s at 52 °C, and extension for 30 s at 72 °C. Amplified products were directly sequenced using CpG_S primer. Reverse Transcription-PCR—Total RNAs were extracted from Alexander, HepG2, Hep3B, and PLC/PRF5 hepatoma cells using the TRIzol according to the manufacturer's instructions. First-strand cDNAs were synthesized from 5 μg of total RNA in 20 μl of reaction volume using SuperScript II reverse transcriptase. Each reverse transcription mixture (1 μl) was used as the template for amplifying ACCβ cDNA. The sense primers for each ACCβ transcript, spanning exon 1o (hexon1o_S), exon 1b (hexon1b_S), and exon 2 (hexon2_S), and antisense primer-containing sequence of exon 2 (hexon2_AS) were used in this experiment. The sizes of the PCR products were determined on 1% agarose gel. Changes of ACCβ Expression Levels in Rat Liver, Heart, and Skeletal Muscle by Diet—ACCβ is expressed predominantly in skeletal muscle and in the heart, where the β-oxidation of fatty acid actively occurs, constituting a major energy source. Sterol regulatory element-binding protein-1 was previously reported to induce ACCβ gene expression in the liver as a response to the intake of a high carbohydrate diet. We attempted to ascertain whether or not ACCβ levels in the heart and skeletal muscle changed in response to feeding status, as did ACCβ levels in the liver. The levels of pyruvate carboxylase, detected with streptavidin-horseradish peroxidase conjugate as a control, were almost the same between fasted and refed groups in liver, heart, and skeletal muscle extracts. The nutritional control had no significant effect on ACCβ expression in heart and skeletal muscle, whereas hepatic ACCβ levels were drastically increased by food intake (Fig. 1). This result is consistent with the findings of many previous reports, in that the posttranslational regulation of ACCβ was a much more important regulatory mechanism in skeletal muscle rather than were changes in enzyme levels (10Rasmussen B.B. Winder W.W. J. Appl. Physiol. 1997; 83: 1104-1109Crossref PubMed Scopus (129) Google Scholar, 11Dean D. Daugaard J.R. Young M.E. Saha A. Vavvas D. Asp S. Kiens B. Kim K.H. Witters L. Richter E.A. Ruderman N. Diabetes. 2000; 49: 1295-1300Crossref PubMed Scopus (102) Google Scholar, 12Boone A.N. Rodrigues B. Brownsey R.W. Biochem. J. 1999; 341: 347-354Crossref PubMed Scopus (44) Google Scholar, 33Ahn Y.H. Kim J.W. Han G.S. Lee B.G. Kim Y.S. Arch. Biochem. Biophys. 1995; 323: 387-396Crossref PubMed Scopus (19) Google Scholar). This also suggests that the transcriptional controls for the expression of ACCβ are quite different between cardiac/skeletal muscle and the liver. Differences of Promoter Usage in Rat Cardiac/Skeletal Muscles and Livers—It was reported that human and rat ACCβ gene expression could be derived from two types of promoters, designated as P-I and P-II (9Lee J.J. Moon Y.A. Ha J.H. Yoon D.J. Ahn Y.H. Kim K.S. J. Biol. Chem. 2001; 276: 2576-2585Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Differences in the regulation of ACCβ gene expression between tissues led us to perform RNase protection assay to determine which promoter is active in the respective organs. Antisense RNA probes used in RNase protection assays contained either exon 1a or exon 1b joined to the exon 2 sequence and were designated probes I and II, respectively (Fig. 2). The total RNA was isolated from the relevant tissues of rats that had fasted for 48 h and refed with a fat-free high carbohydrate diet for 0 or 24 h. Exon 1a and 2 in probe I were fully protected in the rat skeletal muscle and heart, although the exon 1b sequences in probe II were almost digested by RNase, resulting in a band consistent in size with exon 2. Moreover, the intensities of the RNase protected bands were not affected by feeding conditions (Fig. 2A). In contrast, hepatic RNA protected only the exon 1b sequence in probe II from RNase digestion and not the exon 1a sequence of probe I. Food intake caused a marked increase in the level of hepatic ACCβ transcripts (Fig. 2B). These data indicate that rat ACCβ gene expression in heart and skeletal muscle is controlled by P-I, whereas P-II is a major promoter in the liver. The alternative promoter usages would appear to explain the mechanism of different transcriptional regulations of the ACCβ gene between these organs. Determination of Transcription Start Site in Rat ACCβ P-I— In the previous report the size of human exon 1b transcribed by P-II was determined as 67 bp by primer extension analysis using the antisense primer corresponding to exon 2, although the size of exon 1a was not determined due to its large size (9Lee J.J. Moon Y.A. Ha J.H. Yoon D.J. Ahn Y.H. Kim K.S. J. Biol. Chem. 2001; 276: 2576-2585Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). The length of exon 1a was also expected to be much greater than that of exon 1b, judging from sequences of clones obtained from 5′ rapid amplification of cDNA ends (data not shown). To determine the precise transcription start site in P-I, we performed a primer extension analysis using total RNA isolated from rat skeletal and cardiac muscle as the templates and antisense primers corresponding to exon 1a. The size of exon 1a was revealed to be 201 bp in the rat ACCβ gene according to the size of the primer-extended product (Fig. 3A). Interestingly, the sequence of exon 1a shows higher conservation between human and rat than the 5′-flanking region of exon 1a (Fig. 3B). However, we cannot find any conserved promoter element, such as the TATA-, CCAAT-, or GC-box, in the proximal P-I promoter. ACCβ Promoter I Is Activated by MRFs and Retinoic Acid Receptors (RAR and RXR)—The fact that the muscle-specific expression of ACCβ is controlled by promoter P-I, as shown in Fig. 2, led us to check the responsiveness of ACCβ P-I to MRFs and retinoic acid receptors, which are important transcription factors mediating the expression of muscle-specific genes. The luciferase reporter construct, containing the human P-I s

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