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Originally published In Press as doi:10.1074/jbc.M312079200 on February 10, 2004

J. Biol. Chem., Vol. 279, Issue 17, 16954-16962, April 23, 2004
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A Krüppel-like factor KLF15 Contributes Fasting-induced Transcriptional Activation of Mitochondrial Acetyl-CoA Synthetase Gene AceCS2*

Joji Yamamoto,ab Yukio Ikeda,ab Haruhisa Iguchi,b Takahiro Fujino,c Toshiya Tanaka,d Hiroshi Asaba,d Satoshi Iwasaki,b Ryoichi X. Ioka,b Izumi W. Kaneko,c Kenta Magoori,d Sadao Takahashi,e Toshiyuki Mori,f Hiroshi Sakaue,f Tatsuhiko Kodama,d Masashi Yanagisawa,bgh Tokuo T. Yamamoto,c Sadayoshi Ito,a and Juro Sakaibdi

From the aDivision of Nephrology, Endocrinology, and Vascular Medicine, Department of Medicine, the Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan, the bYanagisawa Orphan Receptor Project, Exploratory Research for Advanced Technology, Japan Science and Technology Corporation, Tokyo 135-0064, Japan, the cTohoku University Gene Research Center, Sendai 981-8555, Japan, the dLaboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo 153-8904, Japan, the eThird Department of Internal Medicine, Fukui Medical University, Fukui, 910-1193, Japan, the fDivision of Diabetes and Digestive and Kidney Diseases, Department of Clinical Molecular Medicine, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan, and the gHoward Hughes Medical Institute, Department of Molecular Genetics, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9050

Received for publication, November 4, 2003 , and in revised form, February 4, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Acetyl-CoA synthetase 2 (AceCS2) produces acetyl-CoA for oxidation through the citric acid cycle in the mitochondrial matrix. AceCS2 is highly expressed in the skeletal muscle and is robustly induced by fasting. Quantification of AceCS2 transcripts both in C2C12 and human myotubes indicated that fasting-induced AceCS2 gene expression appears to be independent on insulin action. Characterization of 5'-flanking region of the mouse AceCS2 gene demonstrates that Krüppel-like factor 15 (KLF15) plays a key role in the trans-activation of the AceCS2 gene. Deletion and mutation analyses of AceCS2 promoter region revealed that the most proximal KLF site is a curtail site for the trans-activation of the AceCS2 gene by KLF15. Using Sp-null Drosophila SL2 cells, we showed that the combination of KLF15 and Sp1 resulted in a synergistic activation of the AceCS2 promoter. Mutation analyses of three GC-boxes in the AceCS2 promoter indicated that the GC-box, located 8 bases downstream of the most proximal KLF15 site, is the most important GC-box in the synergistic trans-activation of the AceCS2 gene by KLF15 and Sp1. GST pull-down assays showed that KLF15 interacts with Sp1 in vitro. Quantification of various KLF transcripts revealed that 48 h fasting robustly induced the KLF15 transcripts in the skeletal muscle. Together with the trans-activation of the AceCS2 promoter, it is suggested that fasting-induced AceCS2 expression is largely contributed by KLF15. Furthermore, KLF15 overexpression induced the levels of AceCS2 transcripts both in myoblasts and in myotubes, indicating that AceCS2 gene expression in vivo is indeed induced by KLF15.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Acetyl-CoA is an important intermediate in various metabolic pathways including fatty acid and cholesterol biosynthesis and the energy production by the citric acid cycle. There are several enzymes that generate acetyl-CoA in the mammals, including pyruvate dehydrogenase, which converts pyruvate to acetyl-CoA without generating free acetate. The degradation of fatty acid via {beta}-oxidation system also produces acetyl-CoA as an end product. Although acetate is not an essential source of acetyl-CoA in animals, the enzymatic ligation for the production of acetyl-CoA from acetate and CoA is a key reaction in the catabolism of acetate formed by several conditions including bacterial fermentation in the colon, oxidation of ingested ethanol in the liver. Acetyl-CoA synthetase (AceCS,1 EC 6.2.1.1 [EC] ) is an enzyme that catalyzes the production of acetyl-CoA from acetate and CoA. In particular, this enzyme plays a key role in the nervous system for recycling of acetate released by acetylcholine esterase for the formation and release of acetylcholine in cholinergic nerve terminals.

There are two AceCSs with similar enzymatic properties in mammals: one designated AceCS1 is a cytosolic enzyme and the other, designated AceCS2, is a mitochondrial matrix enzyme (1). Localized in the cytoplasm, AceCS1 provides acetyl-CoA for the synthesis of fatty acids and cholesterol. In contrast, AceCS2 produces acetyl-CoA for oxidation through the citric acid cycle to produce ATP and CO2 in the mitochondrial matrix.

AceCS1 is a member of a family of genes whose transcription is regulated by sterol regulatory element-binding proteins (SREBPs), a basic helix-loop-helix leucine zipper transcription factor that activates multiple genes for cholesterol and fatty acid metabolism (2, 3). The levels of AceCS1 mRNA was induced when cultured cells were deprived of sterols and negatively regulated by sterol addition. In our previous study, we have shown that the AceCS1 promoter region consists of multiple clustered SREBP binding sites and immediately downstream from this region there is a cluster of multiple GC-boxes (3). All these SREBP binding sites are bound with purified SREBP-1a and are required for a maximal response to co-transfected SREBP. The sterol regulation of the AceCS1 gene was critically dependent on three closely spaced SREBP binding sites and an adjacent GC-box. We also showed that SREBP synergistically activated the AceCS1 promoter along with Sp1 or Sp3 but not with nuclear factor Y (NF-Y).

AceCS2 is highly expressed in the cardiac and skeletal muscle and its regulation is completely different from that of AceCS1. A marked induction of AceCS2 mRNA is seen in the heart and skeletal muscle when animals are fasted (1). The levels of AceCS2 mRNA in the skeletal muscle of Zucker diabetic fatty rats were also increased compared with those in the normal littermates. These data indicated that the AceCS2 transcripts are induced in the heart and skeletal muscle under ketogenic conditions including prolonged fasting and diabetes.

During fasting, the expression of more than 94% of genes is not altered in the skeletal muscle. Among genes that are differentially expressed during fasting, genes involved in protein breakdown (components of the ubiquitin-proteasome pathway), fatty acid oxidation, and pyruvate dehydrogenase kinase 4 (PDK4) that suppresses glucose oxidation by inhibiting pyruvate dehydrogenase complex activity, are up-regulated (4, 5). In contrast, genes encoding glycolytic enzymes are down-regulated by fasting. Induction of genes involved in fatty acid oxidation is also seen in the skeletal muscle of diabetic mice (6). Although these changes indicate a complex adaptive program for sparing glucose, the mechanism underlining transcriptional regulation is not fully understood.

To define the mechanism of transcriptional induction of AceCS2 by fasting, we isolated and characterized 5'-flanking region of the mouse AceCS2 gene. In this article, we describe that fasting induced transcriptional activation of the AceCS2 gene in the skeletal muscle is largely contributed by a unique transcription factor, KLF15, a Krüppel-like factor. Our current data indicate a unique role of KLF15 in the activation of genes induced by prolonged fasting.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—We obtained mouse Genome Walker kits and luminescent {beta}-galactosidase reporter system from Clontech; the dual luciferase reporter assay system, pGL3 Basic, pRL-TK, and TNT Quick Coupled Transcription/Translation system from Promega Inc.; pcDNA3, pcDNA3.1, TOPO TA CloningTM kit, TRIzolTM reagent, SuperScript IITM, GeneRacerTM kit, LipofectAMINE PLUS, Dulbecco's modified Eagle's medium (DMEM), and Schneider's medium from Invitrogen; [{gamma}-32P]ATP (6000 Ci/mmol), and L-[35S]methionine (1000 Ci/mmol), GST Gene Fusion System and Bulk GST Purification Module from Amersham Biosciences; QuikChangeTM site-directed mutagenesis kit and MBS mammalian transfection kit from Stratagene; Oligotex-dT30 mRNA purification kit from TaKaRa (Shiga, Japan); nuclear extract kit from Active Motif (Carlsbad, CA); anti-human KLF15 polyclonal antibody from Abcam (catalog no. ab2647; Cambridge, UK); and oligonucleotides from Qiagen. Unless otherwise indicated, all restriction and DNA-modifying enzymes were obtained from Toyobo (Tokyo, Japan).

Expression Plasmids—To create a KLF15 expression plasmid, a full-length cDNA for rat KLF15 (provided by Dr. R. Hiramatsu, Genomics Science Laboratories, Sumitomo Pharmaceuticals Co. Ltd., Takarazuka, Japan) was inserted into pcDNA3. We obtained pcDNA1 vector-based plasmids for human MEF2A, mouse MEF2B, mouse MEF2C, and mouse MEF2D from Dr. E. N. Olson (Department of Molecular Biology, University of Texas Southwestern Medical Center) (7, 8); pSV-SPORT-PGC-1{alpha}, an SV40-driven plasmid containing mouse PGC-1{alpha} from Dr. B. M. Spiegelman (Dana-Farber Cancer Institute and the Department of Cell Biology, Harvard Medical School) (9); pcDNA3-based plasmid for mouse forkhead box O1a (FKHR) and mouse ALL1-fused gene from X chromosome (AFX), and pIRS-MLP-luc, a luciferase reporter plasmid driven by a promoter consisting of three tandem copies of a insulin responsive sequence (IRS) plus the adenovirus major late promoter (MLP) from Dr. A. Fukamizu (Center of Tsukuba Advanced Research Alliance, Institute of Applied Biochemistry, University of Tsukuba) (10, 11); pcDNA3.1-based plasmid for human c-Ets-1 from Dr. T. Minami (Laboratory for Systems Biology and Medicine, University of Tokyo, Japan) (12); and pcDNA3-based plasmids for mouse hepatic nuclear factor 1{alpha} (HNF1{alpha}) and mouse hepatocyte nuclear factor 1{beta} (HNF1{beta}) from Dr. K. Yamagata (Second Department of Internal Medicine, Osaka University Medical School, Suita, Japan) (13, 14). pCMV-MyoD, a CMV-driven plasmid for mouse MyoD, was constructed inserting the reverse transcription-PCR products of C2C12 myotube RNA into the pcDNA3 vector. pPac, a Drosophila actin 5C promoter-driven expression vector, pPac{beta}-gal containing an Escherichia coli {beta}-galactosidase, and pPacSp1 and pPacSp3, respectively, containing Sp1 and Sp3, were provided by Dr. G. Suske (Philipps-Universität Marburg, Germany). pPac-based plasmid encoding rat KLF15 was generated by inserting the KLF15 cDNA into the pPac vector. An Sp1-GST fusion construct, pGEX-Sp1, was prepared by inserting the Sp1 cDNA fragment into the XhoI site of pGEX-4T-2.

Cloning of 5'-Flanking Region of Mouse AceCS2 Gene—The 5'-flanking region of the mouse AceCS2 gene was cloned by PCR using Mouse Genome Walker kits (Clontech) as described previously (3). Briefly, the first PCR was conducted with adaptor primer 1 (provided by the supplier) and AceCS2 primer 1 (5'-TGACCACCCCGATTGTCCAGAG-3') on a mouse genomic library (provided by the supplier). Nested PCR was then carried out with adaptor primer 2 (provided by the supplier) and AceCS2 primer 2 (5'-CGCAGCAGTCGCCCCACACCGCTGC-3') on the first PCR products. The resulting 2.4-kb PCR product was subcloned into the pGEM-T easy cloning vector (Promega) to create pGEM-5'FL-AceCS2. Sequencing of the insert of pGEM-5'FL-AceCS2 was performed in both directions by the PCR cycle sequence method with an automatic sequence analyzer (Beckman Coulter CEQ 2000XL DNA Analysis System).

Rapid Amplification of cDNA End (RACE)—5'-RACE was performed using GeneRacerTM Kit (Invitrogen) according to the manufacturer's protocol. Briefly, 750 ng of poly(A)+ RNA from quadriceps muscles of fasted mice was first treated with calf intestinal phosphatase to remove the 5'-phosphates. This eliminates truncated mRNA and non-mRNA from subsequent ligation with the GeneRacerTM RNA Oligo. The dephosphorylated RNA was then treated with tobacco acid pyrophosphatase to remove the 5' cap structure from intact, full-length mRNA, and was ligated with 0.25 µg of the GeneRacerTM RNA Oligo using T4 RNA ligase at 37 °C for 1 h. The resulting products were then hybridized with random hexamer, reverse transcribed with SuperScript II reverse transcriptase, and subjected to PCR with a primer specific for the RNA Oligo (5'-CGACTGGAGCACGAGGACACTGA-3') and an AceCS2 specific antisense primer (5'-CTGCACATGCTGATCCAGGCAGTTGA-3'). PCR parameters were 94 °C for 30 s and 68 °C for 1 min for 35 cycles. A major product of ~400 bp was cloned into the pCR2.1 vector (Invitrogen): eleven clones were isolated for sequencing analysis.

AceCS2 Promoter-Reporter Constructs—pAceCS2(2347) is the mouse AceCS2 promoter-luciferase reporter gene that spans –2347 to–1 relative to translation initiation site. pAceCS2(1928), pAceCS2(1528), pAceCS2(1383), pAceCS2 (940), pAceCS2 (654), pAceCS2 (110), and pAceCS2 (77), are 5'-deletion mutants of pAceCS2(2347), each contains a deletion with the 5'-end denoted in each parentheses and the same 3'-end point at –1. pAceCS2(2347) was constructed by PCR of pGEM-5'FL-AceCS2 using a forward primer starting from –2347 and a reverse primer OGY3as (5'-CTCGCCCGACGCCCCACGCG-3'). The PCR product was cloned into the SmaI site of pGL3 basic. pAceCS2(1928), pAceCS2(1528), pAceCS2(1383), pAceCS2 (940), pAceCS2 (654), pAce-CS2 (110), and pAceCS2 (77) were constructed in a similar manner to pAceCS2(2347) using respective forward primers starting from the position –1928, –1528, –1383, –940, –654, –110, and –77, respectively, and coupled with a common reverse primer OGY3as. Base substitution mutants were generated in pAceCS2(1928) and pAceCS2 (654) using the QuikChangeTM site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. Oligonucleotides designed to mutate each element were as follows: the KLF site at –91, GGGTGGTG -> GCTGCAGG; GC-box A at –132, AGGGCAGGC -> AGACTAGTC; GC-box B at –76, GGGGCGGAG -> GGAATTCAG; and GC-box C at –65, GGGGCGGGG -> GGATATCGG.

Cells, Cell Culture, and Transfection—HEK293 cells (a line of human embryonic kidney cells) and C2C12 cells (a line of mouse myogenic cells) were obtained from Cell Resource Center for Biomedical Research at Tohoku University (Sendai, Japan). HSkMC (human skeletal muscle cells isolated from limbal skeletal muscle) were purchased from CELL APPLICATIONS, Inc. (San Diego, CA). HEK293 cells were maintained in medium A (DMEM containing 100 units/ml penicillin and 100 µg/ml of streptomycin sulfate, supplemented with 10% fetal bovine serum) at 37 °Cin5%CO2. C2C12 cells and HSkMC were maintained in medium A and differentiated into myotubes in medium B (DMEM containing 100 units/ml penicillin, 100 µg/ml of streptomycin sulfate and 2% horse serum) for 5 days.

Schneider line 2 (SL2) cells, an Sp-null Drosophila cell line, were purchased from Invitrogen, maintained in Schneider's medium containing 100 units/ml penicillin and 100 µg/ml streptomycin sulfate, supplemented with 10% fetal bovine serum, and grown at 23 °C.

HEK293 and SL2 cells were transfected in 24-well plates using LipofectAMINE PLUS as described previously (15). After 24 h, the cells were lysed with 0.1 ml of 1x passive lysis buffer (Promega), and aliquots were used for the measurement of firefly and Renilla luciferase activities as described below.

C2C12 cells were transfected exactly by the same method as that for HEK293 cells, except that the medium was switched to the differentiation medium (medium B) at 24 h post-transfection. After an additional incubation for 48 h, the cells were lysed and subjected to firefly and Renilla luciferase assays.

Enzyme Assays—Firefly and Renilla luciferase activities were measured according to the manufacturer's recommended protocol. Luciferase activities were determined in a Berthold Lumat Flash & Glow LB 955 luminometer. Firefly luciferase activities (relative light unit) were normalized by Renilla luciferase activities (relative light unit). For SL2 cells, {beta}-galactosidase activities were determined using Luminescent {beta}-galactosidase Reporter System (Clontech) and luciferase activities were normalized by the {beta}-galactosidase activities (relative light unit).

Retroviral Vectors and Infection—The plat-E retroviral packaging cell line (16), and the retroviral vector pWZL containing the blasticidin S resistance gene were, respectively, provided by Drs. T. Kitamura (University of Tokyo, Tokyo, Japan) and G. P. Nolan (Stanford University, Palo Alto, CA). KLF15 cDNA was subcloned into the retrovirus vector, pWZL, to generate pWZL-KLF15.

To generate C2C12 cells stably expressing KLF15, the cells were infected with the retroviral vectors pWZL-KLF15 as described by Pear et al. (17) with the following modifications: Plat-E packaging cells were infected with plasmids using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. After 24 h, cells were refed with fresh medium and cultured for an additional 24 h to obtain retroviral supernatants. For retroviral infection, C2C12 cells (50~60% confluence) were incubated with retroviral supernatants in the presence of 8 µg/ml polybrene for 24 h. Cells expressing KLF15 were selected for resistance to blasticidin S hydrochloride (10 µg/ml, Calbiochem, La Jolla, CA) and maintained in medium A. C2C12 cells stably expressing green fluorescent protein (GFP) were generated using a retrovirus vector encoding GFP (pWZL-GFP) and designated as C2C12/GFP.

Animal Manipulation—Male ICR mice were purchased from CLEA Japan, Inc (Tokyo, Japan). The mice were housed in a temperature- and humidity-controlled (26.5 °C and 35%, respectively) facility with a 12 h light/dark cycle (dark cycle was between 20.00–8.00). Mice were allowed to free access of water and a normal chow diet (CE-2, CLEA Japan) before experiments. Mice (7–8 weeks of age) were randomly divided into two groups: one group was fed ad libitum with regular diet and the other group was fasted for 48 h. All mice were sacrificed at the same time between 9.00 and11.00 AM. Quadriceps muscles were removed, weighed, and immediately frozen in liquid nitrogen and stored at –80 °C until RNA was extracted.

Quantitative Real Time PCR—First strand cDNA was synthesized from 5 µg of total RNA and oligo(dT) primers using SuperScript II reverse transcriptase. Specific primers for each gene transcript (listed in Table I) were designed using the Primer Express software (Applied Biosystems). Real time PCR contained, in a final volume of 20 µl, 3.125 ng of reverse-transcribed RNA, 167 nM of each primer, and 10 µl of 2x SYBR Green PCR Master Mix (catalog no. 4309155; Applied Biosystems). PCR was carried out in 384-well plates using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). All reactions were performed in triplicate. The relative amounts of each transcript were calculated using the comparative CT method (18). Mouse cyclophilin or human GAPDH mRNA was used as the invariant control.


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  		TABLE I
Primers for quantitative real-time RT-PCR

 
Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay (EMSA)—For EMSA, nuclear extracts from C2C12 cells stably expressing KLF15 were prepared using Nuclear Extract Kit (Active Motif) according to the manufacturer's protocol. Complementary single-stranded oligonucleotides corresponding to nucleotides –70 to –105 were annealed and end-labeled with [{gamma}-32P]ATP and T4 polynucleotide kinase. The labeled probe was incubated with 10 µg of nuclear extracts in a binding buffer containing 22.5 mM Hepes-KOH, pH 7.9, 2.6 mM MgCl2, 13.3% glycerol, 50 mM KCl, 0.125 mM EDTA, 0.5 mM dithiothreitol, and 0.5 mg/ml poly(dI-dC) for 30 min at room temperature. For competition experiments, excess (100-fold) unlabeled double-stranded oligonucleotide was added to the binding mixture. In the supershift experiment, an antibody for KLF15 (2.5 µg) was added to the binding reaction. Protein-DNA complexes were analyzed by electrophoresis on 6% polyacrylamide gels as described previously (3).

GST Pull-down Analysis—35S-labeled KLF15 was synthesized in vitro using pCMV-KLF15, TNT Quick Coupled Transcription/Translation system (Promega) and L-[35S]methionine (Amersham Biosciences) according to the manufacturer's protocol. GST-Sp1 fusion protein was synthesized by the GST Gene Fusion System (Amersham Biosciences) and purified by the Bulk GST Purification Module (Amersham Biosciences). GST-Sp1 fusion protein and GST expressed in E. coli DH5{alpha} were isolated by using glutathione-Sepharose 4B beads. The immobilized GST protein beads were washed with phosphate-buffered saline and incubated with 35S-labeled KLF15 at 4 °C for 1 h in the binding buffer (20 mM Hepes, pH 7.7, 75 mM KCl, 0.1 mM EDTA, 2.5 mM MgCl2, 0.05% Nonidet P-40, 2 mM dithiothreitol, and 10% glycerol). The beads were washed seven times with the same buffer, and subjected to SDS-polyacrylamide gel electrophoresis and autoradiography.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of Insulin on Levels of AceCS2 Transcripts in Skeletal Muscle—We previously showed that fasting robustly increases the levels of AceCS2 mRNA in the skeletal muscle. There are several known fasting-inducible genes that are negatively regulated by insulin (19). These include genes encoding phosphoenolpyruvate carboxykinase (20), insulin-like growth factor-binding protein-1 (20), insulin receptor substrate-2 (21), and aquaporin adipose (22). To determine whether insulin negatively regulates the expression of AceCS2, we used C2C12 and human myotubes. As a positive control for the insulin response in C2C12 myotubes, we have analyzed the mRNA levels of two well known insulin responsive genes, hexokinase II and PDK4: hexokinase II and PDK4 are, respectively, up- and down-regulated by insulin. Cells were incubated with or without insulin for various time periods and the levels of AceCS2, hexokinase II, and PDK4 mRNAs were determined by quantitative real time PCR. As shown in Fig. 1A, insulin treatment did not alter the levels of AceCS2 mRNA, whereas those of hexokinase II and PDK4 were, respectively, induced and reduced by insulin treatment. Similarly, the levels of AceCS2 mRNA in human skeletal myotubes were not altered (Fig. 1B), suggesting that fasting-induced AceCS2 gene expression is not directly dependent on insulin action.



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  		FIG. 1.
Effects of insulin on the levels of AceCS2 transcripts in cultured myotubes. On day 0, C2C12 cells (A) and HSkMCs (B) were plated in 6-well plates at a density of 1 x 105 cells/well and grown in medium A. On day 2, cells were induced to differentiate into myotubes by incubating in medium B. Medium was changed every other day. On day 6, medium B was replaced by serum-free DMEM supplemented with 0.1% horse serum, 100 units/ml penicillin, and 100 µg/ml of streptomycin sulfate. C2C12 myotubes were treated with 200 nM bovine insulin for the indicated time periods (A), and human myotubes (HSk-MCs) were cultured in the absence or presence of 20 nM bovine insulin for 16 h (B). Total RNA from C2C12 and human myotubes (A and B) were extracted, reverse-transcribed, and subjected to quantitative real time PCR as described under "Experimental Procedures." The levels of AceCS2, Hexokinase II, and PDK4 mRNA treated with insulin are shown in panel A. AceCS2 mRNA levels in HSkMCs cultured in the absence or presence of 20 nM bovine insulin are compared in panel B. Mouse cyclophilin or human GAPDH mRNA was used as the invariant control. Values represent the amount of mRNA in insulin-treated cells relative to that in the cells incubated without insulin, which is arbitrarily defined as 1.

 
5'-Flanking Region of the Mouse AceCS2 Gene—To determine the mechanism regulating fasting-induced expression of the AceCS2 gene, we initially isolated and characterized the 5'-flanking region of the mouse AceCS2 gene. Approximately 2.4-kb upstream of the translation initiation ATG was isolated by PCR (see "Experimental Procedures") and was sequenced (Fig. 2A). The transcription initiation sites were determined by 5'-RACE experiments on poly(A)+ RNA from mouse skeletal muscle using an AceCS2 transcript-specific primer (see "Experimental Procedures"). Sequence analysis of the 5'-RACE products revealed that the AceCS2 gene has multiple transcription start sites: two major points at –23 and –28 and four minor points at –12, –17, –19, and –53 (Fig. 2A); the A of the translation initiation ATG of the gene is designated +1.



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  		FIG. 2.
Mouse AceCS2 Promoter. A, nucleotide sequence of the 5'-flanking region of the mouse AceCS2 gene. Nucleotide position +1 is assigned to the A of the ATG initiator codon (boldface), and the residues preceding it are indicated by negative numbers. The major and minor transcription initiation sites determined by 5'-RACE are indicated by closed and open triangles, respectively. Potential sites for KLF, MyoD, IRS, c-Ets-1, HNF1, MEF2, and GC-boxes are underlined. B, schematic diagram of the mouse AceCS2 promoter. Potential elements are indicated by closed boxes and labeled.

 
Trans-activation of AceCS2 Promoter by KLF15—Potential elements for various transcription factors were identified by computer-assisted search using AliBaba2.1 (www.gene-regulation.com/pub/programs/alibaba2/index.html) and TFSEARCH (www.cbrc.jp/research/db/TFSEARCHJ.html). As summarized in Fig. 2B, the 5'-upstream of the most 5' transcription initiation site (–53) consists of potential sites for MyoD (–1509, –1379, and –698), IRS (–1509 and –1243), c-Ets-1 (–1435 and –1409), HNF1 (–802), MEF2 (–261), and KLF (–1854, –1487, –1143, –540, and –91). There are three GC-boxes located at –65, –76, and –132.

To determine the effects of these transcription factors, the genomic DNA fragment containing the 5'-flanking region (–2347) was ligated with the firefly luciferase gene in pGL3 basic to create pAceCS2(2347). This promoter-reporter was transiently transfected into non-muscle HEK293 cells along with the indicated transcription factor(s) listed in Fig. 3. All the expression plasmids used in this study were well characterized by the original investigators from whom we obtained. All the plasmids were confirmed by restriction enzyme digestions and partial sequencing. For the expression of AFX and FKHR, since they contain a FLAG epitope tag, we performed immunoblot analyses using an anti-FLAG antibody and confirmed that protein were expressed properly (data not shown). For the other transcription (co-) factors, we have performed real time PCR analysis, because of the unavailability of their antibodies. The transfection efficiencies were also examined by Renilla luciferase activities as described under "Experimental Procedure." As shown in Fig. 3, among 13 transcription factors tested, only KLF15 trans-activated the AceCS2 promoter reporter activities: ~5.5-fold induction was detected by co-transfection with KLF15. Other KLFs, KLF6 or KLF9 had almost no effects.



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  		FIG. 3.
Trans-activation of AceCS2 promoter by KLF15. On day 0, HEK293 cells were plated at a density of 2 x 104 cells/well in 24-well plates. On day 1, the cells were transfected with 0.8 µg of pAceCS2(2347) along with 0.01 µg of the indicated expression plasmid(s). To normalize transfection efficiency, Renilla luciferase reporter (pRL-TK, 0.1 µg) was also included. On day 3, the cells were harvested for measurement of firefly and Renilla luciferase activities. Renilla luciferase activity was used as the internal reference to normalize transfection efficiency. The value for pAceCS2(2347)-luciferase activity co-transfected with pcDNA3.1 empty vector was arbitrarily set as 1. The -fold activation (luciferase activity co-transfected with each expression plasmid versus pcDNA3.1) is shown. The error bars represent mean ± S.E. of triplicate incubations. Inset, HEK293 cells were transfected with either 0.01 µg of pcDNA3.1 empty vector, pCMV-FKHR, or pCMV-AFX together with 0.8 µg of pIRS-luc-MLP and 0.1 µg of pRL-TK, and firefly and Renilla luciferase activities were determined as described above.

 
KLF15 is known to form a heterodimer complex with MEF2A and the resulting complex synergistically induces the transcription of GLUT4 gene (23). On the other hand, MEF2 family of transcription factors MEF2A, 2B, 2C, and 2D had no synergistic effects on the AceCS2 reporter activities when coupled with KLF15. Each of the MEF2 family of transcription factors, MEF2A-2D, alone had almost no effects.

We also examined the effects of peroxisome proliferator-activated receptor {gamma} co-activator 1 (PGC-1{alpha}) in the absence or presence of KLF15 or MEF2 family of transcription factors, MEF2A-2D. Neither PGC-1{alpha} alone, PGC-1{alpha} in the presence of KLF15, nor PGC-1{alpha} with each of the MEF2 family of transcription factors had stimulatory effects on the promoter reporter activities. The forkhead box transcription factors FKHR (also known as FOXO1) (24, 25) and AFX (FOXO4) (11) are involved in insulin-responsive gene activation. Thus we examined the effects of FKHR and AFX on the AceCS2 promoter activities. As a positive control experiment, we transfected AFX and FKHR together with a reporter-luciferase under the control of three copies of insulin responsive sequence (IRS) in tandem. This configuration, which is based on the IGFBP-1 promoter/enhancer elements, gave rise to abundant luciferase activity (Fig. 3, inset) (11). Consistent with the lack of insulin-dependence, neither FKHR nor AFX had stimulatory effects on the AceCS2 promoter in the absence or presence of KLF15.

Other factors MyoD, cEts-1, HNF1{alpha}, and HNF1{beta} in the absence or presence of KLF15 were inactive. Although these transcription factors were not active in this reporter assay and we cannot exclude the possibility of their significance in the regulation of AceCS2 gene in vivo, these data indicate that KLF15 plays a key role in the trans-activation of the AceCS2 gene.

Crucial KLF Binding Site in the AceCS2 Promoter—There are five potential KLF15 binding sites within 2.4-kb region of the AceCS2 promoter. Fig. 4A compares the sequences of five potential KLF15 sites and their surroundings in the AceCS2 promoter with that present in the GLUT4 gene. Although four of the five KLF15 sites are inversely orientated, all the five sequences contain an identical sequence of CACCC that is specifically bound by KLF15 (23) and other KLF family of transcription factors (26–28).



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  		FIG. 4.
KLF15 motifs in the AcecS2 promoter. A, alignment of five KLF motifs in the AceCS2 promoter. The sequences of potential KLF motifs at –1854, –1487, –1143, –540, and –91 and their surroundings in the AceCS2 promoter are compared with KLF15 binding sequence in the mouse GLUT4 promoter. The KLF motifs in the inverted orientation are labeled with inv. B, localization of KLF15 responsive element by progressive deletion and mutation analyses. On day 0, HEK293 cells were seeded at a density of 2 x 104 cells/well in 24-well plates. On day 1, the cells were transfected with 0.8 µg of the indicated AceCS2-luciferase reporter and 0.1 µg of pRL-TK together with 0.01 µg of pCMV-KLF15 or pcDNA3.1. On day 2, the cells were harvested, and firefly and Renilla luciferase activities were determined as described under "Experimental Procedures." The -fold activation (luciferase activity co-transfected with pCMV-KLF15 versus pcDNA3.1) is shown. The error bars represent mean ± S.E. of triplicate incubations.

 
To determine the most crucial KLF binding site, a series of 5'-deletions was introduced into the promoter region. Deletion of the sequence from –1928 to –110 did not change the transactivation by KLF15 (Fig. 4B). A further deletion to –77 resulted in almost complete loss of induction by KLF15. Similar results were obtained using a myogenic line, C2C12 cells. Furthermore, the reporter construct containing a mutated KLF binding site lacked inducible activity. Similarly, in the context of pAceCS2 (–1928), the mutation in the most proximal KLF binding site lacked the inducible activity, indicating that the most proximal KLF site is a curtail site for the trans-activation of the AceCS2 gene by KLF15.

EMSA of KLF15-Binding Site in the AceCS2 Promoter—To determine whether KLF15 is capable of binding to the most proximal KLF site of the AceCS2 promoter, we performed EMSA using labeled probe (double-stranded 36-mer corresponding –70 to –105) with the nuclear extracts from C2C12 cells stably expressing KLF15. As shown in Fig. 5, the nuclear extracts containing KLF15 produced a single band of DNA-protein complex. This complex was specific as it can be competed by unlabeled probe. Furthermore, this complex can be supershifted with an antibody against KLF15, indicating that KLF15 is able to bind to the most proximal KLF site to the transcription start point of the AceCS2 gene.



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  		FIG. 5.
EMSA of KLF15-binding to the AceCS2 gene promoter. Nuclear extracts were prepared from C2C12 cells stably expressing KLF15 and incubated with labeled double-stranded oligonucleotide containing a putative KLF binding site (at –91), followed by EMSA as described under "Experimental Procedures." The KLF15-specific shifted band and the supershifted band (S.S) specific to the KLF15 antibody are indicated on the left. Nonspecific band is denoted by an asterisk.

 
Synergistic Transcriptional Activation by KLF15 and Sp1—In contrast to the synergic transcriptional activation of the GLUT4 gene by KLF15 and MEF2 (23), none of MEF family transcription factors in combination with KLF15 synergistically trans-activated the AceCS2 promoter (see Fig. 3). The proximity of the KLF15 binding site and GC boxes raised the possibility that KLF15 and Sp1 may function in a coordinated manner to induce the AceCS2 promoter. To assess this possibility, we performed co-transfection studies using Sp-null Drosophila SL2 cells. As shown in Fig. 6A, we observed that the combination of KLF15 and Sp1 resulted in a synergistic activation of the AceCS2 promoter (lanes 1, 3, and 5): ~10-fold by comparison to Sp1 alone. Although Sp3 recognizes the same sequence as Sp1, and Sp3 alone trans-activated the AceCS2 promoter (Fig. 6A, lane 4), the combination of KLF15 and Sp3 did not synergistically induce the AceCS2 promoter (Fig. 6A, lane 6).



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  		FIG. 6.
KLF15 and Sp1 cooperate to induce AceCS2 promoter activity. A, KLF15 functions synergistically with Sp1. On day 0, SL2 cells were plated at a density of 2 x 105 cells/well in 24-well plates. On day 1, the cells were transfected with 0.5 µg of pAceCS2 (654) and 0.1 µg of pPac-{beta}-gal (internal reference encoding the {beta}-galactosidase gene) together with 0.01 µg of the indicated plasmid. On day 2, the cells were harvested, and luciferase activity was measured and normalized to {beta}-galactosidase activity. Each value represents the average of duplicate incubations. B, mutation analyses of three GC-boxes in AceCS2 promoter. pAceCS2(654) and its mutants, in which each GC-box was mutated, were constructed as described under "Experimental Procedures." On day 0, 2 x 104 cells/well of HEK293 cells were set up in 24-well plates. On day 1, the cells were transfected with 0.8 µg of the indicated luciferase reporter construct and 0.1 µg of pRL-TK together with 0.01 µg of pCMV-KLF15 or pcDNA3.1. On day 2, the cells were harvested, and firefly luciferase activity was measured and normalized to Renilla luciferase activity. The -fold activation (luciferase activity co-transfected with pCMV-KLF15 versus pcDNA3.1) is shown. Each value represents the average of duplicate incubations.

 
There are three GC-boxes, termed as GC-boxes A, B, and C, within 100 bases upstream of the most 5' transcriptional initiation site (–53) of the AceCS2 gene. To further define the role of these GC-boxes in the synergistic trans-activation with KLF15, we introduced mutations in each of the three GC-boxes in pAceCS (654) (see "Experimental Procedures" and Fig. 6B). For this co-transfection experiment with these mutant promoter-reporters, we used HEK293 cells. As shown in Fig. 6B, mutations in GC-box B in the AceCS2 promoter almost completely abolished the trans-activation by KLF15, while other mutants containing mutated GC-boxes A or C retained the trans-activation activity by KLF15. These data indicate that GC-box B, located 8 bases downstream of the most crucial KLF15 site, is the most important site in the synergistic transactivation of the AceCS2 gene by KLF15 and Sp1.

To determine whether KLF15 and Sp1 interact directly to activate the AceCS2 promoter, we carried out GST pull-down assays. As shown in Fig. 7, in vitro transcribed/translated 35S-labeled KLF15 was bound with GST-Sp1 fusion protein, but not with GST, indicating that KLF15 interacts with Sp1 in vitro.



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  		FIG. 7.
KLF15 interacts with Sp1 in vitro. Glutathione beads bound with E. coli cell-expressed GST-Sp1 (10 and 30 µl) or GST (30 µl) were incubated with in vitro transcribed/translated 35S-labeled KLF15 at 4 °C for 1 h. After washing extensively, the proteins bound on the beads were analyzed by SDS-PAGE and visualized by autoradiography.

 
Induction of KLF15 Transcripts in Skeletal Muscle of Fasted Animals—To test whether fasting induces KLF15 expression, like the AceCS2 gene, we carried out quantitative real time RT-PCR analysis of KLF transcripts. As shown in Table II, 48 h fasting robustly induced the KLF15 transcripts in the skeletal muscle of mice: the levels of KLF15 transcripts in fasted animals were ~28-fold higher than those in fed animals. Together with the trans-activation of the AceCS2 promoter, it is suggested that this fasting-induced AceCS2 expression is largely contributed by KLF15.


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  		TABLE II
Quantitative real time PCR of various KLF transcripts in skeletal muscle of fasted and fed mice

Total RNAs from skeletal muscles of fasted (48 h) or fed mice were subjected to quantitative real-time PCR as described under "Experimental Procedures." Cyclophilin mRNA was used as the invariant control. Values represent the relative amount of mRNA in skeletal muscle of fasted mice relative to that of fed mice, which is arbitrarily defined as 1. The values are mean of three mice.

 
Overexpression of KLF15 in C2C12 Myogenic Cells Induces the Expression of AceCS2 Transcripts—We next examined the effects of KLF15 overexpression on the levels of AceCS2 transcripts in C2C12 myogenic cells. C2C12/KLF15 and C2C12/GFP cells were cultured in non-differentiation medium (medium A) or differentiation medium (medium B) and the levels of AceCS2 transcripts were determined by quantitative real time RT-PCR. As shown in Fig. 8, KLF15 overexpression induced the levels of AceCS2 transcripts by severalfold both in myoblasts and in myotubes, indicating that AceCS2 gene expression in vivo is indeed induced by KLF15.



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  		FIG. 8.
Induction of GLUT4 and AceCS2 transcripts in C2C12 cells stably expressing KLF15. On day 0, C2C12 stably expressing KLF15 (C2C12/KLF15) and GFP (C2C12/GFP) were individually plated in 6-well plates at a density of 1 x 105 cells/well and cultured in medium A. On day 2, the cells were either harvested (myoblasts) for RNA extraction, or induced to differentiate into myotubes by incubating in medium B for an additional 7 days. Medium was changed every other day. On day 9, myotubes were harvested for RNA extraction. Total RNA from myoblasts and myotubes were subjected to quantitative real time PCR with specific primers for GLUT4 (A) or AceCS2 (B). Cyclophilin was used as the invariant control. Values represent the amount of mRNA relative to that in C2C12/GFP myoblasts, which is arbitrarily defined as 1. The error bars represent mean ± S.E. of triplicate incubations.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the current study, we provide evidence that fasting-induced transcriptional activation of the AceCS2 gene in the skeletal muscle is contributed largely by a unique transcription factor KLF15. KLF15 is a recently characterized member of the Sp1-like/KLF family and regulates GLUT4 gene expression in both adipose and muscle cells (23). Consistent with the induction of AceCS2 transcripts, the levels of KLF15 transcripts in the skeletal muscle are also robustly induced by fasting. Together with the trans-activation of the AceCS2 promoter by KLF15, this co-induction of KLF15 by fasting may indicate that fasting-induced AceCS2 transcription is largely contributed by KLF15.

KLF15 is a member of the Sp1-like/KLF family, a family of highly related zinc-finger DNA binding proteins that are important regulators of cellular development, differentiation, and activation (29). It is highly expressed in the liver, kidney, adipose tissue, heart, and skeletal muscle (23, 30). Gray et al. demonstrated that KLF15 specifically induces the expression of the insulin-sensitive glucose transporter GLUT4 and glucose uptake in 3T3-L1 cells. It was also shown that KLF15 directly interacts with MEF2, a known activator of the GLUT4 promoter, and activates the GLUT4 promoter in a synergistic manner. On the other hand, our current data show that KLF15 in combination with a general transcription factor Sp1, synergistically activates the AceCS2 promoter. In this context, it is interesting to note that the two AceCSs require Sp1 for their trans-activation; activation of the AceCS1 gene is synergistically regulated by SREBPs and Sp1 (3).

Fasting-induced gene expression is of current interest. Especially, the induction of hepatic gluconeogenic genes during prolonged fasting or starvation is important for survival. Recently, Puigserver et al. (25) showed that insulin-regulated hepatic induction of gluconeogenic genes is mediated through interaction of FKHR and PGC-1{alpha}. In the skeletal muscle, genes involved in fatty acid oxidation and PDK4 are up-regulated during fasting in order to suppress glucose utilization (4, 5). We recently showed evidence that the PPAR{delta} together with PGC-1{alpha} mediates the induction of genes involved in fatty acid transport, {beta}-oxidation, and mitochondrial respiration in the skeletal muscle (31). The gene expression profile induced by PPAR{delta} is very similar to those induced by fasting in the skeletal muscle (4, 5). Therefore, PPAR{delta} together with PGC-1{alpha} may play a part in the muscle expression of genes induced by fasting.

Our current data provide evidence for a unique role of KLF15 in the activation of fasting-induced genes in the skeletal muscle. In the skeletal muscle, KLF15 is the most abundant KLF and its induction by fasting is most potent among 16 KLFs. To our knowledge, AceCS2 and GLUT4 are the only known targets for KLF15. It is therefore important to identify target genes driven by KLF15. These studies are currently in progress.


   FOOTNOTES
 
* This work was supported through Special Coordination Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government, and Exploratory Research for Advanced Technology/Japan Science and Technology Corporation (Yanagisawa orphan receptor project). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

h An Investigator of the Howard Hughes Medical Institute. Back

i To whom correspondence should be addressed: Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo 153-8904, Japan. Tel.: 81-3-5452-5472; Fax: 81-3-5452-5429; E-mail: jmsakai-tky{at}umin.ac.jp.

1 The abbreviations used are: AceCS, acetyl coenzyme A synthetase; AFX, ALL1-fused gene from X chromosome; CMV, cytomegalovirus; FKHR, forkhead box O1a; GFP, green fluorescent protein; GLUT4, glucose transporter 4; GST, glutathione S-transferase; HSkMC, human skeletal muscle cell; HNF, hepatocyte nuclear factor; KLF, Krüppel-like factor; MEF2, myocyte enhancer factor 2; PGC-1{alpha}, peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha}; PPAR, peroxisome proliferator-activated receptor; RACE, rapid amplification of cDNA end; SREBP, sterol regulatory element-binding protein; DMEM, Dulbecco's modified Eagle's medium; EMSA, electrophoretic mobility shift assay; GFP, green fluorescent protein. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Akiyoshi Fukamizu, Ryuji Hiramatsu, Takashi Minami, G. P. Nolan, Eric N. Olson, Bruce M, Spiegelman, Guntram Suske, and Kazuya Yamagata for plasmid constructs, Toshio Kitamura for a retroviral packaging cell line, Kazuya Yamada for helpful discussion, Aoi Uchida and Yasuyo Urashima for technical assistance.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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