NFY interacts with the promoter region of two genes involved in the rat peroxisomal fatty acid β-oxidation: the multifunctional protein type 1 and the 3-ketoacyl-CoA B thiolase
© Desaint et al; licensee BioMed Central Ltd. 2004
Received: 20 February 2004
Accepted: 26 March 2004
Published: 26 March 2004
β-oxidation of long and very long chain fatty acyl-CoA derivatives occurs in peroxisomes, which are ubiquitous subcellular organelles of eukaryotic cells. This pathway releases acetyl-CoA as precursor for several key molecules such as cholesterol. Numerous enzymes participating to cholesterol and fatty acids biosynthesis pathways are co-localized in peroxisomes and some of their encoding genes are known as targets of the NFY transcriptional regulator. However, until now no interaction between NFY transcription factor and genes encoding peroxisomal β-oxidation has been reported.
This work studied the interactions between NFY factor with the rat gene promoters of two enzymes of the fatty acid β-oxidation, MFP-1 (multifunctional protein type 1) and ThB (thiolase B) and their involvement in the cholesterol dependent-gene regulation. Binding of this nuclear factor to the ATTGG motif of the MFP-1 and of the ThB promoters was demonstrated by EMSA (Electrophoretic Mobility Shift Assay) and super shift assay. In contrast, in spite of the presence of putative Sp1 binding sites in these promoters, competitive EMSA did not reveal any binding. The promoter-dependent luciferase gene expression was downregulated by cholesterol in MFP-1 and ThB promoters harbouring constructs.
This work describes for the first time a NFY interaction with promoter sequences of the peroxisomal β-oxidation encoding genes. It suggests that cholesterol would negatively regulate the expression of genes involved in β-oxidation, which generates the initial precursor for its own biosynthesis, via at least the NFY transcription factor.
The current study reports that NFY binds to the proximal promoter regions of both the rat MFP-1 and the rat ThB genes. It suggests that cholesterol would negatively regulate the expression of genes involved in β-oxidation, which generates the initial precursor for it own biosynthesis, via at least the NFY transcription factor.
Results and discussion
Identification of trans-acting factors recognizing ATTGG motifs of the MFP-1 and ThB promoters
For the ThB promoter, incubation of two oligonucleotides corresponding to the NFY-1 or to NFY-2 motifs with nuclear extract leads to the formation of one main complex (Figure 2B). Despite a difference in complex intensity, similar mobilities were obtained for the probes corresponding to NFY-1 or NFY-2 sites. This labelled complex was displaced by adding unlabelled NFY-1 (lanes 2 to 4) or NFY-2 (lanes 7 to 9) competitors. However, it was unaffected with the corresponding mutated oligonucleotides NFY-1 mut (lane 5) and NFY-2 mut (lane 10).
Does Sp1 bind to the MFP-1 promoter (-114/+18 region)?
Involvement of NFY binding site in the regulation of the MFP-1 and ThB gene expression by cholesterol
Further transfection assays of the MFP-1 gene were conducted to locate the fragment involved in the cholesterol-dependent regulation in the -114/+18 promoter region. Two fragments were inserted upstream of the minimal β-globin promoter of the pGLuc vector ; the 132 pGLuc for -114/+18 fragment and the 58 pGLuc for -114/-56 fragment in pGLuc modified plasmid. Sterols induced a downregulation of the 58 and 132 bp fragments by 56%, and -49 %, respectively. From these results, it appears that the NFY sequence located in the 58 bp fragment is probably directly responsible for the repression by sterols, but other factors could be essential too. Among them, the SREBP transcription factor could be also involved due to its essential role in the cholesterol homeostasis. Indeed, the inhibition is more important with the complete promoter region (-3400/+20 fragment) (-70%) than with the 132 bp fragment (-49%), suggesting presence of cholesterol response element upstream to this region. In fact, seven sequences corresponding to a putative E-box are present in the first 500 bp of the promoter. Moreover, the binding of the SREBP-1 factor is 20 fold increased when the NFY factor is present [24, 25]. Such interaction may be involved in the transcriptional regulation of the MFP-1 gene by cholesterol.
This work describes for the first time a NFY interaction with the promoter regions of genes encoding for peroxisomal β-oxidation enzymes and suggests its involvement in the cholesterol-mediated downregulation of the MFP-1 and the ThB genes. Control of the peroxisomal fatty acid β-oxidation would be involved in the peroxisomal cholesterol homeostasis.
Materials and methods
HepG2 cells (ATCC, Manassas, VA, USA) were grown as monolayers in Dulbecco's modified Eagle's medium (Gibco Life Technologies, France) supplemented with 10% (v/v) fetal calf serum (Sigma), penicillin (125 IU/ml) and streptomycin (125 µg/ml). Cells were grown at 37°C in a 5% CO2 atmosphere.
Preparation of nuclear extract for EMSA
Nuclear extracts from normal rat liver were prepared as previously described . Briefly, livers were freezed and crushed in liquid nitrogen. Tissue was lysed with buffer A (0.6% Nonidet P-40, 150 mM NaCl, 10 mM Hepes pH 7.9, 1 mM EDTA, 0.5 mM PMSF), homogenized with a Dounce homogenizer (Pestle B) and centrifuged at 1700 g for 30 s. The supernatant was incubated for 5 min on ice and centrifuged at 5000 g for 5 min. The pellet was resuspended in buffer B (25% glycerol, 20 mM Hepes pH 7.9, 420 mM NaCl, 1.2 mM MgCl2, 0.2 mM EDTA, 0.5 mM DDT, 0.5 mM PMSF, 2 mM benzamide, 5 µg/ml pepstatin A) and incubated for 20 min on ice. Sample was centrifuged at 13000 g for 15 s, and the supernatant was stored at -70°C. The protein concentration of nuclear extract was determined by Bradford assay  by using assay reagent (Bio-Rad Laboratories, France) according to the manufacturer's recommendations.
DNA fragment preparation and construction of reporter plasmid
The -3400/+20 pGLuc plasmid containing the promoter of the rat MFP-1 gene  was digested with Aat II and Bgl I endonucleases (Promega, France) to obtain a 115 bp fragment (-116/-1 region), with a putative NFY binding site. DNA fragments of different proximal promoter regions of the MFP-1 gene were also obtained by PCR with a combination of four primers, 115S (sense) (5'-GTAGATCTGCAGAGCACGAAGT-3'), 115A (antisense)(5'-AGAAGCTTAAGGTATCCTGCACCT-3'), P76S (sense) (5'ATAGATCTAGCGCGCGCCCC T-3') and NF60A (antisense) (5'-ATAAGCTTCGCTGGGCCAAT-3'). To obtain a 132 bp fragment (-114/+18 region) containing putative NFY and 2 Sp1 binding sites, PCR was run for 35 cycles (94°C for 30 s, 50°C for 45s and 72°C for 3 min) with primers 115S/115A. For the 78 bp fragment (-60/+18 region) containing 2 putative Sp1 binding sites and the 58 bp fragment (-114/-56 region) containing a putative NFY binding site fragments, PCR was run for 35 cycles (94°C for 30 s, 47°C for 45 s and 72°C for 3 min) with primers P76S/115A and 115S/NF60A respectively. Amplification was performed using the 3400/+20 pGLuc plasmid as template in 10 mM Tris-HCl buffer, pH 9, 50 mM KCl, 0,1% Triton X-100, 3.12 mM MgCl2, 50 pmol of each primer, 2 mM dNTPs (Promega) and 1 U of Taq DNA polymerase (Promega) in PTC-100™ thermocycler (MJ Research, Reno, NV, USA). PCR fragments were inserted into pGLuc modified plasmid  between Bgl II and Hind III sites to obtain the 132 pGLuc and 58 pGLuc plasmids. The ThB promoter (-2800 base pairs upstream the transcription initiation site, Hind III / Eco T22I promoter region) was inserted at the Hind III site of the pGVB plasmid upstream of the luciferase gene (-2800 pTBLuc) . For the farnesyl diphosphate synthase promoter, a 723 bp sequence (-770 to -47 region upstream the translation initiation site) was inserted upstream to the luciferase gene in the reporter vector pGL2 (pFPPSGLuc) according to C. Le Jossic-Corcos (unpublished results).
Oligonucleotides competitors (Table 1)
Sequences of the oligonucleotides competitors
Name of oligonucleotides
NFY25 mut (-76/-56)
NFY-1 mut (-70/-46)
NFY-2 mut (-118/-95)
Oligonucleotides (Invitrogen, Cergy Pontoise, France) used as competitors in the electrophoretic mobility shift assays include two complementary 27 bp wild-type oligonucleotides (NFY25) corresponding to the -76/-56 region of the MFP-1 promoter containing the ATTGG motif with additional nucleotides representing of 5'Bgl II and 3'Hind III half sites and two complementary 27 bp oligonucleotides (NFY mut) corresponding to the same region with a mutated ATTGG motif to CGGTT. For the NFY binding sites of the thiolase B gene, four wild type oligonucleotides, NFY-1 (30 bp) and NFY-2 (28 bp), corresponding respectively to the -70/-46 and -118/-95 region of the thiolase B promoter with the ATTGG motif with additional nucleotides representing of 5' Hind III and 3' BamH I half sites and four oligonucleotides NFY-1 mut (30 bp) and NFY-2 mut (28 bp) sense and antisense corresponding to the original sequences of the thiolase B promoter with a mutated ATTGG motif (CGGTT) were used. Moreover, two complementary 22 bp oligonucleotides (Sp1) containing the consensus binding-site of the Sp1 factor were purchased from Promega.
Radiolabelling of DNA probes with [a-32P]dCTP
Labelling was performed with 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.05 mg/ml BSA, 5 pmol DNA (132 or 115 bp fragment), 10 µCi [a-32P] dCTP (3000 Ci/mmol, Amersham, Saclay, France), 20 U DNA polymerase I (Klenow fragment, Promega) and 500 µM each of dATP, dGTP and dTTP (Promega). Reaction was incubated for 45 min at 30°C and then stopped by addition of 0.5 M EDTA, pH 8. Radiolabelled probes were purified on Sephadex 50 column (Pharmacia) .
Electrophoretic mobility shift assay (EMSA)
DNA-protein binding reaction contained 10 mM β-mercaptoethanol, 5 mM EDTA, migration buffer (0.01% bromophenol blue, 30% glycerol and 5 mg/ml BSA), 1 µg poly [dI-dC], 1 µg sonicated DNA salmon sperm, 20 µg of rat liver nuclear extract and buffer (0.01 M Tris-HCl, pH 7.1, 0.1 mM EDTA pH 8, 0.08 M NaCl, 3 mM MgCl2, 0.1% Triton X-100, 5% glycerol). The radiolabelled probe (20000 cpm) was added and reaction was incubated for 30 min at 4°C. Reaction was separated on 5% native polyacrylamide gel electrophoresed for 15 min at 5 mA and 40 min at 10 mA. Gel was incubated for 15 min in 5% glycerol, dried for 30 min at 80°C, and exposed overnight at -70°C to a Kodak film (X-OMAT AR).
Transient transfections and reporter gene assays
HepG2 cells (7.104 cells/well) were plated on 24 wells plate in DMEM with 10% lipoproteins-deficient fetal calf serum (Sigma). Cells were transiently transfected using lipofectine (Gibco) with reporter constructs (-3400/+20 pGLuc, 132 pGLuc, 58 pGLuc, -2800 pTBLuc or pFPPSGLuc) and an internal control (pCH110 plasmid encoding β-galactosidase, Pharmacia). Cells were incubated for 5 h in optimem (Gibco) and then for 48 h in the absence or presence of sterols (10 µg/ml of cholesterol and 1 µg/ml 25-hydroxycholesterol). Cell extracts were prepared with lysis buffer of the luciferase assay kit (Promega) and assayed for β-galactosidase (Galactolight kit Tropix, Applied Biosystems, Courtaboeuf, France) and luciferase activities (luciferase assay kit, Promega). The protein concentration (Bradford assay) and β-galactosidase activity were measured in each sample and values were used to normalized luciferase activities.
We thank Dr. Mantovani (University of Milano, Italy) for providing NFY-A and NFY-B antibodies. We thank Dr. Laurent Corcos for his valuable discussions. This work has been supported by the Regional Council of Burgundy, IFR n°92 and by the GDR-CNRS n°2583.
- Reddy JK, Hashimoto T: Peroxisomal beta-oxidation and peroxisome proliferator-activated receptor alpha: an adaptive metabolic system. Annu Rev Nutr. 2001, 21: 193-230. 10.1146/annurev.nutr.21.1.193View ArticlePubMedGoogle Scholar
- Thompson SL, Krisans SK: Rat liver peroxisomes catalyze the initial step in cholesterol synthesis. The condensation of acetyl-CoA units into acetoacetyl-CoA. J Biol Chem. 1990, 265 (10): 5731-5735.PubMedGoogle Scholar
- Kovacs WJ, Olivier LM, Krisans SK: Central role of peroxisomes in isoprenoid biosynthesis. Prog Lipid Res. 2002, 41 (5): 369-391. 10.1016/S0163-7827(02)00002-4View ArticlePubMedGoogle Scholar
- Wanders RJ, Romeijn GJ: Differential deficiency of mevalonate kinase and phosphomevalonate kinase in patients with distinct defects in peroxisome biogenesis: evidence for a major role of peroxisomes in cholesterol biosynthesis. Biochem Biophys Res Commun. 1998, 247 (3): 663-667. 10.1006/bbrc.1998.8836View ArticlePubMedGoogle Scholar
- Maity SN, Golumbek PT, Karsenty G, de Crombrugghe B: Selective activation of transcription by a novel CCAAT binding factor. Science. 1988, 241 (4865): 582-585.View ArticlePubMedGoogle Scholar
- Sinha S, Maity SN, Lu J, de Crombrugghe B: Recombinant rat CBF-C, the third subunit of CBF/NFY, allows formation of a protein-DNA complex with CBF-A and CBF-B and with yeast HAP2 and HAP3. Proc Natl Acad Sci U S A. 1995, 92 (5): 1624-1628.PubMed CentralView ArticlePubMedGoogle Scholar
- Hooft van Huijsduijnen R, Li XY, Black D, Matthes H, Benoist C, Mathis D: Co-evolution from yeast to mouse: cDNA cloning of the two NF-Y (CP-1/CBF) subunits. Embo J. 1990, 9 (10): 3119-3127.PubMed CentralPubMedGoogle Scholar
- Taira T, Sawai M, Ikeda M, Tamai K, Iguchi-Ariga SM, Ariga H: Cell cycle-dependent switch of up-and down-regulation of human hsp70 gene expression by interaction between c-Myc and CBF/NF-Y. J Biol Chem. 1999, 274 (34): 24270-24279. 10.1074/jbc.274.34.24270View ArticlePubMedGoogle Scholar
- Mantovani R: A survey of 178 NF-Y binding CCAAT boxes. Nucleic Acids Res. 1998, 26 (5): 1135-1143. 10.1093/nar/26.5.1135PubMed CentralView ArticlePubMedGoogle Scholar
- Dorn A, Bollekens J, Staub A, Benoist C, Mathis D: A multiplicity of CCAAT box-binding proteins. Cell. 1987, 50 (6): 863-872.View ArticlePubMedGoogle Scholar
- Chodosh LA, Baldwin AS, Carthew RW, Sharp PA: Human CCAAT-binding proteins have heterologous subunits. Cell. 1988, 53 (1): 11-24.View ArticlePubMedGoogle Scholar
- Maity SN, de Crombrugghe B: Role of the CCAAT-binding protein CBF/NF-Y in transcription. Trends Biochem Sci. 1998, 23 (5): 174-178. 10.1016/S0968-0004(98)01201-8View ArticlePubMedGoogle Scholar
- Maity SN, Sinha S, Ruteshouser EC, de Crombrugghe B: Three different polypeptides are necessary for DNA binding of the mammalian heteromeric CCAAT binding factor. J Biol Chem. 1992, 267 (23): 16574-16580.PubMedGoogle Scholar
- Kim IS, Sinha S, de Crombrugghe B, Maity SN: Determination of functional domains in the C subunit of the CCAAT-binding factor (CBF) necessary for formation of a CBF-DNA complex: CBF-B interacts simultaneously with both the CBF-A and CBF-C subunits to form a heterotrimeric CBF molecule. Mol Cell Biol. 1996, 16 (8): 4003-4013.PubMed CentralView ArticlePubMedGoogle Scholar
- Maity SN, Vuorio T, de Crombrugghe B: The B subunit of a rat heteromeric CCAAT-binding transcription factor shows a striking sequence identity with the yeast Hap2 transcription factor. Proc Natl Acad Sci U S A. 1990, 87 (14): 5378-5382.PubMed CentralView ArticlePubMedGoogle Scholar
- Vuorio T, Maity SN, de Crombrugghe B: Purification and molecular cloning of the "A" chain of a rat heteromeric CCAAT-binding protein. Sequence identity with the yeast HAP3 transcription factor. J Biol Chem. 1990, 265 (36): 22480-22486.PubMedGoogle Scholar
- Becker DM, Fikes JD, Guarente L: A cDNA encoding a human CCAAT-binding protein cloned by functional complementation in yeast. Proc Natl Acad Sci U S A. 1991, 88 (5): 1968-1972.PubMed CentralView ArticlePubMedGoogle Scholar
- Li XY, Mantovani R, Hooft van Huijsduijnen R, Andre I, Benoist C, Mathis D: Evolutionary variation of the CCAAT-binding transcription factor NF-Y. Nucleic Acids Res. 1992, 20 (5): 1087-1091.PubMed CentralView ArticlePubMedGoogle Scholar
- McNabb DS, Xing Y, Guarente L: Cloning of yeast HAP5: a novel subunit of a heterotrimeric complex required for CCAAT binding. Genes Dev. 1995, 9 (1): 47-58.View ArticlePubMedGoogle Scholar
- Olesen J, Hahn S, Guarente L: Yeast HAP2 and HAP3 activators both bind to the CYC1 upstream activation site, UAS2, in an interdependent manner. Cell. 1987, 51 (6): 953-961.View ArticlePubMedGoogle Scholar
- Jackson SM, Ericsson J, Osborne TF, Edwards PA: NF-Y has a novel role in sterol-dependent transcription of two cholesterogenic genes. J Biol Chem. 1995, 270 (37): 21445-21448. 10.1074/jbc.270.37.21445View ArticlePubMedGoogle Scholar
- Lopez JM, Bennett MK, Sanchez HB, Rosenfeld JM, Osborne TE: Sterol regulation of acetyl coenzyme A carboxylase: a mechanism for coordinate control of cellular lipid. Proc Natl Acad Sci U S A. 1996, 93 (3): 1049-1053. 10.1073/pnas.93.3.1049PubMed CentralView ArticlePubMedGoogle Scholar
- Bennett MK, Lopez JM, Sanchez HB, Osborne TF: Sterol regulation of fatty acid synthase promoter. Coordinate feedback regulation of two major lipid pathways. J Biol Chem. 1995, 270 (43): 25578-25583. 10.1074/jbc.270.43.25578View ArticlePubMedGoogle Scholar
- Ericsson J, Jackson SM, Kim JB, Spiegelman BM, Edwards PA: Identification of glycerol-3-phosphate acyltransferase as an adipocyte determination and differentiation factor 1 – and sterol regulatory element-binding protein-responsive gene. J Biol Chem. 1997, 272 (11): 7298-7305. 10.1074/jbc.272.11.7298View ArticlePubMedGoogle Scholar
- Ericsson J, Jackson SM, Edwards PA: Synergistic binding of sterol regulatory element-binding protein and NF-Y to the farnesyl diphosphate synthase promoter is critical for sterol-regulated expression of the gene. J Biol Chem. 1996, 271 (40): 24359-24364. 10.1074/jbc.271.40.24359View ArticlePubMedGoogle Scholar
- Mantovani R, Pessara U, Tronche F, Li XY, Knapp AM, Pasquali JL, Benoist C, Mathis D: Monoclonal antibodies to NF-Y define its function in MHC class II and albumin gene transcription. Embo J. 1992, 11 (9): 3315-3322.PubMed CentralPubMedGoogle Scholar
- Kilsdonk EP, Morel DW, Johnson WJ, Rothblat GH: Inhibition of cellular cholesterol efflux by 25-hydroxycholesterol. J Lipid Res. 1995, 36 (3): 505-516.PubMedGoogle Scholar
- Bardot O, Aldridge TC, Latruffe N, Green S: PPAR-RXR heterodimer activates a peroxisome proliferator response element upstream of the bifunctional enzyme gene. Biochem Biophys Res Commun. 1993, 192 (1): 37-45. 10.1006/bbrc.1993.1378View ArticlePubMedGoogle Scholar
- Deryckere F, Gannon F: A one-hour minipreparation technique for extraction of DNA-binding proteins from animal tissues. Biotechniques. 1994, 16 (3): 405-PubMedGoogle Scholar
- Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976, 72: 248-254. 10.1006/abio.1976.9999View ArticlePubMedGoogle Scholar
- Nicolas-Frances V, Dasari VK, Abruzzi E, Osumi T, Latruffe N: The peroxisome proliferator response element (PPRE) present at positions -681/-669 in the rat liver 3-ketoacyl-CoA thiolase B gene functionally interacts differently with PPARalpha and HNF-4. Biochem Biophys Res Commun. 2000, 269 (2): 347-351. 10.1006/bbrc.2000.2249View ArticlePubMedGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T: Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press.;. 1989Google Scholar
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