- Open Access
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.
- Electrophoretic Mobility Shift Assay
- Proximal Promoter Region
- Mevalonate Kinase
- Zellweger Syndrome
- Electrophoretic Mobility Shift Assay Experiment
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.
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.
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.
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