fatty acid -oxidation is with glycolysis one of the two major pathways providing cellular energy . beta - oxidation of long and very long chain fatty acyl - coa derivatives occurs in peroxisomes , which are ubiquitous subcellular organelles in eukaryotic cells . although it is not linked with oxidative phosphorylation , peroxisomal -oxidation of fatty acids releases acetyl - coa , a precursor for several pathways such as cholesterol intermediate biosynthesis , including farnesyl pyophoshate and geranyl - geraniol pyrophosphate ( required for ras protein membrane anchor ) and for cholesterol production as well . for example , data showing that a peroxisomal acetoacetyl - coa thiolase which catalyzes the conversion of acetyl - coa to acetoacetyl - coa , known as a first step in cholesterol biosynthesis , has been reported earlier by thompson and krisans . following this , several enzymes participating to cholesterol pathway have been co - localized to peroxisomes ( i.e. hmg - coa synthase , hmg - coa reductase , mevalonate kinase and farnesyl pyrophosphate synthase ) . in addition , defects in peroxisomes biogenesis , associated with zellweger syndrome and rhizomelic chondrodysplasia punctata , reveal that in human liver mevalonate kinase and phosphomevalonate kinase are peroxisomal enzymes . three enzymes are required for peroxisomal -oxidation , the acyl - coa oxidase , the enoyl - coa hydratase/3-hydroxyacyl - coa dehydrogenase , so called multifunctional enzyme-1 ( mfp-1 ) and 3-ketoacyl - coa thiolase ( th a and b ) . in contrast with numerous eukaryotic genes containing a ccaat box at around -70 bp from the transcription start site , the in silico sequence analysis does not locate any ccaat box in the proximal promoters regions of either rat mfp-1 or rat thb genes . however , the rat mfp-1 promoter contains an attgg sequence at -92/-88 , which corresponds to an inverted ccaat box ( figure 1 ) . in addition , the rat thb promoter contains two inverted ccaat boxes located at nucleotides -60/-56 and-109/-105 , respectively ( figure 1 ) . the inverted ccaat motif is a functional cis - acting element present in promoters and enhancer regions of many eukaryotic genes including those encoding for two type i collagens , albumin , hsp70 , major histocompatibility complex ( mhc ) class ii and numerous cell - cycle - related proteins such as thymidine kinase and cyclins [ 5 - 9 ] . although many nuclear proteins have been described to bind ccaat elements , the heteromeric inverted - ccaat binding transcription factor cbf or cp1 , also called nfy   appears to be the major factor recognizing a true inverted ccaat box located in the -60 to -130 region of promoters . nfy is an ubiquitous transcription factor of 70 kda resulting from the interaction of the three subunits a , b and c , all of which are required to form a nfy - dna complex . tight association of nfy - b and nfy - c subunits is a prerequisite before recruitment of subunit a to the b - c complex leading to the binding of this heterotrimer complex to a specific dna sequence . primary structures of nfy subunits are conserved in mammals as deduced from the cdna sequences of nfy - a , nfy - b and nfy - c among several species [ 6,7,15 - 17 ] . human , mouse , and rat nfy subunits show more than 95% amino acid sequence identity . portions of the amino acid sequences of mammalian nfy - a , nfy - b , and nfy - c are also evolutionarily conserved , with a high degree of sequence identity with segments of yeast hap3 , hap2 , and hap5 , respectively . these polypeptides are components of a saccharomyces cerevisiae ccaat binding transcription factor which controls several nuclear genes involved in the mitochondrial function . the role of nfy has been demonstrated in the transcriptional regulation of genes encoding enzymes implicated in two major synthetic pathways : 3-hydroxy-3-methylglutaryl - coa reductase and farnesyl diphosphate synthase for cholesterol biosynthesis , and acetyl - coa carboxylase and fatty acid synthase for fatty acids biosynthesis [ 21 - 25 ] . nucleotide sequences of the 5'-flanking regions of the multifunctional protein-1 ( mfp-1 ) and thiolase - b ( thb ) genes respectively . each transcription start site is numbered + 1 and the sequence of exon i are shadowed . 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 -oxidation , which generates the initial precursor for it own biosynthesis , via at least the nfy transcription factor . in silico sequences analysis of rat peroxisomal genes encoding mfp-1 and thb reveals the presence of inverted ccaat boxes in both promoters , which are positioned from the transcription start sites at : -68/-64 for the mfp-1 gene and at -56/-60 and -105/-109 for the thb gene ( figure 1 ) . emsa experiments were performed to determine whether liver nuclear factors interact with these inverted ccaat boxes . the 115 bp probe corresponding to the positions -116 to -1 of the mfp-1 promoter region containing a putative nfy binding site and two putative sp1 binding sites ( figure 1 ) was used in emsa experiments . incubation of the 115 bp probe with nuclear extracts resulted in the formation of two close protein - dna complexes , a major one , complex i and a minor one , complex ii ( figure 2a , lanes 1 and 6 ) . these labelled complexes were competed by the unlabeled dna competitor . indeed , increased quantities of nfy25 oligonucleotide corresponding to the -76/-56 region ( figure 2a , lanes 2 to 5 ) completely abolished complexes formation for an excess of 500-fold . in contrast , complexes formation was unaffected by the use of the mutated oligonucleotide ( nfy mut ) over the same excess range ( figure 2a , lanes 7 to 10 ) confirming specificity of the nucleoprotein complexes association . these experiences suggested that the two shifted bands correspond to complexes joining of the -116/-1 region of the mfp-1 rat promoter , by binding to the pentanucleotide attgg sequence . these two bands could result from the existence of two isoforms of nfy - a subunit in the rat liver . unlabelled competitor nfy25 ( lanes 2 to 5 ) or nfy mut ( lanes 7 to 10 ) was added to the radiolabelled dna probe and the rat liver nuclear extract . lanes 1 and 6 contain only a 115 bp radiolabelled fragment with the nuclear extract . b. competition between the oligonucleotides nfy-1 or nfy-2 of thb and different competitors . unlabelled competitor nfy-1 ( lanes 2 to 4 ) or nfy-1 mut ( lane 5 ) was added to the radiolabelled dna probe and the rat liver nuclear extract . unlabeled competitor nfy-2 ( lanes 6 to 9 ) or nfy-2 mut ( lane 10 ) was added to the radiolabelled dna probe and the nuclear extract . lanes 1 and 6 contain only radiolabelled oligonucleotides nfy-1 and nfy-2 respectively with the nuclear extract . 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 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 ) . super - shift experiment using a 132 bp pcr probe ( -114 to + 18 ) corresponding to the mfp-1 promoter was conducted to confirm the presence of nfy transcription factor in nucleoprotein complexes ( figure 3 ) . the addition of the anti - nfy - b polyclonal antibody ( lane 3 ) decreased the intensity of the two complexes observed in lane 2 . due to a weak concentration , the purified anti - nfy - b polyclonal antibody was less effective ( lane 5 ) . however the addition of the anti - nfy - a monoclonal antibody ( lane 4 ) leads to the disappearance of the complexes . this latter monoclonal antibody targets the epitope localized at the c - terminus region of nfy - a known to be a conditional requirement for dna binding activity . the lack of complexes with anti - nfy - b antibodies possibly resulted either in the antibody binding to the nfy - dna binding domain or in the formation of too large a complex to enter in the gel . these data suggest that the inverted ccaat box of mfp-1 interacts , at least in vitro , with nfy transcription factor . super - shift experiments . lane 1 : 132 bp fragment without nuclear extract ; lane 2 : 132 bp fragment + nuclear extract ; lane 3 : 132 bp fragment + nuclear extract + anti - nfy - b polyclonal antibody ; lane 4 : 132 bp fragment + nuclear extract+ anti - nfy - a monoclonal antibody ; lane 5 : 132 bp fragment + nuclear extract + purified anti - nfy - b polyclonal antibody ; lane 6 : 132 bp fragment + pre - immuneserum . the nfy binding site of the mfp-1 promoter is flanked by three putative sp1 binding sites . to test the sp1 protein binding , a 78 bp fragment overlapping two putative sp1 binding sites at -55/-50 and at -37/-32 was used in competition with the 132 bp probe as well as a 22 bp probe containing a sp1 consensus binding site . therefore , the lack of competitive effect in shift assay of the 78 bp fragment or the sp1 probe reveals the absence of nuclear proteins displacement towards the two complexes ( complex i and ii ) that binds the 132 bp fragment ( figure 4 , lanes 2 to 4 and 5 to 7 respectively ) . these results suggest that the sp1 protein is not involved in formation of these complexes and the factors involved in the complexes observed ( figure 2a ) especially bind to the region -114 to -56 . competition experiments between the 132 bp probe and the 58 bp fragment ( -114/-56 region ) confirmed this result ( not shown ) . unlabelled competitor 78 bp fragment ( -60/+18 region of the mfp-1 ) ( lanes 2 to 4 ) or sp1 ( lanes 5 to 7 ) was added to the radiolabelled 132 bp dna fragment ( -114/+18 region of the mfp-1 ) and the rat liver nuclear extract . since nfy is involved in the transcriptional regulation of genes implicated in cholesterol synthesis , such as the farnesyl diphosphate synthase , we used this gene construct as a positive control for the repressive effect of 25-hydroxycholesterol and cholesterol , which gives 94% inhibition ( figure 5 ) . thus , we checked for a regulation of the mfp-1 and the thb gene expression by adding cholesterol into the medium . luciferase reporter constructs containing the -3400 bp mfp-1 or the -2800 bp thb promoter sequences upstream of the transcription initiation site ( -3400/+20 pgl2 and ptbluc ) were transfected into hepg2 cells in presence or in absence of sterols for 48h . in addition to cholesterol , 25-hydroxycholesterol was used since it increases the intracellular cholesterol concentration and acts by blocking cholesterol efflux . figure 5 shows that the promoter activity was down - regulated by cholesterol for the mfp-1 and thiolase b constructs ( -70% ) . effect of cholesterol on regulation of the mfp-1 , thb and fpps promoter reporter genes . the hepg2 cells were transiently cotransfected ( 5 hours ) with -3400/+20 pgluc , 132 pgluc , 58 pgluc , -2800 ptbgluc or pfppsgluc luciferase reporter plasmid and pch110 -galactosidase reporter plasmid and mock treated ( filled bars ) or treated ( open bars ) with 25-hydroxycholesterol+cholesterol ( mixture ) for 48 hours . relative luciferase activities are given , taking as 1 the activity obtained for each plasmid in the absence of sterols . 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 -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 . such interaction may be involved in the transcriptional regulation of the mfp-1 gene by cholesterol . in silico sequences analysis of rat peroxisomal genes encoding mfp-1 and thb reveals the presence of inverted ccaat boxes in both promoters , which are positioned from the transcription start sites at : -68/-64 for the mfp-1 gene and at -56/-60 and -105/-109 for the thb gene ( figure 1 ) . emsa experiments were performed to determine whether liver nuclear factors interact with these inverted ccaat boxes . the 115 bp probe corresponding to the positions -116 to -1 of the mfp-1 promoter region containing a putative nfy binding site and two putative sp1 binding sites ( figure 1 ) was used in emsa experiments . incubation of the 115 bp probe with nuclear extracts resulted in the formation of two close protein - dna complexes , a major one , complex i and a minor one , complex ii ( figure 2a , lanes 1 and 6 ) . these labelled complexes were competed by the unlabeled dna competitor . indeed , increased quantities of nfy25 oligonucleotide corresponding to the -76/-56 region ( figure 2a , lanes 2 to 5 ) completely abolished complexes formation for an excess of 500-fold . in contrast , complexes formation was unaffected by the use of the mutated oligonucleotide ( nfy mut ) over the same excess range ( figure 2a , lanes 7 to 10 ) confirming specificity of the nucleoprotein complexes association . these experiences suggested that the two shifted bands correspond to complexes joining of the -116/-1 region of the mfp-1 rat promoter , by binding to the pentanucleotide attgg sequence . these two bands could result from the existence of two isoforms of nfy - a subunit in the rat liver . unlabelled competitor nfy25 ( lanes 2 to 5 ) or nfy mut ( lanes 7 to 10 ) was added to the radiolabelled dna probe and the rat liver nuclear extract . lanes 1 and 6 contain only a 115 bp radiolabelled fragment with the nuclear extract . b. competition between the oligonucleotides nfy-1 or nfy-2 of thb and different competitors . unlabelled competitor nfy-1 ( lanes 2 to 4 ) or nfy-1 mut ( lane 5 ) was added to the radiolabelled dna probe and the rat liver nuclear extract . unlabeled competitor nfy-2 ( lanes 6 to 9 ) or nfy-2 mut ( lane 10 ) was added to the radiolabelled dna probe and the nuclear extract . lanes 1 and 6 contain only radiolabelled oligonucleotides nfy-1 and nfy-2 respectively with the nuclear extract . 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 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 ) . super - shift experiment using a 132 bp pcr probe ( -114 to + 18 ) corresponding to the mfp-1 promoter was conducted to confirm the presence of nfy transcription factor in nucleoprotein complexes ( figure 3 ) . the addition of the anti - nfy - b polyclonal antibody ( lane 3 ) decreased the intensity of the two complexes observed in lane 2 . due to a weak concentration , the purified anti - nfy - b polyclonal antibody was less effective ( lane 5 ) . however the addition of the anti - nfy - a monoclonal antibody ( lane 4 ) leads to the disappearance of the complexes . this latter monoclonal antibody targets the epitope localized at the c - terminus region of nfy - a known to be a conditional requirement for dna binding activity . the lack of complexes with anti - nfy - b antibodies possibly resulted either in the antibody binding to the nfy - dna binding domain or in the formation of too large a complex to enter in the gel . these data suggest that the inverted ccaat box of mfp-1 interacts , at least in vitro , with nfy transcription factor . super - shift experiments . lane 1 : 132 bp fragment without nuclear extract ; lane 2 : 132 bp fragment + nuclear extract ; lane 3 : 132 bp fragment + nuclear extract + anti - nfy - b polyclonal antibody ; lane 4 : 132 bp fragment + nuclear extract+ anti - nfy - a monoclonal antibody ; lane 5 : 132 bp fragment + nuclear extract + purified anti - nfy - b polyclonal antibody ; lane 6 : 132 bp fragment + pre - immuneserum . the nfy binding site of the mfp-1 promoter is flanked by three putative sp1 binding sites . to test the sp1 protein binding , a 78 bp fragment overlapping two putative sp1 binding sites at -55/-50 and at -37/-32 was used in competition with the 132 bp probe as well as a 22 bp probe containing a sp1 consensus binding site . therefore , the lack of competitive effect in shift assay of the 78 bp fragment or the sp1 probe reveals the absence of nuclear proteins displacement towards the two complexes ( complex i and ii ) that binds the 132 bp fragment ( figure 4 , lanes 2 to 4 and 5 to 7 respectively ) . these results suggest that the sp1 protein is not involved in formation of these complexes and the factors involved in the complexes observed ( figure 2a ) especially bind to the region -114 to -56 . competition experiments between the 132 bp probe and the 58 bp fragment ( -114/-56 region ) confirmed this result ( not shown ) . unlabelled competitor 78 bp fragment ( -60/+18 region of the mfp-1 ) ( lanes 2 to 4 ) or sp1 ( lanes 5 to 7 ) was added to the radiolabelled 132 bp dna fragment ( -114/+18 region of the mfp-1 ) and the rat liver nuclear extract . since nfy is involved in the transcriptional regulation of genes implicated in cholesterol synthesis , such as the farnesyl diphosphate synthase , we used this gene construct as a positive control for the repressive effect of 25-hydroxycholesterol and cholesterol , which gives 94% inhibition ( figure 5 ) . thus , we checked for a regulation of the mfp-1 and the thb gene expression by adding cholesterol into the medium . luciferase reporter constructs containing the -3400 bp mfp-1 or the -2800 bp thb promoter sequences upstream of the transcription initiation site ( -3400/+20 pgl2 and ptbluc ) were transfected into hepg2 cells in presence or in absence of sterols for 48h . in addition to cholesterol , 25-hydroxycholesterol was used since it increases the intracellular cholesterol concentration and acts by blocking cholesterol efflux . figure 5 shows that the promoter activity was down - regulated by cholesterol for the mfp-1 and thiolase b constructs ( -70% ) . effect of cholesterol on regulation of the mfp-1 , thb and fpps promoter reporter genes . the hepg2 cells were transiently cotransfected ( 5 hours ) with -3400/+20 pgluc , 132 pgluc , 58 pgluc , -2800 ptbgluc or pfppsgluc luciferase reporter plasmid and pch110 -galactosidase reporter plasmid and mock treated ( filled bars ) or treated ( open bars ) with 25-hydroxycholesterol+cholesterol ( mixture ) for 48 hours . relative luciferase activities are given , taking as 1 the activity obtained for each plasmid in the absence of sterols . 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 -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 . 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 -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 -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 g / ml ) . 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 g / 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 -70c . 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 . 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 ( 94c for 30 s , 50c for 45s and 72c 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 ( 94c for 30 s , 47c for 45 s and 72c 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 ) . sequences of the oligonucleotides competitors 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 . 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 ci [ a - p ] dctp ( 3000 ci / mmol , amersham , saclay , france ) , 20 u dna polymerase i ( klenow fragment , promega ) and 500 m each of datp , dgtp and dttp ( promega ) . reaction was incubated for 45 min at 30c and then stopped by addition of 0.5 m edta , ph 8 . dna - protein binding reaction contained 10 mm -mercaptoethanol , 5 mm edta , migration buffer ( 0.01% bromophenol blue , 30% glycerol and 5 mg / ml bsa ) , 1 g poly [ di - dc ] , 1 g sonicated dna salmon sperm , 20 g 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 4c . 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 80c , and exposed overnight at -70c to a kodak film ( x - omat ar ) . hepg2 cells ( 7.10cells / 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 -galactosidase , pharmacia ) . cells were incubated for 5 h in optimem ( gibco ) and then for 48 h in the absence or presence of sterols ( 10 g / ml of cholesterol and 1 g / ml 25-hydroxycholesterol ) . cell extracts were prepared with lysis buffer of the luciferase assay kit ( promega ) and assayed for -galactosidase ( galactolight kit tropix , applied biosystems , courtaboeuf , france ) and luciferase activities ( luciferase assay kit , promega ) . the protein concentration ( bradford assay ) and -galactosidase activity were measured in each sample and values were used to normalized luciferase activities . 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 g / ml ) . 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 g / 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 -70c . 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 . 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 ( 94c for 30 s , 50c for 45s and 72c 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 ( 94c for 30 s , 47c for 45 s and 72c 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 ) . sequences of the oligonucleotides competitors 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 . 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 ci [ a - p ] dctp ( 3000 ci / mmol , amersham , saclay , france ) , 20 u dna polymerase i ( klenow fragment , promega ) and 500 m each of datp , dgtp and dttp ( promega ) . reaction was incubated for 45 min at 30c and then stopped by addition of 0.5 m edta , ph 8 . dna - protein binding reaction contained 10 mm -mercaptoethanol , 5 mm edta , migration buffer ( 0.01% bromophenol blue , 30% glycerol and 5 mg / ml bsa ) , 1 g poly [ di - dc ] , 1 g sonicated dna salmon sperm , 20 g 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 ) 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 80c , and exposed overnight at -70c to a kodak film ( x - omat ar ) . hepg2 cells ( 7.10cells / 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 -galactosidase , pharmacia ) . cells were incubated for 5 h in optimem ( gibco ) and then for 48 h in the absence or presence of sterols ( 10 g / ml of cholesterol and 1 g / ml 25-hydroxycholesterol ) . cell extracts were prepared with lysis buffer of the luciferase assay kit ( promega ) and assayed for -galactosidase ( galactolight kit tropix , applied biosystems , courtaboeuf , france ) and luciferase activities ( luciferase assay kit , promega ) . the protein concentration ( bradford assay ) and -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 . this work has been supported by the regional council of burgundy , ifr n92 and by the gdr - cnrs n2583 .
background-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 -oxidation has been reported.resultsthis work studied the interactions between nfy factor with the rat gene promoters of two enzymes of the fatty acid -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.conclusionsthis work describes for the first time a nfy interaction with promoter sequences of the peroxisomal -oxidation encoding genes . 
 it suggests that cholesterol would negatively regulate the expression of genes involved in -oxidation , which generates the initial precursor for its own biosynthesis , via at least the nfy transcription factor .