HUMANMUTATION 29(1),123^129,2008

RESEARCH ARTICLE

Functional Analysis of Promoter Variantsin the Microsomal Triglyceride Transfer Protein(MTTP) Gene

Diana Rubin,1,2� Alexandra Schneider-Muntau,1 Maja Klapper,3 Inke Nitz,3 Ulf Helwig,1,2

Ulrich R. Folsch,2 Jurgen Schrezenmeir,1 and Frank Doring3

1Institute of Physiology and Biochemistry of Nutrition, Federal Research Center for Nutrition and Food, Kiel, Germany; 2Department of GeneralInternal Medicine, University Clinic Schleswig-Holstein, Campus Kiel, Kiel, Germany; 3Department of Molecular Nutrition, Institute of HumanNutrition and Food Science, Christian-Albrechts University of Kiel, Kiel, Germany

Communicated by David S. Rosenblatt

The microsomal triglyceride transfer protein (MTTP) is required for the assembly and secretion ofapolipoprotein B (apoB)-containing lipoproteins from the intestine and liver. According to this function,polymorphic sites in the MTTP gene showed associations to low-density lipoprotein (LDL) cholesterol andrelated traits of the metabolic syndrome. Here we studied the functional impact of common MTTP promoterpolymorphisms rs1800804:T4C (�164T4C), rs1800803:A4T (�400A4T), and rs1800591:G4T(�493G4T) using gene-reporter assays in intestinal Caco-2 and liver Huh-7 cells. Significant results wereobtained in Huh-7 cells. The common MTTP promoter haplotype �164T/�400A/�493G showed about two-fold lower activity than the rare haplotype �164C/�400T/�493T. MTTP promoter mutant constructs �164T/�400A/�493T and �164T/�400T/�493T exhibited similar activity than the common haplotype. Activities ofmutants �164C/�400A/�493G and �164C/�400A/�493T resembled the rare MTTP promoter haplotype.Electrophoretic mobility shift assays (EMSAs) revealed higher binding capacity of the transcriptional factorSterol regulatory element binding protein1a (SREBP1a) to the �164T probe in comparison to the �164Cprobe. In conclusion, our study indicates that the polymorphism �164T4C mediates different activities ofcommon MTTP promoter haplotypes via SREBP1a. This suggested that the already described SREBP-dependent modulation of MTTP expression by diet is more effective in �164T than in �164C carriers.Hum Mutat 29(1), 123–129, 2008. rr 2007 Wiley-Liss, Inc.

KEY WORDS: microsomal triglyceride transfer protein; MTTP; promoter; metabolic syndrome

INTRODUCTION

The human microsomal triglyceride transfer protein (MTTP;MIM] 157147) is a heterodimer of the large and unique 97-kDasubunit and the 55-kDa protein disulfide isomerase. The MTTPsubunit possesses lipid transfer activity and plays a crucial role inthe assembly and secretion of apolipoprotein B (apoB)-containinglipoproteins in the intestine and liver [White et al., 1998; Levyet al., 2002]. It catalyzes the transfer of neutral lipids to thenascent apoB molecule. This stabilizes the newly synthesized apoB[Leung et al., 2000], and facilitates further processing, leading tosecretion of chylomicrons and very low-density lipoprotein(VLDL). Since MTTP is a key protein in lipid metabolism,associations between MTTP variants and lipid parameters orrelated phenotypes have been investigated. There is evidence fromseveral studies that the minor alleles of linked [Ledmyr et al.,2002] promoter rs1800591:G4T/rs1800804:T4C (�493G4T/�164T4C; numbering relative to transcriptional start site) andrs3816873:T4C (Ile128Thr) polymorphisms correspond to lowerLDL levels and protect against other traits of the metabolicsyndrome [Bernard et al., 2000; Bjorn et al., 2000; Garcia-Garciaet al., 2005; Karpe et al., 1998; Ledmyr et al., 2002, 2004; Lundahlet al., 2006; Phillips et al., 2004; Rubin et al., 2006; St-Pierre et al.,2002; Yamada et al., 2006]. No associations or oppositeassociations were found in other studies [Berthier et al., 2004;

Chen et al., 2003; Couture et al., 2000; Herrmann et al., 1998; Juoet al., 2000; Li et al., 2005; Lundahl et al., 2002; Stan et al., 2005].

The functional impact of associated polymorphic sites in theMTTP gene has been studied to some extent. Recently, Ledmyret al. [2006] demonstrated that the minor allele ofrs3816873:T4C (Ile128Thr) confers reduced stability and LDLcholesterol particle binding of the MTTP. The impact of theMTTP promoter polymorphism rs1800591:G4T (�493G4T,relative to the transcription start site) was studied in liver HepG2cells using minimal promoter reporter assays. This study showedtwo-fold lower activity of the construct containing the commonallele (G) in comparison to the minor (T) [Karpe et al., 1998].Since polymorphism rs1800804:T4C (�164T4C, relative to the

Published online 13 September 2007 in Wiley InterScience (www.interscience.wiley.com).

DOI10.1002/humu.20615

Received12 April 2007; accepted revisedmanuscript 24 June 2007.

Grant sponsor: Federal Ministry for Education and Research (Bun-desministerium fuº r Bildung und Forschung [BMBF]), Germany; Grantnumber: AZ 0312823A/B.

�Correspondence to: Diana Rubin, Federal Research Center forNutrition and Food, Institute of Physiology and Biochemistry ofNutrition, Hermann-Weigmann-Str.1,24103 Kiel, Germany.E-mail: diana.rubin@bfel.de

rr 2007 WILEY-LISS, INC.

transcription start site) and other described MTTP promoter SNPs[Berthier et al., 2004; Ledmyr et al., 2002] could also influence thetranscriptional activity of the MTTP promoter, we studied thecombined effects and relative importance of promoter SNPs in thecontext of large MTTP promoter segments. For this, we constructedand determined the transcriptional activity of MTTP promoterhaplotypes and mutants in intestinal Caco-2 and liver Huh-7 cellsusing promoter reporter assays. In addition, we searched fortranscriptional factors which bind with different binding capacityto MTTP promoter variants. Based on these findings, a hypothesisregarding variant specific modulation of MTTP promoter activityhas been provided. The following polymorphisms were used in thisstudy: rs1800804:T4C (�164T4C, relative to the transcriptionstart site), rs1800803:A4T (�400A4T), rs6532821 (�410A4G),rs1800591:G4T (�493G4T), and c.�466A4T (relative to thetranslational start site, refer to NM_000253.1). The latterpolymorphism is �388 bp upstream from the transcription start site.

MATERIALSANDMETHODSSequencing

DNA was isolated from buffy coat (100 ml) using E.Z.N.A.s

Blood DNA MiniKits (Peqlab Biotechnologie GmbH, Erlangen,Germany) according to the manufacturer’s instructions. Thepromoter of the MTTP gene was sequenced from�690 to �1relative to the transcriptional start site. All PCR and sequencingprimers were purchased from MWG Biotech AG (Ebersberg,Germany). PCR was carried out in a volume of 25 ml containing15 ng DNA, 0.5 ml of each primer [10 pmol/ml], 0.75 ml MgCl2[50 mM], 0.2 ml Taq Polymerase [5 u/ ml], 0.125ml of each dNTP[10 mM], 2.5 ml of PCR buffer (10x), and sterile water to a finalvolume of 25 ml (Taq Polymerase, PCR buffer, and MgCl2 fromInvitrogen, Karlsruhe, Germany; dNTPs from Fermentas GmbH,St. Leon-Rot, Germany). To get signals in the linear range of thePCRs, 25 amplification cycles were used. Sequencing wasperformed by terminator cycle sequencing using Big Dye chemistryand ABI 3700 capillary DNA sequencer (Applied Biosystems,Foster City, CA). Sequences of PCR primers and TaqMan assayprimers and probes are available on request.

Cell Culture

Caco-2 and HeLa cells were purchased from DSMZ GmbH(Braunschweig, Germany). Huh-7 cells were a kind gift fromDr. Bartenschlager (Heidelberg, Germany). The cells were culturedin either DMEM (Huh-7) or MEM (Caco-2) supplemented with10% (HeLa, Huh-7) or 20% (Caco-2) newborn calf serum and1 mM nonessential amino acids in a humidified incubator at 371Cunder an atmosphere of 5% CO2. Cells were passaged atpreconfluent densities by use of 0.05% trypsin/0.02% EDTAsolution (Biochrom AG, Berlin, Germany) every 2–3 days.

MTTP (GeneID:4547) Promoter-Reporter Constructs

The dual luciferase system was used (Promega, Madison, WI).Genomic DNA from one �493G/G- and one �493T/Tcarrier wasused to amplify an MTTP promoter segment from �966 to �1relative to the transcriptional start site with the following primers:50-CACCGCCA AAACATTCCCACAG-30 (f), 50-GACCCTCTTCAGAACCTG-30 (r). All amplifications were performed for 30cycles at 951C for 45 seconds, a touchdown from 581C to 541C for30 seconds, and 721C for 1 minute in a buffer containing 2 mMMgSO4, 10 mM dNTP, and 0.5 U Pfu polymerase. Cloningprocedures were performed using Gateway Technology (Invitro-gen, Karlsruhe, Germany), described previously [Li et al., 2006].

attR-sites were inserted into an EcoRV (Promega, Mannheim,Germany) blunt-end site of the reporter plasmid pGL4.10[luc2]encoding Firefly luciferase. The 966-bp fragment of the MTTPgene promoter was cloned by recombination with LR Clonase(Invitrogen, Karlsruhe, Germany) into attR-sites of pGL4.10[luc2]from vector pENTR-hFABP2-Prom-attL containing attL-sitesresulting in final pGL4.10-MTTP promoter-reporter constructs.The empty pGL4.10[luc2] vector served as a negative control.Introduction of mutation into MTTP promoter constructs wasperformed using Quick ChangeTM in vitro mutagenesis Kit(Stratagene, La Jolla, CA) using a complementary set of one ofthe following primers: �493G-T: 50-CTATCTACTTTAACATTATTTTGAAGTGATTGGTTGTGGTATGAATTAACAG-30; and �164T-C: 50-CATTTAAAGTTTCCTCATTGGGTGAAAAAAATTAAAAAGAGTGAGAGACTG-30.

TransientTransfections and ReporterAssays

At 24 hr before transfection, 1� 104cells were seeded on96-well plates. Media was changed before transfection and every48 hr. Transfections were done using FuGENE 6 (Roche,Mannheim, Germany) according to the manufacturer’s instruc-tions. Cells were cotransfected with 47.5 ng pGL4.10[luc2]-MTTPconstructs, or pGL4.10[luc2] as negative control and 2.5 ngpGL4.74[hRluc/TK] vector encoding Renilla luciferase as internalcontrol. Luciferase activities were measured 4 days (Huh-7) or 6days (Caco-2) after transfection by Dual-Luciferases ReporterAssay System (Promega, Germany). A total of 4� 103 HeLa cells/well on a 96-well plate were used for transfections with expressionplasmid SREBP1a. The open reading frame of SREBP1a wasamplified by PCR and subcloned in expression vector pDest40using Gateway technology as described [Nitz et al., 2005].

Electromobility Shift Assay and Supershift

For electromobility shift assay (EMSA), sets of complimentaryoligonucleotides containing the alleles of polymorphism�164T4C were designed: 50-CATTTAAAGTTTCCTCAT/CTGGGTGAAAA-30. Nuclear extracts were prepared from HeLacells transfected with expression plasmids SREBP1a with the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce,Rockford, IL). The EMSAs were performed with the LightShiftChemiluminescent EMSA Kit (Pierce) according to manufac-turer’s instructions. The reaction mixture (20 ml) contained 1 mMTris, 50 mM KCl, 1 mM 1,4-dithiothreitol (DTT), 1mg Poly-Desoxyinosin/Desoxycytosin [Poly(dI-dC)], 2.5% glycerol, and0.1 mM EDTA. DNA-transcription factor complexes wereresolved on a native 4% Tris-borate-EDTA polyacrylamide gelin 0.5� TBE buffer and transferred to a nylon membrane(Amersham Bioscience, Piscataway, NJ). Biotinylated DNA wasdetected with streptavidin-horseradish peroxidase conjugate andchemiluminescent substrate luminol using a ChemiluminescenceNucleic Acid Detection Module (Pierce, Milwaukee, WI). For asupershift, 2mg of SREBP1a antibody (Santa Cruz Biotechnology,Santa Cruz, CA) was added to the reaction mixture before thenuclear extract and preincubated for 10 minutes.

Statistical Analysis

Using HaploView 3.32-Software [Barrett et al., 2005], haplo-type analysis and linkage disequilibrium analysis were performed.All results were controlled for their statistical significance by one-way analysis of variance (ANOVA) followed by a Newman-Keulspost hoc test. A value of Po0.05 was considered to be statisticallysignificant.

124 HUMANMUTATION 29(1),123^129,2008

Human Mutation DOI 10.1002/humu

RESULTSPolymorphisms and Haplotypes inMTTP Promoter

Identification of polymorphisms was accomplished by resequen-cing the proximal promoter region of MTTP using DNA samplesfrom 32 unrelated persons obtained from the MICK cohort[Lindner et al., 2005, 2006; Rubin et al., 2006]. Variants werefound at position �164T4C (relative to the transcription startsite, minor allele frequency 0.234), �388G4A (0.047),�400A4T (0.281), �410A4G (0.016), and �493G4T(0.234) upstream from the transcription start site of MTTP. Allfive polymorphisms were reported in public databases andliterature [Berthier et al., 2004; Karpe et al., 1998]. Polymorph-isms �164T4C and �493G4T were in complete linkagedisequilibrium (D05 1.0) as described [Kel et al., 2003; Ledmyret al., 2002]. Next, haplotypes were computed with the identifiedpolymorphisms. As shown in Table 1, two haplotypes accountedfor more than 90% of the haplotypes observed. These haplotypesare defined by the major and minor alleles of SNPs �164T4C,�400A4T, and �493G4T. The major alleles of polymorphism�388G4A and �410A4G are present in both haplotypes. Based

on these findings, we focused on the two MTTP promoterhaplotypes.

MTTPmRNA Expression and PromoterActivity inIntestinal Caco-2 and Liver Huh-7 Cells

For analysis of MTTP promoter constructs intestinal Caco-2and liver Huh-7 cells were selected. As shown in Fig. 1a, RT-PCRrevealed high MTTP mRNA expression in postconfluent CaCo-2cells. In Huh-7 cells, the MTTP mRNA level is higher underpostconfluent conditions but is also detectable in preconfluentcells (Fig. 1b). According to these results, activity obtained from aMTTP promoter-reporter construct reached a maximum inpostconfluent CaCo-2 (Fig. 1c) and Huh-7 cells (Fig. 1d).Therefore, further experiments were done under postconfluentcell culture conditions.

Activity ofMTTP Promoter-Reporter ConstructsHarboring Haplotypes and Alleles of Polymorphisms�164, �400, and �493

To study the functional influence of MTTP promoter polymorph-isms, activities of haplotypes and derived mutants were determinedby promoter-reporter assays. The constructs contained functional966-bp segments of the MTTP promoter were transiently expressedin Caco-2 (Fig. 2a) and HuH-7 cells (Fig. 2b). For all constructs, weobtained similar results in both cell lines, but only in Huh7 cells diddifferences in promoter activities reach significance. The commonMTTP haplotype �164T/�400A/�493G (construct no. 1) leads toabout two-fold lower reporter activity relative to the minor haplotype�164C/�400T/�493T (no. 6). Mutants �164T/�400T/�493T(no. 5) and �164T/�400A/�493T (no. 3) showed similar activitythan the common haplotype. Activities of mutants �164C/�400A/�493T (no. 4) and �164C/�400A/�493G (no. 2) resembled the

TABLE 1. Haplotypes (HT) CreatedWithMTTP Promoter Polymorphisms�

HT ^493 ^410 ^400 ^388 ^164 %

I G A A G T 71.90II T A T G C 18.70III G A T G T 4.65IV T A T A C 4.65

�rs1800591:G4T (�493G4T, relative to the transcription start site), rs1800803:A4T(�400A4T), c.�466A4T (�388G4A), and rs1800804:T4C (�164T4C). The fourmost common haplotypes are given. Haplotypes were estimated based on sequencingdata obtained from 32 unrelated persons of theMICK cohort. Note: c.�466A4T is re-lative to the translational start site, refer toNM___000253.1.This polymorphism is �388bp upstream from the transcription start site.

FIGURE 1. Expression of MTTP mRNA (a,b) and activity of MTTP promoter constructs (c,d) in Caco-2 (a,c) and Huh-7 (b,d) cells.a,b:Total RNAwas isolated fromCaco-2 (a) and Huh-7 cells (b) 1^9 days after seeding. Levels of mRNA ofMTTP andGAPDHwereanalyzed by RT-PCR.The resulting cDNA products with predicted length of 428bp (MTTP) and 509bp (GAPDH) were separated on1%agarose gel. Marker:GeneRulerTM100-bpDNA Ladder Plus (Fermentas, St. Leon-Rot,Germany). c,d:Caco-2 (c) andHuh-7 (d) cellswere transiently transfected with MTTP luciferase reporter gene construct.The nucleotide sequence �966^�1 relative to transcrip-tional start ofMTTPwas used.ThisMTTP gene promoter segment contains common haplotypes with alleles �164T, �400A, �493G(seeTable1).The activity ofFire£y luciferase reporter wasmeasured 2^6 days after transfection and normalized for transfection e⁄-ciency (Renilla luciferase). Relative luciferase activities of negative control pGl4.10[luc2] were determined for each condition andwere subtracted from corresponding activities ofMTTP promoter constructs.The relative reporter activity 2 days after transfectionwas set to100% and the other activitieswere referenced to it. Bars indicatemeanvalue7standarderrorof themean (SEM) for at leastsix experiments, each one performed in triplicate.

HUMANMUTATION 29(1),123^129,2008 125

Human Mutation DOI 10.1002/humu

rare haplotype. Taken together, the polymorphism �164T4C playsa decisive role in determining different activities of MTTP promoterhaplotypes.

Di¡erences in Binding of SREBP1a toMTTP Probes�164Tand �164C

To see if MTTP promoter regions containing the alleles �164Tor �164C show differences in binding capacity, we tested

SREBP1a using EMSA. This transcriptional factor was selectedbecause MTTP expression is negatively regulated by SREBP [Satoet al., 1999] and the sequence AGTTTCCTCAT/CTT in theregion �174 to �162 contains a putative sterol regulatory element[Hagan et al., 1994; Sato et al., 1999]. As shown in Fig. 3, withthe �164T probe we obtained a stronger shift when using thenuclear extract from HeLa cells transfected with SREBP1a (Fig. 3,lanes 5 and 9) in comparison to the control reaction with an emptyplasmid (Fig. 3, lane 10). No signal was obtained with the �164Cprobe (Fig. 3, lane 2). The shift obtained by the labeled �164Tprobe could be partially competed by excess of unlabeled �164T(Fig. 3, lane 6) but not with the �164C (Fig. 3, lane 7)oligonucleotide. A supershift of the signal was not detected. Thismay be due to the antibody against SREBP1a, which may beimpaired in EMSA experiments as it functions in Western blots.Based on EMSA experiments, we conclude that SREBP1a bindsstrongly and specifically to MTTP promoter segment containingthe major �164T allele.

DISCUSSION

In the recent years, phenotyping of intestinal- and liver-specificMTTP knockout mice [Raabe et al., 1999; Xie et al., 2006]demonstrated that MTTP is a key protein for assembly and

FIGURE 2. Activity ofMTTP promoter-reporter constructs harbor-ing haplotypes and alleles of polymorphisms �164, �400, and�493 in Caco-2 (a) and Huh-7 (b) cells. Caco-2 (a) and Huh-7(b) cellswere transiently transfectedwithMTTPpromoter-reporterconstructs. Nucleotide sequences �966 to �1 relative to tran-scriptional start ofMTTPwereused.TheMTTPgenepromoter seg-ments contain the following alleles at position �164, �400, and�493:TAG (construct 1), CAG (2),TAT (3), CAT (4),TTT (5), andCTT (6). The activity of Fire£y luciferase reporter wasmeasured 4 days (Huh-7) or 6 days (Caco-2) after transfectionand normalized for transfection e⁄ciency (Renilla luciferase).Relative luciferase activities of negative control pGl4.10[luc2]were determined for each condition andwere subtracted fromcor-respondingactivities ofMTTPpromoterconstructs.The relative re-porter activity of construct 1 was set to 100% and theother activities were referenced to it. Bars indicate mean value7SEM for at least six experiments, each one performed in triplicate(�Po0.05; ANOVA, Newman-Keuls post test).

FIGURE 3. Di¡erences in binding of SREBP1a to MTTP probes�164Tand �164C. An EMSAwas performed with labeled oligo-nucleotide containingMTTPpromoter segmentswith the�164Cor �164Tallele. Probes were incubated with nuclear protein ex-tracts fromHeLa cells transfected with SREBP1a plasmid (lanes1^9) or control plasmid (lanes 10 and 11). Lanes 1 and 4: Nega-tive reactions with labeled probes �164C (1) or �164T (4) andwithout nuclear extracts. Lanes 2, 5, and 9: Positive reactionswith labeled probes �164C (2) or �164T (5,9) and1.5 ml of HeLanuclear protein extract transfected with SREBP1a plasmid.Lanes 3, 6, and 7: Speci¢c competitor reaction with same com-ponents as lane 2 and 5 addedwith100-fold excess of unlabeledoligonucleotides �164C (3,7) or �164T (6). Lane 8: Supershiftreactionwith same components as lane 5 and 0.2 mg of antibodyagainst SREBP1a. Lanes 10 and 11: Control reactions withlabeled probe �164T,with (11) or without (10) 100-fold excess ofunlabeled oligonucleotides �164T and 1.5 ml of HeLa nuclearprotein extract transfectedwith control plasmid.

126 HUMANMUTATION 29(1),123^129,2008

Human Mutation DOI 10.1002/humu

secretion of chylomicrons and VLDL. In humans, loss-of-functionmutations of MTTP resulted in hypo- and abetaliproteinemiaassociated with hypocholesterolemia, mild fat malabsorption, andhepatic steatosis [Linton et al., 1993; Scanu et al., 1974]. Thephysiological importance of MTTP was also emphasized byassociation studies, which provide evidence that a haplotype,defined by the minor alleles of polymorphisms Ile128Thr,�164T4C, and �493G4T, is associated with a slight decreasein LDL cholesterol and other traits of the metabolic syndrome[Bernard et al., 2000; Bjorn et al., 2000; Garcia-Garcia et al.,2005; Karpe et al., 1998; Ledmyr et al., 2002, 2004; Lundahl et al.,2006; Phillips et al., 2004; Rubin et al., 2006; St-Pierre et al.,2002; Yamada et al., 2006]. Here we provided functional investi-gations of MTTP promoter haplotypes composed of polymorphisms�164T4C, �400A4T, and �493G4T in human hepatic andintestinal cells. By means of reporter assays using MTTP promoterconstructs, we found in both cell lines an almost two-fold loweractivity of the common haplotype than the rare haplotype. Thisresult led to the question of which polymorphism(s) is causal fordifferent activities. Karpe et al. [1998] reported two-fold lowerreporter activity of a 32-bp minimal MTTP promoter constructbearing the common �493G allele in comparison to the minor�493 allele. No difference was found with �400A4T constructs.This study does not take into account that other regulatoryelements and sequences of the MTTP promoter could influencethe impact of a particular polymorphism. Thus, we used a muta-genesis approach and investigated 966-bp MTTP promoterfragments that contain recognition elements for peroxisome proli-ferator-activated receptors (PPARs) [Ameen et al., 2005], hepaticnuclear factor (HNF)-1 and HNF-4 [Hagan et al., 1994; Sheenaet al., 2005], activator protein (AP)-1 [Hagan et al., 1994; Sheenaet al., 2005], insulin signaling pathway [Hagan et al., 1994], andSREBPs in the regions �124 to �109 and �175 to �162 [Haganet al., 1994; Sato et al., 1999]. This strategy revealed that insteadof �493G4T, the polymorphism at position �164 is responsiblefor the different activity of MTTP promoter haplotypes, at least inliver cells. Therefore, in the context of native sequences thepolymorphism �164T4C seems to be of functional relevance.

Additionally, we were able to show with EMSA that SREBP1abinds with higher capacity to the MTTP promoter region bearingthe common �164T allele in comparison to the minor �164Callele. This indicates that the low activity of the common MTTPpromoter haplotype is essentially determined by the �164T allelevia SREBP1a suppression. The downregulation of MTTP bySREBP1-1 and -2 was described a long time ago [Hagan et al.,1994; Sato et al., 1999]. Since oleate [Qiu et al., 2005] as well aspolyunsaturated fatty acids [Sekiya et al., 2003] reduce SREBP-1activity, the resulting abrogate suppression of MTTP could bemore effective in �164T than in �164C carriers. This possiblediet–gene interaction may explain conflicting results of associationstudies [Bernard et al., 2000; Berthier et al., 2004; Bjorn et al.,2000; Chen et al., 2003; Couture et al., 2000; Garcia-Garcia et al.,2005; Herrmann et al., 1998; Juo et al., 2000; Karpe et al., 1998;Ledmyr et al., 2002, 2004; Li et al., 2005; Lundahl et al., 2002,2006; Phillips et al., 2004; Rubin et al., 2006; St-Pierre et al.,2002; Stan et al., 2005; Yamada et al., 2006] and should be testedin intervention studies or case–control cohorts. Notably, poly-morphic sites in the SREBP1 locus are associated with type 2diabetes and obesity [Eberle et al., 2004; Laudes et al., 2004].Therefore, on the basis of functional interaction between SREBP1and MTTP, it seems to be a promising approach to study the effectsof polymorphism combinations in these genes on traits of themetabolic syndrome.

Although our investigations strongly indicate an important roleof �164T4C polymorphism in determining different transcrip-tional activities of MTTP promoter haplotypes, the linkedIle128Thr polymorphism [Ledmyr et al., 2002] has to be alsotaken into account for functional understanding of associationstudies. Recently, the Ile128Thr polymorphism was examined,whereby the Thr128 variant of MTTP has lower stability andligand binding properties than the Ile128 wild-type protein[Ledmyr et al., 2006]. The promoter haplotype with the lowactivity is almost completely coupled with the Thr128 allele asdescribed by Ledmyr et al. [2002]. The resulting MTTP variant iscomposed of a partial loss-of-function and partial gain-of-functionpolymorphism. The relative importance of these opposite effects isnot known and need to be addressed in future studies. It should bealso noted, that there is no evidence in vivo for genotype specificexpression and function of MTTP promoter haplotypes andexon polymorphism, since it is very difficult to obtain healthyprobes from the intestine or liver. However, based on theconvincing studies in vitro, we propose similar effects of MTTPvariants in vivo.

In conclusion, in the present study we showed by a mutagenesisapproach and transient transfections of 966-bp MTTP promoterconstructs, that the polymorphism �164T4C is mainly respon-sible for the different activities of the MTTP promoter haplotypes.We showed the differential binding of SREBP1a to the MTTPpromoter region containing the �164T4C polymorphism,suggesting that already described SREBP1a-dependent upregula-tion of MTTP expression by diet is more effective in �164T thanin �164C carriers. Therefore, we provide a molecular basis ofvariant-specific regulation of MTTP as a susceptibility gene forLDL cholesterol and other traits of the metabolic syndrome.

ACKNOWLEDGMENTS

This work was supported by the BMBF Project ‘‘Fat andMetabolism–Gene Variation, Gene Regulation and Gene Func-tion.’’ We thank F. Repenning and Y. Dignal for excellent technicalassistance.

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