Hepatocyte Nuclear Factor-4 Activates Medium Chain Acyl-CoA ...

Vol 268, No.

T H EJOURNAL OF BIOLOGICAL CHEMISTRY (D 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Issue of July 5,pp. 13805-13810,1993 Printed in U.S.A.

Hepatocyte Nuclear Factor-4 Activates Medium Chain Acyl-CoA Dehydrogenase Gene Transcription by Interacting with a Complex Regulatory Element” (Received for publication, December 1, 1992, and in revised form, March 15, 1993)

M. Eric CarterSg, Tod GulickliII ,Bradley D. Raisher**, Teresa CairaS, John A. A. LadiasSS, David D. Moorelill, and Daniel P. Kelly* $8 From the Departments of $Medicine and **Pediatrics, Washington University, St. Louis, Missouri63110, the $$Department of Medicine, New England Deaconess Hospital, Harvard Medical School, Boston, Massachusetts 02115, and the YDepartmnt of Molecular Biology, Massachusetts General Hospital and the IIGenetics Department, Harvard Medical School, Boston, Massachusetts 02114

We have recently identified a complex transcrip- rate-limiting step in fatty acid &oxidation (1).MCAD defitional regulatory element in the medium chain acyl- ciency is a common inherited metabolic disorder and is an CoA dehydrogenase (MCAD) genepromoterregion important cause of childhood Reye-like syndrome, hypoglythat confers response to retinoids through interaction cemia, and sudden death (2-6). The clinical manifestations of with receptors for all-trans-retinoic acid(RARs) and MCAD deficiency vary but often become evident when oxi9-cis-retinoic acid (RXRs) (Raisher, B. D., Gulick, T., dative energy demands are high (e.g. during the periods of Zhang, Z., Strauss, A. W., Moore, D. D., and Kelly, D. fasting associated with an infectious illness). We have shown P. (1992) J. Biol. Chem. 267,20264-20269). We ex- previously that MCADgene expression is highly regulated amined the interaction of this element (RAREM~AD) among tissues and during development and that thelevel of with hepatocyte nuclear factor-4 (HNF-4), an orphan its expression correlates with tissue-specific oxidative energy receptor with a tissue expression pattern similar to requirements (7, 8). Thus, MCAD mRNA is abundant in rat that of MCAD. Electrophoretic mobility shift assays and cotransfection experiments showed that HNF-4 heart, kidney, and liver, all of which have high oxidative binds with high affinity RAREMcAD to to activate tran- energy demands and a preference for fatty acid energy subscription by an RXR-independent mechanism. Muta- strates. Themechanisms involved in regulating expression of tional analysis revealed that the MCAD HNF-4 re- the MCAD gene and other nuclear genes encoding enzymes sponse element consists of an imperfect direct repeat in mitochondrial oxidative pathways are unknown. As an initial stepin determining the molecular mechanisms homologous to the consensus sequence for binding to the thyroid receptor/RAR/RXR subgroup of receptors involved in transcriptional regulation of nuclear genes inand thatdistinct sequence requirements dictateHNF- volved in mitochondrial fatty acid oxidation andto gain 4 binding and transactivation. Mobility shift assays further insight intothe pathogenesis of inherited MCAD withanti-HNF-4antiserumdemonstrated thatthe deficiency, we have begun to delineate cis-acting regulatory rat elements within the MCAD gene promoter region (9). ReMCAD HNF-4 response element binds endogenous liver HNF-4 supporting its role in the regulation of cently, we identified a novel retinoic acid-responsive element MCAD gene expressionin vivo. Thus, HNF-4 activates (RARE) within this region that confers transcriptional actiMCAD gene transcription via a complex regulatory vation by interacting with retinoid X receptor a (RXRa), a element, the architecture of which carries important member of the nuclear hormone receptor superfamily of tranimplications for the structureof HNF-4 response ele- scription factors (10). RXRs may act ascoregulators by formments in general. ing heterodimers with other members of the nuclear hormone receptor superfamily including receptors for retinoic acid, thyroid hormone, and vitamin D (11-17). The MCAD RARE contains three hexamer sequences that match the thyroid The mitochondrial flavoenzyme medium chain acyl-CoA receptor (TR)/RAR subfamily consensus binding siteardehydrogenase (MCAD,’ EC 1.3.99.3) catalyzes the initial, ranged in a novel motif. The complex architecture of this * This work wassupported by the Markey Charitable Trust andby element suggests that it interactswith multiple transcription National Institutes of Health Grant DK45416. The costs of publica- factors to regulate MCAD gene expression in response to a tion of this article were defrayed in part by the payment of page variety of nuclear receptor signaling pathways. charges. This article must therefore be hereby marked “aduertiseIn the present report, we show that the MCAD RARE ment” in accordance with 18 U.S.C. Section 1734 solely to indicate activates transcription by interacting with HNF-4, amember this fact. of the steroid/thyroid hormone receptor superfamily that has § A Merck Fellow of the American College of Cardiology. “orphan” $5 The recipient of a Lucille P. Markey scholar award. To whom no known ligand and is thus referred toasan correspondence should be addressed Cardiovascular Division, Wash- receptor (18).HNF-4 and other orphan receptors, including ington University School of Medicine, 660 S. Euclid Ave., Box 8086, apoAI regulatory protein-1 (19) and erb-A related factor 3/ St. Louis, MO 63110. Tel.: 314-362-8919; Fax: 314-362-0186. chicken ovalbumin upstream promoter binding transcription ‘The abbreviations used are: MCAD, medium chain acyl-CoA factor (20,21),play a role in the transcriptional regulation of dehydrogenase; HNF-4, hepatocyte nuclear factor-4; RARE, retinoic a variety of genes involved in metabolic pathways including acid response element; CAT, chloramphenicol acetyltransferase; RXR, retinoid X receptor; RAR, all-tram-retinoic acid; pTKCAT, the apoAI, apoAII, apoCIII, and apoB genes (19, 22, 23), the thymidine kinase promoter constructs; RA, retinoic acid; Vit, vitamin; a-antitrypsin gene, and the transthyretin gene (24, 25). Our TR, thyroid receptor. data demonstrate that HNF-4 activates MCAD gene tran-

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HNF-4MCAD Activates

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scription by directly interacting with the MCAD RARE independent of RXRa.Mutational analysis of the MCAD RARE and comparison of its sequence with that of other known HNF-4 response elements reveal that distinct and novelsequence requirements specify HNF-4 binding and function. Therefore, the MCAD gene promoter region contains a complex regulatory element capable of interacting with multiple members of the steroid/thyroid superfamily of transcription factors. MATERIALSANDMETHODS

Gene Transcription to 2 pg) (Fig. 1 and data not shown). The retarded band was not seen with protein extractsderived from cells infected with wild-type vaccinia (data not shown). Formation of the complex was inhibited by a 100-fold molar excess of unlabeled RAREMCAD but not by an identical molar excess of an unlabeled size-matched unrelated DNA fragment, indicating a specific interaction between the probe and HNF-4. Furthermore, formation of the complex was abolished by addition of a 100-fold molar excess of an unlabeled oligonucleotide derived from an HNF-4 response element in the apoCIII gene (RE,,cI~I) (18) originally employed to isolate HNF-4, providing additional evidence that the band represented a specific interaction between RAREMcAD and HNF-4 (Fig. 1, lane 4 ) . HNF-4 Transactivates the MCAD Gene Promoter-Demonstration of HNF-4 binding to the RAREMCAD led us to examine the functional role of the RAREM~AD-HNF-~ interaction. The transcriptional activity of the RAREMCAD upstream of the herpes simplex virus thymidine kinase promoter in a CAT reporter plasmid (PTKCAT-RAREMCAD) was determined in the absence and presence of HNF-4 expression plasmid (pMT2-HNF-4) cotransfected into NIH-3T3 cells. The presence of a single copy of RAREMcADupstream of the thymidine kinase promoter in the antisense orientation inthe absence of HNF-4 resulted in a modest (50%) decrease in CAT activity, suggesting that theMCAD RARE is asuppressor of transcriptional activity in NIH-3T3 cells (Fig. 2 A ) . Cotransfection of pMT2-HNF-4 with PTKCAT-RAREMCAD resulted in a 6-10-fold increase in CAT activity that resulted from both a release of the mild suppressive effect and an induction of transcriptional activity above that of pTKCAT(-). Activation of PTKCAT-RAREMCAD by cotransfection with the HNF-4 expression vector was also observed in other cell lines, including human hepatoma (HepG2) cells, mouse Y-1 adrenal cortical tumor cells, and simian CV-1 cells (data not shown). Activation was not observed upon cotransfection with pMT2(-), andHNF-4 had no effect on the transcription of pTKCAT(-) (Fig. 2 A ) . Therefore, the MCAD RARE functions as anHNF-4-responsive element. Cotransfection of a 5' deletion series of MCADCAT reporter plasmids with pMT2-HNF-4was performed to evaluate

Plasmids-Construction of full-length MCADCAT(-1193/+178) (with reference to the transcription start site = +l),5' deletional MCADCAT constructs, and thymidine kinase promoter constructs (pTKCAT) have been described (9,lO). pTKCAT-A1 was constructed in a similar manner, by ligating a double-stranded oligonucleotide fragment (see Fig. 5, A l ) intotheBamHI site of thepTKCAT plasmid. DNA sequencing by the dideoxy method confirmed the location and orientation of the insert. Isolation of rat HNF-4 cDNA and construction of the HNF-4 expression construct in the vector pMT2 was described previously (22). Eukaryotic expression vectors for human RARp and human RXRa were constructed in the vector CDM8 containing a cytomegalo-virus promoter (26) or in the vector pMT2 (27). pMT2 without acDNA insert, orpMT2(-), was used as a negative control in transfections. Transfection of Mammalian Cells in Culture-Mouse NIH-3T3 fibroblasts and simian CV-1 cells were employed for transfection. The cells were maintained in either minimal essential medium (NIH3T3) or Dulbecco's modified Eagle's medium (CV-1) supplemented with 10% fetal calf serum. Cotransfection was performed by the calcium phosphate coprecipitation technique(28), with 15 pg of MCADCAT or pTKCAT plasmid and 1-3 pg of receptor expression plasmid with or without the receptor cDNA inserts. One pgof an RSV or SV40 &galactosidase expression vector was also included to correct for transfection efficiency. In CV-1 experiments involving retinoic acid (RA), stimulation cells were incubated in medium supplemented with charcoal-stripped fetalcalf serum inthe presence and M, absence of all-tram-RA (Sigma) a t afinalconcentration of as described previously (10). CAT assays were performed with butyryl-CoA and ["C]chloramphenicol substrate for 60-90 min with separation of butyrylated chloramphenicol by xylene extraction (29) followed by quantitation on a Beckman LS-3801 liquid scintillation counter. Electrophoretic Mobility Shift Assays-Mobility shift assays were performed as described (10, 30). Human RARP, human RXRa, and rat HNF-4 were overexpressed in recombinantvaccinia virus-infected HeLa cells (31). Complementary single-stranded oligonucleotides were synthesized on an Applied Biosystems PCRmate synthesizer, annealed, and gel-purified before fill-in labeling with the Klenow fragment of DNA polymerase I with [(u-~'P]~CTP or [c~-~'P]~ATP. The oligonucleotide sequence (sense strand) for the RARE of the mousePRAR gene (32) is 5'-gatccAAGGGTTCACCGAG'M" CACTCGCATAggatc-3' (lower case letters represent nucleotides added for cloning and labeling and are not partof the element). The remaining oligonucleotide sequences (sense-strand only) are shown (see Fig. 5). Antibody Supershift Assays-Crude rat liver nuclear extract was prepared from adult male Wistar rats by the protocol of Roy et al. (33). The electrophoretic mobility supershift assays were performed as previously described (18)with anti-HNF-4 antiserum kindly provided by Dr. Frances Sladek (University of California, Riverside). Three pl of a 1:lO dilution of antiserum in 3%bovine serum albumin was added to the incubation after addition of the probe and nuclear extract.

Competitor

-

.~.

NS

M A

RESULTS

Binding of HNF-4 with the MCAD RARE-To determine whether the retinoid-responsive element in the MCAD pro1 2 3 4 moter region interacts directly with HNF-4, electrophoretic mobility shift assays were performed with a 32-base pair 32P- FIG.1. Binding of HNF-4 with the MCAD RARE. Mobility shift assays were performed with vaccinia virus-produced HNF-4 and labeled DNAfragment containing the entireRARE as a probe 32P-labeled R A R E M c probe ~ in all lanes. The labels above each lam (RAREMcAD)(10) and HNF-4 overexpressed in a vaccinia/ denote unlabeled competitor added tothe incubation a t 100-fold HeLa cell system. A single prominent complex was observed molar excess as follows: minus sign, no competitor; NS, nonspecific over a wide range of nuclear protein extract amounts (50 ng size-matched DNA fragment; M,RAREMcAD; A, RE,,wlll.

HNF-4 Activates MCAD Gene Transcription A 0 (-) HNF-4 (+) HNF-4

ITK ICAT pTKCAT(-)

0

5 10 15 20 normalized CATactivity (cpm XIO-~)

B

1

II

ICAT

1

pMCADCAT(-1193/+178)

D l

ICAT

pMCADCAT(-361/+178)

1

1pMCADCAT(-311/+178) 0

20 40 60 80 100 120 % normalized activation

FIG. 2. Cotransfection of ~ T K C A T - R A R E Mand C ~ MCADCAT plasmids with HNF-4. A, HNF-4 activation of pTKCATRAREMcAD.Diagrams a t left represent the pTKCAT chimeric constructs pTKCAT(-) and ~TKCAT-RAREMC~.stippled The box represents the MCAD RARE insert. The graph a t right represents CAT activity (normalized to @-galactosidaseactivity). White bars denote CAT activity in the absence of HNF-4 (cotransfected with pMT2(-)), and black bars represent CAT activity in cells with cotransfected pMT-2-HNF-4. The results are representative of a t least three experiments. B , HNF-4activation of pMCADCAT constructs. A series of chimeric constructs containing either full-length pMCADCAT(-1193/+178) or 5' deletions of the upstream flanking region of the MCAD gene linked to a CAT reporter gene, represented on the left, were cotransfected with pMT-2-HNF-4. The stippled box represents the MCAD RARE a t -341 to -308 upstream of the transcription start site. Bars on the right represent CAT activity as a percentof the maximal activation of pMCADCAT(-1193/+178) by HNF-4.

HNF-4-mediated transcriptional activation by the RARE in the context of the MCAD gene promoter and to search for any additional HNF-4-responsive elements within the promoter region (Fig.2B). pMCADCAT(-1193/+178) transcription was activated 4-8-fold by HNF-4 compared with cotransfection of pMT2(-). Deletion of sequences from -1193 to -361 had no appreciable effect on the HNF-4-stimulated transcription, but deletion of the region from -360 to -311, which contained the RARE, abolished HNF-4 responsiveness. Thus, a single HNF-4 response element is present within the MCAD gene promoter region. Determination of the Role of RXRa in the MCAD RAREHNF-4 Interaction-We have previously shown that the RA responsiveness of the MCAD RARE is greater with RXRa compared with either RARa or RARP (10). Recent studies from several laboratories have shown that RXRs function, in part, ascoregulators by heterodimerizing with other members of the nuclear hormone receptor superfamily of transcription factors (12-17). We therefore investigated the possible coregulatory role of RXRa in the binding and activation of the MCAD RARE by HNF-4. As a positive control, the prototypical RAR binding element of the PRAR gene (RARE@& (32) wasused as a probe in mobility shift assays with nuclear

13807

extract containing overexpressed RARP and RXRaalone and mixed in equal amounts. Whereas at low receptor concentrations a complex was barely observed with RAREBRAR in the presence of either RARP or RXRa alone (Fig. 3, lanes 1 and 2), dramatic enhancement of the DNA-protein interaction occurred upon mixing the two extracts (Fig. 3, lane 3 ) . This marked augmentation ofRAR binding by RXRa has been previously demonstrated for this RARE (14-16). In contrast, formation of the HNF-4.RAREwxD complex was not significantly enhanced by the addition of equimolar amounts of overexpressed RXRa (Fig.3, lanes 4-6). Interestingly, the combination of HNF-4- andRXR-containing extracts at concentrations that saturated the probe produced a moderate diminution in both bound complexes compared with that seen with eitherextract alone (Fig.3, lane 6), suggesting that HNF-4 homodimers share at least one site with complexes containing RXR. The mixing experiments with RAREMcAD were also performed with RXRa- and HNF-4-containing extracts over a wide range of extract amounts (0.05-2.5pg) without evidence of heterodimerization on the RARE as determined by radioisotopic scanning analysis of the resultant complexes (data not shown). Furthermore, augmentation of HNF-4 binding to the RAREMCAD was not observed despite addition of increasing amounts of RXRa to the mixture up to a 5:l ratio of RXRa:HNF-4 (Fig. 3, lanes 7-9).Accordingly, binding of HNF-4 to the MCAD element does not require RXRa. To evaluate further therole of RXR in the HNF-4-mediated activation of the MCAD promoter, functional mixing experiments were performed. Previous cotransfection studies have shown that activation of the herpes simplex virus thymidine kinase promoter by R A R E Bwith ~ ~ RARP in the presence of RA is enhanced by RXRa (14). ~TK-RAREMcAD was cotransfected with constructs expressing RXRa andHNF-4 into CV1 cells. In the absence of RA, activation of PTK-RAREMCAD by HNF-4 was blunted rather than enhanced by cotransfection with RXRa (Fig. 4). Conversely, transcriptional activation of pTK-RAREMcAD by RA in the presence of RXRa was diminished by HNF-4 cotransfection. These functional data are consistent with the results of the DNA binding experiments and indicate that RXRa exerts a negative effect on HNF-4-mediated activation of the MCAD promoter. Delineation of the HNF-4 Binding Sites inthe RAREMcADWe have previously describedthe MCAD retinoid-responsive element (10) as containing three potential hexamer binding sequences homologous to the (A/G)G(G/T)TNA consensus binding site recognizedbymembers of the T3/RA/Vit D receptor subfamily (34). Hexamer site 1 is present in the antisense orientation 8 base pairs upstream of the adjacent sense strand hexamers 2 and 3(Fig. 5 A ) . Mutational analysis

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Probe: RARE D R ~ R

RARO:

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RAREMCAD I

HNF-4:

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FIG. 3. Binding of HNF-4 with RAREmcADin the presence and absence of RXRcr. Electrophoretic mobility shift assays with RAREMcADand RAREBMR (as a positive control) probes were performed with receptors as denoted at the top of each lane. Extract amounts used are as follows. Lanes 1-3, RARP and RXRa (0.15 pg each); lanes 4-6, HNF-4 and RXRa(0.15 pg each); lanes 7-9,HNF4 (0.25 pg) and RXRa (1.25 pg). The labels to the right of lane 9 denote the independent bands specific to RXRa and HNF-4, based on the bandshift pattern in lanes 7 and 8 and parallel competition experiments.

HNF-4MCAD Activates

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Gene Transcription

upstream of the thymidine kinasepromoter were performed in CV-1 cells. HNF-4-mediated transcriptional activation of a TKCAT reporter promoter plasmidcontaining a single copy of the A 1 mutant oligonucleotide in the reverse orientation (pTKCAT-A1) was similar to that of ~ T K C A T - R A R E M ~ A D 0 5 (6-8-fold, data not shown). These data togetherwith the 0 LL results of the binding studies confirm that the MCAD gene 0 HNF-4 response element consists of the imperfect direct HNF-4 + + repeat containingbinding half-sites 2 and 3. + + + + RXRa " + + RA HNF-4 binding to theRAREMCAD was compared with bindFIG. 4. Cotransfection of HNF-4 and RXRawith pTKCAT- ing with known HNF-4 elements found in the apolipoprotein (18).In each ) RAREMCD. CV-1 cells were cotransfected with pTKCAT-RAREMcAD, CIII (RE,,c~ll) and transthyretin ( R E ~ Rgenes and eitherpMT2-HNF-4 and/or CDMRXRa (with and without RA case, as with the MCAD element, adjacent hexamer sites in a t 1O"j M) as denoted below the graph. The ordinate represents -fold the same orientation were present. As shown in Fig. 5B, HNFactivation of CAT activity over unstimulated pTKCAT-RAREMcAD, 4 binding to these three elements was approximately equivanormalized to @-galactosidaseactivity. Experiments with RA were performed in media containing charcoal-stripped serum. Activation lent. To evaluate the importance of hexamer spacing, HNF-4 of pTK-RAREMcm by pMT-2-HNF-4 was no different in charcoal- binding to oligonucleotide probes containing insertion mutations between sites 2 and 3 of the RAREMcAD were examined. treated compared with untreated serum (data notshown). Addition of 1 or 2 guanine residues (Alg and Algg, respectively) completely abolished binding. These data suggested A I . RARE^^^^ q a C c G G G T T ~ = C T C T C C G ~ A ~ A A G G g ( l a t c 3' . that HNF-4 binds to elements containing adjacent half-sites 2. A1 galccTTCTCTCCGGGTAAAGGlGA4GGggalc without intervening bases. Surprisingly, however, an oligogalccGGGllTGACCllTCTCTCCCg3jTAAAGGTGAAGGggalc 3. m2 galccGGGTllGACCllTCTCTCCGGGTAAKZTGAAGGggalc 4. m3 nucleotide probe containing a direct repeat of the AGGTCA 5. m2m3 galccGGGmGAcCmCTCTCC~T~TGAAGGggalc 1 2 3 4 5 consensus hexamer without intervening base pairs (Fig. 5B, B DR-0) failed to bind HNF-4. Thus, adjacent half-sites may 5'. IcgaGCGCTGGGCAAAGGTCACCTGClcq~ - 3' 1. RE,c, constitute an HNF-4response element, but there arespecific IcgaCCCTAGGCAAGGTTTCATATGGCClcga 2. R E ~ D galccTTCTCTCCGGGTAAAGGTGAAGGggalC 4-b 3. A1 sequence requirements that appear to dictate a novel consengalccTTCTCTCCGGGTAbQ4GGTGAAGGggalc 4. Alg sus sequence. galurrCTCTCCGGGTAPyi(Y\GGTGAAGGpgalc 5.Algg 6. DR.0 galcIAGGTCAAGGTCAlclag 1 2 3 4 5 6 Interaction of the MCAD HNF-4 Response Element with is a complex FIG. 5. Mutational analysis of HNF-4 binding. A , mobility Endogenous Rat Liver HNF-4"The RAREMCAD shift analysis of HNF-4 binding t o R A R E M cmutant ~ probes. DNA element thatlikely interacts with a varietyof nuclear receptor sequences of the R A R E M c and ~ mutant oligonucleotides used as transcription factors in vivo. Therefore, mobility shift assays probes are shown on the left. The number preceding each probe were performed with crude rat liver nuclear extract to detersequence corresponds to the lane in the assay shown on the right. mine if the MCAD HNF-4 response element bound endogeThe nucleotides shown in lower case letters are restriction sites added for convenience. The sequence shown on the top is the full MCAD nous HNF-4 in the presence of other nuclear receptors. MoRARE with potential receptor binding sites and their orientation bility shift assays with the MCAD HNF-4 response element denoted by arrows (numbered 1-3 for reference). The pointmutations probe (Al) and crude rat liver nuclear extract revealed a are boxed. The probes were labeled to approximatelyequivalent complex of lowmobility (Fig. 6, arrow) that migrated similarly I

15 1

f

-

5'.

"

specific activities, and binding conditions and amount of nuclear protein extract used were identical for each lane. B, analysis of sequence requirements for HNF-4 binding. The mobility shift assay shown on the right was performed as described in A with previously described HNF-4-responsive elementprobes from the apolipoprotein ) (lanes I and 2 ) , CIII (RE.,clll) andtransthyretin ( R E ~ Rgenes mutant oligonucleotides of the MCAD RARE (lanes 3-5), and an artifical direct repeat probe (lane6,DR-0).

CRL

Extract: v.HNF-4 Anti-HNF-4: immunecomplex

"

- +

PI -

u_ .

+

PI

has previously demonstrated that site 1 is crucial for RXRa and RARP binding to this element(10). T o delineate HNF-4 binding sites withinRAREMcAD,electrophoretic mobility shift assays were performed with oligonucleotide probes containing the wild-type element and several mutant sequences. Surprisingly, mutation of theinvariant G residue in the second position of site 1 (data notshown) or complete absenceof site 1produced only a minor decrease in HNF-4binding compared with binding to the wild-type sequence probe (Fig. 5A, lanes 1 and 2). In contrast, G to C mutations in the invariant position in site 2, 3, or both resulted in markedly diminished to HNF-4 binding (Fig. 5A, lanes 3-5). Thus, in contrast RAR 1 2 3 4 5 6 and RXR, hexamer site 1 is not required for HNF-4 binding. Furthermore, the two adjacent hexamer binding sites appear FIG. 6. Binding of the MCAD HNF-4 response elementwith to constitute an HNF-4 response element, consistent with endogenous rat liver HNF-4. Electrophoretic mobility shift assays were performed with "P-labeled A1 probe, in the presence of HNF-4 previous reports that this factor binds to DNA as a dimer produced in vaccinia-infected HeLa cells (lanes 1-3, u.HNF-4) or 3 (18). of crude rat liver nuclear extract (lanes 4-6, CRL).Anti-HNF-4 To determine whether the imperfect repeat consisting of pl (+) or pre-immune serum(PZ) were added to those lanesas indicated adjacent sites 2 and 3 is sufficient for a functional HNF-4 at thetop. The arrow denotes the location of the complex containing response element, cotransfections with the mutant element HNF-4 as defined by the complex in lane 1. The location of the 1 (Fig. 5), supershifted immune complex is indicated. Al, which containssites 2 and 3 butnotsite

HNF-4MCAD Activates

Transcription Gene

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to that observed with HNF-4 produced in vaccinia-infected vation of the MCAD promoter, perhaps by competing for a HeLa cells (Fig. 6, lanes 1 and 4 ) . This complex was almost binding half-site. The distinct but overlapping half-site pair within the element described completely supershifted with the addition of HNF-4 anti- preferences for RXR and HNF-4 serum but not with pre-immune serum confirming the pres- here are also consistent with this conclusion. However, we ence of endogenous HNF-4 (Fig. 6, lanes 2 , 3 , 5 , and 6). The cannot exclude the possibility that RXR-HNF-4heterodimer majority of the additional complexes of higher mobility pres- interactions occur on other response elements. Analysis of other known HNF-4 response elements and the ent in the A1 probe lane were markedly diminished by addition of 100-fold molar excess of an unlabeled unrelated DNA mutational studies shown here allow several conclusions to fragment indicating that these interactions were nonspecific be drawn concerning the sequence requirements for HNF-4 (data not shown). In parallel experiments, similar anti-HNF- binding. The MCAD element and several other reported 4 supershifting patterns were observed with the RAREMCADHNF-4 response elements including those present in genes resemble and REapa~lll probes and rat liver nuclear extract, whereas no encoding transthyretin, apoCIII, and al-antitrypsin supershift was observed with the mutantprobe Alg (data not the recognized binding site for the TR/RAR/Vit DR subgroup shown) consistent with the mutational binding results shown of the nuclear receptor superfamily in containing half-site in Fig. 5. Therefore, the MCAD HNF-4 response element sequences homologous to the recognized consensus (Fig. 7) binds to endogenous liver HNF-4 in the presence of other, ((A/G)G(G/T)TNA). Indeed, mutation of either of the invariant guanines present in the second position of the hexamers potentially competing nuclear receptors. within the MCAD element markedly diminished HNF-4 binding. Each of the elements shown in Fig. 7 contain adjacent DISCUSSION half-sites without intervening base pairs. However, the 5' The MCAD gene is ubiquitously expressed, yet its expres- half-site sequence differs from the 3' half-site (and from the sion is highly modulated in accordance with fatty acid oxida- TR/RAR consensus) in the preferred 4th and 5th base pair tive metabolic rates among tissues, during developmental ((G/A)GG(C/T)AA). Furthermore, HNF-4 did not bind to stages, and in response to fasting and refeeding (7).2 Further- the artificial repeat AGGTCA probe (Fig. 5B, DR-0), whereas more, the age-related and episodic occurrence of the clinical a single substitution of adenine for the fifth position cytidine manifestations of inherited MCAD deficiency suggest that in the 5' hexamer of the DR-0 probe restores HNF-4 binding MCAD expression is regulated in accordance with fatty acid (latter data not shown). Thus, HNF-4may bind to elements oxidative energy demands in uiuo. As an initial step in eluci- arranged asimperfect direct repeatswithout intervening base dating mechanisms of regulation of nuclear genes encoding pairs (D-0 motif). mitochondrial enzymes in fatty acid oxidative pathways, we The distinct sequence of the 5' half-site present in the D-0 have begun to delineate the regulatory regions of the MCAD HNF-4 response elements may be due to structural requiregene and have recently identified a novel and complex reti- ments of HNF-4 dimer binding. The amino acid sequence of noid-responsive element within the MCAD promoter region the region of the first zinc finger in the HNF-4 protein that (10). recognizes the DNA half-site (referred to as the P box) (37) Here we report the regulation of MCAD gene expression by is unusual compared with other reported mammalian tranHNF-4, an orphan member of the nuclear receptor superfam- scription factors in the superfamily. However, it most closely ily of transcription factors, by interaction with this element. resembles the P box present in the TR/RAR/Vit DR subgroup This finding is particularly intriguing in view of the limited in that itdiffers by only a single conservative amino acid (Asp tissue distribution of HNF-4. HNF-4 mRNA levels are high for Glu). This P box homologyis consistent with the homology in liver, kidney, and small intestine and are absentor present of the 3' half-site to theTR/RAR/Vit DR binding consensus. at low levels in brain, spleen, lung, and heart (18, 35). The The specific sequence requirements of the 5' half-site may be tissue expression pattern of MCAD is remarkably similar due to structural featuresof the HNF-4.DNA complex, such with the exception of heart where MCAD is highly expressed as steric constraints imposed by the close proximity of the (8).This similarity in expression patterns suggests a physio- adjacent half-sites. logical role for our findings in vivo and raises the possibility Analysis of the sequences of several other published HNFof a cardiac HNF-4 analogue. Regulation of MCAD expression 4 response elements, including those in the apoAI, apoAII, by HNF-4 is also consistent with the evolving understanding and factor 1X genes (22,38,39),reveals that HNF-4may bind of this transcription factor as a participant in tissue-specific modulation of genes involved in metabolism. HNF-4-responHNF4 sive elements have been identified in several other genes E!mm !ss!awB Relerence involved in metabolic pathways including transthyretin (25) MCAD C T C T C C G G G T A A A G G T G A A G G + lh15Sludy C T C T C C G C G T A A A G G T G A A G G IhhSSIudy and the apolipoprotein genes apoCIII, apoAI, apoAII, and MCAD m2 MCAD m3 C T C T C C G G G T A A A t G T G A A G G . Ih~ssludy apoB (22, 23). C C C T m - A T A T + 18 The MCAD RARE responds preferentially to RXRa over "1lranslhyretln -anlllrypsm A A C A G G G G C T A A G T C C A C T G + 18 RARa and RARp and thus may be considered an RXRE (IO). apoClll C T - m A C C T + rg 2 2 2 3 Recently it has been demonstrated that RXRs act ascoregu- DR-0 A G G T C A A G G T C A lators with other members of the nuclear receptor family consensus 0 - 0 G G G G A A A E E T C A including TR, Vit DR, RAR, and erb-A related factor 3 Dmding w e A T T G T G (chicken ovalbumin upstream promoterbinding transcription FIG. 7. DNA sequence requirements for D-0 HNF-4 refactor) by forming heterodimers that bind to cognate DNA sponse elements. The sequences of known HNF-4 response eleresponse elements (11-17, 36). However, our studies indicate ments with the D-0 motif are shown to the right of the gene of origin. that HNF-4 binds and activates transcription independent of The half-site sequences are delineated by arrows and boldface letterRXR, at least in the context of binding to theMCAD element. ing. The pertinent sequences of several mutant probes are also shown Furthermore, the mixing experiments shown here indicate (MCADm2, MCADm3, and DR-0). The derived consensus half-site that RXR exerts a negative effect on HNF-4-mediated acti- sequences are shown below the elements. A second nested sequence "

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* M. E. Carter and D. P. Kelly, unpublished results.

(underlined) present in allknown HNF-4 response elements as discussed in text is also noted. HNF-4 binding and reference source are shown to the right of the sequences.

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HNF-4 Activates MCAD Gene Transcription

to elements that contain nonadjacent half-sites. Interestingly, each of these elements sharea common sequence ((C/ T)AA(A/G)G(G/T)))present within the original HNF-4 binding consensus derived by Darnell and co-workers (18). An identical motif (CAAAGTG) is also conserved inthe promoter region of a variety of liver-expressing cytochrome P450 genes (40). This sequence is present inthe centralregion of the D-0 repeat consensus formed by the juxtaposition of the imperfect direct repeat (Fig. 7, underlined region). Therefore, in addition to the presence of binding half-sites, additional sequence requirements exist for HNF-4 binding. Further studies will be necessary to determine the precise role of this sequence in HNF-4 binding and function. The structural and functional featuresof the MCAD RARE indicate that it is capable of interacting with multiple members of the nuclear hormone receptor superfamily. In addition to interaction and activation by HNF-4, we have shown previously that the MCAD RARE activates transcription in response to RARa, RARP, and RXRa in thepresence of RA (10).The sequence of this element (Fig. 5A) reveals an unusual orientationand spacing of threepotential hexamer binding sites consisting of a sense-direct repeat 8 base pairs downstream of a single antisense-oriented hexamer. This pattern of half-siteshasnot been reported for any other known hormone-responsive element. Furthermore,HNF-4 binding to the MCAD RARE involves a different pair of hexamers (sites 2 and 3) than that reported previously for RAR or RXR (sites 1 and 2 or 3) (10). Therefore, the novel structural featuresof this regulatory element appear to allow its interaction with a variety of transcription factors within the nuclear hormone receptor superfamily, perhapsin response to various cellular metabolic conditions among tissues. Consistent with this hypothesis, it has recently been demonstrated thatapoCIII and apoB gene transcription is regulated bidirectionally by HNF-4 and two additional orphan receptors, apoAI regulatory protein-1 and erb-A related factor 3 (chicken ovalbumin upstream promoterbinding transcription factor) (22, 23). Preliminary experiments in our laboratory indicate that apoAI regulatory protein-1 and erb-A related factor 3 both suppress MCAD gene transcription via interaction with the MCAD RARE. We speculate that the relative cellular levels of these various nuclear receptors in part confer bidirectional regulation of MCAD gene expression in uiuo. In summary, we conclude that HNF-4 regulates MCAD gene expression througha complex regulatory element at -311 to -322 relative to the transcriptional start site. Furthermore, HNF-4 response elements can consist of adjacent binding half-sites with novel sequence requirements. It is likely that HNF-4 and additional orphan receptors are involved in the regulation of this and other genes involved in mitochondrial oxidative metabolism. Acknowledgments-We give special thanks to Dr. Arnold Strauss for critical reading of this manuscript and Barbara Donnelly for secretarial assistance. We thank Drs. Frances Sladek and James Darnell, Jr. for the anti-HNF-4antiserum.

REFERENCES 1. Beinert, H. (1963) in The Enzymes (Bover. P. D.. Lardv. H.. and Mvrback. K.. e&) 2nl Ed., Vol. 7, p .-447-476"ACadem