Feb 9, 1994 - Expression of the apolipoprotein AI (apoAI) gene in the liver is ...... Mietus-Snyder, M., Sladek, F. M., Ginsburg, G. S., Kuo, C. F., Ladias, J. A. A.,.
Vol. 269, No.
THEJOURNAL.OF BIOLWICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.
Issue of May 6 , PP. 13185-13192, 1994 Printed in U.S.A.
Transcriptional Repression of Apolipoprotein AI Gene Expression by Orphan Receptor ARP-1” (Received for publication, February 9, 1994)
Ruowen Ge*§, Myungchull €thee$ Sohail MalikS, and SotiriosK. KsrathanasisSII From the Wepartment of Cardiovascular Molecular Biology, Lederle Laboratories, Pearl River, New York 10965 and the Wivision of Genetics. Department of Medicine, Brigham and Women’s Hospital, Howard Hughes Medical Institute, Harvard Medical School; Boston, Massachusetts 02115
Expression of the apolipoprotein AI (apoAI) gene in the liver is controlled by a liver-specific enhancer. The function of this enhancer depends on synergistic interactions between transcription factors bound to at least three sites (designated A, B, and C) located within this enhancer. We have previously shownthat anapoAI gene reporter construct containing the entireenhancer is expressed efficiently in a hepatoma cell line and that its activity is repressed by the orphan receptor ARP-1. Moreover, repression by ARP-1is overcome by the retinoid X receptor RXRa in thepresence of retinoic acid. In this study, we showthat ARP-1 represses the apoAI promoter by binding to site A of the apoAI liver-specific enhancer, the repression being a promoter context-specific event. Mapping analysis of ARP-1indicated that its DNA binding domain is essential but not sufficient for repression. Two separate repression domains locatedat the amino- and carboxyl-terminalhalves of ARP-1were found to individually complement the DNA binding domain for efficient repression. We also demonstrate the reversibility of ARP-1 repression by transcription factors C/EBP and Egr-1, which might also be involved in apoAI gene expression.Significantly, repression by ARP-1 was found to be a prerequisite for C/EBP-mediated transactivation. We interpret our results in terms of a model in which ARP-1 repression via itainteraction with site A is an obligatory intermediate step in switching from one activated state of the apoAIgene to another. Regulated tissue-specific and developmental expression of many genes resultsfrom a n interplay of a variety of transcription factors (reviewed in Ref. 1).This is a dynamic process involving both activators and repressors that exert effects their through interactions with their cognate cis-regulatory DNA elements. It is believed that combinations of cis-elements arranged in unique configuration confer on a given gene an individual spatial and temporal transcriptional program. Apolipoprotein AI (apoAI)’ is the major protein component of the plasma high density lipoprotein (reviewed in Ref. 2) and is expressed predominantlyin liver and intestine(3-7). Previous studies have shown that apoAI gene transcriptionis modulated by a diverse number of signals, including developmental mes-
* The costs of publication of this article were defrayed in part by the be hereby marked payment of page charges. This article must therefore “advertisement”in accordance with 18 U.S.C.Section1734 solely to indicate this fact. 5 Present address: Roche Inst. of MolecularBiology,Nutley, NJ 07110. 1) To whom correspondence should be addressed. The abbreviations used are: apoAI, apolipoprotein AI; RA, retinoic acid; CAT, chloramphenicol acetyltransferase; EMSA,electrophoretic mobility shift assay.
sages and hormones as well as various pharmacological and toxic substances (Ref. 2 and references therein). However, the precise mechanisms by which relevant transcriptional signals are sorted out and assimilated by the apoAI gene transcriptional apparatus are notunderstood. The liver-specific expression of apolipoprotein A1 gene is mediated by a powerful liver-specific transcriptional enhancer located at positions -222 to -110 relative to the transcription initiation site (6, 7). This enhancer is primarily comprised of three factor binding sites (site A, -214 to -192; site B, -169 to -146; site C, -134 to -119). In hepatoma HepG2 cells, factors bound to these sites function synergistically to control transcription rates of the apoAI gene (7). Among the factorsimplicated in apoAI gene control are members of the steroidthyroidhormone nuclear receptor superfamily (8-101, including the retinoid X receptor RXRa and the orphan receptor ARP-1. Site A, which displays significant homology to steroid hormone response elements, wasfound to be necessary andsufficient forligand-dependent RXRa activation of transcription from apoAI core promoter elements in hepatic cells. Moreover, ARP-1, whose cDNA was originally cloned by screening an expression library for binding to site A (91, was found to repress transcription via the intact enhancer (10) as well as to repress RXRa-dependent activation via site A. However, the activation potential of RXRa was lost when siteA was situated in its natural context (i.e. within the liver-specific enhancer) (10) indicating the importance of the particularconfiguration of various transcription factors prevailing at this enhancer. Interestingly, repression of apoAI natural promoter by ARP-1 could be overcome by RXRa in thepresence of retinoic acid, indicating that the ARP-l-repressed apoAI natural promoter has regained the ability to respond to RXRa and the ligand. As part of our ongoing studies on the mechanism of repression by ARP-1 and its involvement in the complex regulatory networks controlling apoAI gene expression, we have now undertaken a systematic analysisof the following: 1)the minimal determinants for ARP-1binding andfunction, and 2) the influence of ARP-1 on the function of other transcription factors implicated in apoAI gene regulation. EXPERIMENTALPROCEDURES Plasmid Construction and Purification-The apoAI promoter constructs -256AI.CAT,-192AI.CAT,[AI-41AI.CAT,-41AI.CAT,[AITKCAT, and [Al,,TKCAT have beendescribed before (8,10). Plasmids pMT2-ARP-1,pMT2-ARF’AAl,pMT2-ARPAA6, and pMT2-ARPAA7 employed for expressing ARP-1 and various deletion mutants from the mammalian expression vector pMT2 have also been described (9,10). The plasmid pMT2-COUP was constructed by cloning the Ear3/COUPTF1 cDNA (giR of Dr. M.-J. Tsai) into the EcoRI site of pMT2 vector. Similarly, the chimeric receptor COUP-PR cDNA (gift of Dr. 0. Connelly), which encodes the amino- and carboxyl-terminal regions of human COUP-TF1 and the DNA binding domain of the chicken progesteronereceptor, was subcloned into the EcoRI site of pMT2vector
13185
13186
Regulation of Danscription by Nuclear Hormone Receptors
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FIG.1. ARP-1represses apoAI expression in HepG2 cells through site A of the enhancer. An increasing amount of ARP-1 expression vector (pMT2-ARP-1)was cotransfected with each apoAI.CAT construct (as indicated above of each graph) into HepG2 cells (see “Experimental Procedures”).Within each graph, the CAT activity is expressed as the percentage of the CAT activity exhibited by the same construct in the absence of ARP-1 (designated as 100%).For graphs B-D, the strength of the assayed construct relative to that of -256AI.CAT in the absence of ARP-1is also indicated. A , -256AI.CAT; B , -192AI.CAT; C, [Al-41AI.CAT; and D, -4lAI.CAT. (pMT2-COUP-PR)(11).Plasmid pMT2-ARP-ogala was made as follows. Polymerase chain reaction-amplified p-galactosidase a subunit (pgala) DNA fragment was inserted in-frame into the BamHI site in pGEMARP-AA7, which contains the ARP-1 DNA binding domain in pGEM4 vector (9). The resulting plasmid pGEM-ARP-pgalacontained the pgala subunit fused a t the carboxyl terminus of the ARP-1 DNA binding domain. The insert containing ARP-pgala was excised out by EcoRI digestion of this plasmid and subcloned into pMT2 vector. The C/EBP expression vectorpMT2-CIEBP was constructed by inserting a 1.5kilobase CIEBPa cDNA (gift ofDr. S. McKnight) into the PstI site of pMT2 vector. The Egr-1 expression vector pCMV5-Egr-1 in which the Egr-1 cDNA is under the control of the cytomegalovirus immediate early promoter was a gift from Dr.V.P. Sukhatme (12). All plasmids were isolated on &lagen columns as recommended by the manufacturer. Cell Culture and Dansient Dansfection Assays-Human hepatoma HepG2 cells and monkey kidney CV-1 cells were grown in Dulbecco’s modified Eagle’s mediumsupplemented with 10%fetal calf serum. lo6 cells were seeded onto each 100-mm dish 24 h prior to transfection by the calcium phosphate coprecipitation method as described before (6). The dishes used for HepG2 cells wereprecoated with 50 pg/ml type I11 collagen, whichwas dissolved in 1%acetic acid and sterilized by filtration. 20 pg oftotal plasmid DNA, containing 10 pg of CAT reporter, 3 pg of an internal standard, pRSV-&gal, and varying amounts of receptor expression vector plus a control vector pMT2-UT (to correct for DNA amounts) were added onto eachdish. 16 h later, the cells were shocked by 15% glycerol and re-fed with fresh Dulbecco’s modified Eagle’s medium containing 10%fetal calf serum. For experiments involving 9-cisretinoic acid (9-cis-RA), the serum was treated with dextran-coated charcoal. 9-cis-RA(synthesizedby the chemistry department at Lederle Y,was added Laboratories, Pearl River, NY),dissolved in ethanol at to the transfected cells after glycerol shock to a final concentration of lo4 M. Cells were harvested 48 h after glycerol shock, and CAT and p-galactosidase assays were performed as before (6). EMSA and Determination of Dissociation Constants-10 pg of pMT2-
ARP-1,pMT2-ARPAA1,pMT2-ARPAA6, and pMT2-ARPAA7 were transfected into Cos-1 cells by the DEAE-dextran procedure as described previously (lo), and whole cell extracts containing ARP-1 protein or its mutant versions were made by lysing cells by multiple freezethawing. EMSA employing radiolabeled site A oligonucleotideas probe and the determination ofK, values ofARP-1proteins were performedas described (10). The ARP-pgala fusion protein was made by in vitro transcription and translation using the coupled TNT system from Promega. RESULTS ARP-1-mediated Repression of the apoAI Gene Is Effected through Site A of the Enhancer-We have previously reported
that in transient cotransfection experiments, overexpression of ARP-1 in the hepatoma cell line, HepG2, results in diminished activity of the apoAI promoter/CAT reporter construct, -256Al.CAT, which contains the entire apoAI liver-specific enhancer (9).As a first step toward understanding the molecular mechanism by which ARP-1represses apoAI gene expression in HepG2 cells, we now determinedthe cis-acting elements in the apoAI gene transcriptional control region that are required for ARP-1-mediated repression. CAT reporters containing various apoAI promoter deletions were cotransfected into HepG2 cells with vectors expressing ARP-1 (pMT2-ARP-1)(Fig. 1) (9, 10). The resulting relative CAT activities for each construct were expressed as a percentage of the activity observed in the absence of ARP-1 and then plotted against the amounts of ARP-1 expression plasmid employed in each cotransfection. Consistent with previous results, construct -256AI.CAT, which spans site A (nucleotides -214 to -192), was efficiently repressed by ARP-1 (Fig. M).In contrast, construct -192AI.CAT, which
13187
Regulation of Panscription by Nuclear Hormone Receptors 600
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FIG.2 Effect of ARP-1 on the expression of thymidine kinase promoter. Thymidine kinase promoter with (squares) or without (circles)four copies of site A was tested for its response to ARP-1 expressed from pMT2-ARP-1(open symbols) and RXRa from pMT2-RXRa (filled symbols). CAT assay data are presented as in Fig. 1.
lacked the site A sequences, was not repressed by ARP-1 eficiently (Fig.1B). In addition,when site A was directly linkedto the apoAI basal promoter (construct [Al-41AI.CAT), CAT activity from this reporter was also efficiently repressed (Fig. 1C). Finally, the basal promoter construct -4lAI.CAT was not affected by ARP-1 at all (Fig. lo).In conclusion, site A of the apoAI gene regulatory sequence is necessary and sufficient for the ARP-1-mediated repression. Repression of apoAI Gene Expression by ARP-1 Depends upon the Promoter Context in Which Its Binding Site Is SituatedNext, we asked whether ARP-1 could repress transcription from a heterologous promoter. For this purpose, we conducted cotransfection experiments using a construct containing four copies of site A upstream of the thymidine kinasepromoter in the reporter TK.CAT ([A],TK.CAT) (8). The results in Fig. 2 show that overexpression of ARP-1 in HepG2 cells had no influence on the expression of [A],TK.CAT. Control experiments indicated that this construct retained the potentialto be activated by R X R a (in the presence of 9-cis-RA) (Fig. 2). Similar results were obtained when one copy of site A was placed in front of the thymidine kinase promoter (data not shown). As expected, neither ARP-1 nor RXRa had any influence on the thymidine kinase promoter alone. These observations indicated that the site A-mediated repression of apoAI promoter by ARP-1 is a promoter contextdependent event. Thus, although ARP-1 could diminish transcription mediated by theactivatorsacting via the apoAI enhancer (-256.CAT construct) (Fig. lA ), it failed to counteract the effect of transcription factors (e.g. SP1) driving the thymidinekinase promoter. These resultstherebysuggestthat ARP-1 binding to theapoAI enhancer site A most likely interferes with transcriptional events involving apoAI promoterspecific activators in HepG2 cells accounting for the observed reduction in transcription activity. The DNA Binding Domain of ARP-1 Is Necessary but Not Sufficient for the Repression Function-In order to understand how ARP-1 represses apoAI gene expression, we mapped the ARP-1 domain(s) responsible for this effect. Given the high degree of amino acid sequence conservationin the Ear subfamily of orphan nuclear receptors (9), which includes ARP-1 and COUP-TF1, we have also included COUP-TF1in our analysis. Moreover, both ARP-1and COUP-TF1 have been implicated in repressing apolipoprotein CIII gene expression through a com-
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FIG.3. DNA binding is necessary for ARP-1 repression function. The effect of wild-type COUF"TF1 (A), a COUP-TF1-progesterone receptor chimera ( B ) (see text), and an ARP-1-truncated derivative (ARP-nA7)( C ) (see text) on transcription activity of -256Al.CAT construct was determined as described in the legend to Fig. 1.
mon cis-acting element (13). Thus, as expected, expression of COUP-TF1 resulted inrepression of transcription activity from the -256Al.CAT construct (Fig. 3A). These results are also consistentwithbinding studiesindicating highaffinity of COUP-TF1 for site A (data not shown). For our mapping studies, we examined a COUP-TF1 fusion protein andvarious ARP-1 deletionsin DNA binding and functional assays. To test whether the DNA bindingdomain of is important for their repression ARP-1and/orCOUP-TF1 function, we employed a chimeric derivative of COUP-TF1 receptor. In this chimeric protein, the DNA binding domain of human COUP-TF1 was replaced by the DNA binding domainof
Regulation of Danscription by Nuclear Hormone Receptors
13188
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FTG. 4. DNA binding affinities of ARP-1 and ita deletion mutants. Saturation curves showing binding of AFtP-1and its deletion mutants. A, wild-type ARP-1;B , deletion of residues amino-terminal to the putative DNA binding domain (ARP-AA1); C, deletion of residues carboxylterminal to the DNA binding domain (ARP-AAG);D , the DNA binding domain alone (ARP-AA7).The structure of each protein is shown above each graph frame. At the upper left corner of each graph are the autoradiograms from EMSA showing the retarded gel complexes formed with an increasing amount (left to right) of a radiolabeled oligonucleotide site A probe anda fixed amount of whole cell extract from Cos-1cells transiently transfected with the indicated expression vectors. The Scatchard plots are shown as the small insert graph. The Kd value for each protein is shown.
chicken progesterone receptor(see "Experimental Procedures") (11)whose DNA binding specificity is different from that of the Ear family members.As a result, this COUP-PR fusion protein cannot bind to site A (11)(data not shown). When the COUP-PR chimericprotein was expressed in HepG2 cells, it did not repress the -256AI.CAT reporter expression (Fig. 3B). This result indicated that bindingto site A by ARP-1 or COUP-TF1 proteins through their DNA binding domain is essential for their ability to repress apoAI gene expression in HepG2 cells. To assess whether the DNA binding domain itself can repress apoAI expression, we examined the DNA binding ability of wild-type ARP-1 as well as truncated derivativesof the protein, which include the following: 1)ARP-AA1, with the putative DNA binding domain of ARP-1 and the entirecarboxyl-terminal segment (Fig. 4B); 2) ARP-AA6, with the putative DNA binding domain of ARP-1 and the entire amino-terminal segment (Fig. 4C); and 3) ARP-AA7, with only the DNA binding domain (Fig. 40).'Ib directly compare the DNA binding potential of each polypeptide, we measured their Kd values in an EMSA using siteA as the probe. By comparing results shown in Fig. 4, A and D , it is clear that the polypeptide containing just the DNA binding domain(ARPAA7) could bind to site A with a n affinity (Kd = 6 m)comparable with that of the full-length ARP-1 protein (Kd= 4.2 m). To
test whether binding by the DNA binding domain is sufficient for repression, we then examined the effect of this polypeptide on the expression of -256AI.CAT. An increasing amountof the plasmid pMT2-ARPAA7, which expresses the ARP-1 DNA binding domain polypeptide, was cotransfectedwith reporter -256AI.CAT into HepG2 cells. As shown in Fig. 3C,the DNA binding domain alonehad no effect on -256AI.CAT expression in HepG2 cells. The above results indicated that theability of ARP-1 to bind to site A, although necessary, is notsufficient for repression of apoAI gene expressionin hepaticcells. They are also consistent with the well documented modularity of transcription factors (including nuclear receptors), which display distinct DNA binding and functional domains (14-17). lluo Separate Repression Domains Mediate Repression of apoAI Gene by ARP-I-Comparison of the ARP-1 derivatives ARP-AA1 and ARP-AA6 with respect to their DNA binding dissociation constants and theability to repress transcription further allowed us to map the repression domaids) of ARP-1. Results from the EMSA assay indicated that both ARP-AA1 (Kd = 2.8 m)and ARP-AA6 (Kd= 4.6 I")bind to site A with affinities comparable with thatof the full-length ARP-1 (Kd= 4.2 ILM) (see Fig. 4, A X ) . CAT activity expressed by the -256AI.CAT reporter in HepG2 cells is repressed by increasing amountsof ARP-AA1 and ARP-AA6 as effectively as the wild-type ARP-1
Regulation of Danscription by Nuclear Hormone Receptors
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FIG.5. Existence of two repression domains in ARP-1. Results from cotransfection experiments with an increasing amount of ARP-1 mutants and a constant amount of -256AI.CAT. Data presentation is similar to that in Fig. 1.A, ARP-aA1; B , ARP-aA6; C, ARP-pgala (see text). (Fig. 5,A and B ). These resultsindicated that either the aminoor the carboxyl-terminal end of ARP-1 can complement the DNA binding domain in repression function. There are two possible interpretations of our results as follows. 1)Two distinct repressiondomains located on either side of the DNA binding domain of ARP-1 could account for the observed functions of ARP-1. 2) Alternatively, specific amino acid sequences flanking theARP-1 DNA binding domainper se are not involved in the repression phenomenon. Rather, the presence of bulk peptide at site A interferes with events required for transcription stimulation of the apoAI gene basal promoter somewhat reminiscent of the mechanism of repres-
13189
sion of the chorionic gonadotropin a-subunit promoter by the glucocorticoid receptor (18). To distinguish between these two possibilities, we fused the ARP-1 DNA binding domain to an unrelated polypeptide, the a-peptide of P-galactosidase (ARP-pgala).As shown in Fig. 5C, this fusion protein could not repressapoAI promoter activityin HepG2 cells in a dose-dependent manner. EMSA analysis revealed that the fusion protein could bind to site A (data not shown). These results strongly argue for the presence of two ARP-1 repressiondomains, one located in the carboxyl-terminal segment and the other located in the amino-terminal segment. CIEBP and Egr-1 Can Overcome ARP-1-mediated Repression of apoAI Gene Expression in HepG2 Cells, but HNF4 Cannot-We have previously reported that in the presence of itsligand, RXRa can overcome ARP-1-mediated repression (10). As an extension of these studies and in view of the fact that ARP-1 does not function in isolation within thecontext of the natural enhancer,we have now examined the involvement, vis a vis repression by ARP-1, of transcription factors C/EBP (19, 20), Egr-1 (21), and HNF-4 (221, each of which has been implicated in hepaticgene expression (23-25). Our selection of these transcriptionfactors was further dictated by the presence of potential cognate sites for these factors in the apoAI enhancer as follows: C/EBP, -152 to -178 (site B) ( 7 ) ;Egr-1, -174 to -196 (site E) (12,21); and HNF-4, -214 to -192 (site A) (13). The reporter -256AI.CAT was cotransfected with a constant amount (2 pg) of ARP-1 expression vector together with increasing amounts (2,5, and 10 pg) of either theC/EBP expression vector or the Egr-1 expression vector (Fig. 6, A and B ) . Both C/EBP and Egr-1 could completely overcome ARP-l-mediated repression on -256AI.CAT in HepG2 cells. Note that in the absence of ARP-1, the -fold stimulation of transcription by C/EBP above that observed with -256AI.CAT alone was very low (up to 2-fold stimulation) andoccurred only in thepresence of a very high amount of C/EBP-expressing plasmid (10 pgl plate) (Fig. 6A). In thepresence ofARP-1, the -fold stimulation of C/EBP above the repressed -256AI.CAT expressionwas much more dramatic (up to10-fold) (Fig. 6 A ) and was observable at low C/EBP levels (from 2 pdplate). Egr-1 activated -256AI.CAT expression to very high levels irrespective of the presence or absence ofARP-1(note thescale difference between Fig. 6, A and B 1. As described previously, RXRa,which bound to site A, also stimulated the ARP-1-repressed -256AI.CAT expression in HepG2 cells in the presence of RA (10) (Fig. 6C), although, unlikeC/EBP and Egr-1, RxRa could not activate the ARP-1-repressed apoAI promoter to levels beyond those observed for the non-ARP-1-repressed levels. HNF4 could not activate -256AI.CAT expression in HepG2 cells (see Fig. 6D), although its ability to stimulate the apoAI promoter via site A placed upstream of the core promoter elements (LA1-41Al.CAT) (Fig. 6 E )was unimpaired. In this sense, site A functions as a conditional HNF4-responsive element depending uponthe promoter contextin which it is placed. This is analagous to the conditional reponsiveness of RXRa (10). However, HNF4 differed from RXRa (and C/EBP and Egr-l) in that it could not activate ARP-1-repressed apoAI promoter (-256AI.CAT). These resultsindicatethat ARP-1-repressed apoAI gene in HepG2 cells respond to selective transcriptional signals. Lack of apoAI Gene Expression in Non-hepatic Cells Is Unlikely Due to the Simple Presence of ARP-1 in These CellsGiven the ubiquitous distributionof ARP-1 in non-hepatic but not in hepatic tissues and thehigh apoAI expression levels in the latter (9),we wondered whether there was also a correlation between cell type and theability of a transcription activator (e.g. RXRa) to overcome the imposed repression. Thus, if
Regulation of ll-anscription by Nuclear Hormone Receptors
13190 Im
Calls: Raportar:
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FIG.6. CIEBP, Egr-1, and RXRa can overcome ARP-1-mediatedrepression on -256AI.CAT, but HNF4 cannot. An increasing amount of pMT2-C/EBP or pCMV5-Egr-1 and pMT2-HNF4 (as indicated a t the bottom of each histogram) or a constant amount of pMT2-RXRa (5pg) was cotransfected in the presence (2 pg pMT2-ARF"1)or absence ofARP-1 expression plasmid and 10 pg of reporter -256AI.CAT into HepG2 cells. In E , 10 pg of pMT2-HNF4 was used together with the reporter LA1-41.CAT. The CAT activity values shown are theaverage of at least threedifferent experiments. They are all shown as a relative value to that of the activity of -256AI.CAT in the absence of pMT2-ARP-1. The S.D.is indicated by error bars.
ARP-1 is also responsible for apoAI repression in non-hepatic cells (e.g. CV-1 cells), the expression of CAT activity from the reporter -256AI.CAT in non-hepatic cells would be stimulated by cotransfection with RXRa-expressing plasmid (pMT2RXRa) plus 9-cis-RA. As shown in Fig. 7A, the absence ofCAT activity from -256AI.CAT in CV-1 cells could not be overcome by cotransfecting pMT2-RXRa in thepresence of 9-cis-RA. This was thecase whether or not exogenous ARP-1 was introduced into thecells. Analogous results were also obtained with various other cell lines of non-hepatic origin (data not shown). Apparently, these results could not havebeen causedby the inability of the R X R a expression vector to express R X R a in CV-1 cells, since the [A]-41AI.CAT construct showed a dramatic increase in CAT activity in the presence of RXRa and 9-cis-RA (see Fig. 7 B ) . This is consistent with the previous result that site A is a conditional retinoic acid-responsive element depending on the promoter context it is in (10). In conclusion, repression imposed upon apoAI promoter in non-hepatic cells must differ from that experimentally mediated by AFtP-1 in HepG2 cells. Therefore, the lack of apoAI gene expression in non-hepatic cells is unlikely to resultsolely from the presence of ARP-1 in thesecells. DISCUSSION
In this work, we have studied the repression of apoAI gene transcription by the orphan receptor ARP-1. We have mapped the minimal enhancer element(siteA) required for ARP-1 DNA binding (and function) and defined the ARP-1 domains respon-
sible for its repression function. Our results also indicate that repression of apoAI transcription by ARP-1 is subject to other influences such asthose manifested through the transcription factors CEBP andEgr-1. Repression of lFanscription by ARP-1-How does binding of ARP-1 to site A (in an appropriatepromoter context) result in reduced transcription from the apoAI core promoter? At least two distinct mechanisms can be envisaged as follows. 1)What is observed as repression by ARP-1 could be the resultofARP-1 counteracting the effect of a n activator(perhaps working through site A) that in itsabsence plays a role in maintaining elevated apoAI transcription levels. This could be achieved either by forming inactive heterodimers with this factor or by replacing it on the DNA by ARP-1, which lacks any activation potential. 2) Alternatively, ARP-1 could possess an active silencing domain,whereby it interferes with the basal transcription machinery directly. Although our data do not permit us to unequivocally distinguish between the two possibilities, the following lines of evidence tend to favor the first. First, the direct observation that transcription levels from the construct containing site A upstream of the core promoter elements ([A]-41Al.CAT) (Fig. 1C) are not reduced to levels below those observed with a construct under the control of only the core promoter elements (-41Al.CAT) (Fig. ID) seemingly argues againsttargeting of thebasaltranscription machinery by ARP-1. However, it should be pointed out that the low sensitivity of the assay makes this conclusion somewhat premature. Second, ARP-1-mediated repression is strongly promoter context-dependent. Thus, ARP-1 failed to repress transcription
Regulation of Danscription by Nuclear Hormone Receptors 0
19 13
1
analagous manner cannot be ruled out (29). It should also be borne in mind that ARP-1 remains an orphan member of the nuclear receptor family, and its properties in the presence of any ligand may require reevaluation. Possible Role of ARP-1-mediated Repression in ReprogrammingApolipoprotein A1 Gene Danscription-What cellular need is fulfilled by ARP-1-mediated repression? Not onlyis the ARP-1-inducedrepressed state reversible, it is in fact a prerequisite for activation by R X R a (in thepresence of its ligand) and C/EBP. Our working hypothesis, which provides a framework for interpreting these apparently paradoxical observations, is that the repression state imposed by ARP-1 is an obligatory A R P - 10 RXRa + + + intermediate step in the switch from one transcriptionally acRA + + tivated state t~ another. This could potentially provide the cells with an opportunity to sort and fine tune various incoming signals. We thus imagine that the enhancer is in a constant C a l l : cv-1 state of flux; the complement of transcription factors regulating Raportar: W [A]-4lAl.CAT -4lAI.CAT apoAI gene expression is continually changing as the cell responds and adapts to incoming external signals. Accordingly, for different cell types or cells under different physiological . s f 40 conditions, distinct activation strategies are predicted. Thus, as the cell gears up to respond to 9-cis-retinoic acid, its cognate 30 receptor RXRa is mobilized. But, given the prevailing tranc 0 scription factor distribution at the enhancer, RXRa, which is otherwise quite competent to activate through site A, is unable to do so unless prior (or concurrent) repression by ARP-1 is also induced. We suggest that this requirement reflects a factor exchange that isfacilitated by ARP-1,perhaps by virtue of its ARP.1 + ability to form heterodimers (10). RXRo + + + Nevertheless, we wish to emphasize that theproposed ARPRA + + 1-mediated factor exchange (in response to changes in external F'IG. 7. Response of -266AI.CAT to RxRa and 9-cis-RAin a nonhepatic cell line. A, the reporters (10 pg) -256AI.CAT (shaded) or stimuli) isnot restricted to nuclear hormone receptors, i.e. fac4lAl.CAT (unshaded)were cotransfectedwith 2 pg of pMT2-ARP-1and tors that primarily function through site A as evidenced by an 5 pg of pMT2-RXRainto CV-1 cells in the presence or absence of 10" M ARP-1 requirement for CBBP (Fig. 6). Thus, in this particular 9-cis-RA(as indicated). CAT activities were normalized by the cotransfected &gal activity. The values shown are the average of at least three instance, ARP-1 is hypothesized to recruit C/EBP to its activatexperiments, and the S.D. is shown as error bars in the histogram. B , ing site by facilitating factor exchange via protein-protein inthe reporters [A1-41Al.CAT(shaded)and -4lAl.CAT (unshaded)were teractions with factors bound to neighboring sites. At the same cotransfected with pMT2-ARP and pMT2-RXRa into CV-1 cells and time, the model presupposes a heirarchy for the activation poanalyzed as in A. tential of incoming transcription factors, whereby somefactors fail to overcome ARP-1-imposedrepression (HNF-4) (Fig. 6D), from the heterologous thymidine kinase promoter, which was while others are oblivious to the presence or absence of ARP-1 placed downstream of multiple copies of site A. The simplest in their vicinity (Egr-1) (Fig. 6B). interpretation of this result is that ARP-1 cannot counteract Acknowledgments-We thank Drs. 0. Connelly, S. McKnight, V. P. the effect of transcription activators (most likely SP1) driving Sukhatme, and M. J. Tsai for gifts of plasmids containing c D N h for the thymidine kinase promoter, whereas, with the complement some of the transcriptionfactors used in this work. We also thank Dr. A. of transcription factors assembled at the apoAI enhancer, it Lizonova for assistance with cell culture work and N. Papanicolaou for preparation of plasmid DNA. Can. These conclusions are in apparent variance with the hypothREFERENCES esized mechanism of repression by COUP-TF1,which was 1. Mitchell, P. J., and Tjian, R. (1989)Science 245, 371478 shown to directly target thebasal transcription machinery (26). 2. Karathanasis, S. K (1992)Monogr: Hum. Genet. 14, 140-171 However, this effect of COUP-TF1 is presumably secondary to 3. Zannis, V. I., Cole, F.S., Jackson, C. L., Kurnit, D. M., and Karathanasis, S.K. other mechanisms by which it reduces transcription levels (27) (1985)Biochemistry 24,445Cl-4455 Pm. 4. Elshourbagy, N. A,, Liao, W. S., Mahley, R. W., and Taylor, J. M. (1985) that include competition, for the target site, with members of Natl. Acad. Sci. U.S. A. 82, 203-207 the nuclear receptor family that can activate andalso by form5. Elshourbagy, N. A., Boguski, M. S.,Liao, W. S., Jefferson, L. S., Gordon, J. I., and Taylor, J. M. (1985) P m . Natl. Acad. Sci. U.S. A. 82, 8242-8246 ing inactive heterodimers with them. The same may therefore 6. Sastry, K.N., Seedorf, U., and Karathanasis, S. K.(1988)Mol. Cell. Biol. 8, in fact be true forARP-1whose repression effectsmay be 605414 achieved a t a variety of levels. Clearly, the situation at the 7. Widom, R.L., Ladias, J. A. A,, Kouidou, S.,and Karathanasis, S. K (1991)Mol. Cell. Biol. 11, 677-687 apoAI enhancer is much more complicated,because, in addition 8. Rottman, J. N., Widom, R. L., Nadal-Ginard, B., Mahdavi, V., and Karathato the nuclear receptors, the naturalmilieu ofARP-1 subjects it nasis, S. K (1991)Mol. Cell. Biol. 11, 3814-3820 9. Ladias, J. A,, and Karathanasis, S.K (1991)Science 251, 561565 to influences mediated by unrelated transcription factors (see 10. Widom, R. L., Rhee, M., and Karathanasis, S. K (1992)Mol. Cell. Bwl. 12, below). 3380-3389 The existence of repression domains in ARP-1 is analagous to 11. Power, R.F.,Lydon, J. P., Connelly, 0. M., and O'Malley, B. W. (1991) Science 252, 1546-1548 what is observed for the thyroid hormone and retinoic acid 12. Cao, X.,Koski, R. A., Gashler, A,, McKieman, M., Moms, C. G., Gaffiney, R., ( m a )receptors (28). Since these nuclear receptors have been Hay, R. V., and Sukhatme, V. P. (1990)Mol. Cell. Biol. 10, 1931-1939 shown to have transferable silencing (i.e. repression) domains 13. Mietus-Snyder,M., Sladek, F. M., Ginsburg, G. S., Kuo, C. F.,Ladias, J. A. A., Damell, J. E,,and Karathanasis, S. K (1992)Mol. Cell. Biol.12, 170%1718 that have the clear potential for interacting with basal tran14. Green, S., and Chambon, P. 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-256AI.CAT -4lAI.CAT
+
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