Estrogen Modulation of Apolipoprotein(a) Expression
IDENTIFICATION OF A REGULATORY ELEMENT*

Dario BoffelliDagger , Deborah A. Zajchowski§, Zhuoying YangDagger , and Richard M. LawnDagger

From the Dagger  Falk Cardiovascular Research Center, Stanford University School of Medicine, Stanford, California 94305-5246 and the § Cancer Research Department, Berlex Biosciences, Richmond, California 94804

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Elevated plasma levels of the lipoprotein particle Lp(a) are a major risk factor for cardiovascular disease. Lp(a) plasma levels are determined by the level of expression of its characteristic protein component, apo(a). Apo(a) expression is modulated by several hormones, of which estrogens are the best known. The chromosomal region responsible for estrogen response was identified within an apo(a) enhancer located at ~26 kilobases from the apo(a) promoter. Although the estrogen-responsive unit contains a potential estrogen response element, binding of estrogen receptor-alpha to DNA was not necessary. The receptor, activated by bound estradiol, interacts through its transactivation domains with a transcription factor necessary for the function of the enhancer, preventing its binding to DNA.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Apolipoprotein(a) is the characteristic protein component of the lipoprotein particle Lp(a). Lp(a) consists of a low density lipoprotein covalently linked to apo(a) through its protein moiety, apolipoprotein B-100 (1, 2). Most prospective studies and a recent meta-analysis have identified Lp(a) excess as a major risk factor of premature atherosclerotic vascular disease (3-6), although there are clearly some exceptions (7). Apo(a) is a highly polymorphic glycoprotein with a close sequence homology to plasminogen that contains from 12 to 50 copies of a domain homologous to plasminogen kringle 4 (8, 9). These sequence similarities constitute a basis to explain the correlation between high Lp(a) concentration and atherosclerosis. Through its apo(a) moiety, Lp(a) competes with plasminogen for binding to fibrin. This prevents plasminogen conversion to plasmin, which in turn hinders fibrinolysis and activation of latent transforming growth factor-beta , contributing to an atherogenic phenotype (10-16).

In contrast to other lipoproteins, Lp(a) plasma concentration varies widely (<0.1 to >100 mg dl-1) in individual humans, and at least 90% of this variation is attributable to the apo(a) genetic locus (17, 18), indicating that the regulation of apo(a) expression is significant in controlling pathological levels of Lp(a). Generally, Lp(a) plasma concentration is inversely proportional to the number of apo(a) kringle repeats (19). However, other features of the apo(a) locus contribute to determine Lp(a) plasma levels since Lp(a) plasma concentration can vary several hundredfold in individuals with the same number of kringles (20, 21). Sex steroid hormones and particularly estrogens are known to lower Lp(a) levels. Population studies have shown that Lp(a) levels increase 8-13% in postmenopausal women relative to premenopausal controls (22, 23). Longitudinal studies have reported that hormone replacement therapy lowers Lp(a) concentration as much as 50% (24-27). Data from transgenic mice containing the human apo(a) gene locus on a yeast artificial chromosome corroborate these results (28). Pharmacological doses of 17beta -estradiol lowered plasma apo(a) protein concentration and hepatic apo(a) mRNA concentration by ~80% in these mice (29).

In this study, we describe the identification of the site responsible for estrogen regulation of apo(a) gene expression. Previous work from this laboratory has shown that a DNA region located from -98 to +130 relative to the apo(a) transcription start site is sufficient to drive apo(a) transcription in a reporter vector (30). This region binds to the liver-enriched transcription factor hepatocyte nuclear factor-1alpha and partially accounts for the liver-specific synthesis of apo(a). The homologous apo(a) and plasminogen genes are organized in a tandem head-to-head configuration, with 35 kb1 of genomic DNA separating their 5'-ends (31, 32). Sequences in this "intergenic" region contribute to the regulation of apo(a) expression. Its deletion in the above-mentioned genomic yeast artificial chromosome severely curtails apo(a) expression in the transgenic mouse.2 Several DNase I-hypersensitive sites are located in this region ("DHI-DHIV") (33), two of which correspond to apo(a) gene regulatory sequences. An enhancer contained within a LINE retrotransposon lies 18 kb 5' of the apo(a) promoter and corresponds to hypersensitive site DHIII (34, 35), whereas an enhancer requiring the Sp1 and PPAR transcription factors corresponds to the DHII element, located 26 kb upstream of the apo(a) promoter (34). Here we report that the DHII element is also responsible for estrogen inhibition of apo(a) expression. Estrogen receptor-alpha does not appear to bind directly to the DNA sequence element, but to interfere with transcription factor(s) necessary for the DHII enhancer activity.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Plasmid Construction-- The pGL3a reporter plasmid (35) contains the Photinus pyralis (firefly) luciferase cDNA driven by the apo(a) -98/+130 minimal promoter (30). The apo(a)/plasminogen intergenic region was isolated from a bacterial artificial chromosome clone containing ~150 kb of the apo(a)/plasminogen locus, and restriction fragments were subcloned into the pGL3a plasmid (35). The DHII fragment was generated by polymerase chain reaction synthesis from the appropriate subclone with primers DHII/L (5'-AAGGAGCCCTGAGCCTGAA-3') and DHII/R (5'-TGCCATAAATATACAAGTCCCT-3') containing either an EcoRI or a SacI restriction site. The polymerase chain reaction product was subcloned into the EcoRI site of pGL3a or into the SacI site of either the pGL3-Promoter vector (Promega), which contains the SV40 early promoter region, or the hsvTK vector. Constructs ee-I + ee-II and ee-II were generated by polymerase chain reaction synthesis with primers derived from the sequence shown in Fig. 2A containing the EcoRI restriction site, followed by ligation into the EcoRI site of pGL3a. Constructions of the hsvTK vector, containing the -105/+10 herpes simplex virus thymidine kinase promoter fragment, and of the expression vectors for wild-type and mutant ER-alpha have been described and were kindly provided by Sotirios Karathanasis (36). The vitERE plasmid contains a copy of the vitellogenin gene ERE cloned in front of the thymidine kinase promoter in the hsvTK vector. Site-specific mutagenesis was carried out according to the mismatch primer protocol (37) with oligonucleotides DHII/L and DHII/R as the external primers. The internal primers contained the mutations described in Fig. 3. The polymerase chain reaction products were digested with EcoRI, subcloned into pGL3a, and confirmed by sequencing prior to the transfection assay.

Generation of HepG2-ER Cells-- To generate the HepG2-ER cells, human HepG2 hepatoma cells (ATCC HB8065) were transfected with the pSV2neo/CMV-ER-alpha expression vector as described (38), except that the ER-alpha cDNA corresponds to the HEGO sequence (39). Cells were cultured in Eagle's minimal essential medium (Life Technologies, Inc.) supplemented with 1 mM HEPES, 2 mM glutamine, 0.1 mM minimal essential medium non-essential amino acids, 1.0 mM sodium pyruvate, 50 µg/ml gentamycin, 10% fetal bovine serum, and 10 nM ICI 164,384 during selection in 1000 µg/ml G418 (Life Technologies, Inc.). Stable ER-alpha -expressing clones were identified by Western blotting (38) using anti-ER-alpha antibodies (kindly provided by G. Greene).

Cell Culture and Transfection Assays-- HepG2-ER cells were maintained Eagle's minimal essential medium supplemented with 1 mM HEPES, 2 mM glutamine, 0.1 mM minimal essential medium non-essential amino acids, 1 mM sodium pyruvate, 1000 µg/ml G418, 50 µg/ml gentamycin, and 10% charcoal/dextran-treated fetal bovine serum (Hyclone Laboratories). Approximately 2 × 105 cells/well were seeded in a 12-well cell culture plate 24 h prior to transfection by a calcium phosphate precipitation method according to the manufacturer's protocol (Promega) as we described previously (30). Transfections were carried out in triplicates. Briefly, 1.5 µg of pGL3a-based plasmid and 0.15 µg of either pSV-beta -galactosidase (Promega) or pRL-TK (Promega) control plasmid were added to each well, covered with 2 ml of maintenance medium. Following a 6-h transfection, fresh medium was added containing either 100 nM 17beta -estradiol (Sigma) in ethanol or ethanol alone to a final concentration of 0.01%. After a 36-h incubation with one medium change, the cells were harvested and lysed, and expression activity was determined using either the Dual Light kit (Tropix Inc.) or the Dual Luciferase Reporter kit (Promega) according to the protocols provided. To account for transfection efficiency, results are reported as the ratio of the sample to control plasmid activity.

Gel-shift Analysis-- Nuclear extracts from HepG2 or HepG2-ER cells were prepared as described (30). The following oligonucleotide probes were used: ee-II, 5'-CATGTTGACACAGGTCAAATCCTtgaacTCTGTTGCCCAAATA-3'; and ee-IImut, where the nucleotides indicated in lowercase in ee-II were changed to CCTAG. A complementary oligonucleotide was synthesized, and the probe was end-labeled with [gamma -32P]ATP. The procedure for gel mobility shift assay was as described (40), except that the binding buffer contained 60 mM KCl and 10% (w/v) Ficoll. Human recombinant estrogen receptor-alpha was from PanVera (Madison, WI). Polyclonal antibodies against human ER-alpha and ARP-1 (apoA-I regulatory protein) were from Santa Cruz Biotechnology (Santa Cruz, CA). The sequence of the ARP-1 oligonucleotide used for gel-shift competition was 5'-CTAGCGATATCATGACCTTTGTCCTAGGCCTC-3' (41).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Identification of an Apo(a) Gene Estrogen-responsive Element-- The 35-kb region between the apo(a) and plasminogen genes was isolated in a bacterial artificial chromosome clone, and fragments were subcloned in front of the apo(a) minimal promoter and luciferase cDNA as reported (35). The plasmids were tested in HepG2 cells that had been modified to produce estrogen receptor-alpha (HepG2-ER cells). Following transfection, the cells were treated with 100 nM 17beta -estradiol (E2) for 36 h, and luciferase activity was determined. The expression level of the minimal apo(a) promoter reporter (construct pGLa3) (Fig. 1) was not affected by E2 treatment. No constructs showed significant up-regulation by E2. Only the construct spanning the DHII enhancer showed significant reduction of luciferase activity in the presence of E2 (construct 20). This genomic region was tested in both orientations (constructs 20+ and 20-) (Fig. 1) because its enhancer activity had been found to be orientation-dependent (34). The enhancer activity and E2 response were larger with the insert cloned in the same orientation relative to the apo(a) minimal promoter as it is in the chromosomal locus (defined as the positive orientation). It is noteworthy that expression from the reporter plasmids containing the two other regions previously shown to have enhancer properties, constructs 14 and 24, was not significantly affected by E2 (34, 35, 42).


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Fig. 1.   Apolipoprotein(a)/plasminogen intergenic region. The chromosomal region separating the apo(a) and plasminogen (plg) genes is shown at the top. Their respective direction of transcription is indicated by the arrows at the extremities of the map. The numbering indicates the fragments subcloned into the luciferase reporter vector. Fragments 1 and 24 contain the first EcoRI site upstream of the apo(a) and plasminogen gene transcription start sites, respectively (cf. Ref. 33). DNase I-hypersensitive sites are indicated by vertical arrows. Below is shown the transcriptional activity of the reporter constructs in HepG2-ER cells. HepG2-ER were transfected with 1.5 µg of reporter construct/well (of a 12-well plate) together with 0.15 µg of pSV-beta -galactosidase. Following transfection, the cells were grown in the absence (stippled bars) or presence (black bars) of 100 nM 17beta -estradiol for 36 h. Transcriptional activities, normalized to pSV-beta -galactosidase activity, are expressed relative to the activity of the pGL3a construct, the plasmid containing the luciferase reporter driven by the -98/+130 apo(a) minimal promoter. The data represent the mean ± S.D. of three transfections.

Identification of the Minimal Estrogen-responsive Unit-- To define the minimal estrogen-responsive unit (ERU) located within fragment 20, we characterized the KpnI-SduI subfragment shown in Fig. 2. When transfected into HepG2-ER cells, this fragment was capable of increasing by 10-fold the luciferase expression driven by the apo(a) minimal promoter in a orientation-independent fashion. After E2 treatment, luciferase expression was reduced by ~70%. Fig. 2A highlights the DNA sequences required for the enhancer activity (ee-I and ee-II) (34). Reporter vectors containing both enhancer elements I and II exhibited significant enhancer activity, which was sharply reduced by E2. Enhancer element II alone had a small effect compared with enhancer elements I + II, but was still E2-responsive. However, when enhancer element II was cloned as a tandem duplicate in the reporter vector, enhancer activity and E2 response were the most dramatic. All the responses were essentially independent of the orientation of the cloned construct.


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Fig. 2.   Identification of the minimal estrogen-responsive unit. A shows a blow-up of region 20 of Fig. 1. The KpnI-SduI fragment is the DHII region containing the essential elements of the enhancer, shown in the boxes underneath. B shows the transcriptional activity of the reporter constructs in HepG2-ER cells. The cloning orientation is shown in the construct diagram on the left. Details of the transfection and data analysis are as described in the legend to Fig. 1. Stippled and black bars indicate that the cells were grown in the absence and presence of 100 nM 17beta -estradiol, respectively. plg, plasminogen; luc, luciferase.

The relationship between the ERU and the apo(a) minimal promoter was studied by subcloning the DHII enhancer in both orientations in front of two heterologous promoters, the SV40 early promoter and the hsvTK promoter (data not shown). Upon transfection of HepG2-ER cells, the DHII enhancer increased 3-fold the transcription driven by the SV40 promoter, but had no effect on the hsvTK promoter. The SV40 promoter construct showed a small reduction in luciferase activity following E2 treatment. A similar reduction was observed for the DHII/SV40 construct, suggesting that there is no additional contribution by the DHII insert to E2 response mediated by the SV40 promoter. These results with heterologous promoters suggest that the DHII enhancer/ERU requires a synergistic interaction with factors bound to the apo(a) minimal promoter.

The estrogen responsiveness induced by the DHII element is dependent on both ER-alpha and E2. In fact, when normal HepG2 cells (lacking ER-alpha ) were transfected with the DHII/apo(a) minimal promoter reporter vector, there was no change in luciferase activity in response to E2 treatment. Cotransfection with an expression vector for ER-alpha made normal HepG2 cells estrogen-responsive. This responsiveness was partially competed by the estrogen receptor antagonist ICI 182,780 at a concentration of 1 µM (data not shown).

Mutation Analysis of Enhancer Element II-- Inspection of the sequence of ee-II reveals a very close match to the consensus ERE (shown in boldface in Figs. 2A and 3), the DNA-binding site of ER-alpha (43). An ERE is characterized by a 6-base pair palindromic repeat separated by three nucleotides. The palindromic repeat in ee-II is separated by six nucleotides. We carried out extensive mutagenesis of this site (shown in Fig. 3) and tested the constructs by transfecting HepG2-ER cells. Mutations in the right arm of the ERE reduced the enhancer activity, but retained estrogen responsiveness, as did inserting six nucleotides between the left and right arms of the potential binding site. Mutations in the left arm eliminated both the enhancer activity and estrogen response. The observation that only mutations in the left arm of the potential ERE are effective in impairing estrogen response suggests that ER-alpha might not be bound to the full palindromic site in a canonical manner. However, binding of some trans-acting factor to the left arm sequence is suggested. Furthermore, the observation that estrogen response impairment is accompanied by enhancer activity impairment suggests that ligand-bound ER-alpha interferes with the function of factors necessary to ee-II activity.


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Fig. 3.   Mutation analysis of the estrogen-responsive unit. The sequence at the top highlights the potential ERE in ee-II (cf. Fig. 2). The nucleotide changes in the mutated constructs are shown underneath. Details of the transfection and data analysis are as described in the legend to Fig. 1. Stippled and black bars indicate that the cells were grown in the absence and presence of 100 nM 17beta -estradiol, respectively.

ER-alpha Domain Requirement for E2 Responsiveness-- To gain more insight on the mechanism of E2-dependent repression of the DHII enhancer, we carried out an analysis of which ER-alpha domains are required to that effect. To this end, normal HepG2 cells were cotransfected with the DHII/apo(a) minimal promoter reporter vector or a control vector carrying the luciferase gene driven by the vitellogenin ERE/hsvTK promoter (Fig. 4, shaded and white bars, respectively) plus either wild-type or mutant ER-alpha . As the first group of bars reiterates, estradiol treatment increases activity from the vitellogenin ERE, but reduces activity from the apo(a) gene element. ER-alpha is composed of one DNA-binding domain (DBD), responsible for binding to the ERE (44), and two transactivation domains, N-terminal AF1 and C-terminal AF2 (39). Changing the DBD binding selectivity from an ERE to a glucocorticoid receptor element by a point mutation (mutant AF1-X-AF2) completely destroys E2 responsiveness from the ERE/hsvTK plasmid as expected, but does not eliminate the inhibitory effect of E2 on the DHII enhancer. Deletion of the AF1 domain also nearly fully impairs response from the ERE/hsvTK plasmid, but not the E2 inhibitory response from the DHII enhancer (mutant X-DBD-AF2). Inactivation of the AF2 domain reduces ERE/hsvTK plasmid activation, but retains the E2 effects the DHII enhancer (mutant AF1-DBD-X). Finally, inactivation of both transactivation domains reduces ERE/hsvTK plasmid activation and completely abolishes DHII enhancer repression (mutant X-DBD-X). The requirement for intact transactivation domains supports the hypothesis that ER-alpha interacts with other transcription factors involved in the DHII enhancer activity, thus interfering with their function.


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Fig. 4.   Analysis of the ER-alpha domain requirement for estrogen responsiveness. Normal HepG2 cells were cotransfected with 1.5 µg of either the DHII/pGL3a construct (shaded bars) or the vitERE/hsvTK construct (white bars) together with 0.15 µg of pRL-TK (expressing Renilla reniformis luciferase) and 0.15 µg of expression vector encoding ER-alpha with the indicated truncations and point mutations (indicated by X). Following transfection, the cells were grown in the absence (unhatched bars) or presence (hatched bars) of 100 nM 17beta -estradiol for 36 h. Growth medium was changed once. Transcriptional activities, normalized to pRL-TK activity, are expressed relative to the activity of the vitERE/hsvTK construct, the activity of which (cut off in the figure) was set to 100%. The data represent the mean ± S.D. of three transfections.

That a functional ER-alpha DNA-binding domain is not necessary for E2 responsiveness is a strong indication that ER-alpha exerts its activity without a direct interaction with DNA. To confirm this hypothesis, we subjected an oligonucleotide probe encompassing the sequence of ee-II (cf. Fig. 2A) to electrophoretic mobility shift assay. The formation of a complex between the DNA probe and nuclear protein extracts made from HepG2 cells (Fig. 5, lane 2) was competed by the addition of purified human recombinant ER-alpha (lane 5). Similar results were obtained by incubating the probe with nuclear as well as whole cell protein extracts made from HepG2-ER cells in the presence or absence of 100 nM E2 (data not shown). The addition of polyclonal antibodies against ER-alpha to the incubation mixture failed to supershift any band in the assay (lane 7). These findings support the conclusion that ER-alpha is not bound to ee-II DNA. Similar results were obtained by using as a probe an oligonucleotide containing the GTTCA right-arrow CTAGG mutation, which abolishes the enhancer and estrogen response activity of the ERU (cf. Fig. 3). Inspection of the sequence around this site reveals a potential binding site for the nuclear orphan receptor ARP-1, a member of the steroid receptor superfamily (41). Competition gel-shift experiments with a consensus ARP-1-binding sequence (Fig. 5, lane 4) and gel-supershift experiments with polyclonal antibodies against ARP-1 (lane 8) imply that ARP-1 does not bind the probe sequence either.


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Fig. 5.   Gel-shift analysis of the ERU. An oligonucleotide spanning the ee-II sequence (see Fig. 2A) was used as a probe for a gel-shift assay (lanes 1-8). The same probe was used in lanes 9-16 except for a GTTCA right-arrow ctagg mutation, which inactivates enhancer/estrogen response activity. Lanes 1 and 9 contain probe only, whereas all other lanes contain 5 µg of HepG2 nuclear extract. Lanes 3 and 11 and lanes 4 and 12 contain a 100-fold excess of unlabeled probe and ARP-1 oligonucleotide competitor, respectively. Lanes 5 and 13 contain 0.3 µg of purified human recombinant ER-alpha . Lanes 6 and 14 contain 0.2 µg of goat immunoglobulin G. Lanes 7 and 15 and lanes 8 and 16 contain 0.2 µg of goat anti-ER-alpha and anti-ARP-1 immunoglobulin G, respectively.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Sex steroid hormones are the most potent known regulators of apo(a) gene expression and concomitant plasma concentration of Lp(a). Previous studies have described chromosomal elements involved in apo(a) expression, a 300-base pair promoter encompassing the apo(a) transcription start site and putative apo(a) enhancers located 18 and 26 kb upstream (30, 34, 35). Control elements located at large distances from the promoter are involved in the regulation of numerous other genes, including other apolipoproteins (45-48). Here we report that the DNA region containing the enhancer element at 26 kb from the apo(a) promoter also promotes 17beta -estradiol responsiveness in a reporter vector in transfected hepatic cells. Its position coincides with a DNase I-hypersensitive site (33). Such sites are normally associated with an open chromatin structure, an indication that the DNA region is in a favorable conformation to interact with transcription factors (49). The activity of the 700-base pair fragment containing the DHII enhancer (construct 20 in Fig. 1) was orientation-dependent, the activity being stronger when the fragment was cloned in the same orientation relative to the apo(a) promoter as it is in the chromosomal locus. A larger 2-kb fragment was reported to lack enhancer activity when cloned in the opposite orientation to the apo(a) promoter (34). For this reason, we failed to detect this enhancer in our previous screening for apo(a) regulatory elements. It is not clear what gives rise to this phenomenon. It is possible that additional negative regulatory or insulator elements are present downstream of the DHII enhancer, to ensure proper separation and differential regulation of the apo(a) and plasminogen genes.

The orientation dependence is essentially lost when the enhancer/estrogen-responsive region is deleted down to its core elements. The DHII enhancer was shown to require a 186-base pair region encompassing two footprints (ee-I and ee-II in Fig. 2). The Sp1 and PPAR transcription factors are required for ee-I activity, and the PPAR for ee-II activity (43). (Such PPAR sites could be involved in the reported effect of retinoids on apo(a) expression (50).) We found that the ee-II region alone was sufficient to confer E2 responsiveness to the apo(a) minimal promoter reporter vector. Although this region contains a potential ERE sequence, our data indicate that direct binding of ER-alpha to this DNA site does not seem to be involved. Mutations in the right arm of the ERE do not impair E2 responsiveness. ER-alpha mutants lacking a functional ERE DNA-binding domain are still efficient, and gel shifts and supershifts fail to yield any evidence of ER-alpha binding. On the other hand, the requirement for functional transactivation domains suggests that ER-alpha interacts with a transcription factor necessary for the enhancer activity, interfering in this way with its function. The gel-shift data indicate that the presence of ER-alpha prevents the interaction between other nuclear proteins and the ERU. This interaction is crucial to its function. In fact, an oligonucleotide bearing a mutation that destroys enhancer and estrogen response activity is impaired in its ability to form the same complexes that are inhibited by ER-alpha .

Interaction of ER-alpha with other transcription factors has been reported for a number of gene regulatory units. In some cases, no direct binding of the receptor to a canonical ERE sequence is involved. For example, in erythroid precursor cells, ER-alpha binds to the transcription factor GATA-1 through its AF2 domain without direct or indirect DNA binding, preventing the activation of GATA-1-requiring genes (51, 52). Another well characterized mechanism by which nuclear receptors have been shown to modulate gene expression is competition for common non-DNA-binding cofactors with other transcription factors (36, 53). This possibility is unlikely due to the observation that binding of a transcription factor to a DNA target seems to be involved in the DHII enhancer/ERU activity. In fact, mutations at a specific site of the ERU destroy all DHII activity. This observation also argues against the possibility that ER-alpha exerts its effect indirectly by inhibiting the expression of factors required for DHII activity.

The identity of the factor interacting with ER-alpha is not determined. The sequence spanning the nucleotides whose mutation was crucial contains a potential binding site for the nuclear orphan receptor ARP-1, but gel-shift competition and gel-supershift experiments did not yield evidence to support its role in ERU function.

The physiological relevance of the DHII enhancer/ERU remains to be established in vivo. Experiments are under way to delete this region from a yeast artificial chromosome transgenic mouse carrying the apo(a) genomic locus. It is interesting to note that, although apo(a) expression is responsive to estrogen, plasminogen expression is not. The homologous apo(a) and plasminogen genes are separated by only ~35 kb of genomic DNA. Their minimal promoter regions have significant sequence and functional similarities, including the requirement for hepatocyte nuclear factor-1alpha . It will be of interest to discover how the two genes are differentially regulated. It can be suggested that some form of insulator element (54, 55) separates the last apo(a) control region (probably the DHII region) from the plasminogen promoter.

    ACKNOWLEDGEMENTS

We thank Rhonda Humm for tissue culture assistance, G. Greene for anti-ER-alpha antibodies, and P. Chambon for the HEGO cDNA. We are greatly indebted to Dr. Sotirios Karathanasis for providing vitERE and the wild-type and mutant ER-alpha expression vectors, in addition to many helpful discussions.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL50590, Berlex Biosciences (to D. A. Z.), and a post-doctoral fellowship from the American Heart Association (to Z. Y.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: CV Therapeutics, 3172 Porter Dr., Palo Alto, CA 94304. Tel.: 650-812-9514; Fax: 650-858-2694; E-mail: lawn{at}cvt.com.

2 S. Hughes and E. M. Rubin, personal communication.

    ABBREVIATIONS

The abbreviations used are: kb, kilobase(s); DH, DNase I-hypersensitive; ee, enhancer element; ER-alpha , estrogen receptor-alpha ; ERE, estrogen response element; E2, 17beta -estradiol; ERU, estrogen-responsive unit; DBD, DNA-binding domain.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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