©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification and Characterization of a Second Retinoic Acid Response Element in the Phosphoenolpyruvate Carboxykinase Gene Promoter (*)

(Received for publication, November 6, 1995; and in revised form, January 11, 1996)

Donald K. Scott (§) John A. Mitchell Daryl K. Granner (¶)

From the Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee 37232

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A previously characterized retinoic acid response element (RARE1) in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter confers approximately 50% of the response of this gene to retinoic acid (RA). Transient transfection experiments were performed using constructs containing progressive 5` deletions of the PEPCK promoter to locate other elements that contribute to the RA response. A second RARE (RARE2) was located between -402 and -306. Methylation interference and mobility gel shift assays indicated that RAR/RXR bound specifically to a segment of DNA located between -337 and -321. This region contains consensus and degenerate half-sites for receptor binding separated by 5 bp. Mutations in either half-site selectively decreased the RA response and diminished RAR/RXR binding in mobility gel shift assays. When both RARE1 and RARE2 were mutated, 80% of the RA response was lost. Finally, RARE2 conferred a RA response in a heterologous promoter context. We conclude that RAR/RXR binds to RARE2, and that this DR5-type element is a major contributor to the response of the PEPCK gene to RA.


INTRODUCTION

The induction of transcription in response to retinoic acid is mediated by retinoic acid receptors (RARs), (^1)members of the steroid/thyroid/retinoid superfamily of nuclear hormone receptors. RARs, as well as thyroid hormone receptors (TRs) and 1,25-dihydroxyvitamin D(3) receptors (VDRs) are members of a subfamily of the nuclear hormone receptors that share structural and functional similarities. These receptors bind specifically to their cognate response elements and alter the transcription rates of target genes in a ligand-dependent manner (for reviews, see (1, 2, 3) ). RAR, TR, and VDR bind DNA with much greater affinity when complexed as a heterodimer with the 9-cis-retinoic acid receptor (RXR). The response elements through which these receptors act are closely related and are usually arranged as two half-sites of the sequence RG(G/T)TCA ( (4) and (5) and references therein). The spacing between the half-sites is an important determinant of nuclear receptor binding specificity. Typically, VDR/RXR and TR/RXR heterodimers bind to direct repeat (DR) half-sites separated by 3 or 4 base pairs (designated DR3 and DR4), respectively. RAR/RXR heterodimers are less discriminating since they bind DR1, DR2, and DR5 retinoic acid response elements (RAREs)(6, 7, 8, 9) . RAR/RXR heterodimers also bind to (and trans-activate from) inverted (10, 11) or everted repeats (12, 13) . It is also important to note that RAREs often contain at least one degenerate half-site(5) .

Phosphoenolpyruvate carboxykinase (PEPCK, EC 4.1.1.32) catalyzes the conversion of oxalacetate to phosphoenolpyruvate, the rate-limiting step of gluconeogenesis, and is, therefore, a focal point for hormonal regulation of blood glucose homeostasis (for reviews, see Refs. 14 and 15). The PEPCK gene product is apparently not allosterically or post-translationally modified to alter its activity. Rather, the activity of PEPCK is directly related to the amount of protein, which is predominantly affected by the rate of transcription of the gene (16) . PEPCK gene transcription is positively regulated by glucagon (via cAMP), glucocorticoids, and retinoic acid(17, 18, 19) . Insulin rapidly inhibits PEPCK gene transcription and is dominant over the positive effectors(20) . Thus, PEPCK is an excellent model system for the studies concerning the integration of multihormonal signals. The cis-acting elements that confer these responses, as well as developmental and liver-specific expression, all lie within the 460 bp upstream of the transcription start site of the PEPCK gene(21, 22) .

We previously demonstrated that retinoic acid (RA) increases the transcription rate of the endogenous PEPCK gene in H4IIE rat hepatoma cells. Utilizing PEPCK promoter-chloramphenicol acetyltransferase (CAT) fusion constructs in transient transfection assays, an imperfect DR1 RARE (RARE1) was localized to a segment between -451 and -433 bp relative to the transcription start site(23) . This core sequence confers approximately 50% of the PEPCK RA response in the intact PEPCK promoter and mediates a RA response when inserted upstream of a minimal thymidine kinase promoter(23, 24) . RAR/RXR binds to RARE1 with high affinity and comprises the functional trans-activating complex that mediates the RA response from this element(24) . The core sequence of the PEPCK RARE is coincident with the minimal functional boundaries of the accessory factor 1 (AF1) element, which is required for a complete glucocorticoid response and is a component of the complex PEPCK glucocorticoid response unit (GRU(19, 24, 25, 26) ). Any mutation in RARE1/AF1 that abolishes the RA response from this element also diminishes the glucocorticoid response and vice versa. Thus, the RARE1/AF1 element is required for at least two distinct hormone response pathways.

In the present paper, we report the identification of a second RARE (RARE2) within the PEPCK promoter. Mutations that decrease the binding of RAR/RXR to RARE2 also reduce the RA response of PEPCK promoter-CAT fusion constructs. When RARE2 is ligated to a thymidine kinase promoter, it confers a RA response to an associated reporter gene.


EXPERIMENTAL PROCEDURES

Plasmid Construction

The construction of a series of reporter constructs containing 5` deletion mutations of the PEPCK promoter ligated to the CAT gene has been described(27) . Site-directed mutations of pPL32 were made using either the oligonucleotide-mediated mutagenesis method (28) or the polymerase chain reaction megaprimer method(29) , as described previously(30) . The thymidine kinase-CAT fusion constructs were made by digesting the plasmid pTKCAT (containing the thymidine kinase promoter from -105 to +51 ligated to CAT(31) ) with BamHI and inserting double-stranded oligonucleotides containing RARE2. The oligonucleotide for RARE2 was also used in methylation interference experiments (see below). The sequence of RARE2 was 5`-GATCCGTCCCGGCCAGCCCTGTCCTTGACCCCCACCTGACAATTAAGGCAAGAGCCT-3`. The sequence of all constructs was verified by the dideoxynucleotide sequencing method.

Transient Transfections

The maintenance and transfection of H4IIE cells has been previously described(24, 27) , as has the measurement of CAT activity(32) . The mammalian expression vector pRShRARalpha was provided by Ronald Evans (Salk Institute, San Diego, CA).

Gel Mobility Shift Assays

The electrophoretic gel shift assays were performed as described previously(25) . RXR antiserum was obtained from Jackie Dyke (Salk Institute, San Diego, CA). The bacterial expression vector pET8CRARalpha was generously provided by Herbert Samuels (New York University Medical Center, New York, NY). The construction of the vector pET3aRXRalpha was described previously(30) . RAR and RXR were both expressed in bacteria and purified according to the method of Forman et al.(33) .

Methylation Interference

Methylation interference was performed essentially as described by Hall et al.(24) . Instead of using a labeled restriction fragment as the probe, single-stranded oligonucleotides containing RARE2 sequences listed above were end-labeled with [-P]ATP and T4 polynucleotide kinase (New England Biochemical Corp.) and then annealed to the unlabeled complimentary strand.


RESULTS

Identification of a Second PEPCK RARE

Since the RARE1 element only contributes approximately 50% of the RA response to the PEPCK gene(23, 24) , we searched for other elements that could account for the remainder of the RA response. To locate a putative second RARE in the PEPCK promoter region, we tested progressive 5` deletions of PEPCK promoter-CAT fusion constructs in transient transfection experiments (Fig. 1). A 15-fold RA response was observed in H4IIE cells transfected with constructs containing a promoter end point of -2100 or -467. Removal of the sequence between -467 and -437 of the PEPCK promoter resulted in about a 50% decrease in the RA response, confirming previous observations(23) . This deletion removes the RARE1 element (located between -451 and -439(23, 24) ). Truncation of the PEPCK promoter to -402 did not further reduce the RA response. Deletion of the sequence between -402 and -306 resulted in an additional reduction in RA activity, which suggested that a second functional RARE was located in this region. Inspection of these 100 bp revealed a consensus half-site for RAR/RXR binding(4, 5) , adjacent to 2 overlapping potential degenerate half-sites (from -337 to -321, see Fig. 2A). The region of DNA from -337 to -321 is referred to as RARE2. The three half-sites are referred to as the alpha, beta, and half-sites, where the latter is the consensus half-site. When the half-site was mutated, the RA response was reduced to the same level exhibited by a block mutation in the RARE1 (Fig. 2B). Furthermore, when block mutations were introduced in both the RARE1 and the half-site, greater than 80% of the PEPCK response to RA was lost.


Figure 1: Localization of a second RARE within the PEPCK gene promoter. The complex glucocorticoid regulatory unit (GRU) is shown with its constituent accessory factor elements (AF1 and AF2) positioned upstream of two glucocorticoid response elements (GRE). Included are elements required for minimal basal transcription (NF1, CRE, and TATA(49) ). A series of constructs containing progressively less of the 5` end of the PEPCK promoter fused to CAT were co-transfected (10 µg each) with pRShRARalpha (5 µg) into H4IIE cells. CAT activity was measured in cell lysates 18 h after treatment with or without 2 µM RA.




Figure 2: Mutations in RARE1 and RARE2 reduce the retinoic acid response of a PEPCK promoter-CAT fusion gene. A, the sequence of RARE2 contains two overlapping degenerate RAR/RXR half-sites (alpha and beta) and a consensus half-site (). The half-site is underlined by an arrow to indicate the conventional orientation of nuclear hormone receptor half-sites(4, 5) . The RARE2m construct contains a block mutation of the half-site as outlined with a box. Also shown is the sequence of RARE1 and the block mutation in the B half-site, RARE1m, which is also highlighted with a box. B, the plasmid pPL32 contains the wild type PEPCK promoter from -467 to +69, relative to the initiation site, ligated to CAT. The construct RARE1m is identical with pPL32 except for a block mutation in the RARE1 element (referred to as SDM B in (26) ). The plasmid RARE2m is identical with pPL32 except that it contains a block mutation in RARE2, as shown in A. The construct RARE1m/RARE2m contains both block mutations. Results are expressed as the average fold induction of CAT activity (with RA/without RA) ± S.E. of geq4 separate determinations.



RAR and RXR Heterodimers Bind Specifically to RARE2

Mobility gel shift assays were utilized to test if RAR/RXR heterodimers bind specifically to RARE2. Two shifted bands were observed when an RARE2 probe was incubated with rat liver nuclear extracts (bands I and II, Fig. 3A). A similar observation was noted previously with an RARE1 probe(24) . Hepatic nuclear factor 4 (HNF-4) and chicken ovalbumin upstream promoter transcription factor (COUP-TF) from rat liver nuclear extracts bind RARE1, and RAR/RXR complexes are seen only when exogenous RAR is added to the reaction mixture. Thus, RAR is limiting and RXR is abundant in rat liver nuclear extracts(24, 25) . In light of this, RAR or mock bacterial extract was added to an incubation mixture containing rat liver nuclear extract and an RARE2 probe. Under these conditions, a third band that migrates between bands I and II appeared in the lane containing RAR (Fig. 3A). Antiserum directed against RXR supershifted this RAR-dependent complex, which suggests that the intermediate migrating band is an RAR/RXR heterodimer. An oligonucleotide containing RARE1, which binds with high affinity to RAR/RXR(24) , efficiently competed for the binding of the RAR/RXR complex (Fig. 3B). In addition, a 100-fold molar excess of an oligonucleotide containing RARE2 effectively prevented formation of the RAR/RXR complex, whereas the same molar excess of an oligonucleotide containing a block mutation of RARE2 (RARE2m, see Fig. 2A for the sequence) or a nonspecific oligonucleotide (AF2) did not (Fig. 3B). Thus, RAR/RXR heterodimers bind specifically to RARE2. The identification and potential functions of bands I and II are currently being examined. (^2)


Figure 3: RAR/RXR heterodimers bind specifically to RARE2. Mobility gel shift assays were performed using a double-stranded, end-labeled RARE2 oligonucleotide as probe. I and II denote complexes formed when rat liver nuclear extract was incubated with RARE2. A, the binding reaction mixture included 5 µg of rat liver nuclear extract (NE) and the indicated additional components: antiRXR, 1 µl of RXR antiserum; NS, 1 µl of nonspecific antiserum; RAR, 100 fmol of partially purified, bacterially expressed hRARalpha; Mock, an equivalent volume of mock bacterial extract. B, the indicated double-stranded competitor oligonucleotides were added in a 100 molar excess prior to the addition of 100 fmol of bacterially expressed hRARalpha and 5 µg of rat liver nuclear extract. RARE1 (PEPCK -460 to -425); AF2, accessory factor 2 (PEPCK -433 to -396); RARE2 (PEPCK -346 to -304); RARE2m, same as RARE2 except for a block mutation in the half-site (see Fig. 2A).



Methylation Interference Defines an Upstream Half-site for the RAR/RXR Heterodimer

We next performed methylation interference assays using a double-stranded oligonucleotide containing RARE2 to more precisely define the binding site for RAR/RXR (Fig. 4A). As expected, methylated guanine residues within the half-site interfered with the binding of RAR/RXR. In addition, two methylated guanine residues within the alpha half-site interfered to some extent with RAR/RXR binding. As shown in Fig. 4B, these interfering residues defined two potential half-sites for RAR/RXR binding within RARE2. The alpha half-site is degenerate since it contains 4 of the 6 bp consensus nuclear receptor half-site sequence, RG(G/T)TCA(4, 5) . The alpha and half-sites are separated by 5 bp, which is consistent with known RAREs(4, 5) . By contrast, methylated G residues within the beta half-site did not interfere with the binding of RAR/RXR (Fig. 4).


Figure 4: Methylation interference defines a second half-site for RAR/RXR binding within RARE2. A, mobility gel shift assays were performed using approximately 300 fmol each of bacterially expressed RAR and RXR with an end-labeled and partially methylated RARE2 element as probe. DNA was isolated from the free and RAR/RXR-bound bands and subjected to piperidine cleavage and separation on a 6% denaturing polyacrylamide gel. The methylated G residues that interfered with RAR/RXR binding are indicated with an asterisk. TS, top strand; BS, bottom strand; F, free DNA; B, RAR/RXR-bound DNA. B, a diagram of RARE2 showing the methylated G residues that interfere with RAR/RXR binding. The arrows underscore potential half sites for RAR/RXR binding.



The alpha and Half-sites Are Both Required for a Complete Retinoic Acid Response

Based on the methylation interference data, constructs containing double point mutations were made wherein selected guanine residues demonstrated to make contacts with RAR/RXR were changed ( Fig. 4and Fig. 5A). Transient transfection experiments utilizing these constructs were used to test whether the alpha and half-sites were necessary for a complete RA response (Fig. 5A). When either the alpha or half-site was mutated, the RA response was decreased by 40-60% (RARE2alpham and RARE2m, Fig. 5A). Most of the remainder of the RA response was conferred by RARE1. These data indicated that mutations in the alpha and half-sites virtually eliminated the RA response from RARE2. In contrast, mutations of the beta half-site did not affect the RA response of cells transfected with the chimeric PEPCK promoter-CAT constructs (RARE2betam, Fig. 5A).


Figure 5: Detailed mutational analysis of RARE2. A, a series of constructs were made with mutations in each of the potential binding motifs within RARE2, as described under ``Materials and Methods.'' Apart from the nucleotides underlined, all of the mutations are identical with pPL32 (the full-length PEPCK promoter fused to CAT). Ten µg of the reporter constructs and 5 µg of pRShRARalpha were cotransfected into H4IIE cells. After 18 h of treatment with or without 2 µM RA, CAT activity was measured from cell lysates. The results are expressed as the mean fold induction of CAT activity (with RA/without RA) ± the S.E. of geq4 separate experiments. B, mobility gel shift assays were performed with a double-stranded, end-labeled oligonucleotide containing RARE2 as probe. Double-stranded competitor oligonucleotides were added in 100 molar excess prior to the addition of 100 fmol each of bacterially expressed RAR and RXR.



Mobility gel shift assays were performed with RAR, RXR, and an RARE2 probe (Fig. 5B). Double-stranded oligonucleotides containing wild type sequences or mutations within the beta half-site (RARE2betam) effectively competed for the retinoid receptor complex. However, oligonucleotides containing the RARE2alpham or RARE2m mutations failed to compete completely for the binding of RAR/RXR heterodimers. Indeed, RARE2alpham competed for RAR/RXR binding more efficiently than RARE2m, which is in close agreement with the observed methylation interference data (Fig. 4). Taken together, these data demonstrate that this element is a DR5-type RARE.

RARE2 Functions in a Heterologous Context

A construct consisting of RARE2 placed upstream of a minimal thymidine kinase promoter and ligated to the CAT reporter gene was tested for its ability to mediate a RA response in H4IIE cells. RARE2 mediated a 3-fold RA response in either orientation, and an additive, 6-fold response was observed when two RARE2 elements were placed in tandem upstream of a thymidine kinase promoter (Fig. 6). The RA response in this context was due to the binding of RAR/RXR to RARE2 since the RA response was lost when the half-site was mutated (compare RARE2TK with RARE2mTK in Fig. 6).


Figure 6: RARE2 confers a RA response in the context of a heterologous promoter. Plasmids were made wherein RARE2 was ligated either in the correct (RARE2TK) or inverse (RARE2RTK) orientation, or in tandem ((RARE2)2TK) upstream of the minimal thymidine kinase promoter-CAT reporter construct (TK). In addition, the half-site was mutated in the context of the RARE2TK construct (RARE2mTK). These constructs (10 µg each) were cotransfected with 5 µg of pRShRARalpha and CAT activity was measured 18 h after treatment with or without 2 µM RA. The data are expressed as an average fold induction of CAT activity (with RA/without RA) ± S.E. of geq3 separate experiments.




DISCUSSION

We have previously shown that RA increases the rate of transcription of the endogenous PEPCK gene and of PEPCK promoter-CAT fusion constructs(23) . This response is mediated, in part, by a pleiotropic element, termed RARE1, that is required for complete RA and glucocorticoid responses(19, 23, 24, 25, 26) . A second RARE in the PEPCK promoter region has now been located between -337 and -321 in relation to the transcription start site ( Fig. 1and Fig. 2). RAR/RXR heterodimers bind specifically to this sequence (Fig. 3B and 5B) which is a degenerate DR5-type RARE ( Fig. 4and Fig. 5). Furthermore, RARE2 is active in the context of a heterologous promoter, and in either orientation (Fig. 6), which is consistent with previous observations of other RAREs ( (4) and (5) and references therein).

It is worth noting that most RAREs in their natural context, including both RAREs in the PEPCK promoter ( (23) and (24) and this study), are not perfect direct repeats(5) . Additionally, RAR/RXR binds to (and transactivates through) palindromes (10, 11) and everted repeats(12, 13) . Furthermore, the mouse laminin B1(34) , rat growth hormone(34) , and human oxytocin (35) gene promoters contain complex RAREs composed of 3 or 4 repeats in various orientations that can span up to 80 bp. The work of many investigators has increased our understanding of the role of protein/DNA interactions in RA signaling(6, 7, 8, 36, 37, 38) , yet it is still vitally important to continue to identify and characterize natural RAREs so that we might understand RA signal transduction in physiologically relevant contexts.

The physiological role of RA in the regulation of PEPCK gene transcription is unclear. However, it is known that RA-deficient rats have impaired gluconeogenesis (39) and, since PEPCK is the rate-limiting enzyme of gluconeogenesis(14) , it is possible that RA is required for maintenance of basal PEPCK expression in hepatocytes. In fact, recent data from transgenic mice support this idea. Transgenic mice expressing a PEPCK-promoter driven transgene and put on a RA-deficient diet, expressed 50% less of the transgene mRNA than control mice or RA-deficient mice supplemented with RA(40) . Since RA levels apparently do not change appreciably in healthy adult animals (41) , RA probably does not acutely affect PEPCK transcription. As we have proposed before, RA may be permissive for both generating relatively high basal levels of transcription and elevating hormonal responsiveness of the PEPCK gene(42) . Indeed, when RA is added together with glucocorticoids or cAMP, there is an additive or even synergistic effect on PEPCK gene expression (43) . (^3)

Giralt et al.(44) identified a thyroid hormone response element (TRE) in the PEPCK promoter. The TRE is localized between -334 and -318 and mediates a modest 3-fold response to thyroid hormone (T(3)) in human HepG2 hepatoma cells, which incidentally do not express the endogenous PEPCK gene. The element is also required for the synergism observed when these cells are treated with both cAMP and T(3)(44) . Recently, Park et al.(45) characterized this TRE in much more detail. The sequences to which TR/RXR binds are contained within the RARE described here. Using our nomenclature, TR/RXR binds to the beta and half-sites of RARE2, whereas RAR/RXR binds to the alpha and half-sites. The functional significance of the observed overlap of RAR/RXR and TR/RXR binding is not clear and we have been able to demonstrate only a very weak thyroid hormone response in H4IIE cells, in which the endogenous PEPCK gene is expressed(42) . However, when TR and RAR are co-expressed with PEPCK promoter-CAT constructs, the RA response is decreased(42) . TR may affect the RA response by sequestering RAR in nonproductive RAR/TR heterodimers which bind to RARE1(42) . Alternatively, TR may bind to RXR in solution, thus preventing the formation of RAR/RXR heterodimers (46) . The competition of TR/RXR and RAR/RXR for RARE2 could contribute to the blunting of the RA response.

It is perhaps not surprising that the second PEPCK RARE is required for more than one hormone response. Genes requiring the integration of multiple environmental cues often contain complex regulatory domains with overlapping and multiple functions (for review, see (17) ). Within the PEPCK gene promoter, the complex GRU serves as a prototypical example of a metabolic control domain. The GRU is composed of two glucocorticoid receptor binding sites (GRE1 and GRE2) and two accessory factor elements, AF1 and AF2, located just upstream of the GREs (see Fig. 1). AF1 and AF2 are both required for a complete glucocorticoid response; the GREs are essentially silent without the accessory factor activity(19) . AF1 activity is mediated by the binding of either COUP-TF or HNF-4. Both of these proteins are members of the RAR/TR/VDR subfamily of the steroid hormone superfamily and are orphan receptors lacking known ligands. AF2 activity is mediated by the binding of hepatic nuclear factor 3 (HNF-3), a member of the fork-head family of transcription factors. (^4)Remarkably, AF1 and AF2 are also required for other hormone signaling pathways. Thus, AF1 is a RARE (via RAR/RXR binding(23, 24) ) and AF2 is an insulin-responsive sequence (via an unidentified insulin responsive factor, distinct from HNF-3(47, 48) ). A similar situation may exist for RARE2. We have evidence that the second PEPCK RARE is also an accessory factor element required for a complete glucocorticoid response.^2


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants DK35107 and DK20593 from the Vanderbilt Diabetes Research and Training Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported, in part, by Training Grant DK07061.

To whom correspondence should be addressed. Tel.: 615-322-7004; Fax: 615-322-7236.

(^1)
The abbreviations used are: RAR, retinoic acid receptor; TR, thyroid hormone receptor; VDR, 1,25-dihydroxyvitamin D(3) receptor; RXR, 9-cis-retinoic acid receptor; DR, direct repeat; RARE, retinoic acid response element; PEPCK, phosphoenolpyruvate carboxykinase; RA, all-trans-retinoic acid; CAT, chloramphenicol acetyltransferase; RARE1, the first RARE characterized in the PEPCK gene promoter; AF1, accessory factor 1; GRU, glucocorticoid regulatory unit; RARE2, the second RARE within the PEPCK promoter; HNF-4, hepatic nuclear factor 4; COUP-TF, chicken upstream ovalbumin promoter transcription factor; PPAR, peroxisome proliferator activated receptor; TRE, thyroid hormone response element; T(3), thyroid hormone; GRE, glucocorticoid receptor binding site; AF2, accessory factor 2; HNF-3, hepatic nuclear factor 3; bp, base pair(s).

(^2)
D. K. Scott and D. K. Granner, manuscript in preparation.

(^3)
J. A. Mitchell, unpublished observations.

(^4)
W. Wang and D. K. Granner, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Cathy Caldwell for her excellent technical assistance and members of the Granner laboratory and Gary Robinson for their critical reading of the manuscript.


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