The Caudal Homeobox Protein cdx-2/3 Activates Endogenous Proglucagon Gene Expression in InR1-G9 Islet Cells

Tianru Jin1, D. K. Y. Trinh, Feng Wang and Daniel J. Drucker

Department of Medicine Banting and Best Diabetes Centre The Toronto Hospital University of Toronto Toronto, Ontario, M5G 2C4, Canada


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The proglucagon gene is expressed in a highly cell-specific manner in islet and enteroendocrine cells. DNA sequences within the proximal proglucagon G1 promoter region bind the homeobox protein cdx-2/3, and cdx-2/3 activates the proglucagon promoter in fibroblasts. We show here that cdx-2/3 activates the proglucagon promoter in both islet (InR1-G9) and enteroendocrine (STC-1 and GLUTag) cell lines. Furthermore, transfected cdx-2/3 increased the levels of endogenous proglucagon mRNA transcripts in both transient and stable transfections of InR1-G9 islet cells. The cdx-2/3-dependent induction of endogenous proglucagon mRNA transcripts in stable islet lines was associated with a corresponding increase in the transcriptional activity of proglucagon promoter-luciferase plasmids. An amino-terminally truncated cdx-2/3 derivative containing the homeodomain and carboxy-terminal region of the molecule inhibited both the cdx-2/3 activation of the proglucagon promoter and the induction of endogenous proglucagon mRNA transcripts. These observations demonstrate that cdx-2/3, acting through the proximal G1 element, is a major transcriptional determinant of cell-specific proglucagon gene expression in pancreatic islet cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The proglucagon gene encodes a number of biologically active peptide hormones important for the control of plasma glucose in vivo. Glucagon, released from the pancreatic A cell, regulates glycogenolysis and gluconeogenesis, whereas a truncated form of GLP-1, liberated from the intestinal L cell, stimulates glucose-dependent insulin secretion from the islet ß-cell (1). Accordingly, the molecular factors important for the control of proglucagon gene transcription in the endocrine pancreas and enteroendocrine cells of the intestine are of considerable interest and importance.

Experiments using transgenic mice and gene transfer in vitro have identified several regions within the first few kilobases of the rat proglucagon promoter that appear to be important for control of proglucagon gene transcription (2, 3). Expression of proglucagon gene-SV40 T antigen transgenes in mice led to the delineation of an upstream element, designated the proglucagon gene upstream enhancer, that is necessary for proglucagon gene transcription in enteroendocrine cells in vivo (4). A number of control elements in the more proximal proglucagon promoter have been identified that function as islet cell-specific enhancers (5). DNA sequences within the first 100 bp of the promoter are functionally important for restricting proglucagon gene transcription to islet and intestinal endocrine cell types (6). This proximal promoter element, termed G1, contains a number of AT-rich sequences characteristic of the binding sites for homeobox genes.

A combination of transfection and electrophoretic mobility shift assay experiments have suggested that the homeobox transcription factor isl-1 binds to the proglucagon G1 element and activates the proglucagon promoter (7). Reduction of isl-1 levels in InR1-G9 islet cells was associated with decreased levels of proglucagon mRNA transcripts, providing further support for the importance of isl-1 in the control of proglucagon gene expression (7). The caudal homeobox protein cdx-2/3, present in both islet and intestinal nuclear extracts, also binds to the AT-rich sequences in the proglucagon G1 element (8), and transfected cdx-2/3 activates the proglucagon promoter in BHK fibroblasts. To delineate a role for cdx-2/3 in the control of proglucagon gene transcription in endocrine cells, we have now examined the cdx-2/3-dependent regulation of proglucagon gene transcription in islet and enteroendocrine cell lines.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Because cdx-2/3 activated the proglucagon promoter in BHK fibroblasts (that do not express cdx-2/3) (8), we wished to determine whether cdx-2/3 introduced into islet or intestinal cell lines also increased the activity of the proglucagon promoter. Cotransfection of cdx-2/3 and a series of 5'-deleted proglucagon-luciferase plasmids into GLUTag and STC-1 intestinal cells and InR1-G9 islet cells demonstrated a cell-specific activation pattern of proglucagon promoter activity (Fig. 1Go). The cdx-2/3-dependent activation of the proglucagon promoter plasmids containing the largest amounts of 5'-flanking sequences, such as (-2292)GLU-Luc, was somewhat greater in InR1-G9 cells compared with GLUTag cells. In contrast, cdx-2/3 did not function as an activator of the longer proglucagon-luciferase plasmids in STC-1 cells until proglucagon gene 5'-flanking sequences upstream of -155 were deleted. Further deletion of promoter sequences to -60 eliminated the cdx-2/3-dependent activation (data not shown), consistent with the known location of the cdx-2/3-binding site in the rat proglucagon promoter (8). Taken together, the differential cdx-2/3-dependent activation of the proglucagon promoter in STC-1 vs. InR1-G9 and GLUTag cells suggests that the cell-specific expression of DNA-binding proteins, as previously documented for the upstream proglucagon enhancer sequences (4), may modify the transcriptional activity of the proglucagon promoter, and its response to cdx-2/3 in different cell types. Furthermore, the transfection data shown here clearly establish that cdx-2/3 activates the proglucagon promoter not only in fibroblasts (8), but also in islet and intestinal cell lines.



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Figure 1. Cdx-2/3-Dependent Transcriptional Activation of the Proglucagon Promoter in Different Cell Types

GLUTag (a), STC-1 (b), and InR1-G9 (c) proglucagon-producing cell lines were transfected with 5 µg (InR1-G9) or 7.5 µg (STC-1 and GLUTag) of luciferase reporter genes driven by different proglucagon promoter fragments in the presence (+) or absence (-) of 5 µg hamster cdx-3 under the control of a CMV promoter (8). The data are expressed as mean relative luciferase activity ± SEM normalized to the activity obtained after transfection of the promoterless luciferase plasmid in the same experiment.

 
The finding that exogenous cdx-2/3 activated proglucagon promoter plasmids in endocrine cells prompted us to determine whether cdx-2/3 transfected into InR1-G9 cells was also capable of activating the endogenous proglucagon gene. The results of this experiment are shown in Figs. 2Go and 3Go. Increasing amounts of cdx-2/3 transiently transfected into InR1-G9 cells resulted in a corresponding increase in the levels of proglucagon mRNA transcripts. In contrast, no increase in the levels of tubulin mRNA transcripts was detected in the same experiment. The transcription factor isl-1 also increased proglucagon gene expression in transfected InR1-G9 cells, consistent with previous findings that depletion of isl-1 in InR1-G9 cells caused a reduction in the basal levels of proglucagon mRNA (7).



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Figure 2. Cdx-2/3 Activates Endogenous Proglucagon Gene Expression in InR1-G9 Cells

a, InR1-G9 cells were either mock-transfected (WT) or transfected with the CMV expression vector alone (pBAT7), with cdx-3 in the antisense orientation (AS), or with various amounts (in micrograms) of isl-1 or cdx-3. RNA was extracted for Northern analysis with cDNA probes for glucagon (G) or tubulin (T) 16 h after the transfection. The ethidium bromide stain of the RNA gel showing the migration positions of 28 and 18S ribosomal RNA is shown. b, GLUTag cells were transfected with cdx-3 or pBAT7 in the amounts indicated (micrograms DNA in parentheses). RNA was extracted 18 h after the transfection, and Northern blots were hybridized with the cDNA probes for proglucagon (G) and tubulin (T). The relative densitometric values for the RNA signals are shown below.

 


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Figure 3. Northern Analysis

Northern analysis of InR1-G9 G418-resistant stable islet cell lines (wt, wild type), transfected with the expression vector alone (pSR1neo- V1 and V2), a pool of 50–100 clones (M1), or individual clones (designated C). The blots were probed with radiolabeled cDNAs for glucagon (G), cdx-2/3, or rat chromogranin (C). The ethidium bromide-stained gel before transfer, along with the integrity of 28S and 18S ribosomal RNA, is shown. Various exposures of the blots were scanned with a laser densitometer, and the relative densitometric units, normalized to the values obtained for the signals in the InR1-G9 wild type lane, are shown below.

 
To determine whether cdx-2/3 was capable of activating endogenous proglucagon gene expression in various cell types, we transfected increasing amounts of cdx-2/3 into mouse intestinal GLUTag cells. No increase in the levels of proglucagon mRNA transcripts was observed in GLUTag cells (Fig. 2bGo), suggesting that the activation of endogenous proglucagon gene expression by cdx-2/3 is highly cell-specific. Furthermore, we did not observe activation of proglucagon gene transcription (as assessed by Northern analysis) after transfection of cdx-2/3 into STC-1 enteroendocrine cells, or BHK fibroblasts (data not shown).

To provide complementary evidence correlating increased expression of cdx-2/3 with activation of endogenous proglucagon gene expression, we transfected either isl-1 or cdx-2/3 [ligated in the plasmid pSR1neo (7)] into InR1-G9 cells and selected transfectants with the antibiotic G418. Analysis of a few randomly selected stably transfected G418-resistant InR1-G9 clones that expressed cdx-2/3 displayed increased basal levels of proglucagon mRNA transcripts in three of four lines examined (data not shown). Accordingly, to correlate more precisely the levels of cdx-2/3 expression with potential changes in expression of the endogenous proglucagon gene, a larger number of clones were isolated and examined (Fig. 3Go). Increased cdx-2/3 expression was generally associated with induction of proglucagon gene expression in InR1-G9 cells. Of 21 clones examined, 18 had increased levels of both cdx-2/3 and proglucagon mRNA, whereas three clones had normal levels of cdx-2/3 and proglucagon (Fig. 3Go and data not shown). Nevertheless, we did not observe a perfect quantitative correlation between cdx-2/3 and proglucagon gene expression, as some clones, e.g. C4, had very high levels of cdx-2/3 mRNA, but comparatively modest induction of proglucagon gene expression. In contrast to the activation of the proglucagon gene detected in association with increased cdx-2/3 expression, the levels of mRNA transcripts for tubulin (Fig. 3Go) or the neuroendocrine gene chromogranin (Fig. 3Go) were not induced by cdx-2/3 in the G418-resistant cell lines. The results of these experiments strongly suggest that cdx-2/3 activates the endogenous proglucagon gene in InR1-G9 islet cells.

The increased levels of proglucagon mRNA transcripts were likely attributable in part to an increase in proglucagon promoter activity. To examine this directly, we transfected proglucagon-luciferase plasmids into wild type InR1-G9 cells, and InR1-G9 clones C2 and C4, that exhibited normal and increased levels of endogenous proglucagon mRNA transcripts, respectively. Proglucagon promoter activity was clearly increased in the C4 InR1-G9 clone with all three proglucagon promoter plasmids that contained the cdx-2/3-binding site, consistent with the increased levels of both cdx-2/3 and proglucagon mRNA transcripts detected in the C4 cell line (Fig. 4Go). In contrast, no increase in luciferase activity was observed after transfection of the same plasmids in the C2 clone, which exhibited wild type levels of proglucagon mRNA transcripts. Furthermore, the increased activity of proglucagon promoter plasmids was not seen after deletion of 5'-flanking sequences to -60, consistent with the elimination of the cdx-2/3-binding site. Taken together, these experiments clearly demonstrate a correlation between expression of the cdx-2/3 gene and activation of both the endogenous proglucagon gene and the transfected proglucagon promoter in InR1-G9 islet cells.



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Figure 4. Proglucagon Promoter Activity in InR1-G9 Cells Expressing cdx-3

Five micrograms of the promoterless control plasmid (pBLUC) or the different proglucagon-luciferase reporter plasmids were transfected into wild type InR1-G9 cells, (WT), clone C2 (a G418-resistant cdx-3-transfected InR1-G9 cell clone showing no increase in either proglucagon or cdx-3 expression), and clone C4 (a G418-resistant cdx-3-transfected InR1-G9 cell clone that exhibits increased expression of both proglucagon and cdx-3). The cells were harvested 16 h after transfection, and reporter gene activity was measured as described in Fig. 2Go.

 
The results of recent experiments using RT-PCR for the analysis of cdx-2/3 gene expression suggested that an amino-terminally-truncated cdx-2/3 protein (that contains the homeodomain and carboxy-terminal cdx-2/3 sequences), binds the G1 promoter element but fails to activate transcription from a G1-linked promoter in InR1-G9 cells (9). To determine the putative importance of the N-terminally-deleted cdx-2/3 protein for proglucagon promoter activity, we cotransfected increasing amounts of cdx-2/3 alone or cdx-2/3 in the presence of {Delta}NT-cdx-2/3, an internally deleted cdx-2/3 cDNA (that we generated by PCR), which lacks amino acids 8–180 (Fig. 5Go). Activation of the proglucagon promoter by transfected cdx-2/3 in InR1-G9 cells was found to be maximal at concentrations of transfected cdx-2/3 approximating 100 ng, and cotransfection with the {Delta}NT-cdx-2/3 plasmid completely abrogated the cdx-2/3 induction of proglucagon promoter activity, despite increasing amounts of cdx-2/3 in the transfection experiments (Fig. 5aGo). The {Delta}NT-cdx-2/3 plasmid also attenuated the basal activity of the proglucagon promoter in InR1-G9 cells (Fig. 5bGo), consistent with the recently described competition of this protein with wild type cdx-2/3 for binding to the G1 promoter site (9). To ascertain whether the N-terminal cdx-2/3 deletion might also influence the levels of endogenous proglucagon mRNA transcripts, the {Delta}NT-cdx-2/3 plasmid was transiently transfected into InR1-G9 cells alone or in the presence of cdx-2/3, after which the levels of proglucagon mRNA transcripts were examined (Fig. 5cGo). The results of this experiment demonstrate that the {Delta}NT-cdx-2/3 plasmid abrogates the cdx-2/3-dependent induction of proglucagon mRNA transcripts in InR1-G9 islet cells.



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Figure 5. Effect of {Delta}[8–180] cdx-2/3 on the Transcriptional Activity of the Proglucagon Promoter in InR-1-G9 Cells

a, Proglucagon promoter plasmids (5 µg) containing 2,292, 329, 217, or 82 bp of rat proglucagon gene 5'-flanking sequences fused to the luciferase cDNA were transfected into InR1-G9 cells along with 10 µg of pBAT8 (the promoterless expression vector) or 10 µg of {Delta}N[8–180] cdx-2/3. The values are depicted as the mean ± SEM of three different transfections. *, P < 0.05;**, P < .01. b, Inhibition of the cdx-2/3 induction of proglucagon promoter activity by {Delta}N[8–180] cdx-2/3. All transfections contained 5 µg of -82 proglucagon-luciferase as the reporter and varying amounts of cdx-2/3 (100–400 ng) in the presence of 15 µg {Delta}N[8–180] cdx-2/3, or 15 µg pBAT7. The values are depicted as the mean ± SEM of three different transfections. *, P < 0.05. c, Effect of cdx-2/3 plasmids on proglucagon gene expression in InR1-G9 islet cells. Ten micrograms of each of the various plasmids shown were transfected into InR1-G9 cells, and RNA was harvested 24 h after transfection for Northern analysis. G, Glucagon; T, tubulin. The relative densitometric values for the RNA signals are shown below.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cdx-2/3-binding sites have been identified in the sucrase-isomaltase, carbonic anhydrase 1, apolipoprotein B, and proglucagon gene promoters (8, 10, 11, 12). Although cdx-2/3 activates the sucrase-isomaltase and proglucagon promoters in transfection of heterologous cell types (8, 10), cdx-2/3-binding sites in the third intron of the human apoB gene mediate repression of gene transcription, possibly through interaction with members of the CCAAT/enhancer binding protein transcription factor family (11). Despite increasing evidence supporting a role for cdx-2/3 as a regulator of intestinal gene transcription, the only genetic target identified for cdx-2/3, in extraintestinal tissues such as the endocrine pancreas, is the proglucagon gene. Furthermore, no experiments to date have demonstrated that cdx-2/3 activates the expression of the mRNAs for sucrase-isomaltase, carbonic anhydrase 1, or apolipoprotein B.

Establishment of stable intestinal epithelial IEC-6 cell lines expressing high levels of mouse cdx-2 was followed by proliferation arrest and differentiation in association with expression of cdx-2. The proliferation arrest was released after several days, despite the continuing expression of cdx-2. Although no induction of the cdx-2-responsive SI gene was observed after 40 days of culture, IEC-6 cells expressing cdx-2 for more than 50 days did express SI gene transcripts (13). These observations suggest an indirect link between prolonged expression of cdx-2 and the activation of endogenous target gene expression.

The cdx-2/3-dependent activation of endogenous proglucagon gene expression reported here was observed in InR1-G9 islet cells but not in enteroendocrine or fibroblast cell lines. In contrast, cdx-2/3 activated the proglucagon promoter in transfection experiments in fibroblasts and intestinal endocrine cells. The mechanisms determining the cell-specific cdx-2/3 responsiveness of the endogenous proglucagon gene promoter remain unknown. We have demonstrated by RT-PCR that InR1-G9 and GLUTag cells express a different profile of homeobox genes (data not shown), and it is possible that homeobox proteins with different affinities for the G1-binding site may modify the effect of transfected cdx-2/3 on activation of the endogenous proglucagon promoter in GLUTag cells. Similar cell-specific differences in target gene responsiveness have recently been reported for the IPF-1 induction of insulin mRNA transcripts (14). The insulin gene homeobox transcription factor IPF-1 induced the expression of the insulin and amylin genes in stably transfected, glucagon-producing AN 697 islet cells, but not in rat embryo fibroblasts. Furthermore, transient transfection of IPF-1 into either {alpha}TC1.9 or AN697 glucagon-producing islet cell lines was not associated with induction of endogenous insulin gene expression (14). These observations differ from our demonstration that cdx-2/3 activates the proglucagon promoter and endogenous proglucagon gene in both transient transfections and in stable cell lines expressing increased levels of cdx-2/3. The mechanisms for the inability of IPF-1 to induce insulin gene expression in transient transfections remain unknown, but it has been suggested that IPF-1 action may require the expression of other factors necessary for activation of the insulin gene promoter, and induction of these factors may require more than the 48–72 h duration of a transient transfection (14).

The data reported here demonstrate that increased expression of cdx-2/3 correlates with the increased expression of a specific endogenous mRNA and not simply a transfected fusion gene. The LIM domain homeobox gene isl-1 was previously shown to bind to an adjacent site in the proglucagon gene G1 promoter region, and reduced levels of isl-1 were associated with a decrease in the levels of proglucagon mRNA transcripts (7). The results of our experiments demonstrate that transfected isl-1 is also associated with an increase in endogenous proglucagon mRNA transcripts, consistent with the postulated role for isl-1 as a positive activator of proglucagon gene transcription.

Although cdx-2/3 activated the expression of proglucagon-luciferase reporter genes in three different endocrine cell lines, considerable heterogeneity was observed with respect to the relative degree and pattern of reporter gene activation in the different cell types. We have previously documented that the relative profile of basal proglucagon promoter transcriptional activation differs after transfection of STC-1, InR1-G9, and GLUTag cell lines (4). Whereas the latter two cell types appear to more accurately represent the A and L cell phenotype, respectively, STC-1 cells are plurihormonal and much less representative of a pure population of differentiated glucagon-producing cells (15). Furthermore, we have recently shown that the pattern of DNA-protein interaction using STC-1 extracts and proglucagon promoter sequences clearly differs from the profile of binding events detected with extracts from InR1-G9 and GLUTag cells (4). Taken together, these observations suggest that cell heterogeneity and differential expression of transcription factors may account for the observed differences in proglucagon gene promoter activity in various cell types.

The recent report that an amino-terminally truncated cdx-2/3 RNA transcript was detected in InR1-G9 cells by RT-PCR (9) prompted us to examine the potential significance of such a molecule for the control of proglucagon promoter activity. Although the relative degree of inhibition of promoter activity varied somewhat with the various proglucagon-promoter plasmids analyzed, we consistently observed an inhibition of the cdx-2/3-dependent induction of proglucagon promoter activity in the presence of the {Delta}NT-cdx-2/3 plasmid. Furthermore, the {Delta}NT-cdx-2/3 plasmid diminished the ability of the wild type cdx-2/3 plasmid to activate endogenous proglucagon mRNA transcripts in InR1-G9 islets, but the {Delta}NT-cdx-2/3 plasmid alone did not reduce the basal levels of proglucagon mRNA. This may be due to competition for binding to the proglucagon G1 promoter site (by other homeobox proteins), or to other mechanisms that remain to be elucidated.

The results of our experiments with the {Delta}NT-cdx-2/3 plasmid suggest that such an N-terminally-truncated protein, if expressed in islet cells, might possibly modulate the activity of cdx-2/3 on the proglucagon promoter, and therefore the ratio of wild type and truncated cdx-2/3 proteins could be an important determinant of cdx-2/3 activity. Nevertheless, we have not observed a smaller cdx-2/3 RNA transcript by Northern blotting, and Western blot analysis of InR1-G9 cells using antisera detected against the carboxy-terminal region of cdx-2/3 did not detect the presence of any smaller immunoreactive cdx-2/3 proteins that might correspond in size to the predicted N-terminal cdx-2/3 deletion (8). Accordingly, the biological significance of the N-terminal cdx-2/3 deletion in the control of proglucagon gene expression awaits the definitive detection of this protein in islet and enteroendocrine cells and remains uncertain.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
The plasmid pBAT7.cdx-3 was kindly provided by M. S. German (San Francisco, CA) (16). The expression of hamster cdx-3 (313 amino acids) in this plasmid is under the control of a cytomegalovirus (CMV) promoter. The series of 5'-deleted proglucagon-luciferase plasmids used here has been previously described (4, 8). The rat isl-1 expression plasmid was reported previously (7). The plasmid {Delta}NT-cdx-2/3, which contains a deletion from amino acids 8–180 in hamster cdx-3, was constructed by PCR, and the sequence of the N-terminal-deleted cdx-3 cDNA was verified by DNA sequencing. For generation of stable G418-resistant cell lines, hamster cdx-3 was ligated into the psR1neo expression vector, which contains the neo resistance cassette (7).

Cell Lines, Northern Analysis, and Transfections
The InR1-G9, STC-1, and GLUTag cell lines were propagated and transfected as previously described (15, 17, 18, 19). RNA isolation used the acid phenol extraction method (20), and Northern blotting was carried out using nylon membranes as described (21, 22). The luciferase activity was analyzed by normalizing each transfection relative to the protein concentration in each transfected extract, and luciferase activity was measured as described (23). The reporter gene activity was expressed relative to the activity obtained using the promoterless luciferase plasmid pBluc in the same experiment. All transfections were carried out in triplicate on at least three separate occasions, and statistical significance was assessed by Student’s t test.


    FOOTNOTES
 
Address requests for reprints to: Dr. D. Drucker, The Toronto Hospital, 200 Elizabeth Street CCRW3-838, Toronto M5G 2C4, Canada.

This work was supported by an operating grant from the Medical Research Council of Canada. T.J. was supported by a fellowship award from the Ontario Ministry of Health. D.T. was supported by a fellowship award from the Banting and Best Diabetes Centre. D.J.D. is a Scientist of the Medical Research Council of Canada.

1 Present address: Oncology Research Laboratories, The Toronto Hospital, University of Toronto, 67 College Street, Toronto, Ontario M5G 2M1, Canada. Back

Received for publication October 4, 1996. Revision received October 29, 1996. Accepted for publication November 4, 1996.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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