Characterization of the Aldolase B Intronic Enhancer
1998; Elsevier BV; Volume: 273; Issue: 39 Linguagem: Inglês
10.1074/jbc.273.39.25237
ISSN1083-351X
AutoresClaudine Grégori, Arlette Porteu, Soledad López, Axel Kahn, Anne‐Lise Pichard,
Tópico(s)Protein Structure and Dynamics
ResumoThe aldolase B gene is transcribed at a high level in the liver, kidney, and small intestine. This high level of gene expression results from cooperation between a weak but liver-specific promoter and an intronic activator. A deletional study of this activator present in the first intron allowed us to ascribe the maximal enhancer function to a 400-base pair (bp) fragment (+1916 to + 2329). This enhancer is highly liver-specific and enhances the activity of heterologous minimal promoters in a position and distance-independent fashion in transiently transfected Hep G2 hepatoma cells. The aldolase B enhancer is composed of two domains, a 200-bp module (Ba) inactive by itself but which synergizes with another 200-bp module (Bb) that alone retains 25% of the total enhancer activity. The Bb sequence is 76% homologous between human and rat genes and contains several binding sites for liver-enriched nuclear factors. By electrophoretic mobility shift assays, we demonstrated that elements 5 and 7 bind hepatic nuclear factor 1 (HNF1), whereas element 2 binds hepatic nuclear factor 4 (HNF4). A functional analysis of the enhancer whose elements have been mutated demonstrated that mutation of any of the HNF1 sites totally suppressed enhancer activity, whereas mutation of the HNF4-binding site reduced it by 80%. The aldolase B gene is transcribed at a high level in the liver, kidney, and small intestine. This high level of gene expression results from cooperation between a weak but liver-specific promoter and an intronic activator. A deletional study of this activator present in the first intron allowed us to ascribe the maximal enhancer function to a 400-base pair (bp) fragment (+1916 to + 2329). This enhancer is highly liver-specific and enhances the activity of heterologous minimal promoters in a position and distance-independent fashion in transiently transfected Hep G2 hepatoma cells. The aldolase B enhancer is composed of two domains, a 200-bp module (Ba) inactive by itself but which synergizes with another 200-bp module (Bb) that alone retains 25% of the total enhancer activity. The Bb sequence is 76% homologous between human and rat genes and contains several binding sites for liver-enriched nuclear factors. By electrophoretic mobility shift assays, we demonstrated that elements 5 and 7 bind hepatic nuclear factor 1 (HNF1), whereas element 2 binds hepatic nuclear factor 4 (HNF4). A functional analysis of the enhancer whose elements have been mutated demonstrated that mutation of any of the HNF1 sites totally suppressed enhancer activity, whereas mutation of the HNF4-binding site reduced it by 80%. base pair(s) kilobase pair(s) chloramphenicol acetyltransferase thymidine kinase hepatic nuclear factor chicken ovalbumin upstream promoter-transcription factor CAAT/enhancer binding protein L-type pyruvate kinase. Aldolase B, one of the three known aldolase isoenzymes, is the only expressed isoform in highly differentiated hepatocytes (1Schapira F. Hatzfeld A. Weber A. Markert C.L. Isozymes. 3. Academic Press, New York1975: 987-1003Crossref Google Scholar) and is also found in kidney and small adult intestine where it is associated with aldolases A or C (2Guder W.G. Ross B.D. Kidney Int. 1984; 26: 101-111Abstract Full Text PDF PubMed Scopus (396) Google Scholar). Aldolase B catalyzes the reversible cleavage of fructose 1-phosphate into dihydroxyacetone phosphate and glyceraldehyde; therefore, it is involved in both glycolytic and gluconeogenic pathways. In human, hereditary fructose intolerance is a potentially fatal autosomal recessive disease resulting from aldolase B deficiency. In addition aldolase B gene transcription is regulated by hormones and diet; it is partially repressed by glucagon and cyclic AMP and stimulated 4-fold by glucose and insulin (3Munnich A. Besmond C. Darguy S. Reach G. Vaulont S. Dreyfus J.C. Kahn A. J. Clin. Invest. 1985; 75: 1045-1052Crossref PubMed Scopus (39) Google Scholar). The aldolase B gene proximal promoter was shown to be liver cell-specific as judged from transient transfection experiments in the hepatoma cell line Hep G2 and in hepatocytes in primary culture (4Grégori C. Ginot F. Decaux J.F. Weber A. Berbar T. Kahn A. Pichard A.L. Biochem. Biophys. Res. Commun. 1991; 176: 722-729Crossref PubMed Scopus (21) Google Scholar). However the promoter activity of the −192-bp1 5′-flanking fragment was always very low in these cells. Recently we explained this result by a dominant restriction of the transcriptional activity due to binding of the hepatic nuclear factor 3 (HNF3) to the PAB element of the promoter (5Grégori C. Kahn A. Pichard A.L. Nucleic Acids Res. 1994; 22: 1242-1246Crossref PubMed Scopus (33) Google Scholar). This PAB element binds in a mutually exclusive fashion either hepatic nuclear factor 1 (HNF1), which stimulates promoter activity, or HNF3 that, on the contrary, restrained the aldolase B promoter activity (6Grégori C. Kahn A. Pichard A.L. Nucleic Acids Res. 1993; 21: 897-903Crossref PubMed Scopus (63) Google Scholar). In transgenic mice transgenes directed by the −232-bp proximal promoter fragment were totally silent. The addition of 1.8 kb of sequences located in the first intron of the aldolase B gene (+685 to +2514) led to a 50-fold stimulation of the promoter activity ex vivo in Hep G2 cells and allowed for a correct, tissue-specific expression in transgenic mice (7Sabourin J.-C. Kern A.-S. Gregori C. Porteu A. Cywiner C. Chatelet F.-P. Kahn A. Pichard A.-L. J. Biol. Chem. 1996; 271: 3469-3473Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The purpose of this work was to delineate the minimal intronic fragment responsible for the enhancer activity and to characterize DNA elements and cognate trans-acting factors involved in both activity and tissue specificity of this enhancer. Among various DNA elements detected in a 400-bp fragment endowed with a liver cell-specific enhancer activity, two HNF1-binding sites were shown to be indispensable for this activity. In addition, a conserved HNF4-binding site also behaved as a positive cis-acting element of the enhancer. For generation of the internal deletion, we started with the −232 A100B/CAT construct previously described (7Sabourin J.-C. Kern A.-S. Gregori C. Porteu A. Cywiner C. Chatelet F.-P. Kahn A. Pichard A.-L. J. Biol. Chem. 1996; 271: 3469-3473Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). The −232A100B1200/CAT and −232A100B600/CAT plasmids were obtained by excision of a fragment between sites StuI (located within the B fragment) and KpnI or BamHI (located within the plasmid linkers). The −232A100Ba/CAT, −232A100Bb/CAT, −232A100Bc/CAT, and −232A100Ba+b/CAT plasmids were obtained by cloning in both orientations, into the SmaI site of the −232A100/CAT plasmid (7Sabourin J.-C. Kern A.-S. Gregori C. Porteu A. Cywiner C. Chatelet F.-P. Kahn A. Pichard A.-L. J. Biol. Chem. 1996; 271: 3469-3473Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), the fragment of interest generated by polymerase chain reaction. The a+b polymerase chain reaction fragment was also subcloned in the AflIII site (located upstream from the promoter) or in the ClaI site (located downstream of the CAT gene) of the previously described pECAT vector (4Grégori C. Ginot F. Decaux J.F. Weber A. Berbar T. Kahn A. Pichard A.L. Biochem. Biophys. Res. Commun. 1991; 176: 722-729Crossref PubMed Scopus (21) Google Scholar). Then the various promoter fragments −232 A100 (7Sabourin J.-C. Kern A.-S. Gregori C. Porteu A. Cywiner C. Chatelet F.-P. Kahn A. Pichard A.-L. J. Biol. Chem. 1996; 271: 3469-3473Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), −194 to + 14 (4Grégori C. Ginot F. Decaux J.F. Weber A. Berbar T. Kahn A. Pichard A.L. Biochem. Biophys. Res. Commun. 1991; 176: 722-729Crossref PubMed Scopus (21) Google Scholar) of the aldolase B gene, or −183 to + 11 of the pyruvate kinase gene (8Cognet M. Bergot M.O. Kahn A. J. Biol. Chem. 1991; 266: 7368-7375Abstract Full Text PDF PubMed Google Scholar), or −105 to +51 of the herpes simplex thymidine kinase (9Luckow B. Schütz G. Nucleic Acids Res. 1987; 15: 5490Crossref PubMed Scopus (1401) Google Scholar) gene were excised and subcloned in one or both of these two plasmids. Plasmids with block mutations in elements 5 and 7 or deletions in elements 2 and 4 were constructed by inserting the mutated fragments, obtained by a two-step polymerase chain reaction procedure (5Grégori C. Kahn A. Pichard A.L. Nucleic Acids Res. 1994; 22: 1242-1246Crossref PubMed Scopus (33) Google Scholar, 10Zaret K.S. Liu J.K. DiPersio M.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5469-5473Crossref PubMed Scopus (69) Google Scholar), in the SmaI site of the −232A100 CAT plasmid. Sequence details on the block mutations are given in Fig. 4. All constructs were checked by DNA sequencing. The primer sequences used are available upon request. Hep G2 cells were grown in Dulbecco's modified medium in the presence of 10% (v/v) fetal calf serum, 1 μml-triiodothyronin, 1 μm dexamethasone, 10 nm insulin, at 37 °C in 5% (v/v) CO2. Mouse 3T6 cells were grown under the same conditions without hormones. Transfection were carried out by the calcium phosphate method (11Graham F.L. Van der Eb A.J. Virology. 1973; 52: 456-467Crossref PubMed Scopus (6499) Google Scholar), in experimental conditions previously described (5Grégori C. Kahn A. Pichard A.L. Nucleic Acids Res. 1994; 22: 1242-1246Crossref PubMed Scopus (33) Google Scholar). In each experiment 7.5 μg of the CAT plasmids and 2 μg of the luciferase plasmid were cotransfected. The pRSV luciferase standardization plasmid was used to monitor variations in transfection efficacy. Chloramphenicol acetyltransferase (CAT) assay (12Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5292) Google Scholar) and luciferase assay (13De Wet J.R. Wood K.V. De Luca M. Helinski D.R. Subramani S. Mol. Cell. Biol. 1987; 7: 725-737Crossref PubMed Scopus (2482) Google Scholar) were performed as described (5Grégori C. Kahn A. Pichard A.L. Nucleic Acids Res. 1994; 22: 1242-1246Crossref PubMed Scopus (33) Google Scholar). Nuclear extracts from adult rat liver and brain were purified according to Gorski et al. (14Gorski K. Carneiro M. Schibler U. Cell. 1986; 47: 767-776Abstract Full Text PDF PubMed Scopus (973) Google Scholar). The double-stranded oligonucleotides used as probes or competitors were as follows: element 2, +2146, 5′ TAAAGGAGTAAAGTTCATTATTGTTAAGTATTCCAGGCT 3′, +2184; element 4, +2195, 5′ TCCCAGTGACAAACATTGACCTGTGA 3′, 2220; element 5, +2212, 5′ GACCTGTGACTCTGTTTTATGATTAACTGAGGGGC 3′, +2246; element 7, +2275, 5′ TTAGTCCCTTTGTAGAAGTTTAACTTCCTG 3′, +2304; HNF1, rat L-PK L1 (15Vaulont S. Puzenat N. Cognet M. Kahn A. Raymondjean M. J. Mol. Biol. 1989; 209: 205-219Crossref PubMed Scopus (113) Google Scholar), −106 AAGAGAGATGCTAGCTGGTTATACTTTAACCAGGACTCATCTCATCT −60; rat albumin PE56 (16Cereghini S. Raymondjean M. Carranca A.G. Herbomel P. Yaniv M. Cell. 1987; 50: 627-638Abstract Full Text PDF PubMed Scopus (215) Google Scholar), −63 TGTGGTTAATGATCTACAGTTA −41; HNF3, mouse transthyretin (17Costa R.H. Grayson D. Darnell J. Mol. Cell. Biol. 1989; 9: 1415-1425Crossref PubMed Scopus (428) Google Scholar), −111 GTTGACTAAGTCAATAATCAGA −90; HNF4, rat L-PK L3 (15Vaulont S. Puzenat N. Cognet M. Kahn A. Raymondjean M. J. Mol. Biol. 1989; 209: 205-219Crossref PubMed Scopus (113) Google Scholar), −150 TGGTTCCTGGACTCTGGCCCCCAGTGTACA −121; rat phosphoenolpyruvate carboxykinase (18Hall R.K. Scott D.K. Noisin E.L. Lucas P.C. Granner D.K. Mol. Cell. Biol. 1992; 12 (-): 5527Crossref PubMed Scopus (77) Google Scholar), −452 GGCCCACGGCCAAAGGTCATGACCG −432; human α1-antitrypsin (19Monaci P. Nicosia A. Cortese R. EMBO J. 1988; 7: 2075-2087Crossref PubMed Scopus (126) Google Scholar), −128 CCAGCCAGTGGACTTAGCCCCTGTTTGCTC −99; synthetic DR1 (DR1) (20Nakshatri H. Nakshatri B.-N. Nucleic Acids Res. 1998; 26: 2491-2499Crossref PubMed Scopus (83) Google Scholar), GGGGATCCCCCAGGTCAGGTCGAGGTCATTAGA; C/EBP, rat albumin DE1 (21Tronche F. Rollier A. Herbomel P. Bach I. Cereghini S. Weiss M. Yaniv M. Mol. Biol. & Med. 1990; 7: 173-185PubMed Google Scholar), −109 GGTATGATTTTGTAATGGGGTAGG −86; AP1 (22Xanthopoulos K.G. Prezioso V.R. Chen W.S. Sladek F.M. Cortese R. Darnell J.E. Proc. Natl. Acad. Sci U. S. A. 1991; 88: 3807-3811Crossref PubMed Scopus (142) Google Scholar), AGGGGCCATGTGACTCATTACACCAG. Labeling and gel shift assays in 6% (w/v) polyacrylamide gel were performed as described (23Raymondjean M. Cereghini S. Yaniv M. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 757-761Crossref PubMed Scopus (142) Google Scholar) in the presence of poly(dI·dC) (2 μg/sample, as a mean) as a nonspecific competitor. Supershift analysis was performed by incubating the reaction mix with anti-HNF1 or anti-HNF4 antisera (a generous gift from F. Reigeisen and M. Yaniv) at room temperature for 20 min. The DNA constructs were digested with restriction enzymes ClaI (cutting in 3′ in the vector) and HindIII (cutting in 5′ in the plasmid linker). The fragments of interest were isolated by electrophoresis, electroeluted, and purified by using elutip-d columns (Schleicher & Schuell), and then microinjected into fertilized mouse eggs according to Gordon and Ruddle (24Gordon J.W. Ruddle F.H. Methods Enzymol. 1983; 101: 411-433Crossref PubMed Scopus (160) Google Scholar). The progeny was analyzed for the presence of the transgene by Southern blot. In our previous studies we identified a 1.8-kb B region (+685 to +2514) localized in the first intron of the aldolase B gene that was absolutely required for transgene expression in the liver of transgenic mice (7Sabourin J.-C. Kern A.-S. Gregori C. Porteu A. Cywiner C. Chatelet F.-P. Kahn A. Pichard A.-L. J. Biol. Chem. 1996; 271: 3469-3473Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In transient transfection experiments in hepatoma Hep G2 cells, this B fragment stimulated about 50-fold the basal activity of a 232-bp proximal aldolase B promoter (4Grégori C. Ginot F. Decaux J.F. Weber A. Berbar T. Kahn A. Pichard A.L. Biochem. Biophys. Res. Commun. 1991; 176: 722-729Crossref PubMed Scopus (21) Google Scholar). To determine the cis-active sequences in this region, a series of deleted mutants were constructed, and their activity was tested in transient transfection experiments in Hep G2 cells (Fig. 1). Taking advantage of a uniqueStuI restriction site in the B fragment, we first analyzed the effects of the upstream 1200-bp (+685 to +1915) and of the downstream 600-bp (+1916 to +2514) parts of the B fragment. The B1200 subfragment did not change expression of the reference construct devoid of the B element (−232A100CAT construct) whereas, in contrast, the B600 downstream subfragment led to a 120-fold stimulation of the basal activity. This subfragment seemed to be more efficacious than the complete B fragment, perhaps due to its closer position with respect to the minimal promoter. Further subdivision of these 600 bp in three short DNA fragments of 200 bp each, designated fragments Ba, Bb, and Bc, showed that fragments Ba and Bc alone were totally inactive, whereas the Bb fragment (+2118 to +2329) retained 25% of the activation observed with the B600 subfragment. Finally association of the fragments B (a+b) restored the full activation reached with the B600 fragment (Fig. 1). These results indicated that the 400-bp region, spanning from +1916 to +2329 bp, is able to recapitulate the enhancer activity of the intronic B element of the aldolase B gene. This 400-bp enhancer can be divided into downstream 200 bp, conferring by themselves part of the enhancer activity, and upstream 200 bp by themselves inactive but cooperating with the downstream part to confer a full enhancer activity. To verify in vivo the relevance of results obtained ex vivo, we generated transgenic mice harboring the constructs studied above (Table I). The transgene bearing the upstream B1200 subfragment was totally inactive in all 7 lines obtained. In contrast, transgenes including either the downstream B600 subfragment or parts B(a+b) of this subfragment were detectably active in 8 out of 10 lines studied and were specifically expressed in the liver and kidney but not in the spleen and brain. As previously reported (7Sabourin J.-C. Kern A.-S. Gregori C. Porteu A. Cywiner C. Chatelet F.-P. Kahn A. Pichard A.-L. J. Biol. Chem. 1996; 271: 3469-3473Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), transgene expression is highly dependent on a position effect, thus explaining the various levels of transgene activity and their total inactivity in two lines (once with each construct). In any case, these results confirm in vivo the delineation of the enhancer region of the aldolase B gene established from ex vivo experiments.Table IExpression of the aldolase B/CAT transgenes in different tissuesTransgeneMouseCopy numberLiverKidneyIntestineSpleenBrainCAT activity (cpm/μg wet tissue/45 min)232A100/CAT4016<0.1<0.1<0.1<0.1<0.1232A100B/CAT91075690.81.10.4 18004535.23.70.10.52.452150.34.80.44.60.417100.350.9<0.1<0.1<0.17200.398.25<0.1<0.1<0.1232A100B/CAT528<0.1<0.1NDNDND 120017100<0.1<0.1NDNDND163<0.1<0.1NDNDND381<0.1<0.1NDNDND40100.160.13NDNDND478<0.1<0.1NDNDND3570<0.1<0.1NDNDND232A100B/CAT661001016ND<0.1<0.1 600(a + b +621207.62.18ND<0.1<0.1 c)645<0.1<0.1ND<0.1<0.1232A100B/CAT831070.91<0.1 400(a + b)145110.40.20.20.22121265110.2261<0.1<0.1<0.1<0.1<0.133182.30.7<0.1<0.1<0.160500.170.25<0.1<0.1<0.141300.250.3<0.1<0.1<0.1Transgene expression is expressed by CAT activity, in cpm/mg wet tissue/45 min; all results correspond to the means of three independent measurements (in the organs of the founders mice).ND, not determined. Open table in a new tab Transgene expression is expressed by CAT activity, in cpm/mg wet tissue/45 min; all results correspond to the means of three independent measurements (in the organs of the founders mice). ND, not determined. The next question was whether the B(a+b) fragment had all canonical properties of an enhancer and whether it was liver cell-specific. To answer this question we placed the a+b fragment in both orientations in its normal intronic position or in a distal position, 1.2 kb upstream from the promoter or 1 kb downstream of the CAT gene. The activity of all these constructs was tested by transient transfections in Hep G2 cells, and the results are reported in Table II. The fold activation observed was totally independent of the forward or backward orientation of the (a+b) fragment and almost independent of its position with respect to the cap site, either upstream from the promoter or downstream of the CAT gene or in its natural intronic position, in agreement with canonical enhancer properties (25Banerji J. Rusconi S. Schaffner W. Cell. 1981; 27: 299-308Abstract Full Text PDF PubMed Scopus (935) Google Scholar). However, when the enhancer strength was tested on a construct consisting of the aldolase B promoter spanning from −194 to + 14 bp, and lacking the first 100 bp and the last 120 bp of the intronic sequence (i.e. both splice sites), the level of activation was reduced. We do not know if this reduction results from a specific cooperation between the enhancer and intronic sequences located in the extreme 5′ and 3′ parts of the first intron or from a general increase in the level of transgene expression linked to the presence of a functional intron, already documented in mice but not in transient transfection experiments (26Whitelaw B. Archibald A.L. Harris S. McClenaghan M. Simons J.P. Clark A.J. Transgenic Res. 1991; 1: 3-13Crossref PubMed Scopus (109) Google Scholar).Table IIProperties of the B(a + b) intronic enhancer fragment in transiently transfected cellsConstructsHep G23T6-fold enhancement−232A100CAT0−232A100 (a + b) CAT120 ± 37−232A100 (b + a) CAT118 (4)(a + b)–––– −232A100CAT78 (4)−190CAT00(a + b) –––– −190CAT25 (3)−190 CAT ––––(a + b)30 ± 8:4 (2)−183 PK CAT00−183 PK CAT ––––(a + b)18 (3):1.6 (2)−105 TK CAT00−105 TK CAT –––– (a + b)15 (3)1.1 (2)Constructs with different promoters, with or without the B(a + b) fragment in different positions, were transfected into either Hep G2 hepatoma cells or 3T6 fibroblasts; the CAT activity was measured and normalized by the luciferase activity generated by the cotransfected pRSV luciferase plasmid. The −232A100 CAT construct contains 232 bp of the aldolase B promoter plus 100 bp of the intronic element A (see Fig. 1). The −190 CAT construct contains 190 bp of the aldolase B promoter without splice sites. The −183 PK CAT construct is driven by the L-type pyruvate kinase promoter. The −105 tk CAT construct is driven by the HSV thymidine kinase promoter. The position of the B(a + b) enhancer fragment present in the first intron of the aldolase B gene is indicated with respect to the promoter and to the CAT gene. In an intronic position in −232A100 CAT constructs, this fragment was studied in both orientations (a + b) and (b + a). The results are expressed as fold enhancement with respect to the enhancerless constructs. When more than four independent experiments were performed, the results are given as means ± S.E.; otherwise, the means are given, and in parentheses are the number of experiments. Open table in a new tab Constructs with different promoters, with or without the B(a + b) fragment in different positions, were transfected into either Hep G2 hepatoma cells or 3T6 fibroblasts; the CAT activity was measured and normalized by the luciferase activity generated by the cotransfected pRSV luciferase plasmid. The −232A100 CAT construct contains 232 bp of the aldolase B promoter plus 100 bp of the intronic element A (see Fig. 1). The −190 CAT construct contains 190 bp of the aldolase B promoter without splice sites. The −183 PK CAT construct is driven by the L-type pyruvate kinase promoter. The −105 tk CAT construct is driven by the HSV thymidine kinase promoter. The position of the B(a + b) enhancer fragment present in the first intron of the aldolase B gene is indicated with respect to the promoter and to the CAT gene. In an intronic position in −232A100 CAT constructs, this fragment was studied in both orientations (a + b) and (b + a). The results are expressed as fold enhancement with respect to the enhancerless constructs. When more than four independent experiments were performed, the results are given as means ± S.E.; otherwise, the means are given, and in parentheses are the number of experiments. The activity of the B(a+b) enhancer fragment was also tested on heterologous promoters, either the liver-specific −183-bp proximal promoter of the L-type pyruvate kinase gene (8Cognet M. Bergot M.O. Kahn A. J. Biol. Chem. 1991; 266: 7368-7375Abstract Full Text PDF PubMed Google Scholar) or the ubiquitous 105-bp promoter of the thymidine kinase (tk) gene (9Luckow B. Schütz G. Nucleic Acids Res. 1987; 15: 5490Crossref PubMed Scopus (1401) Google Scholar). The B(a+b) fragment enhanced the activity of these promoters by 18- and 15-fold, respectively (Table II). It is noteworthy that stimulation of the L-type pyruvate kinase promoter by the aldolase B enhancer was approximately similar to that by the SV 40 enhancer, previously reported (8Cognet M. Bergot M.O. Kahn A. J. Biol. Chem. 1991; 266: 7368-7375Abstract Full Text PDF PubMed Google Scholar). To determine whether the enhancer activity of the a+b fragment was by itself specific to liver cells, the constructs containing either the aldolase B or the L-type pyruvate kinase or the tk promoter, with or without the B(a+b) enhancer, were transiently transfected in mouse 3T6 cells that do not express the aldolase B gene. The enhancer was unable to turn on the liver-specific aldolase B orL-type pyruvate kinase promoters as well to activate the ubiquitous tk promoter. These results indicate that the aldolase B(a+b) enhancer was clearly cell-specific. A computer analysis of the a+b enhancer sequence using the recently published Matinspector program (27Paca-Uccaralertkun S. Zhao L.-J. Adya N. Cross J.V. Cullen B.R. Boros I.M. Giam C.-Z. Mol. Cell. Biol. 1994; 14: 456-462Crossref PubMed Google Scholar) was performed. Only the analysis of the Bb (+2118 to +2329) enhancer fragment gave relevant information indicating potential binding sites for liver-enriched nuclear factors such as HNF1, HNF3, HNF4, and CAAT/enhancer binding protein (C/EBP) and for the ubiquitous AP1 complex. Since this Bb short fragment alone also retained part of the enhancer function and is 76% conserved between human and rat aldolase B genes, we focused our attention on these 200 bp. To confirm that elements of the Bb fragment actually interact with DNA-binding proteins, we first used in vivo DNase I footprinting experiments. The in vivo footprint revealed protein occupancy all over the fragment (not shown), such that it was rather difficult to deduce from this pattern well delineated windows. However, we used this experiment together with the identification of potential binding sites to design seven oligonucleotides that were used for gel shift assay experiments. Fig. 2summarizes features of sequence analysis and in vivofootprinting experiments and shows the elements whose binding activity was then individually analyzed by gel shift assays. We found that elements 1 and 3 bind factors present in both liver and brain nuclear extracts; in contrast, elements 2, 4, 5, and 7 have a different binding activity in liver and brain (Fig. 3). To determine whether elements 2, 4, 5, and 7 bind previously identified liver-specific transcription factors, we used oligonucleotides of known binding specificity as competitor in the gel retardation experiments. Binding to element 2 was only competed for by the HNF4 oligonucleotide (Fig. 3, a), whereas binding to elements 5 and 7 were highly competed for by the HNF1 oligonucleotide and by each other (Fig. 3,c and d). In addition, binding activity of element 2 was specifically supershifted by anti-HNF4 antibodies (Fig. 3, a), whereas binding activities of elements 5 and 7 were both supershifted by anti-HNF1 antibodies (Fig. 3, c and d).Figure 3Gel shift analysis of elements B2, B4, B5, and B7 of the enhancer fragment Bb. The labeled, double-stranded oligonucleotide probes used for nuclear factor binding as well as the competitor oligonucleotides are described under "Materials and Methods." All competition experiments were performed by adding 50-fold excess of unlabeled double-stranded competitors. The lanes (−) in A, B, and D indicate the absence of competitor; the nature of the competitors is indicated abovethe corresponding lanes. The arrows indicate position of specific bands (specifically displaced by cognate competitor) and of nonspecific bands (ns) (either nondisplaced, or nonspecifically displaced). A, gel shift assays with liver or brain nuclear extracts. B, gel shift assays with liver nuclear extracts, competition with various oligonucleotides reproducing known binding sites. C, panels a, b, and c, supershift assays using 1 μl of specific anti-HNF1 or anti-HNF4 antibodies; in the control (−), we used 1 μl of nonimmune serum. D, panel b, gel shift assays using L-PK HNF4 oligonucleotide (−), competition with either itself, or element 2, or element 4. Panel a, gel shift assay characterization of element B2; panel b, gel shift assay characterization of element B4; panel c, gel shift assay characterization of element B5; panel d, gel shift assay characterization of element B7.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The binding activity of element 4 was more difficult to identify. Competition experiments using oligonucleotides with different affinity for either HNF4 and/or COUP-TF were performed (Fig. 3, b), and none of them totally competed for the binding to element 4. The element 4 binding activity was also insensitive to anti-HNF4 antibodies. In contrast, element 4 as well as element 2 were effective in displacing HNF4 bound to the L3 L-PK site (Fig. 3, b). Therefore element 4 could bind factor(s) of the nuclear receptor superfamily different from HNF4 and could bind HNF4 with a low affinity. These element 4-binding factors are not likely to correspond mainly to COUP-TF (28Ladias J.A. Hadzopoulou-Cladaras M. Kardasses D. Cardot P. Cheng J. Zannis V. Cladaras C. J. Biol. Chem. 1992; 267: 15849-15860Abstract Full Text PDF PubMed Google Scholar), which is expressed in the brain as well as in the liver. Moreover element 4, whose sequence was reminiscent of an HNF3 recognition site, failed to bind this nuclear factor in our experiments as judged from competition experiments with an authentic HNF3-binding oligonucleotide. These results establish that the aldolase B enhancer is modular in nature, possessing binding sites for at least two liver-specific transcription factors, HNF1 and HNF4. The relative contribution of elements 2, 4, 5, and 7 to the enhancer strength in Hep G2 cells was tested by transient transfection of mutant constructs in which each of these element were mutated separately or in combination. The mutations were obtained as described under "Materials and Methods," and the a+b-mutated fragments were introduced into an intronic position of the −232 A100 CAT aldolase B vector. We verified by electrophoretic mobility shift assay that mutated element 5 did not bind HNF1 (not shown). Mutation in this element as well as deletion of the other HNF1-binding site, element 7, rendered the enhancer totally inactive. Surprisingly, when both HNF1-binding sites were deleted, we observed an activity of
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