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The Aldolase A Promoter in Proliferating Rat Thymocytes Is Regulated by a Cluster of SP1 Sites and a Distal Modulator
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
225, 997–1005 (1996)
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The Aldolase A Promoter in Proliferating Rat Thymocytes Is Regulated by a Cluster of SP1 Sites and a Distal Modulator Ulrich Hermfisse, Doris Scha¨fer, Roland Netzker, and Karl Brand1 Institut fu¨r Biochemie der Medizinischen Fakulta¨t, Friedrich-Alexander-Universita¨t Erlangen-Nu¨rnberg, Fahrstr. 17, D-91054 Erlangen, Germany Received July 12, 1996 In mitogen-stimulated rat thymocytes the activities and mRNA levels of aldolase A increase remarkably during proliferation pointing to a transcriptional regulation of this enzyme. By DNAse I footprinting and mobility shift competition assays five binding sites for the activating transcription factor Sp1 and one site for an AP-1 like nuclear factor could be identified in the core activating region of the proximal aldolase AH1 promoter downstream of 0400. Transfection data and differences found in nuclear protein binding of resting and proliferating cells to DNA sites suggest that Sp1 is an integral part of the mechanism by which the AH1 promoter achieves high level transcription during proliferation. Moreover we demonstrate that an element between positions 01066/0731 significantly attenuates the AH1 promoter driven transcription as well as transcription regulated by the heterologous SV40 promoter. From this effect a functional linkage between the distal muscle-restricted M1 promoter and the active AH1 promoter can be suggested. q 1996 Academic Press, Inc.
In the S-phase of the cell cycle 48 hours after mitogen-stimulation of rat thymocytes an eight to tenfold increase in the activity of fructose 1,6-bisphosphate aldolase (EC 4.1.2.13) and other glycolytic enzymes has been observed (1, 2). Recently we reported similar increases in the mRNA levels for pyruvate kinase M2 (3) and in an extended study also for glyceraldehyde 3phosphate dehydrogenase, hexokinase I and II and aldolase in rat thymocytes during cell proliferation (4). Three distinct aldolase isozymes have been described in mammals: aldolase A, B and C (5, 6, 7). In tissues, aldolase C is the isoform exclusively expressed in brain, type B is predominantly found in liver, and aldolase A is expressed in skeletal muscle, heart, spleen and several other tissues (8, 9, 10). Apart from this tissue-specific expression, changes in the isozyme pattern have been observed during development (11, 12, 13) and carcinogenesis (14, 15). By Northern blot analyses with isozyme-specific probes we showed that in quiescent as well as in proliferating rat thymocytes type A is the only aldolase isozyme that is expressed (4). For rat and mouse aldolase A two promoter regions have been identified whereas in human cells three distinct aldolase A promoters have been reported (8, 16, 17, 11, 18, 19). Alternative usage of the promoters allows the tissue-specific generation of mRNAs differing only in the 5* untranslated leader exons. In rat, expression from the distal M1 promoter is restricted to skeletal muscle, whereas the proximal AH1 promoter is expressed ubiquitously (9, 20). Transfection experiments of the M1 promoter and analyses of its cis-elements indicated that this promoter is governed by muscle-specific transcription factors in differentiated myotubes (21, 22). Investigation of the AH1 promoter by transient transfection of human HepG2 cells revealed the existence of a positive regulatory element within the proximal AH1 promoter region up 1 Correspondence to Prof. Dr. Karl Brand, Institut fu¨r Biochemie der Med. Fakulta¨t, Universita¨t Erlangen-Nu¨rnberg, Fahrstr. 17, D-91054 Erlangen, Deutschland. Fax: /49 9131 852484. E-Mail: [email protected]. uni-erlangen.de.
997 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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to position 0311 (23, 21). However, until now there are no data available which transcription factors are involved in the expression from the AH1 promoter. We describe a detailed examination of the aldolase A AH1 promoter and its regulatory regions with respect to the observed proliferation-dependent increase in activity and mRNA level of aldolase A in rat thymocytes. In expression assays using promoter-CAT fusion constructs different from that used by Joh et al. (23, 21) we have investigated the proximal activating region (0400/050) and, moreover, have found a new negatively regulating transcriptional module between 01067/0732. The analysis of protein-DNA-interactions with thymocyte nuclear extracts revealed a clustering of activating transcription factor sites on the proximal promoter and a proliferation-dependent binding of the corresponding factors. MATERIALS AND METHODS Cells. Isolation and culture conditions of rat thymocytes were performed according to Brand et al. (1). FTO2B rat hepatoma cells (24) were grown in DMEM/Ham’s F-12 medium supplemented with 5 % heat-inactivated fetal calf serum, 200 U/ml penicillin G and 100 U/ml streptomycin. Transient transfection assays and reporter enzyme activity measurements. The calcium phosphate DNA-coprecipitate method (25) was performed according to Chen and Okayama (26). FTO2B cells were split 24 h before transfection in a density of 2 1 106 per dish. 3 h before adding the plasmids 9 ml fresh medium was applied to each culture dish. 18 mg pCAT vector and 6 mg pGL-3 Control in 200 ml H2O were mixed with 800 ml 5/4 1 HBS (50 mM HEPES, 172.5 mM NaCl, 6.25 mM KCl, 875 mM Na2HPO4 , pH 7.05). 52 ml 2.5 M CaCl2 were added, the solution was vortexed vigorously for 20 s and left at room temperature for 20 min. The mixture was added to the culture dish and incubated at 37 7C for 4 h. After aspiration of the medium, the cells were maintained for 3 min in 15 % (vol/vol) glycerol in DMEM/Ham’s F-12. This solution was replaced by 8 ml medium and the cells were further incubated for 48 h. For reporter enzyme activity measurements, cells were lysed by repeated freeze/thaw cycles in 150 ml 0.25 M Tris-HCl pH 7.8. Each assay was done in duplicate. Supernatants were assayed in a Berthold Lumat LB 9501 luminometer for luciferase activity as described elsewhere (27). After heat inactivation of deacetylases (60 7C, 10 min) CAT activity was measured by incubating the extract in a total volume of 120 ml with 250 nCi D-threo[dichloroacetyl-1-14C]chlor- amphenicol (54 mCi/mmol, Amersham) and 60 nmol n-butyryl coenzyme A (Sigma, Deisenhofen) in 0.25 M Tris-HCl pH 7.8 for 4 h. The reaction mixture was extracted by vortexing for exactly 30 s with 300 ml xylene and the xylene phase was reextracted twice with 100 ml 0.25 M Tris-HCl. 200 ml xylene phase were mixed with 2 ml Rotiszint Eco Plus (Roth, Karlsruhe, Germany) and analyzed in a Wallac 1410 liquid scintillation counter. CAT activities were normalized to equal amounts of luciferase activity. The results are given as the corrected CAT activities relative to the values of the indicated constructs in Fig. 2. Values presented correspond to the mean of at least three individual transfection assays except pCBA50 (2 experiments) which exerted only a 6-fold absolute CAT activity compared to a mock-transfected control. The A- and C-series in Fig. 2 were performed with two separate vector preparations. CAT reporter constructs. The aldolase A promoter corresponding to the region 01075 to /141 with /1 as the first base of exon AH1 was amplified by PCR (GenBank Accession No. U20643). Reaction conditions and sequences of the primers were already published (4). Subfragments of the promoter were cut with the appropriate restriction enzymes, blunt-ended and inserted into commercially available pCAT vectors (PROMEGA). Constructs were ligated 5* upstream of the CAT reporter gene in the pCAT Basic vector. The fragments 0558//141 and 0408//141 were PCR-amplified using Pfu DNA polymerase with fragment 01075//141 as template. Mutants pCBA400-50d and pCBA400-50i were constructed by cutting pCBA400-50 with Xho I, blunt-ending by Mung bean nuclease (d) or filling-in with Klenow polymerase (i) and subsequent ligation. A blunt-ended Pst I site was used for the insertions of the fragment 01067/0732 into the vector pCAT Control. Plasmids were amplified in the E. coli strain DH5a, purified by alkaline lysis and CsCl gradient centrifugation or, alternatively, by QIAGEN anion exchange chromatography. Nuclear extracts, electrophoretic mobility shift assays (EMSA) and DNAse I footprinting. Nuclear extracts from resting and proliferating rat thymocytes and FTO2B cells were prepared according to Dignam et al. (28). Crude nuclear extract was isolated by the micropreparation technique described by Andrews and Faller (29). Protein concentration was quantified with the Bradford reagent (Bio-Rad Laboratories, Richmond, USA). The DNA fragments were labelled with [a-32P]dATP by filling-in with Klenow polymerase (30). For mobility shift assays 10 fmol of the indicated probe, 2 mg double-stranded poly(dIdC•dIdC) and 5 mg nuclear protein were incubated for 30 min at room temperature in 1 1 DBB (20 mM Tris-HCl, 50 mM KCl, 5 mM MgCl2 , 0.5 mM DTE, 5 % (v/v) glycerol, pH 8). The binding complex was separated by 5 % polyacrylamide gel electrophoresis at 4 7C in 1 1 TGE (25 mM Tris-HCl, 190 mM glycine, 1 mM EDTA, pH 8.3). Competition experiments were performed by incubating the nuclear extract for 30 min at 4 7C with the indicated unlabelled duplex oligonucleotide or the anti-Sp1 antibody prior to the addition of the 32Plabelled probe. DNAse I footprinting assays were performed according to Raymondjean et al. (31) with slight modifica998
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FIG. 1. Schematic representation of the rat aldolase A gene promoter. (A) Exon-intron structure of the rat aldolase A gene as published by Joh et al. (16). (B) The 5* end of the gene containing the promoters M1 and AH1 was amplified by PCR and sequenced (GenBank Accession No. U20643). (C) The proximal promoter region 0312//14 was analyzed by footprinting and gel retardation assays with the indicated probes. Five elements could be identified, represented by circles (Sp1 recognition sites) and a triangle (AP-1), respectively. Numbers within the symbols correspond to the DNAse-protected sites shown in Fig. 3. DNA fragments A to E were used in mobility shift competition assays (Fig. 4).
tions. About 5 ng of labelled probe was mixed with up to 100 mg nuclear extract in 1 1 DBB and incubated for 20 min on ice, then transferred to room temperature. DNAse I was added to a final concentration of 50 U/ml and the mixture was further incubated for 2 min. Digestion was stopped by adding 1 vol of 2 1 DSS (1.6 M NH4OAc, 1 % SDS, 100 mM EDTA, 100 mgtRNA per ml). After phenol-CHCl3 extraction and ethanol precipitation, the products were resolved in a 6 % denaturing polyacrylamide gel and visualized by autoradiography.
RESULTS AND DISCUSSION
Transfection Analyses of Rat Aldolase A Promoter Fragments Fused to the CAT Reporter Gene Recently we demonstrated by northern blot hybridisation with an exon AH1 specific probe that aldolase A is the only isozyme expressed in rat thymocytes throughout cell cycle progression (4). For the investigation of transcriptional regulation the rat aldolase A promoter region was isolated by PCR amplification (Fig. 1) and subconstructs were cloned into the expression vectors pCAT Basic or pCAT Control (Fig. 2). CAT expression assays were performed with the rat hepatoma cell line FTO2B which has been proved for expressing the same mRNAs as rat thymocytes by Northern hybridisation and S1 nuclease mapping (data not shown). Our transfection analyses revealed the existence of two regulatory regions upstream of exon AH1. In Fig. 2 the constructs tested and their relative CAT activities are shown as solid bars. Deletion 999
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FIG. 2. CAT expression analyses of the rat aldolase A promoter region. CAT activities were adjusted to equal luciferase activities. Results are given as relative values { SEM with respect to construct pCBA1067 (A), pCBA40050 (B) or the vector pCAT Control (C) set to 100 %. The number of independent transfection experiments for each construct (n) is given on the right. (A) 5* deletion constructs of the AH1 promoter spanning from 01067 to /141 were inserted 5* upstream of the CAT gene into the promoterless vector pCAT Basic. (B) Deletion constructs of the proximal activating region responsible for the aldolase A expression from the AH1 promoter. (C) Relative CAT activities of A1066-731 CAT fusion constructs in transient expression assays with FTO2B cells. The SV40 early promoter and the SV40 enhancer are represented by diagonal and vertical lines, respectively.
from 01067 to 0727 (pCBA727, set to 100 %) resulted in a remarkable increase in CAT activity pointing to a reducing module within 01067/0728 (Fig. 2A). The constructs pCBA558, pCBA408 and pCBA312 (63.0 { 10.7 %) exerted a slower expression than pCBA727, but the activities are remarkably higher than pCBA200. Deletion down to 050 (pCBA50) abolished CAT expression almost completely. From these data we conclude that the region downstream of 0727 is responsible for activated gene expression and that the core promoter is located downstream of 0200. This is in contrast to Joh et al. because they could not detect any changes in expression with promoter fragments extending position 0312 (21). This might be due to differences in the transfected cell line used (human HepG2 vs. rat FTO2B cells) and/or the different 3* end of the promoter region analyzed (position /16 vs. /141). However, our results with the constructs shorter than 0312 correspond with their findings. To investigate the putative cis-element around 0200, the strong activator region 0400/050 (pCBA400-50) was deleted from both sides and mutated by insertion or deletion of 4 bases, respectively (Fig. 4B). In contrast to the almost silent subconstructs pCBA200-50 and pCBA400-200, pCBA400-50d as well as pCBA400-50i yielded moderate relative CAT activities (25.0 { 5.0 and 13.7 { 4.3 %). Combined with the mobility shift data of probe 0213/0167 (Fig. 4C), where only Sp1 was bound, the early assumption of a cis-element at position 0200 could not be supported. 1000
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However, along with the protein-DNA interaction data presented later there is strong evidence that a cluster of activating cis-elements at least between positions 0312//14 governs transcriptional activity of the rat aldolase A promoter. This proximal region resambles by position and by function the activating region of the human aldolase A promoter H analyzed by Concordet et al. (32). A Distal Region of the Rat Aldolase A Promoter Represses Transcription The remarkable 16.5-fold increase in relative CAT activity observed with construct pCBA727 compared to pCBA1067 (Fig. 2A) prompted us to investigate the distal region of the promoter. Sequence 01066/0731 was inserted about 2.7 kb upstream of the heterologous early SV40 promoter in the vector pCAT Control in either orientation (Fig. 2C). Relative CAT activities were 26.2 { 4.5 % for the normal oriented and 96.2 { 15.5 % for the reverse oriented construct (pCAT Control set to 100 %). These data suggest the presence of a reducing module located within the sequence 01066/0731. Recently Lupo et al. (33) characterized a silencer found within the most distal human aldolase A promoter by Constanzo et al. (34). Sequence comparison between the rat and the human silencing region did not yield any homology of interest, moreover there is no AGAGAG motif present in 01066/0731 (33 and ref. therein). The role of these reducing elements for the transcriptional regulation of the housekeeping gene product aldolase A still remains to be elucidated. Proliferation-Dependent Binding of Nuclear Factors to the Activating Module To identify nuclear proteins binding to the activating region revealed from the transfection analyses, DNAse I footprinting has been performed with nuclear extracts from proliferating rat thymocytes (Fig. 3). With probe pr4 (0312/0200) two regions were protected (sites 1 and 2, Fig. 3A). Site 1 contains the sequence GGGGGAGGGGG, which exhibits 80 % homology to the Sp1 consensus binding site (35). Site 2 possesses the core sequence TGAGTCAG, which is fully homologous to the AP-1 binding site of the human collagenase promoter (36). With probe pr1 (084//14) two protected regions were observed (sites 3 and 4, Fig. 3B), both containing the GGGCGG core binding sequence characteristic for GC boxes. It is interesting to note that site 3 and the transcription initiation site of mRNA III are separated by only 6 bases. In gel mobility shift competition assays the binding factors were further analyzed (Fig. 4 and 5). With same protein amounts of nuclear extracts from resting and proliferating rat thymocytes incubated with the indicated probes in Fig. 4 proliferation-dependent protein binding could be observed. Since all the probes contain potential binding sites for Sp1 the extract from proliferating cells was preincubated using an anti-Sp1 antibody (Santa Cruz Biotechnology, USA). The bands with pr1 - containing two GC boxes - and pr2 could be abolished, for pr3 the formation of the upper band was prevented by the anti-Sp1 antibody, indicating the presence of Sp1 within the complex (Fig. 4). The faster migrating band could be either an additional nuclear factor from rat thymocytes which is able to associate with the Sp1 recognition sequence, or it results from proteolytic degradation products of Sp1 which do not react with the antibody (37). In an additional mobility shift experiment with probe 0171/085, containing a GC box at position 0114, we identified the binding protein as being Sp1 (data not shown). Summing up we could identify five GC boxes being recognized by Sp1 in the activating region of the AH1 promoter. Further investigation of the protected site 2 in Fig. 3A, containing sequence 0279 CTGAGTCA 0272, was performed by using different DNA fragments as competitors of probe pr4 (0312/0200) in EMSA. This analysis identified band 2 as an AP-1 related protein complex because the AP-1 oligonucleotide, being homologous to the aldolase A promoter only in the above mentioned core sequence, successfully competed with probe 4 (Fig. 5, lane B). Complex 1001
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FIG. 3. Identification of protein binding sites in the rat aldolase A promoter by DNAse I footprinting. The 5* endlabelled DNA fragments representing the noncoding strands were incubated without (lane 2) or with different protein amounts of proliferating rat thymocyte nuclear extract before DNAse I digestion (lanes 3 and 4). Each lane G contains a Maxam and Gilbert guanine sequencing reaction of the DNA fragment used. (A) DNA, encompassing sequences 0316/0200 (pr4), was incubated with 40 mg (lane 3) or 80 mg (lane 4) of nuclear extract. (B) The probe 084//14 (pr1) of the AH1 promoter was DNAse I digested in the presence of 80 mg (lane 3) or 40 mg (lane 4) nuclear extract. The protected regions are represented by open boxes (sites 1 to 4).
formation has not been found with extract from resting cells (data not shown). The binding site, also known as TPA responsive element (36), can be recognized by a variety of factors including members of the Jun- and Fos-family (38) or factors like NF-ATp (39) or TAP-1 (40). Some of these proteins are known to transactivate immediately early after cell activation (c-Fos, NF-AT), whereas c-Jun homodimers are responsible for sustained activation. Taken together the results of the expression assays and the nuclear protein-DNA interaction analyses, transcriptional activation of the housekeeping gene aldolase A in rat is governed at least by five Sp1 and one AP-1 binding site, positioned between 0279 and 07. However, this activation is influenced positively by upstream sequences up to 0727, but a negative regulator within 01066/0731 is able to attenuate the AH1 promoter driven transcription. Though it is well known that the transcription factor Sp1 is an important regulator of promoter activity (35, 41) here we are presenting additional data for a proliferation-dependent 1002
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FIG. 4. Identification and differential binding of Sp1 to the rat aldolase A promoter region. Electrophoretic mobility shift assays were performed using 10 fmol of the end-labelled fragments of the aldolase A promoter spanning from (A) 084//14 (pr1); (B) 0213/0167 (pr2); (C) 0243/0200 (pr3). Same protein amounts of nuclear extract prepared from quiescent (n) and proliferating (p) rat thymocytes after 48 h of culture were used in the assays. For each probe the nuclear extract from proliferating thymocytes was preincubated with an anti-Sp1 antibody (/). Arrows indicate the positions of the DNA-Sp1 binding complexes.
complex formation between Sp1 and its corresponding GC boxes within the proximal rat aldolase A promoter. Recent work of other groups focused on the contribution of Sp1 to transcriptional activation, i. e. for the human PFKM promoter (42) or the human adenosine
FIG. 5. Mobility shift competition assay identifying an AP-1 like transcription factor present in proliferating rat thymocytes. 10 fmol end-labelled probe 0312/0200 (pr4) was incubated with 5 mg protein of nuclear extract from proliferating rat thymocytes (/) and a 100-fold molar excess of the indicated unlabelled DNA fragments and oligonucleotides shown in Fig. 1C. Competitor B is a commercially available AP-1 oligonucleotide which is homolog to position 0278/0273. The arrow indicates the protein-DNA binding complex which could be efficiently competed with competitors A (0312/0243), B (AP-1 oligo), and C (0287/0265), but not with fragment D (0307/0285) or E (0243/0200). 1003
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deaminase promoter (43). It has been shown that expression of both housekeeping genes is governed by Sp1. The clustering of Sp1 sites in the proximal aldolase A promoter (Fig. 1C) may be required for the formation of higher order Sp1 complexes, and it is known that DNAbound Sp1 molecules can associate with each other (44) to govern synergistic transcriptional stimulation. Further investigation will focus upon the mechanisms or factors involved in the onset of induction of the rat aldolase A gene. ACKNOWLEDGMENTS The authors wish to thank G. Glaser and A. Hartwig for their expert assistance. We also thank G. H. Fey, Institute for Microbiology, Biochemistry, and Genetics, University of Erlangen-Nuremberg, for kindly providing the rat hepatoma cell line FTO2B. We are grateful to Elisabeth Schweins for reviewing the manuscript. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Br 612/15-2).
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The Aldolase A Promoter in Proliferating Rat Thymocytes Is Regulated by a Cluster of SP1 Sites and a Distal Modulator Ulrich Hermfisse, Doris Scha¨fer, Roland Netzker, and Karl Brand1 Institut fu¨r Biochemie der Medizinischen Fakulta¨t, Friedrich-Alexander-Universita¨t Erlangen-Nu¨rnberg, Fahrstr. 17, D-91054 Erlangen, Germany Received July 12, 1996 In mitogen-stimulated rat thymocytes the activities and mRNA levels of aldolase A increase remarkably during proliferation pointing to a transcriptional regulation of this enzyme. By DNAse I footprinting and mobility shift competition assays five binding sites for the activating transcription factor Sp1 and one site for an AP-1 like nuclear factor could be identified in the core activating region of the proximal aldolase AH1 promoter downstream of 0400. Transfection data and differences found in nuclear protein binding of resting and proliferating cells to DNA sites suggest that Sp1 is an integral part of the mechanism by which the AH1 promoter achieves high level transcription during proliferation. Moreover we demonstrate that an element between positions 01066/0731 significantly attenuates the AH1 promoter driven transcription as well as transcription regulated by the heterologous SV40 promoter. From this effect a functional linkage between the distal muscle-restricted M1 promoter and the active AH1 promoter can be suggested. q 1996 Academic Press, Inc.
In the S-phase of the cell cycle 48 hours after mitogen-stimulation of rat thymocytes an eight to tenfold increase in the activity of fructose 1,6-bisphosphate aldolase (EC 4.1.2.13) and other glycolytic enzymes has been observed (1, 2). Recently we reported similar increases in the mRNA levels for pyruvate kinase M2 (3) and in an extended study also for glyceraldehyde 3phosphate dehydrogenase, hexokinase I and II and aldolase in rat thymocytes during cell proliferation (4). Three distinct aldolase isozymes have been described in mammals: aldolase A, B and C (5, 6, 7). In tissues, aldolase C is the isoform exclusively expressed in brain, type B is predominantly found in liver, and aldolase A is expressed in skeletal muscle, heart, spleen and several other tissues (8, 9, 10). Apart from this tissue-specific expression, changes in the isozyme pattern have been observed during development (11, 12, 13) and carcinogenesis (14, 15). By Northern blot analyses with isozyme-specific probes we showed that in quiescent as well as in proliferating rat thymocytes type A is the only aldolase isozyme that is expressed (4). For rat and mouse aldolase A two promoter regions have been identified whereas in human cells three distinct aldolase A promoters have been reported (8, 16, 17, 11, 18, 19). Alternative usage of the promoters allows the tissue-specific generation of mRNAs differing only in the 5* untranslated leader exons. In rat, expression from the distal M1 promoter is restricted to skeletal muscle, whereas the proximal AH1 promoter is expressed ubiquitously (9, 20). Transfection experiments of the M1 promoter and analyses of its cis-elements indicated that this promoter is governed by muscle-specific transcription factors in differentiated myotubes (21, 22). Investigation of the AH1 promoter by transient transfection of human HepG2 cells revealed the existence of a positive regulatory element within the proximal AH1 promoter region up 1 Correspondence to Prof. Dr. Karl Brand, Institut fu¨r Biochemie der Med. Fakulta¨t, Universita¨t Erlangen-Nu¨rnberg, Fahrstr. 17, D-91054 Erlangen, Deutschland. Fax: /49 9131 852484. E-Mail: [email protected]. uni-erlangen.de.
997 0006-291X/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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to position 0311 (23, 21). However, until now there are no data available which transcription factors are involved in the expression from the AH1 promoter. We describe a detailed examination of the aldolase A AH1 promoter and its regulatory regions with respect to the observed proliferation-dependent increase in activity and mRNA level of aldolase A in rat thymocytes. In expression assays using promoter-CAT fusion constructs different from that used by Joh et al. (23, 21) we have investigated the proximal activating region (0400/050) and, moreover, have found a new negatively regulating transcriptional module between 01067/0732. The analysis of protein-DNA-interactions with thymocyte nuclear extracts revealed a clustering of activating transcription factor sites on the proximal promoter and a proliferation-dependent binding of the corresponding factors. MATERIALS AND METHODS Cells. Isolation and culture conditions of rat thymocytes were performed according to Brand et al. (1). FTO2B rat hepatoma cells (24) were grown in DMEM/Ham’s F-12 medium supplemented with 5 % heat-inactivated fetal calf serum, 200 U/ml penicillin G and 100 U/ml streptomycin. Transient transfection assays and reporter enzyme activity measurements. The calcium phosphate DNA-coprecipitate method (25) was performed according to Chen and Okayama (26). FTO2B cells were split 24 h before transfection in a density of 2 1 106 per dish. 3 h before adding the plasmids 9 ml fresh medium was applied to each culture dish. 18 mg pCAT vector and 6 mg pGL-3 Control in 200 ml H2O were mixed with 800 ml 5/4 1 HBS (50 mM HEPES, 172.5 mM NaCl, 6.25 mM KCl, 875 mM Na2HPO4 , pH 7.05). 52 ml 2.5 M CaCl2 were added, the solution was vortexed vigorously for 20 s and left at room temperature for 20 min. The mixture was added to the culture dish and incubated at 37 7C for 4 h. After aspiration of the medium, the cells were maintained for 3 min in 15 % (vol/vol) glycerol in DMEM/Ham’s F-12. This solution was replaced by 8 ml medium and the cells were further incubated for 48 h. For reporter enzyme activity measurements, cells were lysed by repeated freeze/thaw cycles in 150 ml 0.25 M Tris-HCl pH 7.8. Each assay was done in duplicate. Supernatants were assayed in a Berthold Lumat LB 9501 luminometer for luciferase activity as described elsewhere (27). After heat inactivation of deacetylases (60 7C, 10 min) CAT activity was measured by incubating the extract in a total volume of 120 ml with 250 nCi D-threo[dichloroacetyl-1-14C]chlor- amphenicol (54 mCi/mmol, Amersham) and 60 nmol n-butyryl coenzyme A (Sigma, Deisenhofen) in 0.25 M Tris-HCl pH 7.8 for 4 h. The reaction mixture was extracted by vortexing for exactly 30 s with 300 ml xylene and the xylene phase was reextracted twice with 100 ml 0.25 M Tris-HCl. 200 ml xylene phase were mixed with 2 ml Rotiszint Eco Plus (Roth, Karlsruhe, Germany) and analyzed in a Wallac 1410 liquid scintillation counter. CAT activities were normalized to equal amounts of luciferase activity. The results are given as the corrected CAT activities relative to the values of the indicated constructs in Fig. 2. Values presented correspond to the mean of at least three individual transfection assays except pCBA50 (2 experiments) which exerted only a 6-fold absolute CAT activity compared to a mock-transfected control. The A- and C-series in Fig. 2 were performed with two separate vector preparations. CAT reporter constructs. The aldolase A promoter corresponding to the region 01075 to /141 with /1 as the first base of exon AH1 was amplified by PCR (GenBank Accession No. U20643). Reaction conditions and sequences of the primers were already published (4). Subfragments of the promoter were cut with the appropriate restriction enzymes, blunt-ended and inserted into commercially available pCAT vectors (PROMEGA). Constructs were ligated 5* upstream of the CAT reporter gene in the pCAT Basic vector. The fragments 0558//141 and 0408//141 were PCR-amplified using Pfu DNA polymerase with fragment 01075//141 as template. Mutants pCBA400-50d and pCBA400-50i were constructed by cutting pCBA400-50 with Xho I, blunt-ending by Mung bean nuclease (d) or filling-in with Klenow polymerase (i) and subsequent ligation. A blunt-ended Pst I site was used for the insertions of the fragment 01067/0732 into the vector pCAT Control. Plasmids were amplified in the E. coli strain DH5a, purified by alkaline lysis and CsCl gradient centrifugation or, alternatively, by QIAGEN anion exchange chromatography. Nuclear extracts, electrophoretic mobility shift assays (EMSA) and DNAse I footprinting. Nuclear extracts from resting and proliferating rat thymocytes and FTO2B cells were prepared according to Dignam et al. (28). Crude nuclear extract was isolated by the micropreparation technique described by Andrews and Faller (29). Protein concentration was quantified with the Bradford reagent (Bio-Rad Laboratories, Richmond, USA). The DNA fragments were labelled with [a-32P]dATP by filling-in with Klenow polymerase (30). For mobility shift assays 10 fmol of the indicated probe, 2 mg double-stranded poly(dIdC•dIdC) and 5 mg nuclear protein were incubated for 30 min at room temperature in 1 1 DBB (20 mM Tris-HCl, 50 mM KCl, 5 mM MgCl2 , 0.5 mM DTE, 5 % (v/v) glycerol, pH 8). The binding complex was separated by 5 % polyacrylamide gel electrophoresis at 4 7C in 1 1 TGE (25 mM Tris-HCl, 190 mM glycine, 1 mM EDTA, pH 8.3). Competition experiments were performed by incubating the nuclear extract for 30 min at 4 7C with the indicated unlabelled duplex oligonucleotide or the anti-Sp1 antibody prior to the addition of the 32Plabelled probe. DNAse I footprinting assays were performed according to Raymondjean et al. (31) with slight modifica998
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FIG. 1. Schematic representation of the rat aldolase A gene promoter. (A) Exon-intron structure of the rat aldolase A gene as published by Joh et al. (16). (B) The 5* end of the gene containing the promoters M1 and AH1 was amplified by PCR and sequenced (GenBank Accession No. U20643). (C) The proximal promoter region 0312//14 was analyzed by footprinting and gel retardation assays with the indicated probes. Five elements could be identified, represented by circles (Sp1 recognition sites) and a triangle (AP-1), respectively. Numbers within the symbols correspond to the DNAse-protected sites shown in Fig. 3. DNA fragments A to E were used in mobility shift competition assays (Fig. 4).
tions. About 5 ng of labelled probe was mixed with up to 100 mg nuclear extract in 1 1 DBB and incubated for 20 min on ice, then transferred to room temperature. DNAse I was added to a final concentration of 50 U/ml and the mixture was further incubated for 2 min. Digestion was stopped by adding 1 vol of 2 1 DSS (1.6 M NH4OAc, 1 % SDS, 100 mM EDTA, 100 mgtRNA per ml). After phenol-CHCl3 extraction and ethanol precipitation, the products were resolved in a 6 % denaturing polyacrylamide gel and visualized by autoradiography.
RESULTS AND DISCUSSION
Transfection Analyses of Rat Aldolase A Promoter Fragments Fused to the CAT Reporter Gene Recently we demonstrated by northern blot hybridisation with an exon AH1 specific probe that aldolase A is the only isozyme expressed in rat thymocytes throughout cell cycle progression (4). For the investigation of transcriptional regulation the rat aldolase A promoter region was isolated by PCR amplification (Fig. 1) and subconstructs were cloned into the expression vectors pCAT Basic or pCAT Control (Fig. 2). CAT expression assays were performed with the rat hepatoma cell line FTO2B which has been proved for expressing the same mRNAs as rat thymocytes by Northern hybridisation and S1 nuclease mapping (data not shown). Our transfection analyses revealed the existence of two regulatory regions upstream of exon AH1. In Fig. 2 the constructs tested and their relative CAT activities are shown as solid bars. Deletion 999
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FIG. 2. CAT expression analyses of the rat aldolase A promoter region. CAT activities were adjusted to equal luciferase activities. Results are given as relative values { SEM with respect to construct pCBA1067 (A), pCBA40050 (B) or the vector pCAT Control (C) set to 100 %. The number of independent transfection experiments for each construct (n) is given on the right. (A) 5* deletion constructs of the AH1 promoter spanning from 01067 to /141 were inserted 5* upstream of the CAT gene into the promoterless vector pCAT Basic. (B) Deletion constructs of the proximal activating region responsible for the aldolase A expression from the AH1 promoter. (C) Relative CAT activities of A1066-731 CAT fusion constructs in transient expression assays with FTO2B cells. The SV40 early promoter and the SV40 enhancer are represented by diagonal and vertical lines, respectively.
from 01067 to 0727 (pCBA727, set to 100 %) resulted in a remarkable increase in CAT activity pointing to a reducing module within 01067/0728 (Fig. 2A). The constructs pCBA558, pCBA408 and pCBA312 (63.0 { 10.7 %) exerted a slower expression than pCBA727, but the activities are remarkably higher than pCBA200. Deletion down to 050 (pCBA50) abolished CAT expression almost completely. From these data we conclude that the region downstream of 0727 is responsible for activated gene expression and that the core promoter is located downstream of 0200. This is in contrast to Joh et al. because they could not detect any changes in expression with promoter fragments extending position 0312 (21). This might be due to differences in the transfected cell line used (human HepG2 vs. rat FTO2B cells) and/or the different 3* end of the promoter region analyzed (position /16 vs. /141). However, our results with the constructs shorter than 0312 correspond with their findings. To investigate the putative cis-element around 0200, the strong activator region 0400/050 (pCBA400-50) was deleted from both sides and mutated by insertion or deletion of 4 bases, respectively (Fig. 4B). In contrast to the almost silent subconstructs pCBA200-50 and pCBA400-200, pCBA400-50d as well as pCBA400-50i yielded moderate relative CAT activities (25.0 { 5.0 and 13.7 { 4.3 %). Combined with the mobility shift data of probe 0213/0167 (Fig. 4C), where only Sp1 was bound, the early assumption of a cis-element at position 0200 could not be supported. 1000
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However, along with the protein-DNA interaction data presented later there is strong evidence that a cluster of activating cis-elements at least between positions 0312//14 governs transcriptional activity of the rat aldolase A promoter. This proximal region resambles by position and by function the activating region of the human aldolase A promoter H analyzed by Concordet et al. (32). A Distal Region of the Rat Aldolase A Promoter Represses Transcription The remarkable 16.5-fold increase in relative CAT activity observed with construct pCBA727 compared to pCBA1067 (Fig. 2A) prompted us to investigate the distal region of the promoter. Sequence 01066/0731 was inserted about 2.7 kb upstream of the heterologous early SV40 promoter in the vector pCAT Control in either orientation (Fig. 2C). Relative CAT activities were 26.2 { 4.5 % for the normal oriented and 96.2 { 15.5 % for the reverse oriented construct (pCAT Control set to 100 %). These data suggest the presence of a reducing module located within the sequence 01066/0731. Recently Lupo et al. (33) characterized a silencer found within the most distal human aldolase A promoter by Constanzo et al. (34). Sequence comparison between the rat and the human silencing region did not yield any homology of interest, moreover there is no AGAGAG motif present in 01066/0731 (33 and ref. therein). The role of these reducing elements for the transcriptional regulation of the housekeeping gene product aldolase A still remains to be elucidated. Proliferation-Dependent Binding of Nuclear Factors to the Activating Module To identify nuclear proteins binding to the activating region revealed from the transfection analyses, DNAse I footprinting has been performed with nuclear extracts from proliferating rat thymocytes (Fig. 3). With probe pr4 (0312/0200) two regions were protected (sites 1 and 2, Fig. 3A). Site 1 contains the sequence GGGGGAGGGGG, which exhibits 80 % homology to the Sp1 consensus binding site (35). Site 2 possesses the core sequence TGAGTCAG, which is fully homologous to the AP-1 binding site of the human collagenase promoter (36). With probe pr1 (084//14) two protected regions were observed (sites 3 and 4, Fig. 3B), both containing the GGGCGG core binding sequence characteristic for GC boxes. It is interesting to note that site 3 and the transcription initiation site of mRNA III are separated by only 6 bases. In gel mobility shift competition assays the binding factors were further analyzed (Fig. 4 and 5). With same protein amounts of nuclear extracts from resting and proliferating rat thymocytes incubated with the indicated probes in Fig. 4 proliferation-dependent protein binding could be observed. Since all the probes contain potential binding sites for Sp1 the extract from proliferating cells was preincubated using an anti-Sp1 antibody (Santa Cruz Biotechnology, USA). The bands with pr1 - containing two GC boxes - and pr2 could be abolished, for pr3 the formation of the upper band was prevented by the anti-Sp1 antibody, indicating the presence of Sp1 within the complex (Fig. 4). The faster migrating band could be either an additional nuclear factor from rat thymocytes which is able to associate with the Sp1 recognition sequence, or it results from proteolytic degradation products of Sp1 which do not react with the antibody (37). In an additional mobility shift experiment with probe 0171/085, containing a GC box at position 0114, we identified the binding protein as being Sp1 (data not shown). Summing up we could identify five GC boxes being recognized by Sp1 in the activating region of the AH1 promoter. Further investigation of the protected site 2 in Fig. 3A, containing sequence 0279 CTGAGTCA 0272, was performed by using different DNA fragments as competitors of probe pr4 (0312/0200) in EMSA. This analysis identified band 2 as an AP-1 related protein complex because the AP-1 oligonucleotide, being homologous to the aldolase A promoter only in the above mentioned core sequence, successfully competed with probe 4 (Fig. 5, lane B). Complex 1001
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FIG. 3. Identification of protein binding sites in the rat aldolase A promoter by DNAse I footprinting. The 5* endlabelled DNA fragments representing the noncoding strands were incubated without (lane 2) or with different protein amounts of proliferating rat thymocyte nuclear extract before DNAse I digestion (lanes 3 and 4). Each lane G contains a Maxam and Gilbert guanine sequencing reaction of the DNA fragment used. (A) DNA, encompassing sequences 0316/0200 (pr4), was incubated with 40 mg (lane 3) or 80 mg (lane 4) of nuclear extract. (B) The probe 084//14 (pr1) of the AH1 promoter was DNAse I digested in the presence of 80 mg (lane 3) or 40 mg (lane 4) nuclear extract. The protected regions are represented by open boxes (sites 1 to 4).
formation has not been found with extract from resting cells (data not shown). The binding site, also known as TPA responsive element (36), can be recognized by a variety of factors including members of the Jun- and Fos-family (38) or factors like NF-ATp (39) or TAP-1 (40). Some of these proteins are known to transactivate immediately early after cell activation (c-Fos, NF-AT), whereas c-Jun homodimers are responsible for sustained activation. Taken together the results of the expression assays and the nuclear protein-DNA interaction analyses, transcriptional activation of the housekeeping gene aldolase A in rat is governed at least by five Sp1 and one AP-1 binding site, positioned between 0279 and 07. However, this activation is influenced positively by upstream sequences up to 0727, but a negative regulator within 01066/0731 is able to attenuate the AH1 promoter driven transcription. Though it is well known that the transcription factor Sp1 is an important regulator of promoter activity (35, 41) here we are presenting additional data for a proliferation-dependent 1002
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FIG. 4. Identification and differential binding of Sp1 to the rat aldolase A promoter region. Electrophoretic mobility shift assays were performed using 10 fmol of the end-labelled fragments of the aldolase A promoter spanning from (A) 084//14 (pr1); (B) 0213/0167 (pr2); (C) 0243/0200 (pr3). Same protein amounts of nuclear extract prepared from quiescent (n) and proliferating (p) rat thymocytes after 48 h of culture were used in the assays. For each probe the nuclear extract from proliferating thymocytes was preincubated with an anti-Sp1 antibody (/). Arrows indicate the positions of the DNA-Sp1 binding complexes.
complex formation between Sp1 and its corresponding GC boxes within the proximal rat aldolase A promoter. Recent work of other groups focused on the contribution of Sp1 to transcriptional activation, i. e. for the human PFKM promoter (42) or the human adenosine
FIG. 5. Mobility shift competition assay identifying an AP-1 like transcription factor present in proliferating rat thymocytes. 10 fmol end-labelled probe 0312/0200 (pr4) was incubated with 5 mg protein of nuclear extract from proliferating rat thymocytes (/) and a 100-fold molar excess of the indicated unlabelled DNA fragments and oligonucleotides shown in Fig. 1C. Competitor B is a commercially available AP-1 oligonucleotide which is homolog to position 0278/0273. The arrow indicates the protein-DNA binding complex which could be efficiently competed with competitors A (0312/0243), B (AP-1 oligo), and C (0287/0265), but not with fragment D (0307/0285) or E (0243/0200). 1003
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deaminase promoter (43). It has been shown that expression of both housekeeping genes is governed by Sp1. The clustering of Sp1 sites in the proximal aldolase A promoter (Fig. 1C) may be required for the formation of higher order Sp1 complexes, and it is known that DNAbound Sp1 molecules can associate with each other (44) to govern synergistic transcriptional stimulation. Further investigation will focus upon the mechanisms or factors involved in the onset of induction of the rat aldolase A gene. ACKNOWLEDGMENTS The authors wish to thank G. Glaser and A. Hartwig for their expert assistance. We also thank G. H. Fey, Institute for Microbiology, Biochemistry, and Genetics, University of Erlangen-Nuremberg, for kindly providing the rat hepatoma cell line FTO2B. We are grateful to Elisabeth Schweins for reviewing the manuscript. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Br 612/15-2).
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34. Costanzo, P., Lupo, A., Rippa, E., Grosso, M., Salvatore, F., and Izzo, P. (1993) Biochem. Biophys. Res. Commun. 195, 935–944. 35. Kadonaga, J. T., Jones, K. A., and Tjian, R. (1986) Trends Biochem. Sci. 11, 20–23. 36. Angel, P., Imagawa, M., Chiu, R., Stein, B., Imbra, R. J., Rahmsdorf, H. J., Jonat, C., Herrlich, P., and Karin, M. (1987) Cell 49, 729–739. 37. Hagen, G., Muller, S., Beato, M., and Suske, G. (1994) EMBO J. 13, 3843–3851. 38. Angel, P., and Karin, M. (1991) Biochim. Biophys. Acta 1072, 129–157. 39. Emmel, E. A., Verweij, C. L., Durand, D. B., Higgins, K. M., Lacy, E., and Crabtree, G. R. (1989) Science 246, 1617–1620. 40. Gardner, K., Moore, T. C., Davis-Smyth, T., Krutzsch, H., and Levens, D. (1994) J. Biol. Chem. 269, 32963– 32971. 41. Lecointe, N., Bernard, O., Naert, K., Joulin, V., Larsen, C. J., Romeo, P. H., and Mathieumahul, D. (1994) Oncogene 9, 2623–2632. 42. Johnson, J. L., and Mclachlan, A. (1994) Nucleic Acids Res. 22, 5085–5092. 43. Dusing, M. R., and Wiginton, D. A. (1994) Nucleic Acids Res. 22, 669–677. 44. Courey, A. J., Holtzman, D. A., Jackson, S. P., and Tjian, R. (1989) Cell 59, 827–836.
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