VIRUS GENES 6:3,261-271, 1992 © KluwerAcademicPublishers, Manufacturedin The Netherlands
Multiple Cis-Acting DNA Elements that Regulate Transcription of the Adenovirus 12 E1A Gene YOSHIHISA YAMAZAKI, YUKO SHIMADA, HITOMI SHIBATA-SAKURAI, YUKITO MASAMUNE, AND YOSHINOBU NAKANISHI Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Ishikawa, Japan Received August 29, 1991 Accepted October 1, 1991 Requests for reprints should be addressed to Y. Nakanishi, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920, Japan.
Key words: adenovirus E1A gene, transcription regulation, c/s-acting element, autoregulation, DNA transfection, enhancer
Abstract To delineate cis-acting elements for adenovirus (Ad) 12 EIA gene transcription, we transfected HeLa and NIH3T3 cells with DNAs having various deletions in the 5'-upstream region linked to the chloramphenicol acetyltransferase gene. Deletions in the regions between nucleotide (nt) positions 54 and 166, and t67 and 200, with respect to the left end of the viral genome at nt position 1, caused a two- to three-fold reduction in transcription. Transcription decreased to an almost undetectable level with loss of the region between nt positions 201 and 282. The effect of these mutations was almost consistent between both cell lines. The region between nt positions 77 and 94 stimulated transcription when situated upstream of the simian virus 40 early promoter in either orientation. Transcription was stimulated about ninefold in the presence of the DNA that encodes the product of the 13S, but not the 12S mRNA of the Adl2 E1A gene. These results indicate that transcription of the Adl2 E1A gene is regulated by multiple cis-acting elements and is stimulated by its own gene product.
Introduction
The adenovirus (Ad) E1A gene is expressed immediately after viral infection, and its product, the E1A protein, activates other Ad early and cellular genes, bringing about replication of the viral genome (reviewed in 1-4). Moreover, function of the E1A protein is required for the transforming and tumorigenic activities of Ad
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(reviewed in 3-5). It is thus important to elucidate the mechanism by which E1A gene expression is regulated in order to gain a better understanding of transformation and tumor induction. The control mechanism for EIA gene transcription has been well studied with nontumorigenic Ad2 or Ad5 (reviewed in 1,6), but that for the EIA gene of tumorigenic Ad remains to be clarified. We have studied transcription control of the EIA gene of type 12 Ad, one of the most tumorigenic Ad, in a cell-free system and have identified the cis-acting DNA elements and trans-acting factors required for transcription stimulation (7,8). However, some cis-acting elements, such as the enhancer (reviewed in 9-11), are not fully active in a cell-free transcription reaction (12-14). We thus reexamined cis-acting elements for Adl2 EIA gene transcription by a DNA transfection assay and identified elements that were not detected in previous studies performed in a cell-free system. Materials and Methods
Plasmid DNAs Adl2 EIA DNAs linked to the chloramphenicol acetyltransferase (CAT) gene were constructed with DNA fragments containing various lengths of the 5'-end region of the Adl2 EIA gene inserted adjacent to the CAT gene in plasmid pBLCAT3 (15). The insert DNA fragments were prepared from plasmid pE1A21 (16) by digestion with either restriction enzymes or Bal31 nuclease. All DNA constructs were confirmed by sequencing not to have any internal deletions or base changes in the Ad sequences. Plasmids pDBCAT and plBCAT were constructed by inserting a synthetic DNA oligo B into a BgllI site of plasmid pA10CAT2 that contained the simian virus 40 (SV40) early promoter upstream from the CAT gene. Oligo B had the sequence 5'-GTGGAGGTGTGGCTTTGG-3', which corresponds to the region between nucleotide (nt) positions 77 and 94 of the Adl2 DNA with respect to the left end of the viral genome at nt position 1, which is flanked by the sequence 5'-GATC-3' at the 5' ends of both strands. The multiplicity and orientation of the inserted oligo B was determined by sequencing, and two DNA constructs containing a monomer insert in direct (pDBCAT) and inverted (plBCAT) orientation were isolated. Plasmids pl2HI2S and pI2H13S, which were kindly provided by Dr. Y. Sawada, encode the products of the 12S and 13S mRNAs of the Adl2 E1A gene, respectively. They contained the 1594-bp most left end region of the Adl2 DNA with deletions in the region between nt positions 977 and 1143 (pl2H12S), and 1070 and 1143 (pl2H13S) (Y. Sawada, M. Ishino, and K. Fujinaga, unpublished data).
Celt culture and DNA transfection assay HeLa $3 and NIH3T3 cells were maintained in Eagle's medium or Dulbecco's modified Eagle medium (Nissui, Japan) supplemented with 10% fetal bovine se-
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rum (FBS) (Whittaker M.A. Bioproducts) at 37°C under a humidified atmosphere of 5% C O 2 in air. Both cell lines were plated at 5-I0 x 105 cells/dish (Falcon 3003) 1 day before DNA transfeetion. After overnight culture, cells were successively washed with phosphate buffered saline (PBS) and TBS-D solution (25 mM TrisHCI, pH 7.5, 0.137 M NaC1, 5 mM KC1, 0.6 mM Na2HPO 4, 0.7 mM CaCI2, 0.1% glucose). TBS-D (250 txl), containing 10 I~g DNA (purified by two successive centrifugations in CsC1) and 1 mg/ml DEAE-dextran (Pharmacia) was added to the washed cells, which were then cultured at 37°C for 30-90 min. The DNA solution was discarded, and cells were washed successively with TBS-D and PBS. Then 9 ml of medium containing FBS and 1 ml of 2 mg/ml chloroquine (Sigma) was added and cells were incubated at 37°C for 3-4 hr. Cells were washed three times with medium without FBS and then cultured in medium with FBS at 37°C for 48 hr. Cells were collected, washed three times with PBS, and suspended in 0.1 ml of 0.25 M Tris-HC1, pH 7.5. Cell lysates were prepared by three consecutive freeze-thaw cycles. The CAT assay was conducted according to the previously described method (17,18) in an 85-1xl reaction mixture containing 0.1 txCi of ~4C-chloramphenicol (Cm, 50-60 Ci/mMol; New England Nuclear) and 50 ~1 of cell lysates. After I hr incubation at 37°C, Cm was extracted with ethylacetate, dried, and separated on a silica gel plate (Whatman LK6D). The amount of Cm on the plate was determined using an image analyzer (BAS2000, Fuji Photo Film), and the ratio of acetylated Cm to total Cm was calculated for CAT activity. Results
Transfection o f HeLa and NIH3T3 cells with Adl2 E1A/CAT DNAs To identify cis-acting DNA regions for Adl2 E1A gene transcription, plasmid DNAs were constructed that have various lengths of the 5' end region of the Adl2 E1A gene linked to the CAT gene (Fig. 1A). These DNAs were introduced into HeLa and NIH3T3 cells, and were examined for expression of the CAT gene (Fig. 1B). The results of repeated experiments were quantified and are summarized in Fig. 2. The effect of various deletions on transcription was almost the same in both cell lines, suggesting that similar trans-acting factors function to regulate transcription of the Adl2 E1A gene in both human (HeLa) and mouse (NIH3T3) cells. The regions between nt positions 54 and 166, and 167 and 200 appeared to stimulate transcription two- to three-fold. When the region between nt positions 201 and 282 was lost, expression of the CAT gene decreased to an almost undetectable level, suggesting that this region contains a cis-acting element(s) that is most responsible for the maintenance of basal-level transcription of the Adl2 E1A gene.
Effect of the b-region on transcription of the Adl2 EIA gene We previously pointed out that nuclear extracts of Ehrlich ascites tumor cells gave a footprint in the region between nt positions 77 and 94, which we named the
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YAMAZAKI ET AL.
Fig. 1. Transfection of HeLa and NIH3T3 cells with Adl2 EIA/CATDNA. A: Schematic representation of Adl2 E1A/CAT DNA. Numbers indicate the extent of deletion in each DNA construct with
respect to the left end of the viral genome at nt position 1. Letters D and P with arrows show the distal and proximal transcription initiation sites of the Adl2 EIA gene, respectively (16,33,34). Boxes with letters a and b indicate the positions of footprints observed with nuclear extracts of Ehrlich ascites tumor cells (7). B: CAT expression from Adl2 EIA/CAT DNAs. HeLa and NIH3T3 cells were transfected with Adl2 EIA/CAT DNAs, and CAT activity in the cell lysates was determined. Autoradiograms of silica gel plates are shown. The positions of acetylated (Ac-Cm)and unacetylated Cm are indicated.
b-region (7). This region contains a sequence quite similar to the core sequence of
the SV40 enhancer, 5'-GTGGA/TA/TA/TG-3', and has close sequence similarity to the B element of the polyomavirus enhancer. H o w e v e r , the b-region showed almost no effect on transcription o f the A d l 2 E 1 A gene in a cell-free transcription reaction (7). Since the region between nt positions 54 and 166, which includes the b-region, was found to stimulate transcription in a transfection assay, we next examined whether or not the b-region affects transcription from the early promoter of SV40. A synthetic D N A , oligo B, containing the entire b-region was inserted upstream of the SV40 early promoter in both directions, and resulting D N A s were introduced into H e L a cells followed by a C A T assay. The results clearly showed that the b-region stimulated transcription regardless of orientation (Fig. 3). This indicates that the b-region acts as a positive regulatory element for SV40 early transcription and suggests it to be responsible for the stimulatory effect o f the region between nt positions 54 and 166 on A d l 2 E I A gene transcription. H o w ever, the extent of transcription stimulation by the b-region was much less than that by the SV40 enhancer (Fig. 3), although their sequences are homologous.
265
TRANSCRIPTION REGULATION OF Adl2 EIA GENE
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Multiple Cis-Acting DNA Elements that Regulate Transcription of the Adenovirus 12 E1A Gene YOSHIHISA YAMAZAKI, YUKO SHIMADA, HITOMI SHIBATA-SAKURAI, YUKITO MASAMUNE, AND YOSHINOBU NAKANISHI Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Ishikawa, Japan Received August 29, 1991 Accepted October 1, 1991 Requests for reprints should be addressed to Y. Nakanishi, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920, Japan.
Key words: adenovirus E1A gene, transcription regulation, c/s-acting element, autoregulation, DNA transfection, enhancer
Abstract To delineate cis-acting elements for adenovirus (Ad) 12 EIA gene transcription, we transfected HeLa and NIH3T3 cells with DNAs having various deletions in the 5'-upstream region linked to the chloramphenicol acetyltransferase gene. Deletions in the regions between nucleotide (nt) positions 54 and 166, and t67 and 200, with respect to the left end of the viral genome at nt position 1, caused a two- to three-fold reduction in transcription. Transcription decreased to an almost undetectable level with loss of the region between nt positions 201 and 282. The effect of these mutations was almost consistent between both cell lines. The region between nt positions 77 and 94 stimulated transcription when situated upstream of the simian virus 40 early promoter in either orientation. Transcription was stimulated about ninefold in the presence of the DNA that encodes the product of the 13S, but not the 12S mRNA of the Adl2 E1A gene. These results indicate that transcription of the Adl2 E1A gene is regulated by multiple cis-acting elements and is stimulated by its own gene product.
Introduction
The adenovirus (Ad) E1A gene is expressed immediately after viral infection, and its product, the E1A protein, activates other Ad early and cellular genes, bringing about replication of the viral genome (reviewed in 1-4). Moreover, function of the E1A protein is required for the transforming and tumorigenic activities of Ad
262
YAMAZAKI ET AL.
(reviewed in 3-5). It is thus important to elucidate the mechanism by which E1A gene expression is regulated in order to gain a better understanding of transformation and tumor induction. The control mechanism for EIA gene transcription has been well studied with nontumorigenic Ad2 or Ad5 (reviewed in 1,6), but that for the EIA gene of tumorigenic Ad remains to be clarified. We have studied transcription control of the EIA gene of type 12 Ad, one of the most tumorigenic Ad, in a cell-free system and have identified the cis-acting DNA elements and trans-acting factors required for transcription stimulation (7,8). However, some cis-acting elements, such as the enhancer (reviewed in 9-11), are not fully active in a cell-free transcription reaction (12-14). We thus reexamined cis-acting elements for Adl2 EIA gene transcription by a DNA transfection assay and identified elements that were not detected in previous studies performed in a cell-free system. Materials and Methods
Plasmid DNAs Adl2 EIA DNAs linked to the chloramphenicol acetyltransferase (CAT) gene were constructed with DNA fragments containing various lengths of the 5'-end region of the Adl2 EIA gene inserted adjacent to the CAT gene in plasmid pBLCAT3 (15). The insert DNA fragments were prepared from plasmid pE1A21 (16) by digestion with either restriction enzymes or Bal31 nuclease. All DNA constructs were confirmed by sequencing not to have any internal deletions or base changes in the Ad sequences. Plasmids pDBCAT and plBCAT were constructed by inserting a synthetic DNA oligo B into a BgllI site of plasmid pA10CAT2 that contained the simian virus 40 (SV40) early promoter upstream from the CAT gene. Oligo B had the sequence 5'-GTGGAGGTGTGGCTTTGG-3', which corresponds to the region between nucleotide (nt) positions 77 and 94 of the Adl2 DNA with respect to the left end of the viral genome at nt position 1, which is flanked by the sequence 5'-GATC-3' at the 5' ends of both strands. The multiplicity and orientation of the inserted oligo B was determined by sequencing, and two DNA constructs containing a monomer insert in direct (pDBCAT) and inverted (plBCAT) orientation were isolated. Plasmids pl2HI2S and pI2H13S, which were kindly provided by Dr. Y. Sawada, encode the products of the 12S and 13S mRNAs of the Adl2 E1A gene, respectively. They contained the 1594-bp most left end region of the Adl2 DNA with deletions in the region between nt positions 977 and 1143 (pl2H12S), and 1070 and 1143 (pl2H13S) (Y. Sawada, M. Ishino, and K. Fujinaga, unpublished data).
Celt culture and DNA transfection assay HeLa $3 and NIH3T3 cells were maintained in Eagle's medium or Dulbecco's modified Eagle medium (Nissui, Japan) supplemented with 10% fetal bovine se-
TRANSCRIPTION R E G U L A T I O N OF Adl2 EIA GENE
263
rum (FBS) (Whittaker M.A. Bioproducts) at 37°C under a humidified atmosphere of 5% C O 2 in air. Both cell lines were plated at 5-I0 x 105 cells/dish (Falcon 3003) 1 day before DNA transfeetion. After overnight culture, cells were successively washed with phosphate buffered saline (PBS) and TBS-D solution (25 mM TrisHCI, pH 7.5, 0.137 M NaC1, 5 mM KC1, 0.6 mM Na2HPO 4, 0.7 mM CaCI2, 0.1% glucose). TBS-D (250 txl), containing 10 I~g DNA (purified by two successive centrifugations in CsC1) and 1 mg/ml DEAE-dextran (Pharmacia) was added to the washed cells, which were then cultured at 37°C for 30-90 min. The DNA solution was discarded, and cells were washed successively with TBS-D and PBS. Then 9 ml of medium containing FBS and 1 ml of 2 mg/ml chloroquine (Sigma) was added and cells were incubated at 37°C for 3-4 hr. Cells were washed three times with medium without FBS and then cultured in medium with FBS at 37°C for 48 hr. Cells were collected, washed three times with PBS, and suspended in 0.1 ml of 0.25 M Tris-HC1, pH 7.5. Cell lysates were prepared by three consecutive freeze-thaw cycles. The CAT assay was conducted according to the previously described method (17,18) in an 85-1xl reaction mixture containing 0.1 txCi of ~4C-chloramphenicol (Cm, 50-60 Ci/mMol; New England Nuclear) and 50 ~1 of cell lysates. After I hr incubation at 37°C, Cm was extracted with ethylacetate, dried, and separated on a silica gel plate (Whatman LK6D). The amount of Cm on the plate was determined using an image analyzer (BAS2000, Fuji Photo Film), and the ratio of acetylated Cm to total Cm was calculated for CAT activity. Results
Transfection o f HeLa and NIH3T3 cells with Adl2 E1A/CAT DNAs To identify cis-acting DNA regions for Adl2 E1A gene transcription, plasmid DNAs were constructed that have various lengths of the 5' end region of the Adl2 E1A gene linked to the CAT gene (Fig. 1A). These DNAs were introduced into HeLa and NIH3T3 cells, and were examined for expression of the CAT gene (Fig. 1B). The results of repeated experiments were quantified and are summarized in Fig. 2. The effect of various deletions on transcription was almost the same in both cell lines, suggesting that similar trans-acting factors function to regulate transcription of the Adl2 E1A gene in both human (HeLa) and mouse (NIH3T3) cells. The regions between nt positions 54 and 166, and 167 and 200 appeared to stimulate transcription two- to three-fold. When the region between nt positions 201 and 282 was lost, expression of the CAT gene decreased to an almost undetectable level, suggesting that this region contains a cis-acting element(s) that is most responsible for the maintenance of basal-level transcription of the Adl2 E1A gene.
Effect of the b-region on transcription of the Adl2 EIA gene We previously pointed out that nuclear extracts of Ehrlich ascites tumor cells gave a footprint in the region between nt positions 77 and 94, which we named the
264
YAMAZAKI ET AL.
Fig. 1. Transfection of HeLa and NIH3T3 cells with Adl2 EIA/CATDNA. A: Schematic representation of Adl2 E1A/CAT DNA. Numbers indicate the extent of deletion in each DNA construct with
respect to the left end of the viral genome at nt position 1. Letters D and P with arrows show the distal and proximal transcription initiation sites of the Adl2 EIA gene, respectively (16,33,34). Boxes with letters a and b indicate the positions of footprints observed with nuclear extracts of Ehrlich ascites tumor cells (7). B: CAT expression from Adl2 EIA/CAT DNAs. HeLa and NIH3T3 cells were transfected with Adl2 EIA/CAT DNAs, and CAT activity in the cell lysates was determined. Autoradiograms of silica gel plates are shown. The positions of acetylated (Ac-Cm)and unacetylated Cm are indicated.
b-region (7). This region contains a sequence quite similar to the core sequence of
the SV40 enhancer, 5'-GTGGA/TA/TA/TG-3', and has close sequence similarity to the B element of the polyomavirus enhancer. H o w e v e r , the b-region showed almost no effect on transcription o f the A d l 2 E 1 A gene in a cell-free transcription reaction (7). Since the region between nt positions 54 and 166, which includes the b-region, was found to stimulate transcription in a transfection assay, we next examined whether or not the b-region affects transcription from the early promoter of SV40. A synthetic D N A , oligo B, containing the entire b-region was inserted upstream of the SV40 early promoter in both directions, and resulting D N A s were introduced into H e L a cells followed by a C A T assay. The results clearly showed that the b-region stimulated transcription regardless of orientation (Fig. 3). This indicates that the b-region acts as a positive regulatory element for SV40 early transcription and suggests it to be responsible for the stimulatory effect o f the region between nt positions 54 and 166 on A d l 2 E I A gene transcription. H o w ever, the extent of transcription stimulation by the b-region was much less than that by the SV40 enhancer (Fig. 3), although their sequences are homologous.
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