Gene. 109 (1991) 171-176 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/91/$03.50
171
GENE 06201
Transcription stimulation of the adenovirus type-12 Ela gene in vitro by a novel factor bound to a region adjacent to a TATA box (Recombinant DNA; transcription regulation; cell-free transcription; promoter region; DNA-protein interaction)
Hitomi Shibata-Sakurai, Tomomi Ando, Yukito Masamune and Yoshinobu Nakanishi Faculty of Pharmaceutical Sciences. Kanazawa University, Kanazawa, Ishikawa 920 (Japan)
Received by H. van Ormondt: 5 August 1991 Revised/Accepted: 28 August/30August 1991 Received at publishers: 25 September 1991
SUMMARY The E l a gene of adenovirus (Ad) type-12 possesses two transcription start points (tsp) separated by 139 nucleotides (nt). We previously found that transcription from a tsp distal to the coding region is preferentially stimulated in a cell-free reaction by nuclear factor I (NF-I) bound to a region near the left end of the virus genome. We report here on the identification of a cis-acting DNA region and a trans-acting factor for transcription initiated at the proximal tsp of the Adl2 E l a gene. A deletion in the region between nt -50 and -36 relative to the proximal tsp at + I caused a significant decrease in the proximal transcription in a cell-free reaction using nuclear extracts of HeLa cells. A cellular factor binding to this region was shown to be responsible for transcription stimulation. This E IA-stimulating factor (ESF-1) appeared to recognize the sequence 5 ' - T G T C A - 3 ' located immediately upstream from a TATA box. Unlike NF-I, the ESF-1 activity did not seem to be influenced by the E I A protein. Our results indicated that ESF-I stimulates the proximal transcription of the Ad 12 E l a gene by binding to the region adjacent to a TATA box.
INTRODUCTION The Ad EIA protein is essential for the transformation of virus-infected cells as well as the induction of tumors in certain animals (reviewed in Grand, 1987). Presumably, production of E IA protein is a strictly controlled process since it possesses multiple important functions related to virus replication and cell growth (reviewed in Moran and Correspondence to: Dr. Y. Nakanishi, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920 (Japan) TeL/Fax (762)33-4047.
Abbreviations: Ad, adenovirus(es);bp, base pair(s); ESF-I, EIA-stimulating factor 1; DTT, dithiothreitol; Hepes, 4-(2-hydroxyethyl)-l-piperazine ethanesulfonicacid; NF-I, nuclear factor I; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; PMSF, phenylmethylsulfonylfluoride; SV40, simian virus 40; tsp, transcription start point(s); wt, wild type.
Mathews, 1987; Flint and Shenk, 1989; Boulanger and Blair, 1991). Although the mechanism which regulates the E l a gene transcription of nononcogenie Ad2 and Ad5 has been studied (reviewed in Berk, 1986; Nevins, 1987; Jones et al., 1988; Boulanger and Blair, 1991), that of the tumorinducing Ad is yet to be elucidated. Ad 12 belongs to a group of highly oncogenic Ad (Lewis and Cook, 1984) and the mechanism controlling production of the E 1A protein may be involved in the oncogcnicity. We have studied the regulatory mechanism for Adl2 Ela gene transcription which is initiated at two tsp separated by 139 nt (Sawada and Fujinaga, 1980; Saito et ai., 1981). Our previous studies have shown that transcription from a tsp distal to the coding region is stimulated by NF-I, a cellular protein essential for Ad DNA replication (Nagata et al., 1982), which binds to a region located near the left end of the Adl2 genome (Koikeda et al., 1990; H. Kawamura, K. Nagata, Y.M. and Y.N., unpublished data). We have also
172 suggested that NF-I loses its DNA-binding activity in the presence of the Adl2 E1A protein (Koikeda et al., 1990). Transcription initiated at the proximal tsp should thus become dominant as the E IA protein accumulates in Ad 12infected cells. In this aspect, the regulatory mechanism of the proximal transcription of the Ad 12 Ela gene should be clarified for a better understanding of the pathway for tumor induction by Ad 12. In this study, we identified a cis-acting DNA region and a trans-acting cellular factor(s) responsible for maximum proximal transcription of the Ad 12 Ela gene.
RESULTS A N D DISCUSSION
(a) Identification of a c/s-acting element for the proximal transcription of the Adl2 Ela gene Our previous studies demonstrated that a region upstream from nt -67, relative to the proximal tsp at + 1, did not contain an nt sequence which affected the proximal
I
400
|
500
I
bp
e
TATA
ATG
P
I,
= - - . t Jh
65b "--41
.
i
379
i
395
i
i
W
~4
i
transcription of the Ad 12 Ela gene in cell-free transcription using nuclear extracts of Ehrlich ascites tumor cells (Shibata et al., 1989). To examine the existence of any cis-acting nt sequences for proximal transcription, a series of mutant DNAs lacking various portions of the 5'-upstream region including the TATA box were constructed. These were then used as DNA templates in cell-free transcription with nuclear extracts of HeLa cells, and transcripts from the proximal tsp were analyzed by primer extension (Fig. 1). A previous observation with mouse cell extracts, that a region beyond nt -67 neither stimulated nor inhibited the proximal transcription was confirmed with HeLa cell extracts (lanes I and 2). Transcription was unaffected even when a further deletion vas made down to nt -51 (lane 3). However, loss of the adjacent 15-bp region caused a significant decrease in transcription, which became almost equal to that from a DNA template lacking the TATA box (lanes 4 and 5). These results suggested that the region between nt -50 and -36, which we referred to as the C-region, stimulates the proximal transcription of the Ad 12 E l a gene. The C-region included a 9-nt sequence, 5'-GTCAGCTGA, which is well conserved among the Ela genes of Ad types 2, 5, 7, and 12 at a similar distance from the TATA box (Fig. 2). This suggests a general role of this sequence in transcription regulation of the Ad E l a gene. This 9-nt sequence is somewhat similar to the AP-4-binding site, 5'-CAGCTGTGG, which is present within the SV40
....... 410
,,
443
• A7
i
W zx4zxGzx6 A7 I P " " 000,
2 3 4 5 M nt 0
.......67
-26 Fig. 1. Identification of a cis-acting region for the proximal transcription of the Adl2 Ela gene in a cell-free reaction. (Top)The structure of the wt (W) and mutant (A4-A7) DNAs. The location of the proximal TATA box, the proximal tsp (P), a translation start codon (ATG), and a primer (thick bar) are indicated along with a schematic representation of the 5'-end region ofthe Adl2 Ela gene. Numbers at the leR are nt positions of the left ends of the mutant DNAs with respect to the left end of the
virus genome at nt I. The 5'-upstream region of the Adl2 Ela gene was deleted by digesting the BamHl-cleaved pEIA21 DNA (wt) (Nakanishi et al., 1987) with nuclease BAL 31, and resultant DNAs were self-ligated with BglII.linker. The extent of the deletions was determined by sequencing each DNA construct (Sanger et al., 1977). (Bottom) An autoradiogram of an 8% polyacrylamide-7 M urea gel containing the primerextended products. The wt and mutant DNAs were transcribed in vitro with nuclear extracts of HeLa cells and the resultant RNA transcripts were analyzed by primer extension. The arrow indicates the position of a 65-nt product derived from the proximal transcript. Lane M contained the HpaII.cleaved pBR322 DNA as size markers and sizes are shown in nt. HeLa $3 cells were cultured in minimal essential medium No. 4 (Nissui) supplemented with 10% fetal bovine serum. Nuclear extracts were prepared as previously described (Dignam et al., 1983; Nakanishi et al., 1987) at protein concentration of 8-12mg/ml. Cell-free transcription was conducted at 30°C for ! h in a 25-/~1 reaction mixture containing 12 mM Hepes pH 7.9/0.12 mM EDTA/0.3 mM DTT/60 mM KCI/5 mM MgCl~/0.3 mM PMSF/12% glycerol/0.5 mM ATP, GTP, CTP, and UTP/5 mM creatine phosphate/0.5/~g of a closed-circular DNA template/10-15/~l of nuclear extracts. RNA transcripts were extracted and annealed at 65°C for 1 h with 0.2 pmol of a 32P-labeled primer which had been synthesized to represent the stretch between nt 490 and 509 of the Adl2 DNA (Shibata et al., 1989). Reverse transcription was then conducted at 50°C for 1 h in a 35-#1 reaction mixture containing 17 mM Tris. HCI pH 8.8/7 mM MgCi2/70 mM KCI/0.3 mM EDTA/4 mM DTT/0.4 mM dATP, dGTP, dCTP, and TTP/7/~g actinomycin D per ml/6 units of avian myeloblastosis virus reverse transcriptase.
173 A5
Ad12 TC~ 3TCAGCTGA T C G T T T G G G T A T T T A A Ad2 A T A
4ss ~TCAGCTGA
~GCGCAGTGTATTTAT
Ad5 AT~
4~s 9TCAGCTGA
CGTGTAGTGTATTTAT
Ad7 GG~
465 3TCAGCTGA
TCGCTAGGGTATTTAA
Fig. 2. Nucleotide sequence of the cis-acting C-region. The nt sequences ofthe 5'-upstream region of the Eia genes oftypes 2, 5, 7, and 12 Ad are shown. Numbers refer to nt positions with respect to the left end of the Ad genome at nt !. Putative TATA boxes are underlined and the conserved 9-nt sequence is boxed. Arrows indicate the left ends of the t w e deletion mutant DNAs, A5 and A6.
enhancer (Mermod et al., 1988). Previous studies by other investigators did not carefully examine the corresponding DNA regions flanking the Ad2 and Ad5 Eta genes. We proceeded to look for a factor(s) in HeLa cell extracts which interacts with the C-region.
(b) Detection of factor(s) binding to the C-region Oligo 'C' representing the entire C-region was synthesized and used as a probe in a gel-shift assay (Fig. 3A). Incubation of the probe with HeLa cell extracts yielded a clear shift band which disappeared when unlabeled probe was added in excess to the binding reaction (lanes 1 and 2), suggesting this band to be a complex formed between the oligo probe and specific DNA-binding factor(s) present in the extracts. By contrast, the addition of excess amounts of oligos containing binding sites for ATF/CREB (Lin and Green, 1988), AP-4, and AP-l/Jun (Lee etal., 1987; Bohmann et al., 1987; Angel et al., 1988), which were all shown to function as probes in a gel-shift assay (data not shown), did not affect the intensity of the shift-band (lanes 3-5). A DNase I footprint assay revealed that a very short stretch within the C-region was protected from the DNasedigestion by factor(s) present in HeLa extracts (Fig. 3B). The protected sequence 5'-TGTCA (nt -50 to -46) was distinct from the sequence bound by AP-4. We designated the binding factor ESF-I. Next, we examined whether the binding of ESF-I to the C-region is required for transcription stimulation. Oligo 'C' and oligo 'AP4' were added in excess to the transcription reaction and their effects on the proximal transcription of the Adl2 Ela gene were examined (Fig. 4). A control reaction was conducted in the presence of poly(dl-dC)poly(dl-dC), which did not significantly affect transcription (lanes 1 and 2). Oligo 'C' inhibited the proximal transcription from wt pE1A21 DNA down to a level similar to that from ,46 DNA lacking the C-region (lanes 3 and 4), whereas oligo 'AP4' had minimal effect (lanes 5 and 6). These results led us to conclude that a novel factor ESF-I
stimulates the proximal transcription of the Ad 12 Ela gene in vitro by binding to a region adjacent to the TATA box. Although ESF-1 is distinct from ATF/CREB, the fact that both factors bind to a region close to a TATA box allowed us to propose a mechanism for the ESF-I action. It has been suggested that ATF/CREB stimulates transcription of the Ad5 E4 gene by modulating the activity of TFIID (Lee and Green, 1987; Horikoshi et ai., 1988), a general transcription factor bound to a TATA box (reviewed in Sawadogo and Sentenac, 1990). Moreover, a proteinprotein interaction between TFIID and transactivators has recently been demonstrated (Stringer et al., 1990; Horikoshi et al., 1991). ESF-1 might therefore stimulate the proximal transcription of the Ad 12 Ela gene by interacting with TFIID.
(c) ESF-I activity in nuclear extracts of Ela-expressing cells We previously suggested that the Adl2 E IA protein autoregulates the transcription of its own gene by modulating the DNA-binding activity of NF-I which selectively stimulates the distal transcription (Koikeda et al., 1990). We thus examined if stimulation by ESF-I of the proximal transcription is also influenced by the E1A protein. Nuclear extracts were prepared from cell lines 293 and 321, the former of which produces Ad5 (Graham et al., 1977) and the latter both Ad5 and Adl2 E1A proteins (Y. Sawada and T. Shenk, personal communication). These extracts were compared to HeLa cell extracts in gel-shift and cell-free transcription assays (Fig. 5). Both extracts prepared from Ela-expressing cells possessed activities for binding to the C-region and transcribing ,45 DNA more efficiently than ,46 DNA at almost the same level as those of HeLa extracts. It seemed that transcription activity of 321 cell extracts with a ,46 DNA template was higher than that of other extracts. However, a considerable portion of the signal was due to the mRNA for the Adl2 E1A protein that was present in 321 cells and contaminated the extracts (lane 7). These results suggest that the activity of trans-acting ESF-1 was not influenced, either positively or negatively, by the E1A protein and that the mechanism which controls the proximal transcription of the Ad 12 Ela gene is not under EIA autoregulation. Together with the results from our previous studies, a model for transcription regulation of the Adl2 Ela gene is proposed (Fig. 6). The two modes of transcription of the Adl2 Eta gene, i.e., distal and proximal, are individually stimulated by cellular factors NF-I and ESF- 1, respectively. The DNA-binding activity of NF-I might be inhibited by E1A protein (Koikeda et al., 1990), whereas ESF-1 binds to the C-region and stimulates the proximal transcription regardless of the presence of E1A protein. Thus, proximal transcription quite possibly becomes dominant as E1A
174
A
protein accumulates in Adl2-infected cells. However, it remains uncertain whether this mechanism for the regulation of Ad 12 E l a gene transcription is involved in transformation and tumor induction by Adl2 (Linder and Marshall, 1990).
probe" la...qP
I
competitor '
."
¢.~ I . - o.. a top
(d) Conclusions (1) The proximal transcription of the Adl2 Ela gene is stimulated in vitro by a novel cellular factor, ESF-1, which recognizes the sequence 5 ' - T G T C A - 3 ' located just upstream from a TATA box. (2) Transcription regulation of the Adl2 Ela gene by ESF-I does not appear to be under autoregulation.
free " probe
1 2 3 4 5
A HeLa G 1 2 3 4
NE 5
6
[-
[
171
GENE 06201
Transcription stimulation of the adenovirus type-12 Ela gene in vitro by a novel factor bound to a region adjacent to a TATA box (Recombinant DNA; transcription regulation; cell-free transcription; promoter region; DNA-protein interaction)
Hitomi Shibata-Sakurai, Tomomi Ando, Yukito Masamune and Yoshinobu Nakanishi Faculty of Pharmaceutical Sciences. Kanazawa University, Kanazawa, Ishikawa 920 (Japan)
Received by H. van Ormondt: 5 August 1991 Revised/Accepted: 28 August/30August 1991 Received at publishers: 25 September 1991
SUMMARY The E l a gene of adenovirus (Ad) type-12 possesses two transcription start points (tsp) separated by 139 nucleotides (nt). We previously found that transcription from a tsp distal to the coding region is preferentially stimulated in a cell-free reaction by nuclear factor I (NF-I) bound to a region near the left end of the virus genome. We report here on the identification of a cis-acting DNA region and a trans-acting factor for transcription initiated at the proximal tsp of the Adl2 E l a gene. A deletion in the region between nt -50 and -36 relative to the proximal tsp at + I caused a significant decrease in the proximal transcription in a cell-free reaction using nuclear extracts of HeLa cells. A cellular factor binding to this region was shown to be responsible for transcription stimulation. This E IA-stimulating factor (ESF-1) appeared to recognize the sequence 5 ' - T G T C A - 3 ' located immediately upstream from a TATA box. Unlike NF-I, the ESF-1 activity did not seem to be influenced by the E I A protein. Our results indicated that ESF-I stimulates the proximal transcription of the Ad 12 E l a gene by binding to the region adjacent to a TATA box.
INTRODUCTION The Ad EIA protein is essential for the transformation of virus-infected cells as well as the induction of tumors in certain animals (reviewed in Grand, 1987). Presumably, production of E IA protein is a strictly controlled process since it possesses multiple important functions related to virus replication and cell growth (reviewed in Moran and Correspondence to: Dr. Y. Nakanishi, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920 (Japan) TeL/Fax (762)33-4047.
Abbreviations: Ad, adenovirus(es);bp, base pair(s); ESF-I, EIA-stimulating factor 1; DTT, dithiothreitol; Hepes, 4-(2-hydroxyethyl)-l-piperazine ethanesulfonicacid; NF-I, nuclear factor I; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; PMSF, phenylmethylsulfonylfluoride; SV40, simian virus 40; tsp, transcription start point(s); wt, wild type.
Mathews, 1987; Flint and Shenk, 1989; Boulanger and Blair, 1991). Although the mechanism which regulates the E l a gene transcription of nononcogenie Ad2 and Ad5 has been studied (reviewed in Berk, 1986; Nevins, 1987; Jones et al., 1988; Boulanger and Blair, 1991), that of the tumorinducing Ad is yet to be elucidated. Ad 12 belongs to a group of highly oncogenic Ad (Lewis and Cook, 1984) and the mechanism controlling production of the E 1A protein may be involved in the oncogcnicity. We have studied the regulatory mechanism for Adl2 Ela gene transcription which is initiated at two tsp separated by 139 nt (Sawada and Fujinaga, 1980; Saito et ai., 1981). Our previous studies have shown that transcription from a tsp distal to the coding region is stimulated by NF-I, a cellular protein essential for Ad DNA replication (Nagata et al., 1982), which binds to a region located near the left end of the Adl2 genome (Koikeda et al., 1990; H. Kawamura, K. Nagata, Y.M. and Y.N., unpublished data). We have also
172 suggested that NF-I loses its DNA-binding activity in the presence of the Adl2 E1A protein (Koikeda et al., 1990). Transcription initiated at the proximal tsp should thus become dominant as the E IA protein accumulates in Ad 12infected cells. In this aspect, the regulatory mechanism of the proximal transcription of the Ad 12 Ela gene should be clarified for a better understanding of the pathway for tumor induction by Ad 12. In this study, we identified a cis-acting DNA region and a trans-acting cellular factor(s) responsible for maximum proximal transcription of the Ad 12 Ela gene.
RESULTS A N D DISCUSSION
(a) Identification of a c/s-acting element for the proximal transcription of the Adl2 Ela gene Our previous studies demonstrated that a region upstream from nt -67, relative to the proximal tsp at + 1, did not contain an nt sequence which affected the proximal
I
400
|
500
I
bp
e
TATA
ATG
P
I,
= - - . t Jh
65b "--41
.
i
379
i
395
i
i
W
~4
i
transcription of the Ad 12 Ela gene in cell-free transcription using nuclear extracts of Ehrlich ascites tumor cells (Shibata et al., 1989). To examine the existence of any cis-acting nt sequences for proximal transcription, a series of mutant DNAs lacking various portions of the 5'-upstream region including the TATA box were constructed. These were then used as DNA templates in cell-free transcription with nuclear extracts of HeLa cells, and transcripts from the proximal tsp were analyzed by primer extension (Fig. 1). A previous observation with mouse cell extracts, that a region beyond nt -67 neither stimulated nor inhibited the proximal transcription was confirmed with HeLa cell extracts (lanes I and 2). Transcription was unaffected even when a further deletion vas made down to nt -51 (lane 3). However, loss of the adjacent 15-bp region caused a significant decrease in transcription, which became almost equal to that from a DNA template lacking the TATA box (lanes 4 and 5). These results suggested that the region between nt -50 and -36, which we referred to as the C-region, stimulates the proximal transcription of the Ad 12 E l a gene. The C-region included a 9-nt sequence, 5'-GTCAGCTGA, which is well conserved among the Ela genes of Ad types 2, 5, 7, and 12 at a similar distance from the TATA box (Fig. 2). This suggests a general role of this sequence in transcription regulation of the Ad E l a gene. This 9-nt sequence is somewhat similar to the AP-4-binding site, 5'-CAGCTGTGG, which is present within the SV40
....... 410
,,
443
• A7
i
W zx4zxGzx6 A7 I P " " 000,
2 3 4 5 M nt 0
.......67
-26 Fig. 1. Identification of a cis-acting region for the proximal transcription of the Adl2 Ela gene in a cell-free reaction. (Top)The structure of the wt (W) and mutant (A4-A7) DNAs. The location of the proximal TATA box, the proximal tsp (P), a translation start codon (ATG), and a primer (thick bar) are indicated along with a schematic representation of the 5'-end region ofthe Adl2 Ela gene. Numbers at the leR are nt positions of the left ends of the mutant DNAs with respect to the left end of the
virus genome at nt I. The 5'-upstream region of the Adl2 Ela gene was deleted by digesting the BamHl-cleaved pEIA21 DNA (wt) (Nakanishi et al., 1987) with nuclease BAL 31, and resultant DNAs were self-ligated with BglII.linker. The extent of the deletions was determined by sequencing each DNA construct (Sanger et al., 1977). (Bottom) An autoradiogram of an 8% polyacrylamide-7 M urea gel containing the primerextended products. The wt and mutant DNAs were transcribed in vitro with nuclear extracts of HeLa cells and the resultant RNA transcripts were analyzed by primer extension. The arrow indicates the position of a 65-nt product derived from the proximal transcript. Lane M contained the HpaII.cleaved pBR322 DNA as size markers and sizes are shown in nt. HeLa $3 cells were cultured in minimal essential medium No. 4 (Nissui) supplemented with 10% fetal bovine serum. Nuclear extracts were prepared as previously described (Dignam et al., 1983; Nakanishi et al., 1987) at protein concentration of 8-12mg/ml. Cell-free transcription was conducted at 30°C for ! h in a 25-/~1 reaction mixture containing 12 mM Hepes pH 7.9/0.12 mM EDTA/0.3 mM DTT/60 mM KCI/5 mM MgCl~/0.3 mM PMSF/12% glycerol/0.5 mM ATP, GTP, CTP, and UTP/5 mM creatine phosphate/0.5/~g of a closed-circular DNA template/10-15/~l of nuclear extracts. RNA transcripts were extracted and annealed at 65°C for 1 h with 0.2 pmol of a 32P-labeled primer which had been synthesized to represent the stretch between nt 490 and 509 of the Adl2 DNA (Shibata et al., 1989). Reverse transcription was then conducted at 50°C for 1 h in a 35-#1 reaction mixture containing 17 mM Tris. HCI pH 8.8/7 mM MgCi2/70 mM KCI/0.3 mM EDTA/4 mM DTT/0.4 mM dATP, dGTP, dCTP, and TTP/7/~g actinomycin D per ml/6 units of avian myeloblastosis virus reverse transcriptase.
173 A5
Ad12 TC~ 3TCAGCTGA T C G T T T G G G T A T T T A A Ad2 A T A
4ss ~TCAGCTGA
~GCGCAGTGTATTTAT
Ad5 AT~
4~s 9TCAGCTGA
CGTGTAGTGTATTTAT
Ad7 GG~
465 3TCAGCTGA
TCGCTAGGGTATTTAA
Fig. 2. Nucleotide sequence of the cis-acting C-region. The nt sequences ofthe 5'-upstream region of the Eia genes oftypes 2, 5, 7, and 12 Ad are shown. Numbers refer to nt positions with respect to the left end of the Ad genome at nt !. Putative TATA boxes are underlined and the conserved 9-nt sequence is boxed. Arrows indicate the left ends of the t w e deletion mutant DNAs, A5 and A6.
enhancer (Mermod et al., 1988). Previous studies by other investigators did not carefully examine the corresponding DNA regions flanking the Ad2 and Ad5 Eta genes. We proceeded to look for a factor(s) in HeLa cell extracts which interacts with the C-region.
(b) Detection of factor(s) binding to the C-region Oligo 'C' representing the entire C-region was synthesized and used as a probe in a gel-shift assay (Fig. 3A). Incubation of the probe with HeLa cell extracts yielded a clear shift band which disappeared when unlabeled probe was added in excess to the binding reaction (lanes 1 and 2), suggesting this band to be a complex formed between the oligo probe and specific DNA-binding factor(s) present in the extracts. By contrast, the addition of excess amounts of oligos containing binding sites for ATF/CREB (Lin and Green, 1988), AP-4, and AP-l/Jun (Lee etal., 1987; Bohmann et al., 1987; Angel et al., 1988), which were all shown to function as probes in a gel-shift assay (data not shown), did not affect the intensity of the shift-band (lanes 3-5). A DNase I footprint assay revealed that a very short stretch within the C-region was protected from the DNasedigestion by factor(s) present in HeLa extracts (Fig. 3B). The protected sequence 5'-TGTCA (nt -50 to -46) was distinct from the sequence bound by AP-4. We designated the binding factor ESF-I. Next, we examined whether the binding of ESF-I to the C-region is required for transcription stimulation. Oligo 'C' and oligo 'AP4' were added in excess to the transcription reaction and their effects on the proximal transcription of the Adl2 Ela gene were examined (Fig. 4). A control reaction was conducted in the presence of poly(dl-dC)poly(dl-dC), which did not significantly affect transcription (lanes 1 and 2). Oligo 'C' inhibited the proximal transcription from wt pE1A21 DNA down to a level similar to that from ,46 DNA lacking the C-region (lanes 3 and 4), whereas oligo 'AP4' had minimal effect (lanes 5 and 6). These results led us to conclude that a novel factor ESF-I
stimulates the proximal transcription of the Ad 12 Ela gene in vitro by binding to a region adjacent to the TATA box. Although ESF-1 is distinct from ATF/CREB, the fact that both factors bind to a region close to a TATA box allowed us to propose a mechanism for the ESF-I action. It has been suggested that ATF/CREB stimulates transcription of the Ad5 E4 gene by modulating the activity of TFIID (Lee and Green, 1987; Horikoshi et ai., 1988), a general transcription factor bound to a TATA box (reviewed in Sawadogo and Sentenac, 1990). Moreover, a proteinprotein interaction between TFIID and transactivators has recently been demonstrated (Stringer et al., 1990; Horikoshi et al., 1991). ESF-1 might therefore stimulate the proximal transcription of the Ad 12 Ela gene by interacting with TFIID.
(c) ESF-I activity in nuclear extracts of Ela-expressing cells We previously suggested that the Adl2 E IA protein autoregulates the transcription of its own gene by modulating the DNA-binding activity of NF-I which selectively stimulates the distal transcription (Koikeda et al., 1990). We thus examined if stimulation by ESF-I of the proximal transcription is also influenced by the E1A protein. Nuclear extracts were prepared from cell lines 293 and 321, the former of which produces Ad5 (Graham et al., 1977) and the latter both Ad5 and Adl2 E1A proteins (Y. Sawada and T. Shenk, personal communication). These extracts were compared to HeLa cell extracts in gel-shift and cell-free transcription assays (Fig. 5). Both extracts prepared from Ela-expressing cells possessed activities for binding to the C-region and transcribing ,45 DNA more efficiently than ,46 DNA at almost the same level as those of HeLa extracts. It seemed that transcription activity of 321 cell extracts with a ,46 DNA template was higher than that of other extracts. However, a considerable portion of the signal was due to the mRNA for the Adl2 E1A protein that was present in 321 cells and contaminated the extracts (lane 7). These results suggest that the activity of trans-acting ESF-1 was not influenced, either positively or negatively, by the E1A protein and that the mechanism which controls the proximal transcription of the Ad 12 Ela gene is not under EIA autoregulation. Together with the results from our previous studies, a model for transcription regulation of the Adl2 Ela gene is proposed (Fig. 6). The two modes of transcription of the Adl2 Eta gene, i.e., distal and proximal, are individually stimulated by cellular factors NF-I and ESF- 1, respectively. The DNA-binding activity of NF-I might be inhibited by E1A protein (Koikeda et al., 1990), whereas ESF-1 binds to the C-region and stimulates the proximal transcription regardless of the presence of E1A protein. Thus, proximal transcription quite possibly becomes dominant as E1A
174
A
protein accumulates in Adl2-infected cells. However, it remains uncertain whether this mechanism for the regulation of Ad 12 E l a gene transcription is involved in transformation and tumor induction by Adl2 (Linder and Marshall, 1990).
probe" la...qP
I
competitor '
."
¢.~ I . - o.. a top
(d) Conclusions (1) The proximal transcription of the Adl2 Ela gene is stimulated in vitro by a novel cellular factor, ESF-1, which recognizes the sequence 5 ' - T G T C A - 3 ' located just upstream from a TATA box. (2) Transcription regulation of the Adl2 Ela gene by ESF-I does not appear to be under autoregulation.
free " probe
1 2 3 4 5
A HeLa G 1 2 3 4
NE 5
6
[-
[
