Biochimica et Biophysica Acta, 1048 (1990) 85-92
85
Elsevier BBAEXP 92026
Nuclear factor I stimulates transcription of the adenovirus 12 EIA gene in a cell-free system Satoshi Koikeda 1, Rie Ibuki 1, Yukiharu Sawada 2, Kyosuke Nagata 3, Hitomi Shibata 1; Yukito Masamune 1 and Yoshinobu Nakanishi I Faculty ofPharmaceuticalSciences, Kanazawa University, Ishikawa, 2 Department of Molecular Biology, Cancer Research Institute, Sapporo Medical College, Sapporo, and 3 Department of Molecular Genetics, National Institute of Genetics, Yata, Mishima, Shizuoka (Japan)
(Received 28 July 1989)
Key words: Adenovirus EIA gene; Cell free transcription; Nuclear factor I; Transcriptional control Binding to the cis-acting region of NF-l-like protein a n d / o r NF-Ill-like protein was previously suggested to be responsible for the preferential stimulation of transcription from distal start-site of the adenovirus 12 EIA gene in a cell-free system. In this study, nuclear extracts of Ehrlich ascites tumor cells depleted of NF-I-like protein were found to lose activity to stimulate the E1A gene transcription. This activity was recovered when NF-I purified from HeLa cells with no contamination of NF-III was supplemented. It is thus evident that NF-I is involved in stimulating distal transcription of the adenovirus 12 E1A gene. Moreover, activities for both stimulating the E1A gene transcription and binding to a region recognized by NF-I did not apparently exist in nuclear extracts of a cell line expressing the adenovirus 12 E1A gene. These results suggest that transcription of the adenovirus 12 EIA gene may possibly be autoregulated at least in part through modulation of the activity of NF-I.
Introduction Adenovirus E1A protein is essential for transforming virus-infected cells and inducing tumors in certain animals [1]. Cis- and trans-acting factors for transcription of the E I A gene have been studied with non-oncogenic Ad 2 and Ad 5 [2,3]. However, only little is known of the mechanism which regulates transcription of the Ad 12 E1A gene. Ad 12 belongs to a group of highly oncogenic adenoviruses and its E1A protein is required for the induction of tumors in new-born rodents [4-7]. It is thus important to elucidate the mechanism by which E I A gene transcription is controlled for greater clarification not only of cell transformation but the manner by which tumors are induced by Ad 12. The Ad 12 E I A gene has two start-sites for transcription located at positions 306 (distal site) and 445 (proximal site) with respect to the left end of the viral genome as position 1 (see Fig. 1). We previously re-
ported that factors present in nuclear extracts of Ehrlich ascites tumor cells bind to two distinct sequence elements in the 5'-upstream region of the Ad 12 E1A gene. Two elements are situated between positions 19 and 55, designated the a-region, and between positions 77 and 94, designated the b-region. Binding to the a-region of NF-I-like protein a n d / o r NF-III-like protein present in the extracts was suggested to be essential for the preferential stimulation of transcription from distal start-site of the E I A gene in a cell-free system [8]. The focal point of attention in the present study is which of these two proteins is actually responsible for stimulating the transcription. Evidence was obtained demonstrating the involvement of NF-I-like protein in the E1A gene transcription control and that this is possibly related to the autoregulation of the Ad 12 E1A gene.
Materials and Methods DNA-affinity fractionation
Abbreviations: Ad, adenovirus; NF-I, nuclear factor I; NF-III, nuclear factor III, Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid. Correspondence: Y. Nakanishi, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa, 920 Japan.
column preparation
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Oligo A has a sequence corresponding to a region between positions 13 and 48 of the Ad 12 genome and its lower strand contains an additional sequence of 5 ' - A C G A C G A G G G - 3 ' at the 5'-side (see Fig. 1). 5 0 / t g of oligo A were mixed with 0.6 g of CNBr-activated
0167-4781/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
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~-TAT AT AT A+ A/~T AT AC CT'~'AT ACTGGACTAGTGCX; A A T A T T AAAAT(~AAGTGGGCGT AG
b 70 80 90 100 110 120 T GTGTAATT~4LG,ATTGGGTG~;'AGGTGTGGC~TTGGCGTGC'q~TGTAAGTTTGGGCGGATGA~ Fig. 1. Structure of the 5'-end region of the Ad 12 E1A gene. (Top) Locations of the a-region and the b-region (boxes with letters a and b) and two transcription start-sites (arrows with letters D and P) are shown. TA indicates the position of the TATA-box. The coding region for E1A protein is shown with a box. Numbers indicate nucleotide positions relative to the left end of viral DNA. (Bottom) Locations of footprints (brackets with letters a, b and NF-I), sequences of the probes used for gel shift assay (boxes) and presumed binding site for NF-III [28] (waved line) are shown along with nucleotide sequences of a region between positions 1 and 120.
Sepharose 4B (Pharmacia) and the coupling reaction was carried out according to the method of Wu et al. [9]. A trace a m o u n t of 32p-labeled oligo A was added to the initial material and the extent of coupling was monitored throughout the course of the reaction. The final coupling efficiency was 24%. The affinity resin was equilibrated with buffer I consisting of 20 m M Hepes (pH 7.9), 0.2 m M E D T A , 0.1 M KCI, 0.5 m M dithiothreitol and 20% glycerol. 0.5 ml (12 mg protein) of nuclear extracts of Ehrlich ascites tumor cells were loaded onto the column and the flow-through fraction was collected. Bound materials were eluted stepwise from the column with buffer I containing 1 M KC1 and the eluent was dialyzed against buffer I. Protein concentration was determined by the method of Bradford [10].
In uitro transcription, DNase I footprinting, gel shift assay and filter binding assay In vitro transcription of the A d 12 E1A gene was performed with intact plasmids pE1A21 (wild-type D N A ) and dll having no region between positions 1 and 166 as templates [8,11]. R N A was extracted, divided into two and transcripts from proximal and distal start-sites were separately analyzed by primer extension with the P- and D-primers, respectively, as previously described [8]. D N a s e I footprinting was conducted with the upper probe as described [8]. Oligo A and oligo B, the latter containing a sequence corresponding to a region between positions 71 and 100 of the A d 12 genome and an additional sequence of 5 ' - C G A C G A C G A G G G - Y at the 5'-side of the lower strand (see Fig. 1), were labeled with [c~-32p]dCTP by Klenow enzyme and used as probes in gel shift assay. A b o u t 1
ng of each of these probes and protein fractions were incubated at 3 0 ° C for 10 min in 10 /~l of reaction mixture containing 12 m M Hepes ( p H 7.9), 0.12 m M E D T A , 60 m M KCI, 20 m M NaC1, 0.3 m M dithiothreitol, 5 m M MgC12, 12% glycerol and 0.25 m g / m l sonicated salmon testis D N A . The samples were subsequently loaded on a 6% polyacrylamide gel and electrophoresed in a buffer containing 50 m M Tris-borate ( p H 8.3) and 1 m M E D T A at 15 v o l t / c m at r o o m temperature. Filter binding assay was performed according to the described procedure [12], except that 1 /~g of p o l y ( d I - d C ) , poly(dI-dC) was included in reaction. The probe was a 73 bp fragment of the left end of A d 5 D N A that contained binding sites for both N F - I and N F - I I I [13]. Each binding reaction was done with 0.05 ~tg of H e L a N F - I , 2 fmol of the 32p-labeled probe and various amounts of competitors as indicated in Fig. 4. Chemically synthesized oligo I and oligo O C T A were used as competitors that contained the binding sites for N F - ! [13] and N F - I I I [14], respectively. The former had a sequence of 5 ' - C C T T A T T T T G G A T T G A A G C C A A TATGATA-3' and the latter 5 ' - A G A A T C G C T TATGCAAATAAGGTGAAGAG-3'.
Cell culture, RNA analysis and nuclear extract preparation 321 and 293 cells were cultured in D u l b e c c o - m o d ified m i n i m u m essential m e d i u m with 10% fetal bovine serum. H e L a $3 cells were cultured in m i n i m u m essential medium No. 4 (Nissui) with 10% fetal bovine serum. R N A of Ad 12-infected KB cells was prepared as described [15], and 321 cell R N A was prepared by the method of Chomczynski and Sacchi [16]. Poly(A)-containing R N A was enriched by oligo(dT)-cellulose chro-
87
matography, separated in an 1.5% agarose gel containing formaldehyde and blotted onto nitrocellulose paper. Hybridization analysis was performed with a 32P-labeled plasmid containing the entire Ad 12 E1A gene or a human fl-actin pseudogene as the probe under standard conditions [17]. Primer extension analysis was conducted with the P-primer, and 65-base and 204-base cDNAs were synthesized from proximal and distal transcripts of the E1A gene, respectively. For preparation of nuclear extracts, about 10 9 of 321, 293 and HeLa cells were harvested at sub-confluent growth and extracts were prepared by the same procedure as that for Ehrlich ascites tumor cell extracts [11,18]. Usually, about 2 ml extracts (10-15 m g / m l protein) were obtained from 10 9 cells.
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Analysis of in vitro transcription in the extracts depleted of factors that bind to the cis-acting region Nuclear extracts of Ehrlich ascites tumor cells were fractionated by oligo A-specific DNA-affinity chromatography so that the extracts would lose factors that bind to the c/s-acting a-region. The extracts were made to pass through DNA-affinity column and the flowthrough fraction was collected. Elimination of binding activity to the a-region was first analyzed by gel shift assay using synthetic oligonucleotides, oligo A and oligo B as probes that contain sequences corresponding to the a-region and the b-region, respectively (see Fig. 1). The unfractionated nuclear extracts produced specific shifts with both probes, while the flow-through fraction did so only with the oligo B probe, indicating loss of factors that bind to the a-region (Fig. 2A). DNA-binding activity of the flow-through fraction was next analyzed by DNase I footprinting (Fig. 2B). A footprint on the a-region produced by the flow-through fraction was found much weaker than that produced by the nuclear extracts, while protection of the b-region by the two protein fractions was almost the same level (lanes 2 and 3). These results were consistent with those obtained by gel shift assay. However, a region between nucleotide positions 43 and 55, the upper portion of the a-region which is thought to be bound by NF-III-like protein (see Fig. 1), appeared to be still protected by the flowthrough fraction (lane 3). Proteins bound to the column were eluted and characterized by footprint analysis. The eluted materials gave a footprint specifically on the a-region hut the protected area was less than that produced by nuclear extracts (compare lanes 2 and 4 in Fig. 2B). Moreover, the footprint produced by the eluted materials was exactly the same as the one produced by NF-I purified from HeLa cells (lane 5), indicating the eluted materials to contain only NF-I-like protein. These results indicate that the flow-through fraction selec-
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Fig. 2. DNA-binding activity in Ehrlich asciies tumor cell extracts and fractions of DNA-affinity chromatography. (A) 24 /tg of the unfractionated nuclear extracts (NE) and 22 ~g of the flow-through fraction of DNA-affinity chromatography (FT) were analyzed by gel shift assay with the oligo A and oligo B probes. Arrowheads indicate positions of the specific shift bands and free probes. TOP shows the origin of the gel. (B) 6.3 ttg of NE, 8.2 /tg of FT, 2.0 #g of the materials eluted from DNA-affinity column (BOUND) and 0.15 ~tg of HeLa NF-I (denatured DNA-cellulose fraction) [19] were analyzed by DNase I footprinting. Brackets with letters a, b and NF-I indicate the regions of footprints. Numbers show nucleotide positions of the boundary of footprints. Lanes M, and 1 and 6 are HpalI-cleaved pBR322 as markers and no protein control, respectively.
tively lost NF-I-like protein, with NF-III-like protein and factor(s) binding to the b-region remaining intact. In vitro transcription was then analyzed in the flowthrough fraction using wild-type and dll DNAs as templates (Fig. 3). As previously observed [8], transcription from distal start-site of the Ad 12 E1A gene was seen to be selectively stimulated in nuclear extracts depending on a region between positions 1 and 166 (lanes 5 and 6). However, the amounts of distal transcript synthesized from the two D N A templates in the flow-through fraction were almost the same (lanes 7 and 8), and were as small as that synthesized from dll in nuclear extracts (lane 6). On the other hand, the flowthrough fraction contained as much activity as the unfractionated extracts for synthesizing the proximal transcript (lanes 1-4). It thus follows that the flowthrough fraction has been deprived of activity to stimulate distal transcription of the Ad 12 E1A gene from DNA possessing the cis-acting region. NF-I-like protein
88
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II Fig. 3. In vitro transcription of the Ad 12 ElA gene in Ehrlich ascites tumor cell extracts and the flow-through fraction of DNA-affinity chromatography. Wild-type pElA21 (W) and dll (D) DNAs were used as templates for in vitro transcription in nuclear extracts (NE) or the flow-through fraction (F’T). Positions of extension products derived from proximal and distal transcripts are indicated by arrowheads with letters P and D, respectively. Lane M contained HpaII-cleaved pBR322 as size markers, and size is shown in bases. Shown below the panel is a schematic representation of the structure of two DNA templates. Positions of the a-region and the b-region, and distal (D) and proximal (P) transcription start-sites are indicated by boxes and arrows, respectively.
in nuclear extracts of Ehrlich ascites tumor cells was thus concluded responsible, at least in part, for stimulating this transcription. Addition of HeLa NF-I to the extracts depleted of NF-Ilike protein Examination was subsequently made of reconstitution of the stimulatory activity in the extracts depleted of NF-I-like protein. To determine the direct effect of NF-I, it was purified from HeLa cells as described previously [19]. Purified HeLa NF-I was first examined whether it would have contamination of NF-III activity by filter binding assay with the probe containing the binding sites for both NF-I and NF-III. As shown in Fig. 4, binding activity present in the NF-I preparation completely disappeared by addition of oligo I that contains the NF-I site, whereas no competition was observed in the presence of an excess amount of oligo OCTA containing the NF-III site. Binding activity in nuclear extracts of mouse kidney, which had been shown to contain NF-III-like protein referred to as NF-K in addition to NF-I [13], was similarly examined. Binding activity present in an amount of the kidney extracts
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Fig. 4. DNA-binding activity in HeLa NF-I. Filter binding assay was performed with the HeLa NF-I preparation using the probe containing both the NF-I and NF-III sites. Indicated amounts of oligo I (closed circle) and oligo OCTA (open circle) were added to the binding reaction as competitors of NF-I and NF-III, respectively. Amounts of DNA retained on filter in binding reactions with various amounts of competitors were shown relative to that in the reaction with no competitor.
containing the same level of NF-I activity as the HeLa NF-I preparation used in the experiment shown above was completely competed only when oligo I and oligo OCTA were simultaneously added (data not shown). These results clearly indicate that the HeLa NF-I preparation is free of NF-III activity.
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Fig. 5. Effect of HeLa NF-I on in vitro transcription of the Ad 12 ElA gene in the extracts depleted of NF-I-like protein. 0.3 pg of HeLa NF-I were added to in vitro transcription in the flow-through fraction of DNA-affinity chromatography. Symbols are as in Fig. 3. Exposure to X-ray film for the right panel was 4-times longer than that for the left panel.
89 HeLa NF-I was then added to the transcription reaction performed in the flow-through fraction of DNA-affinity chromatography. From the results in Fig. 5, the flow-through fraction supplemented with HeLa NF-I regained activity to stimulate distal transcription of the Ad 12 E1A gene from wild-type DNA (lane 7) while the addition of NF-I had neither this effect nor any on distal transcription from dll DNA (lane 8) or proximal one from either D N A template (lanes 3 and 4). HeLa NF-I thus became a substitute for NF-I-like protein present in Ehrlich ascites tumor cell extracts and NF-I appears quite likely to be a trans-acting factor that stimulates transcription of the Ad 12 EIA gene. Involvement of NF-III-like protein in this transcription was not clear from the present study.
Analysis of in oitro transcription in nuclear extracts of 321 cells expressing the Ad 12 EIA gene Transcription of the E1A gene may possibly be autoregulated by its own E1A protein (Refs. 20 and 21 and Y. Sawada, unpublished data). For examination of this point, distal transcription of the E1A gene in vitro was compared for extracts of Ad 12 E1A-expressing 321 cells and Ehrlich ascites tumor cells. The cell line 321
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D~ Fig. 7. In vitro transcription of the Ad 12 E I A gene in 321 cell extracts. In vitro transcription was conducted with pE1A21 (w) and dll (d) D N A s as templates in nuclear extracts of Ehrlich ascites tumor cells (EAT) and 321 cells (321). Lanes 7 and 10 contained products from control reaction with no D N A template. Other symbols are as in Fig. 3.
has been established (Y. Sawada and T. Shenk, unpublished data) by introducing the E1 region of Ad 12 into 293 cell [22]. Expression of the Ad 12 E1A gene in 321 cells was first analyzed by blot hybridization and primer extension. Transcripts of this gene in 321 cells were found to be only about one-fourth the number in virusinfected KB cells and both distal and proximal start-sites for transcription were used (Fig. 6). Nuclear extracts were then prepared from 321 cells and in vitro transcription was conducted with wild-type and dll DNAs as templates. In Fig. 7, the amount of distal transcript synthesized from either template was almost the same (lanes 8 and 9) and as small as that synthesized from dll D N A in Ehrlich ascites tumor cell extracts (lane 4). 321 cell extracts were thus shown to possess no activity to stimulate distal transcription of the Ad 12 E1A gene from D N A containing the cis-acting region. Transcription of the E1A gene in nuclear extracts of HeLa cells was next examined to determine whether differences in transcriptional activity of Ehrlich ascites tumor cell and 321 cell extracts merely reflect species-specificity of mouse and human. In HeLa cell
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Fig. 6. Analysis of 321 cell RNA. (A) R N A s of 321 cells (lanes 1 and 2) and Ad 12-infected KB cells (lanes 3 and 4) were analyzed in blot hybridization with a probe of the Ad 12 E I A gene or the h u m a n fl-actin pseudogene as a control. KB cell R N A was extracted either 8 or 26 h after virus infection (hpi). Arrowheads indicate positions of ribosomal RNAs. (B) R N A s of Ad 12-infected KB cells (lanes 1 and 2) and 321 cells (lanes 3 and 4) were analyzed in primer extension with the P-primer. Arrowheads with letters D and P indicate positions of extension products derived from distal and proximal transcripts of the Ad 12 E I A gene, respectively. Lane M contained HpaII-cleaved pBR322 and size is in bases.
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Fig. 8. In vitro transcription of the Ad 12 E I A gene in HeLa cell extracts. In vitro transcription was conducted with two D N A templates (W, D) in HeLa cell extracts. Symbols are as in Fig. 3.
90 extracts the a m o u n t of distal t r a n s c r i p t synthesized from w i l d - t y p e D N A was m o r e than that from d l l (lanes 3 a n d 4 in Fig. 8). 321 cell extracts m a y thus be i n c a p a b l e of s t i m u l a t i n g this t r a n s c r i p t i o n owing to the expression o f early viral genes, most likely of the A d 12 E 1 A gene.
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Analysis of DNA-binding activity in 321 and 293 cell extracts T h e a b o v e results are consistent with b i n d i n g activity to the cis-acting a-region in the two extracts. In Fig. 9A, b i n d i n g activity to the a-region in 321 cell extracts a p p e a r e d m u c h less than that in H e L a cell extracts while b o t h extracts showed essentially the same level of f o o t p r i n t s on the b-region. It can be also n o t e d that p r o t e c t i o n of the u p p e r p o r t i o n of the a-region, which is p r e s u m a b l y b o u n d b y N F - I I I , was almost c o m p a r a b l e b e t w e e n the two extracts. Differences in b i n d i n g activity to the a-region of these extracts b e c a m e m o r e evid e n t in gel shift assay. A s seen in Fig. 9B, H e L a cell extracts p r o d u c e d two shift b a n d s (a a n d b) a n d 321 cell
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Fig. 9. DNA-binding activity in HeLa cell and 321 cell extracts. (A) DNase I footprinting was performed with 14/tg of HeLa cell extracts (HELA) or 10 #g of 321 cell extracts (321). Other simbols are as in Fig. 2B. (B) Gel shift assay was done with 14/Lg of HeLa or 321 cell extracts using oligo A and oligo B as probes. 200-fold molar excess of non-labeled oligo A (lanes 2 and 5) and oligo B (lanes 3 and 6) were present as competitors in the binding reaction. Arrowheads with letters a, b, c and d indicate the positions of the shift bands.
Fig. 10. DNA-binding activity in HeLa, 321 and 293 cell extracts. 10-11 /~g of two different preparations of the three extracts were analyzed for binding activity to the a-region (lanes 1-6) and the b-region (lanes 7-12) by gel shift assay. Positions of the specific and non-specific shift bands are shown with arrowheads.
extracts d i d o n l y b a n d b with the oligo A p r o b e , while b o t h showed the same shifts ( b a n d s c a n d d) with the oligo B probe. Bands a a n d c specifically d i s a p p e a r e d in the presence of an excess a m o u n t of n o n - l a b e l e d oligo A a n d oligo B, respectively (lanes 2 a n d 3). However, there was little c o m p e t i t i o n for b a n d s b a n d d by a d d i t i o n of either oligonucleotide. B a n d s a a n d c were thus shown to be specific p r o t e i n - D N A complexes. Thus, quite likely, 321 cell extracts possess no activity for b i n d i n g to the N F - I site while b i n d i n g activity to the N F - I I I site a n d the b - r e g i o n is c o m p a r a b l e to that in H e L a cell extracts. W e next e x a m i n e d b i n d i n g activity to the N F - I site p r e s e n t in n u c l e a r extracts of 293 cells in which o n l y the A d 5 E 1 A gene is expressed [22]. T w o different p r e p a r a tions of H e L a , 321 a n d 293 cell extracts were c o m p a r e d by gel shift assay (Fig. 10). These three extracts showed an obvious difference in b i n d i n g activity to the N F - I site (lanes 1-6), while the level of b i n d i n g activity to the b-region was a l m o s t c o m p a r a b l e a m o n g t h e m (lanes 7 - 1 2 ) . 293 cell extracts were f o u n d to c o n t a i n d e t e c t a ble b u t m u c h less b i n d i n g activity to the N F - I site than H e L a cell extracts, whereas 321 cell extracts s h o w e d no N F - I activity as o b s e r v e d in Fig. 9B. These results indicate that the level of N F - I activity was significantly low in 293 cell extracts c o m p a r i n g to that in H e L a cell extracts a n d was c o m p l e t e l y a b s e n t f r o m 321 cell extracts. This suggests that a d e n o v i r u s E 1 A p r o t e i n w o u l d cause decrease in N F - I activity a n d s i m u l t a n e o u s expression of the A d 5 a n d A d 12 E1A genes w o u l d be responsible for its c o m p l e t e loss in 321 cells. W e hence p r o p o s e that A d 12 E 1 A p r o t e i n a u t o r e g u l a t e s the transcription of its own gene b y m o d u l a t i n g the activity of NF-I.
91
Discussion Transcription regulation of the adenovirus EIA gene has been studied with non-oncogenic types 2 and 5 adenoviruses and several cis-acting elements and the factors that bind to them have been reported [2,3]. In the present study, the control mechanism of transcription of the E1A gene of type 12 adenovirus belonging to a group of highly oncogenic adenoviruses was investigated. The results of various experiments indicate NF-I or NF-I-like protein to stimulate transcription of the Ad 12 E1A gene from distal start-site by binding to a region located near the left end of the viral genome. NF-I is not apparently involved in the regulation of transcription of either the Ad 2 or Ad 5 E1A gene, and thus the control mechanism of E1A gene transcription may possibly differ for Ad 12 and non-oncogenic adenoviruses. Involvement of NF-III-like protein in regulation of the Ad 12 E1A gene transcription has yet to be examined. Binding site of NF-I-like protein was situated quite close to the control region of the viral genome replication [12]. NF-I is thus likely to bind to the same sequence element on the Ad 12 DNA for regulating both transcription of the E1A gene and replication of the viral genome. Binding of NF-I to the same region was recently shown to inhibit promoter activity which allows transcription with an orientation opposite to that of the E1A gene [23,24]. It would thus be quite pertinent to clarify the mechanism by which NF-I becomes functional in one of two different reactions. NF-I obviously stimulates the E1A gene transcription prior to the expression of early viral genes whose products are a requisite to the viral genome replication. With the transcription of early genes as preparation for the viral genome replication, the situation becomes more complex. Further research is required to gain better understanding of the mechanism of NF-I action in transcription and replication of the adenovirus DNA. Factor(s) bound to the b-region has been suggested to interact with those bound to the a-region resulting in mutual inhibition of binding to the target sequences [25]. The trans-acting function of NF-I may thus be controlled by factor(s) that binds to the b-region located adjacent to the NF-I binding site. Activity for both stimulating distal transcription of the Ad 12 E1A gene and binding to the NF-I site has been shown not to be present in nuclear extracts of 321 cells that express both the Ad 5 and Ad 12 E1A genes. Inactivation of NF-I or synthesis of a very small amount of NF-I in 321 cells may account for this. It was suggested that adenovirus E1A protein would cause a decrease in NF-I activity. E1A protein is thought to control transcription of various genes by modulating the apparent activity of cellular transcription factors already existing [1,2]. Involvement of protein phosphor-
ylation was recently suggested in E1A-dependent transcription activation [26,27]. Thus, E1A protein may possibly modify the NF-I molecule to inactivate its binding activity to the cis-acting region. However, more direct evidence supporting E1A-dependent inactivation of NF-I should be obtained since not only the E1A gene but other viral genes are expressed in 321 and 293 cells. It also remains to be clarified whether this proposed mechanism would be involved in E1A-dependent viral transformation and tumorigenesis.
Acknowledgements We thank O. Nikaido and F. Suzuki for their support in the cell culture, C. Wu and H. Ueda for a protocol of oligoDNA-affinity chromatography and their valuable suggestions and T. Kakunaga for providing the actin DNA. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and by grants from The Uehara Memorial Foundation and The Research Foundation for Pharmaceutical Sciences.
References 1 Grand, R.J.A. (1987) Biochem. J. 241, 25-38. 2 Berk, A.J. (1986) Annu. Rev. Genet. 20, 45-79. 3 Jones, N.C., Rigby, P.W.J. and Ziff, E.B. (1988) Genes Dev. 2, 267-281. 4 Huebner, R.J., Rowe, W.P. and Lane, W.T. (1962) Proc. Natl. Acad. Sci. USA 48, 2051-2058. 5 Trentin, J.J., Yabe, Y. and Taylor, G. (1962) Science 137, 835-841. 6 Lewis, A.M., Jr. and Cook, J.L. (1984) Curr. Top. Microbiol. Immunol. 110, 1-22. 7 Sawada, Y., Raska, K., Jr. and Shenk, T. (1988) Virology 166, 281-284. 8 Shibata, H., Zheng, J.-H., Koikeda, S., Masamune, Y. and Nakanishi, Y. (1989) Biochim. Biophys. Acta 1007, 184-191. 9 Wu, C., Tsai, C. and Wilson, S. (1988) in Genetic Engineering (Setlow, J.K. and Hollaender, A., eds.), Vol. 10, Plenum Press, New York, London, in press. 10 Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 11 Nakanishi, Y., Shibata, H., Hase, T. and Masamune, Y. (1987) Biochem. Biophys. Res. Commun. 146, 783-790. 12 Nagata, K., Guggenheimer, R.A. and Hurwitz, J. (1983) Proc. Natl. Acad. Sci. USA 80, 6177-6181. 13 Matsumoto, K., Nagata, K., Hanaoka, F. and Ui, M. (1989) J. Biochem. 105, 927-932. 14 Fletcher, C., Heintz, N. and Roeder, R.G. (1987) Cell 51 773-781. 15 Sawada, Y. and Fujinaga, K. (1980) J. Virol. 36, 639-651. 16 Chomczynski, P. and Sacchi, N. (1987) Anal. Biochem. 162, 156-159. 17 Anderson, M.L.M. and Young, B.D. (1985) in Nucleic Acid Hybridisation, a Practical Approach (Hames, B.D. and Higgins, S.J., eds), pp. 73-111, IRL Press, Oxford, Washington, DC. 18 Dignam, J.D., Lebovitz, R.M. and Roeder, R.G. (1983) Nucleic Acids Res. 11, 1475-1489. 19 Nagata, K., Guggenheimer, R.A., Enomoto, T., Lichy, J.H. a n d Hurwitz, J. (1982) Proc. Natl. Acad. Sci. USA 79, 6438-6442. 20 Hearing, P. and Shenk, T. (1985) Mol. Cell. Biol. 5, 3214-3221. 21 Smith, D.H., Kegler, D.M. and Ziff, E.B. (1985) Mol. Cell. Biol. 5, 2684-2696.
92 22 Graham, F.L., Smiley, J., Russell, W.C. and Martin, R. (1977) J. Gen. Virol. 36, 69-72. 23 Ooyama, S., Imai, T., Hanaka, S. and Handa, H. (1989) EMBO J. 8, 863-868. 24 Matsumoto, K., Nagata, K., Yamanaka, K., Hanaoka, F. and Ui, M. (1989) Biochem. Biophys. Res. Commun., in press. 25 Nakanishi, Y., Koikeda, S., Shibata, H. and Masamune, Y. (1989) Biochem. Biophys. Res. Commun. 158, 685-689.
26 Bagchi, S., Raychaudhuri, P. and Nevins, J.R. (1989) Proc. Natl. Acad. Sci. USA 86, 4352-4356. 27 Raychaudhuri, P., Bagchi, S. and Nevins, J.R. (1989) Genes Dev. 3, 620-627. 28 O'Neill, E.A., Fletcher, C., Burrow, C.R., Heintz, N., Roeder, R.G. and Kelly, T.J. (1988) Science 241, 1210-1213.
85
Elsevier BBAEXP 92026
Nuclear factor I stimulates transcription of the adenovirus 12 EIA gene in a cell-free system Satoshi Koikeda 1, Rie Ibuki 1, Yukiharu Sawada 2, Kyosuke Nagata 3, Hitomi Shibata 1; Yukito Masamune 1 and Yoshinobu Nakanishi I Faculty ofPharmaceuticalSciences, Kanazawa University, Ishikawa, 2 Department of Molecular Biology, Cancer Research Institute, Sapporo Medical College, Sapporo, and 3 Department of Molecular Genetics, National Institute of Genetics, Yata, Mishima, Shizuoka (Japan)
(Received 28 July 1989)
Key words: Adenovirus EIA gene; Cell free transcription; Nuclear factor I; Transcriptional control Binding to the cis-acting region of NF-l-like protein a n d / o r NF-Ill-like protein was previously suggested to be responsible for the preferential stimulation of transcription from distal start-site of the adenovirus 12 EIA gene in a cell-free system. In this study, nuclear extracts of Ehrlich ascites tumor cells depleted of NF-I-like protein were found to lose activity to stimulate the E1A gene transcription. This activity was recovered when NF-I purified from HeLa cells with no contamination of NF-III was supplemented. It is thus evident that NF-I is involved in stimulating distal transcription of the adenovirus 12 E1A gene. Moreover, activities for both stimulating the E1A gene transcription and binding to a region recognized by NF-I did not apparently exist in nuclear extracts of a cell line expressing the adenovirus 12 E1A gene. These results suggest that transcription of the adenovirus 12 EIA gene may possibly be autoregulated at least in part through modulation of the activity of NF-I.
Introduction Adenovirus E1A protein is essential for transforming virus-infected cells and inducing tumors in certain animals [1]. Cis- and trans-acting factors for transcription of the E I A gene have been studied with non-oncogenic Ad 2 and Ad 5 [2,3]. However, only little is known of the mechanism which regulates transcription of the Ad 12 E1A gene. Ad 12 belongs to a group of highly oncogenic adenoviruses and its E1A protein is required for the induction of tumors in new-born rodents [4-7]. It is thus important to elucidate the mechanism by which E I A gene transcription is controlled for greater clarification not only of cell transformation but the manner by which tumors are induced by Ad 12. The Ad 12 E I A gene has two start-sites for transcription located at positions 306 (distal site) and 445 (proximal site) with respect to the left end of the viral genome as position 1 (see Fig. 1). We previously re-
ported that factors present in nuclear extracts of Ehrlich ascites tumor cells bind to two distinct sequence elements in the 5'-upstream region of the Ad 12 E1A gene. Two elements are situated between positions 19 and 55, designated the a-region, and between positions 77 and 94, designated the b-region. Binding to the a-region of NF-I-like protein a n d / o r NF-III-like protein present in the extracts was suggested to be essential for the preferential stimulation of transcription from distal start-site of the E I A gene in a cell-free system [8]. The focal point of attention in the present study is which of these two proteins is actually responsible for stimulating the transcription. Evidence was obtained demonstrating the involvement of NF-I-like protein in the E1A gene transcription control and that this is possibly related to the autoregulation of the Ad 12 E1A gene.
Materials and Methods DNA-affinity fractionation
Abbreviations: Ad, adenovirus; NF-I, nuclear factor I; NF-III, nuclear factor III, Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonicacid. Correspondence: Y. Nakanishi, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa, 920 Japan.
column preparation
and
nuclear
extract
Oligo A has a sequence corresponding to a region between positions 13 and 48 of the Ad 12 genome and its lower strand contains an additional sequence of 5 ' - A C G A C G A G G G - 3 ' at the 5'-side (see Fig. 1). 5 0 / t g of oligo A were mixed with 0.6 g of CNBr-activated
0167-4781/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
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1
190
200
300
ab
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590
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NF'I
w
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10
!
20
30
40
50
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~-TAT AT AT A+ A/~T AT AC CT'~'AT ACTGGACTAGTGCX; A A T A T T AAAAT(~AAGTGGGCGT AG
b 70 80 90 100 110 120 T GTGTAATT~4LG,ATTGGGTG~;'AGGTGTGGC~TTGGCGTGC'q~TGTAAGTTTGGGCGGATGA~ Fig. 1. Structure of the 5'-end region of the Ad 12 E1A gene. (Top) Locations of the a-region and the b-region (boxes with letters a and b) and two transcription start-sites (arrows with letters D and P) are shown. TA indicates the position of the TATA-box. The coding region for E1A protein is shown with a box. Numbers indicate nucleotide positions relative to the left end of viral DNA. (Bottom) Locations of footprints (brackets with letters a, b and NF-I), sequences of the probes used for gel shift assay (boxes) and presumed binding site for NF-III [28] (waved line) are shown along with nucleotide sequences of a region between positions 1 and 120.
Sepharose 4B (Pharmacia) and the coupling reaction was carried out according to the method of Wu et al. [9]. A trace a m o u n t of 32p-labeled oligo A was added to the initial material and the extent of coupling was monitored throughout the course of the reaction. The final coupling efficiency was 24%. The affinity resin was equilibrated with buffer I consisting of 20 m M Hepes (pH 7.9), 0.2 m M E D T A , 0.1 M KCI, 0.5 m M dithiothreitol and 20% glycerol. 0.5 ml (12 mg protein) of nuclear extracts of Ehrlich ascites tumor cells were loaded onto the column and the flow-through fraction was collected. Bound materials were eluted stepwise from the column with buffer I containing 1 M KC1 and the eluent was dialyzed against buffer I. Protein concentration was determined by the method of Bradford [10].
In uitro transcription, DNase I footprinting, gel shift assay and filter binding assay In vitro transcription of the A d 12 E1A gene was performed with intact plasmids pE1A21 (wild-type D N A ) and dll having no region between positions 1 and 166 as templates [8,11]. R N A was extracted, divided into two and transcripts from proximal and distal start-sites were separately analyzed by primer extension with the P- and D-primers, respectively, as previously described [8]. D N a s e I footprinting was conducted with the upper probe as described [8]. Oligo A and oligo B, the latter containing a sequence corresponding to a region between positions 71 and 100 of the A d 12 genome and an additional sequence of 5 ' - C G A C G A C G A G G G - Y at the 5'-side of the lower strand (see Fig. 1), were labeled with [c~-32p]dCTP by Klenow enzyme and used as probes in gel shift assay. A b o u t 1
ng of each of these probes and protein fractions were incubated at 3 0 ° C for 10 min in 10 /~l of reaction mixture containing 12 m M Hepes ( p H 7.9), 0.12 m M E D T A , 60 m M KCI, 20 m M NaC1, 0.3 m M dithiothreitol, 5 m M MgC12, 12% glycerol and 0.25 m g / m l sonicated salmon testis D N A . The samples were subsequently loaded on a 6% polyacrylamide gel and electrophoresed in a buffer containing 50 m M Tris-borate ( p H 8.3) and 1 m M E D T A at 15 v o l t / c m at r o o m temperature. Filter binding assay was performed according to the described procedure [12], except that 1 /~g of p o l y ( d I - d C ) , poly(dI-dC) was included in reaction. The probe was a 73 bp fragment of the left end of A d 5 D N A that contained binding sites for both N F - I and N F - I I I [13]. Each binding reaction was done with 0.05 ~tg of H e L a N F - I , 2 fmol of the 32p-labeled probe and various amounts of competitors as indicated in Fig. 4. Chemically synthesized oligo I and oligo O C T A were used as competitors that contained the binding sites for N F - ! [13] and N F - I I I [14], respectively. The former had a sequence of 5 ' - C C T T A T T T T G G A T T G A A G C C A A TATGATA-3' and the latter 5 ' - A G A A T C G C T TATGCAAATAAGGTGAAGAG-3'.
Cell culture, RNA analysis and nuclear extract preparation 321 and 293 cells were cultured in D u l b e c c o - m o d ified m i n i m u m essential m e d i u m with 10% fetal bovine serum. H e L a $3 cells were cultured in m i n i m u m essential medium No. 4 (Nissui) with 10% fetal bovine serum. R N A of Ad 12-infected KB cells was prepared as described [15], and 321 cell R N A was prepared by the method of Chomczynski and Sacchi [16]. Poly(A)-containing R N A was enriched by oligo(dT)-cellulose chro-
87
matography, separated in an 1.5% agarose gel containing formaldehyde and blotted onto nitrocellulose paper. Hybridization analysis was performed with a 32P-labeled plasmid containing the entire Ad 12 E1A gene or a human fl-actin pseudogene as the probe under standard conditions [17]. Primer extension analysis was conducted with the P-primer, and 65-base and 204-base cDNAs were synthesized from proximal and distal transcripts of the E1A gene, respectively. For preparation of nuclear extracts, about 10 9 of 321, 293 and HeLa cells were harvested at sub-confluent growth and extracts were prepared by the same procedure as that for Ehrlich ascites tumor cell extracts [11,18]. Usually, about 2 ml extracts (10-15 m g / m l protein) were obtained from 10 9 cells.
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22
Analysis of in vitro transcription in the extracts depleted of factors that bind to the cis-acting region Nuclear extracts of Ehrlich ascites tumor cells were fractionated by oligo A-specific DNA-affinity chromatography so that the extracts would lose factors that bind to the c/s-acting a-region. The extracts were made to pass through DNA-affinity column and the flowthrough fraction was collected. Elimination of binding activity to the a-region was first analyzed by gel shift assay using synthetic oligonucleotides, oligo A and oligo B as probes that contain sequences corresponding to the a-region and the b-region, respectively (see Fig. 1). The unfractionated nuclear extracts produced specific shifts with both probes, while the flow-through fraction did so only with the oligo B probe, indicating loss of factors that bind to the a-region (Fig. 2A). DNA-binding activity of the flow-through fraction was next analyzed by DNase I footprinting (Fig. 2B). A footprint on the a-region produced by the flow-through fraction was found much weaker than that produced by the nuclear extracts, while protection of the b-region by the two protein fractions was almost the same level (lanes 2 and 3). These results were consistent with those obtained by gel shift assay. However, a region between nucleotide positions 43 and 55, the upper portion of the a-region which is thought to be bound by NF-III-like protein (see Fig. 1), appeared to be still protected by the flowthrough fraction (lane 3). Proteins bound to the column were eluted and characterized by footprint analysis. The eluted materials gave a footprint specifically on the a-region hut the protected area was less than that produced by nuclear extracts (compare lanes 2 and 4 in Fig. 2B). Moreover, the footprint produced by the eluted materials was exactly the same as the one produced by NF-I purified from HeLa cells (lane 5), indicating the eluted materials to contain only NF-I-like protein. These results indicate that the flow-through fraction selec-
~!~ i~ '~ ~i'~i'~!
Fig. 2. DNA-binding activity in Ehrlich asciies tumor cell extracts and fractions of DNA-affinity chromatography. (A) 24 /tg of the unfractionated nuclear extracts (NE) and 22 ~g of the flow-through fraction of DNA-affinity chromatography (FT) were analyzed by gel shift assay with the oligo A and oligo B probes. Arrowheads indicate positions of the specific shift bands and free probes. TOP shows the origin of the gel. (B) 6.3 ttg of NE, 8.2 /tg of FT, 2.0 #g of the materials eluted from DNA-affinity column (BOUND) and 0.15 ~tg of HeLa NF-I (denatured DNA-cellulose fraction) [19] were analyzed by DNase I footprinting. Brackets with letters a, b and NF-I indicate the regions of footprints. Numbers show nucleotide positions of the boundary of footprints. Lanes M, and 1 and 6 are HpalI-cleaved pBR322 as markers and no protein control, respectively.
tively lost NF-I-like protein, with NF-III-like protein and factor(s) binding to the b-region remaining intact. In vitro transcription was then analyzed in the flowthrough fraction using wild-type and dll DNAs as templates (Fig. 3). As previously observed [8], transcription from distal start-site of the Ad 12 E1A gene was seen to be selectively stimulated in nuclear extracts depending on a region between positions 1 and 166 (lanes 5 and 6). However, the amounts of distal transcript synthesized from the two D N A templates in the flow-through fraction were almost the same (lanes 7 and 8), and were as small as that synthesized from dll in nuclear extracts (lane 6). On the other hand, the flowthrough fraction contained as much activity as the unfractionated extracts for synthesizing the proximal transcript (lanes 1-4). It thus follows that the flowthrough fraction has been deprived of activity to stimulate distal transcription of the Ad 12 E1A gene from DNA possessing the cis-acting region. NF-I-like protein
88
+4-I W
template;
0
W
0
0
0
0.03
0.1
0.3
Compstitor
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II
II Fig. 3. In vitro transcription of the Ad 12 ElA gene in Ehrlich ascites tumor cell extracts and the flow-through fraction of DNA-affinity chromatography. Wild-type pElA21 (W) and dll (D) DNAs were used as templates for in vitro transcription in nuclear extracts (NE) or the flow-through fraction (F’T). Positions of extension products derived from proximal and distal transcripts are indicated by arrowheads with letters P and D, respectively. Lane M contained HpaII-cleaved pBR322 as size markers, and size is shown in bases. Shown below the panel is a schematic representation of the structure of two DNA templates. Positions of the a-region and the b-region, and distal (D) and proximal (P) transcription start-sites are indicated by boxes and arrows, respectively.
in nuclear extracts of Ehrlich ascites tumor cells was thus concluded responsible, at least in part, for stimulating this transcription. Addition of HeLa NF-I to the extracts depleted of NF-Ilike protein Examination was subsequently made of reconstitution of the stimulatory activity in the extracts depleted of NF-I-like protein. To determine the direct effect of NF-I, it was purified from HeLa cells as described previously [19]. Purified HeLa NF-I was first examined whether it would have contamination of NF-III activity by filter binding assay with the probe containing the binding sites for both NF-I and NF-III. As shown in Fig. 4, binding activity present in the NF-I preparation completely disappeared by addition of oligo I that contains the NF-I site, whereas no competition was observed in the presence of an excess amount of oligo OCTA containing the NF-III site. Binding activity in nuclear extracts of mouse kidney, which had been shown to contain NF-III-like protein referred to as NF-K in addition to NF-I [13], was similarly examined. Binding activity present in an amount of the kidney extracts
1
3
lpmoll
Fig. 4. DNA-binding activity in HeLa NF-I. Filter binding assay was performed with the HeLa NF-I preparation using the probe containing both the NF-I and NF-III sites. Indicated amounts of oligo I (closed circle) and oligo OCTA (open circle) were added to the binding reaction as competitors of NF-I and NF-III, respectively. Amounts of DNA retained on filter in binding reactions with various amounts of competitors were shown relative to that in the reaction with no competitor.
containing the same level of NF-I activity as the HeLa NF-I preparation used in the experiment shown above was completely competed only when oligo I and oligo OCTA were simultaneously added (data not shown). These results clearly indicate that the HeLa NF-I preparation is free of NF-III activity.
NE nn template
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Fig. 5. Effect of HeLa NF-I on in vitro transcription of the Ad 12 ElA gene in the extracts depleted of NF-I-like protein. 0.3 pg of HeLa NF-I were added to in vitro transcription in the flow-through fraction of DNA-affinity chromatography. Symbols are as in Fig. 3. Exposure to X-ray film for the right panel was 4-times longer than that for the left panel.
89 HeLa NF-I was then added to the transcription reaction performed in the flow-through fraction of DNA-affinity chromatography. From the results in Fig. 5, the flow-through fraction supplemented with HeLa NF-I regained activity to stimulate distal transcription of the Ad 12 E1A gene from wild-type DNA (lane 7) while the addition of NF-I had neither this effect nor any on distal transcription from dll DNA (lane 8) or proximal one from either D N A template (lanes 3 and 4). HeLa NF-I thus became a substitute for NF-I-like protein present in Ehrlich ascites tumor cell extracts and NF-I appears quite likely to be a trans-acting factor that stimulates transcription of the Ad 12 EIA gene. Involvement of NF-III-like protein in this transcription was not clear from the present study.
Analysis of in oitro transcription in nuclear extracts of 321 cells expressing the Ad 12 EIA gene Transcription of the E1A gene may possibly be autoregulated by its own E1A protein (Refs. 20 and 21 and Y. Sawada, unpublished data). For examination of this point, distal transcription of the E1A gene in vitro was compared for extracts of Ad 12 E1A-expressing 321 cells and Ehrlich ascites tumor cells. The cell line 321
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D~ Fig. 7. In vitro transcription of the Ad 12 E I A gene in 321 cell extracts. In vitro transcription was conducted with pE1A21 (w) and dll (d) D N A s as templates in nuclear extracts of Ehrlich ascites tumor cells (EAT) and 321 cells (321). Lanes 7 and 10 contained products from control reaction with no D N A template. Other symbols are as in Fig. 3.
has been established (Y. Sawada and T. Shenk, unpublished data) by introducing the E1 region of Ad 12 into 293 cell [22]. Expression of the Ad 12 E1A gene in 321 cells was first analyzed by blot hybridization and primer extension. Transcripts of this gene in 321 cells were found to be only about one-fourth the number in virusinfected KB cells and both distal and proximal start-sites for transcription were used (Fig. 6). Nuclear extracts were then prepared from 321 cells and in vitro transcription was conducted with wild-type and dll DNAs as templates. In Fig. 7, the amount of distal transcript synthesized from either template was almost the same (lanes 8 and 9) and as small as that synthesized from dll D N A in Ehrlich ascites tumor cell extracts (lane 4). 321 cell extracts were thus shown to possess no activity to stimulate distal transcription of the Ad 12 E1A gene from D N A containing the cis-acting region. Transcription of the E1A gene in nuclear extracts of HeLa cells was next examined to determine whether differences in transcriptional activity of Ehrlich ascites tumor cell and 321 cell extracts merely reflect species-specificity of mouse and human. In HeLa cell
primer,
p
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template, W D
Fig. 6. Analysis of 321 cell RNA. (A) R N A s of 321 cells (lanes 1 and 2) and Ad 12-infected KB cells (lanes 3 and 4) were analyzed in blot hybridization with a probe of the Ad 12 E I A gene or the h u m a n fl-actin pseudogene as a control. KB cell R N A was extracted either 8 or 26 h after virus infection (hpi). Arrowheads indicate positions of ribosomal RNAs. (B) R N A s of Ad 12-infected KB cells (lanes 1 and 2) and 321 cells (lanes 3 and 4) were analyzed in primer extension with the P-primer. Arrowheads with letters D and P indicate positions of extension products derived from distal and proximal transcripts of the Ad 12 E I A gene, respectively. Lane M contained HpaII-cleaved pBR322 and size is in bases.
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34
Fig. 8. In vitro transcription of the Ad 12 E I A gene in HeLa cell extracts. In vitro transcription was conducted with two D N A templates (W, D) in HeLa cell extracts. Symbols are as in Fig. 3.
90 extracts the a m o u n t of distal t r a n s c r i p t synthesized from w i l d - t y p e D N A was m o r e than that from d l l (lanes 3 a n d 4 in Fig. 8). 321 cell extracts m a y thus be i n c a p a b l e of s t i m u l a t i n g this t r a n s c r i p t i o n owing to the expression o f early viral genes, most likely of the A d 12 E 1 A gene.
PROBE:
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Analysis of DNA-binding activity in 321 and 293 cell extracts T h e a b o v e results are consistent with b i n d i n g activity to the cis-acting a-region in the two extracts. In Fig. 9A, b i n d i n g activity to the a-region in 321 cell extracts a p p e a r e d m u c h less than that in H e L a cell extracts while b o t h extracts showed essentially the same level of f o o t p r i n t s on the b-region. It can be also n o t e d that p r o t e c t i o n of the u p p e r p o r t i o n of the a-region, which is p r e s u m a b l y b o u n d b y N F - I I I , was almost c o m p a r a b l e b e t w e e n the two extracts. Differences in b i n d i n g activity to the a-region of these extracts b e c a m e m o r e evid e n t in gel shift assay. A s seen in Fig. 9B, H e L a cell extracts p r o d u c e d two shift b a n d s (a a n d b) a n d 321 cell
(B) gel s h i f t
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Fig. 9. DNA-binding activity in HeLa cell and 321 cell extracts. (A) DNase I footprinting was performed with 14/tg of HeLa cell extracts (HELA) or 10 #g of 321 cell extracts (321). Other simbols are as in Fig. 2B. (B) Gel shift assay was done with 14/Lg of HeLa or 321 cell extracts using oligo A and oligo B as probes. 200-fold molar excess of non-labeled oligo A (lanes 2 and 5) and oligo B (lanes 3 and 6) were present as competitors in the binding reaction. Arrowheads with letters a, b, c and d indicate the positions of the shift bands.
Fig. 10. DNA-binding activity in HeLa, 321 and 293 cell extracts. 10-11 /~g of two different preparations of the three extracts were analyzed for binding activity to the a-region (lanes 1-6) and the b-region (lanes 7-12) by gel shift assay. Positions of the specific and non-specific shift bands are shown with arrowheads.
extracts d i d o n l y b a n d b with the oligo A p r o b e , while b o t h showed the same shifts ( b a n d s c a n d d) with the oligo B probe. Bands a a n d c specifically d i s a p p e a r e d in the presence of an excess a m o u n t of n o n - l a b e l e d oligo A a n d oligo B, respectively (lanes 2 a n d 3). However, there was little c o m p e t i t i o n for b a n d s b a n d d by a d d i t i o n of either oligonucleotide. B a n d s a a n d c were thus shown to be specific p r o t e i n - D N A complexes. Thus, quite likely, 321 cell extracts possess no activity for b i n d i n g to the N F - I site while b i n d i n g activity to the N F - I I I site a n d the b - r e g i o n is c o m p a r a b l e to that in H e L a cell extracts. W e next e x a m i n e d b i n d i n g activity to the N F - I site p r e s e n t in n u c l e a r extracts of 293 cells in which o n l y the A d 5 E 1 A gene is expressed [22]. T w o different p r e p a r a tions of H e L a , 321 a n d 293 cell extracts were c o m p a r e d by gel shift assay (Fig. 10). These three extracts showed an obvious difference in b i n d i n g activity to the N F - I site (lanes 1-6), while the level of b i n d i n g activity to the b-region was a l m o s t c o m p a r a b l e a m o n g t h e m (lanes 7 - 1 2 ) . 293 cell extracts were f o u n d to c o n t a i n d e t e c t a ble b u t m u c h less b i n d i n g activity to the N F - I site than H e L a cell extracts, whereas 321 cell extracts s h o w e d no N F - I activity as o b s e r v e d in Fig. 9B. These results indicate that the level of N F - I activity was significantly low in 293 cell extracts c o m p a r i n g to that in H e L a cell extracts a n d was c o m p l e t e l y a b s e n t f r o m 321 cell extracts. This suggests that a d e n o v i r u s E 1 A p r o t e i n w o u l d cause decrease in N F - I activity a n d s i m u l t a n e o u s expression of the A d 5 a n d A d 12 E1A genes w o u l d be responsible for its c o m p l e t e loss in 321 cells. W e hence p r o p o s e that A d 12 E 1 A p r o t e i n a u t o r e g u l a t e s the transcription of its own gene b y m o d u l a t i n g the activity of NF-I.
91
Discussion Transcription regulation of the adenovirus EIA gene has been studied with non-oncogenic types 2 and 5 adenoviruses and several cis-acting elements and the factors that bind to them have been reported [2,3]. In the present study, the control mechanism of transcription of the E1A gene of type 12 adenovirus belonging to a group of highly oncogenic adenoviruses was investigated. The results of various experiments indicate NF-I or NF-I-like protein to stimulate transcription of the Ad 12 E1A gene from distal start-site by binding to a region located near the left end of the viral genome. NF-I is not apparently involved in the regulation of transcription of either the Ad 2 or Ad 5 E1A gene, and thus the control mechanism of E1A gene transcription may possibly differ for Ad 12 and non-oncogenic adenoviruses. Involvement of NF-III-like protein in regulation of the Ad 12 E1A gene transcription has yet to be examined. Binding site of NF-I-like protein was situated quite close to the control region of the viral genome replication [12]. NF-I is thus likely to bind to the same sequence element on the Ad 12 DNA for regulating both transcription of the E1A gene and replication of the viral genome. Binding of NF-I to the same region was recently shown to inhibit promoter activity which allows transcription with an orientation opposite to that of the E1A gene [23,24]. It would thus be quite pertinent to clarify the mechanism by which NF-I becomes functional in one of two different reactions. NF-I obviously stimulates the E1A gene transcription prior to the expression of early viral genes whose products are a requisite to the viral genome replication. With the transcription of early genes as preparation for the viral genome replication, the situation becomes more complex. Further research is required to gain better understanding of the mechanism of NF-I action in transcription and replication of the adenovirus DNA. Factor(s) bound to the b-region has been suggested to interact with those bound to the a-region resulting in mutual inhibition of binding to the target sequences [25]. The trans-acting function of NF-I may thus be controlled by factor(s) that binds to the b-region located adjacent to the NF-I binding site. Activity for both stimulating distal transcription of the Ad 12 E1A gene and binding to the NF-I site has been shown not to be present in nuclear extracts of 321 cells that express both the Ad 5 and Ad 12 E1A genes. Inactivation of NF-I or synthesis of a very small amount of NF-I in 321 cells may account for this. It was suggested that adenovirus E1A protein would cause a decrease in NF-I activity. E1A protein is thought to control transcription of various genes by modulating the apparent activity of cellular transcription factors already existing [1,2]. Involvement of protein phosphor-
ylation was recently suggested in E1A-dependent transcription activation [26,27]. Thus, E1A protein may possibly modify the NF-I molecule to inactivate its binding activity to the cis-acting region. However, more direct evidence supporting E1A-dependent inactivation of NF-I should be obtained since not only the E1A gene but other viral genes are expressed in 321 and 293 cells. It also remains to be clarified whether this proposed mechanism would be involved in E1A-dependent viral transformation and tumorigenesis.
Acknowledgements We thank O. Nikaido and F. Suzuki for their support in the cell culture, C. Wu and H. Ueda for a protocol of oligoDNA-affinity chromatography and their valuable suggestions and T. Kakunaga for providing the actin DNA. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and by grants from The Uehara Memorial Foundation and The Research Foundation for Pharmaceutical Sciences.
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