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Activation of the major late promoter in adenovirus transformed cells by 5-azacytidine
VIROLOGY
165, 296-298
(1988)
Activation
of the Major Late Promoter
in Adenovirus
Transformed
Cells by 5-Azacytidine
NORMAND ROBERT,’ MIKLOS TOTH,’ RAYMOND TELLIER,~ JOSEPH CLAUDE V. DERY,~ AND JOSEPH M. WEBER~ Dbpartement
de Microbiologic, Received
Universit6 November
de Sherbrooke, 30, 1987;
accepted
Sherbrooke, March
Quebec
HORVATH,
J 1 H 5N4,
Canada
2, 1988
The adenovirus major late promoter (MLP) is normally not active in transformed cells. We investigated if it could be activated with 5-azacytidine. Three days of treatment with 10 NM 5-azacytidine induced transient activation of the MLP as shown by hybridization with an Ll r-strand-specific probe. The pollll-transcribed VA-RNAs were not activated. Ll activation was not accompanied by detectable changes in methylation of Hpall sites at the promoter or in the body of the transcript. Stably activated cell clones could be obtained at 209/o frequency after long-term drug treatment. o ISSS Academic
Press.
Inc.
About 2-4% of the cytosine bases in mammalian DNA are present as 5-methylcytosine in the dinucleotide 5’-CpG-3’ (3). Although adenovirus DNA is not methylated, integrated viral sequences in transformed cells become methylated. An inverse correlation was observed between the extent of DNA methylation and the transcription of integrated viral genes into mRNA (3). Of the integrated adenovirus genes in transformed cells, only the early genes have been observed to be expressed (4, 8). A curious exception to this general observation is an early mRNA transcribed from the major late promoter encompassing the Ll group of late genes (7, 10). As this RNA has not been observed in any virus transformed cells the reasons for its negative regulation became the object of the experiments reported here. We investigated whether the late promoter could be activated by the demethylating drug 5-azacytidine, and if so, which of the Ll transcripts would be expressed. The adenovirus type 2 transformed rat cell line F4, which contains 16 copies of the sequences from 0 to 62% and from 92 to loo%, but expresses only the El and E4 regions, was chosen for the study (4, 8). F4 cells were incubated for 3 days in 3 or 10 pl\/15-azacytidine (Sigma), the medium was changed, and total cytoplasmic RNA was extracted as described previously after 1, 2, or 3 days of further incubation. The RNA from untreated and treated Fq cells was spotted
on nitrocellulose membranes and hybridized with appropriate nick-translated DNA probes. The probe used to reveal El-specific RNA was the Bg/lI-E fragment (O-9.4) cloned in pBR322. The probe used to reveal RNA from the Ll region of the genome was the Hindill-I fragment (31.5-37.3) cloned in pBR322. To rule out artifacts, such as false negatives or contamination with DNA, in some experiments the same filter was hybridized with the El probe, and then washed and hybridized with the Ll probe. As shown in the top part of Fig. 1, the transcription of Ll-specific RNA was induced with 10 PLM 5-azacytidine. In parallel the transcription of El RNA appeared to be depressed about three- to ninefold on the first day after drug treatment. However, both the El depression and the Ll activation were transitory. RNA transcribed from the late promoter is complementary to the r-strand of the viral genome. To verify that the induced Ll -specific RNA was indeed complementary to the r-strand the hybridizations were repeated with M 13 probes containing inserts from both DNA strands of the Ll region. Positive signal was obtained only with the r-strand probe, showing that only the Ll and not the E2b region was transcribed. This was expected because the E2 promoter is absent from F4 cells (4). To determine whether the observed transcription activation is indeed Ll-specific and not read-through transcription from upstream promoters, total cytoplasmic RNA was subjected to Northern analysis using a small Ll probe (29.7-32.1). 5-Azacytidine treatment induced a single major RNA species of 3.8 kb (Fig. 2) which appears to correspond to the Ll a transcript described previously by Akusjarvi (7, 2). Note that VARNA was not induced. Because splicing is altered in
’ Present address: Departement de Microbiologic, Faculte de Medecine Veterinaire, C.P. 5000, St-Hyacinthe, Que. * Present address: Institute of Biochemistry, Biological Research Center, 6701 Szeged, P.O.B. 521, Hungary. 3 Present address: Departement de Biologie. Universite de Sherbrooke. 4 Present address: lnstitut de Recherches Cliniques, Montreal. 5 To whom requests for reprints should be addressed. 0042-6822/88
$3.00
Copynght 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved
296
SHORT
RNA
>:,
-
c
days after 2 1 -
-7
AzaC 3
probe
-
El
I
1
0 AzaC
297
COMMUNICATIONS
t
10 PM AzaC
on transcription and methylation FIG. 1. The effect of 5-azacytidine of viral genes in an adenovirus 2 transformed rat cell line (F4). Cells were incubated in the presence of 10 &4 5-azacytidine for 3 days, the drug was removed, and total cytoplasmic RNA was extracted from equal members of cells after 1, 2, or 3 days. Dilutions of the RNA were spotted on nitrocellulose membranes and hybridized with El @g/II-E) or Ll (HindIll-I) probes. The control (C) was RNA extracted from untreated F, cells. The DNA was extracted from the nuclear fraction of these cells and digested with HindIll, and then with Smal, Hpall, or Il/lspl. After electrophoresis and blotting, the filter was hybridized with a probe containing the Hindlll-l fragment to light up the Ll region. As indicated, some of the samples digested only with HindIll were diluted two- and fourfold to facilitate the quantitation of digestion by Smal and Hpall.
treatment did not noticeably alter it. Therefore Ll induction is not accompanied by significant demethylation of the coding region. This is in agreement with Doerfler’s suggestion that the critical methylation sites may be located near the promoter region of genes (5, 6). To explore this alternative the DNA from the induction experiment described in Fig. 2 was digested with HindIll, followed by /-/pall or Mspl and subjected to a similar analysis as above. The major late promoter lies within the HindIll-C fragment, proximal to the downstream terminus. The fragment contains 15 Hpall sites. The promoter region is spanned by two /-/pall fragments (241 and 274 nucleotides) which we detected with a Xhol-/-/indIll probe (5778-6231). Figure 3 shows that the promoter proximal region, containing the two Hpall sites, is completely methylated and drug treatment failed to change this. Therefore, the observed drug-activation of transcription from the major late promoter can take place without extensive demethylation of Hpall sites near the promoter or in the coding region of the transcript. These experiments cannot rule out the possibility that demethylation occurred in a small proportion of cells or only in 1 of the 16 integrated copies, or a combination of both. As noted above the 5-AzaC-induced Ll transcription was transitory. Several factors may be involved, such as unstable demethylation or toxicity of the altered gene expression, either viral or cellular (12). The following experiment was performed to determine if stable induction of the Ll region could be obtained with the drug. F4 cells were grown in the presence of 3 &I 5-azacytidine for 4 weeks, and then cloned in the absence of drug. As a control, untreated F4 ceils were a
the absence of VA-RNA this may explain the single Ll a precursor RNA species observed in these cells (11). To determine if Ll induction was correlated with a change in the state of methylation of the DNA of this region, the DNA was extracted from the nuclear fraction of the same cells where induction was observed. The DNA was digested with HindIll, and then samples were digested with Smal, /-/pal I, or Mspl. After electrophoresis and blotting, the filter was hybridized with a probe containing the HindIll-I fragment to light-up the Ll region. Smal is methylation-sensitive and cuts once within the HindIll-I fragment giving rise to a large and a very small fragment. /-/pall and Mspl both recognize 5’-CCGG-3’, but E/pall is refractory to methylation in the CG pair. As evident in the lower part of Fig. 1, the Ll region was extensively methylated and 5-azacytidine
b
c
of the Ll region in 5FIG. 2. Northern analysis of transcription azacytidine-treated transformed cells. F, cells were incubated for 2 days in the presence of 10 pM 5-azacytidine followed by 1 day in the absence of drug. Total cytoplasmic RNA was extracted, electrophoresed in a denaturing agarose gel, blotted, and hybridized with an Ll probe spanning the region 29.7-32.1, as described (9). (a) F, cells treated with drug; (b) untreated F, cells; (c) HEp2 cells infected for 12 hr with 1 PFU/cell of Ad2.
298
SHORT 0
AzaC
IOpM = Q I”
COMMUNICATIONS
AzaC =
n g
I
x
:: 5 -3433
and coding regions were indeed extensively methylated. Although methylation may be a crucial component in keeping the late promoter unexpressed, our results showed that Ll transcription can be induced by 5-azacytidine. Unfortunately we could not determine whether transcription activation occurred through demethylation or some other mechanism. ACKNOWLEDGMENTS This work was funded by the National Cancer Institute of Canada and the Medical Research Council of Canada. C.V.D. was the recipient of a studentship from FCAC. R.T. was the recipient of a Terry Fox Cancer Research Clerkship award, and J.M.W. was Research Associate of the NCIC.
a
b
c
d
e
f
FIG. 3. The state of methylation of the major late promoter region after 5-AzaC induction of RNA synthesis. The DNA was extracted from the nuclear fraction of the cells from the experiment described in Fig. 2, digested with HindIll, and then #pall (b, e) or Mspl (c, f) and Southern blots were probed with anXhol-HindIll fragment spanning two of the /-@all sites in the promoter region (of 241 and 274 bp).
also cloned. Expression of the El and Ll regions was checked by RNA-DNA hybridization as above. Of nine clones from control cells all were positive for El RNA and none for Ll RNA. Of 10 clones from 5-azacytidine-treated cells all were positive for E 1 RNA, but only two for Ll RNA. These results show that stable induction of Ll transcription is possible with 5-azacytidine. As Ll expression is not normally observed in transformed cells we must conclude that its expression is (normally) precluded in such cells. Both the promoter
REFERENCES 1. AKUSJARVI, G., J. Viral. 56, 879-886 (1985). 2. AKUSJARVI, G., and PERSSON, H.. Nature (London) 292,420-426 (1981). 3. DOERFLER, W.. Annu. Rev. Biochem. 52, 93-l 24 (1983). 4. FLINT, S. J., ln “DNATumor Viruses” (J. Tooze, Ed.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1981. 5. KRUCZEK, I., and DOERFLER. W.. Proc. Nafl. Acad. Sci. USA 80, 7586-7590 (1983). 6. LANGNER, K. D., VARDIMON, L., RENZ, D., and DOERFLER, W., Proc. Natl. Acad. Sci. USA 81, 2950-2954 (1984). 7. LEWIS, J. B., and MATHEWS, M. B., Ce//21, 303-313 (1980). 8. LEWIS, J. B., and MATHEWS, M. B., Virology 115, 345-360. 9. MANIATIS, T., FRITSCH, E. F.. and SAMBROOK, J., “Molecular Cloning, a Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 10. MILLER, 1. S., RICCIARDI, R. P.. ROBERTS, B. E., PATERSON, B. M., and MATHEWS. M. B.. J. Mol. Biol. 142, 455-488 (1980). 11. SVENSSON, C., and AKUSJARVI, G., Proc. Nat/. Acad. Sci. USA 83, 4690-4694 (1986). 12. WIGLER, M.. LEVY, D., and PERUCHO, M., Ce//24, 33-40 (1981).
165, 296-298
(1988)
Activation
of the Major Late Promoter
in Adenovirus
Transformed
Cells by 5-Azacytidine
NORMAND ROBERT,’ MIKLOS TOTH,’ RAYMOND TELLIER,~ JOSEPH CLAUDE V. DERY,~ AND JOSEPH M. WEBER~ Dbpartement
de Microbiologic, Received
Universit6 November
de Sherbrooke, 30, 1987;
accepted
Sherbrooke, March
Quebec
HORVATH,
J 1 H 5N4,
Canada
2, 1988
The adenovirus major late promoter (MLP) is normally not active in transformed cells. We investigated if it could be activated with 5-azacytidine. Three days of treatment with 10 NM 5-azacytidine induced transient activation of the MLP as shown by hybridization with an Ll r-strand-specific probe. The pollll-transcribed VA-RNAs were not activated. Ll activation was not accompanied by detectable changes in methylation of Hpall sites at the promoter or in the body of the transcript. Stably activated cell clones could be obtained at 209/o frequency after long-term drug treatment. o ISSS Academic
Press.
Inc.
About 2-4% of the cytosine bases in mammalian DNA are present as 5-methylcytosine in the dinucleotide 5’-CpG-3’ (3). Although adenovirus DNA is not methylated, integrated viral sequences in transformed cells become methylated. An inverse correlation was observed between the extent of DNA methylation and the transcription of integrated viral genes into mRNA (3). Of the integrated adenovirus genes in transformed cells, only the early genes have been observed to be expressed (4, 8). A curious exception to this general observation is an early mRNA transcribed from the major late promoter encompassing the Ll group of late genes (7, 10). As this RNA has not been observed in any virus transformed cells the reasons for its negative regulation became the object of the experiments reported here. We investigated whether the late promoter could be activated by the demethylating drug 5-azacytidine, and if so, which of the Ll transcripts would be expressed. The adenovirus type 2 transformed rat cell line F4, which contains 16 copies of the sequences from 0 to 62% and from 92 to loo%, but expresses only the El and E4 regions, was chosen for the study (4, 8). F4 cells were incubated for 3 days in 3 or 10 pl\/15-azacytidine (Sigma), the medium was changed, and total cytoplasmic RNA was extracted as described previously after 1, 2, or 3 days of further incubation. The RNA from untreated and treated Fq cells was spotted
on nitrocellulose membranes and hybridized with appropriate nick-translated DNA probes. The probe used to reveal El-specific RNA was the Bg/lI-E fragment (O-9.4) cloned in pBR322. The probe used to reveal RNA from the Ll region of the genome was the Hindill-I fragment (31.5-37.3) cloned in pBR322. To rule out artifacts, such as false negatives or contamination with DNA, in some experiments the same filter was hybridized with the El probe, and then washed and hybridized with the Ll probe. As shown in the top part of Fig. 1, the transcription of Ll-specific RNA was induced with 10 PLM 5-azacytidine. In parallel the transcription of El RNA appeared to be depressed about three- to ninefold on the first day after drug treatment. However, both the El depression and the Ll activation were transitory. RNA transcribed from the late promoter is complementary to the r-strand of the viral genome. To verify that the induced Ll -specific RNA was indeed complementary to the r-strand the hybridizations were repeated with M 13 probes containing inserts from both DNA strands of the Ll region. Positive signal was obtained only with the r-strand probe, showing that only the Ll and not the E2b region was transcribed. This was expected because the E2 promoter is absent from F4 cells (4). To determine whether the observed transcription activation is indeed Ll-specific and not read-through transcription from upstream promoters, total cytoplasmic RNA was subjected to Northern analysis using a small Ll probe (29.7-32.1). 5-Azacytidine treatment induced a single major RNA species of 3.8 kb (Fig. 2) which appears to correspond to the Ll a transcript described previously by Akusjarvi (7, 2). Note that VARNA was not induced. Because splicing is altered in
’ Present address: Departement de Microbiologic, Faculte de Medecine Veterinaire, C.P. 5000, St-Hyacinthe, Que. * Present address: Institute of Biochemistry, Biological Research Center, 6701 Szeged, P.O.B. 521, Hungary. 3 Present address: Departement de Biologie. Universite de Sherbrooke. 4 Present address: lnstitut de Recherches Cliniques, Montreal. 5 To whom requests for reprints should be addressed. 0042-6822/88
$3.00
Copynght 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved
296
SHORT
RNA
>:,
-
c
days after 2 1 -
-7
AzaC 3
probe
-
El
I
1
0 AzaC
297
COMMUNICATIONS
t
10 PM AzaC
on transcription and methylation FIG. 1. The effect of 5-azacytidine of viral genes in an adenovirus 2 transformed rat cell line (F4). Cells were incubated in the presence of 10 &4 5-azacytidine for 3 days, the drug was removed, and total cytoplasmic RNA was extracted from equal members of cells after 1, 2, or 3 days. Dilutions of the RNA were spotted on nitrocellulose membranes and hybridized with El @g/II-E) or Ll (HindIll-I) probes. The control (C) was RNA extracted from untreated F, cells. The DNA was extracted from the nuclear fraction of these cells and digested with HindIll, and then with Smal, Hpall, or Il/lspl. After electrophoresis and blotting, the filter was hybridized with a probe containing the Hindlll-l fragment to light up the Ll region. As indicated, some of the samples digested only with HindIll were diluted two- and fourfold to facilitate the quantitation of digestion by Smal and Hpall.
treatment did not noticeably alter it. Therefore Ll induction is not accompanied by significant demethylation of the coding region. This is in agreement with Doerfler’s suggestion that the critical methylation sites may be located near the promoter region of genes (5, 6). To explore this alternative the DNA from the induction experiment described in Fig. 2 was digested with HindIll, followed by /-/pall or Mspl and subjected to a similar analysis as above. The major late promoter lies within the HindIll-C fragment, proximal to the downstream terminus. The fragment contains 15 Hpall sites. The promoter region is spanned by two /-/pall fragments (241 and 274 nucleotides) which we detected with a Xhol-/-/indIll probe (5778-6231). Figure 3 shows that the promoter proximal region, containing the two Hpall sites, is completely methylated and drug treatment failed to change this. Therefore, the observed drug-activation of transcription from the major late promoter can take place without extensive demethylation of Hpall sites near the promoter or in the coding region of the transcript. These experiments cannot rule out the possibility that demethylation occurred in a small proportion of cells or only in 1 of the 16 integrated copies, or a combination of both. As noted above the 5-AzaC-induced Ll transcription was transitory. Several factors may be involved, such as unstable demethylation or toxicity of the altered gene expression, either viral or cellular (12). The following experiment was performed to determine if stable induction of the Ll region could be obtained with the drug. F4 cells were grown in the presence of 3 &I 5-azacytidine for 4 weeks, and then cloned in the absence of drug. As a control, untreated F4 ceils were a
the absence of VA-RNA this may explain the single Ll a precursor RNA species observed in these cells (11). To determine if Ll induction was correlated with a change in the state of methylation of the DNA of this region, the DNA was extracted from the nuclear fraction of the same cells where induction was observed. The DNA was digested with HindIll, and then samples were digested with Smal, /-/pal I, or Mspl. After electrophoresis and blotting, the filter was hybridized with a probe containing the HindIll-I fragment to light-up the Ll region. Smal is methylation-sensitive and cuts once within the HindIll-I fragment giving rise to a large and a very small fragment. /-/pall and Mspl both recognize 5’-CCGG-3’, but E/pall is refractory to methylation in the CG pair. As evident in the lower part of Fig. 1, the Ll region was extensively methylated and 5-azacytidine
b
c
of the Ll region in 5FIG. 2. Northern analysis of transcription azacytidine-treated transformed cells. F, cells were incubated for 2 days in the presence of 10 pM 5-azacytidine followed by 1 day in the absence of drug. Total cytoplasmic RNA was extracted, electrophoresed in a denaturing agarose gel, blotted, and hybridized with an Ll probe spanning the region 29.7-32.1, as described (9). (a) F, cells treated with drug; (b) untreated F, cells; (c) HEp2 cells infected for 12 hr with 1 PFU/cell of Ad2.
298
SHORT 0
AzaC
IOpM = Q I”
COMMUNICATIONS
AzaC =
n g
I
x
:: 5 -3433
and coding regions were indeed extensively methylated. Although methylation may be a crucial component in keeping the late promoter unexpressed, our results showed that Ll transcription can be induced by 5-azacytidine. Unfortunately we could not determine whether transcription activation occurred through demethylation or some other mechanism. ACKNOWLEDGMENTS This work was funded by the National Cancer Institute of Canada and the Medical Research Council of Canada. C.V.D. was the recipient of a studentship from FCAC. R.T. was the recipient of a Terry Fox Cancer Research Clerkship award, and J.M.W. was Research Associate of the NCIC.
a
b
c
d
e
f
FIG. 3. The state of methylation of the major late promoter region after 5-AzaC induction of RNA synthesis. The DNA was extracted from the nuclear fraction of the cells from the experiment described in Fig. 2, digested with HindIll, and then #pall (b, e) or Mspl (c, f) and Southern blots were probed with anXhol-HindIll fragment spanning two of the /-@all sites in the promoter region (of 241 and 274 bp).
also cloned. Expression of the El and Ll regions was checked by RNA-DNA hybridization as above. Of nine clones from control cells all were positive for El RNA and none for Ll RNA. Of 10 clones from 5-azacytidine-treated cells all were positive for E 1 RNA, but only two for Ll RNA. These results show that stable induction of Ll transcription is possible with 5-azacytidine. As Ll expression is not normally observed in transformed cells we must conclude that its expression is (normally) precluded in such cells. Both the promoter
REFERENCES 1. AKUSJARVI, G., J. Viral. 56, 879-886 (1985). 2. AKUSJARVI, G., and PERSSON, H.. Nature (London) 292,420-426 (1981). 3. DOERFLER, W.. Annu. Rev. Biochem. 52, 93-l 24 (1983). 4. FLINT, S. J., ln “DNATumor Viruses” (J. Tooze, Ed.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1981. 5. KRUCZEK, I., and DOERFLER. W.. Proc. Nafl. Acad. Sci. USA 80, 7586-7590 (1983). 6. LANGNER, K. D., VARDIMON, L., RENZ, D., and DOERFLER, W., Proc. Natl. Acad. Sci. USA 81, 2950-2954 (1984). 7. LEWIS, J. B., and MATHEWS, M. B., Ce//21, 303-313 (1980). 8. LEWIS, J. B., and MATHEWS, M. B., Virology 115, 345-360. 9. MANIATIS, T., FRITSCH, E. F.. and SAMBROOK, J., “Molecular Cloning, a Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 10. MILLER, 1. S., RICCIARDI, R. P.. ROBERTS, B. E., PATERSON, B. M., and MATHEWS. M. B.. J. Mol. Biol. 142, 455-488 (1980). 11. SVENSSON, C., and AKUSJARVI, G., Proc. Nat/. Acad. Sci. USA 83, 4690-4694 (1986). 12. WIGLER, M.. LEVY, D., and PERUCHO, M., Ce//24, 33-40 (1981).