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In VitroTranscriptional Activation of p21 Promoter by p53
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
234, 300–302 (1997)
RC976637
In Vitro Transcriptional Activation of p21 Promoter by p53 Tae Kook Kim Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138
Received April 11, 1997
The tumor suppressor p53 can control cell growth by inducing cell cycle arrest. This is effected, at least in part, by transcriptional activation of p21 gene, a cell cycle inhibitor. Here in vitro transcription assay for p21 has been established. The assay recapitulates activation of p21 promoter mediated by p53 in vitro. Thus this in vitro assay will allow to study how transcriptional regulation of p21 can be linked to cell cycle controls during development and neoplastic transformation processes. q 1997 Academic Press
Transcriptional regulation is critical in cellular proliferation and differentiation processes (1). The development of multicellular organisms requires precise control of cell division. A lot of evidence has now demonstrated important connections between transcriptional regulation and cell cycle controls during development (2). Irregulations of this interplay can lead to the abnormal development and neoplastic transformation (3). Examination of the expression of p21 gene (4, 5), a cell cycle inhibitor, shows a nice correlation between p21 transcription and the withdrawal of cells from the division cycle during differentiation (6, 7, 8, 9). And ectopic expression of p21 induces the differentiation (7). In addition, transcription of the p21 gene is induced by a wide range of cell growth regulatory signals including DNA damage from a p53-dependent mechanism (5). The p53 protein acts as part of a checkpoint pathway which regulates cell proliferation in response to many signals including DNA damage (10). Thus appropriate regulation of p21 levels by transcription is important for normal cell division, developmental decisions and checkpoint controls. Loss of these controls can contribute to neoplastic transformation. To gain insights into detailed molecular mechanisms in p21 activation, in vitro transcription assay system has been set up in the present study. The established assay readily recapitulates p21 promoter activation by p53 in vitro. MATERIALS AND METHODS Preparation of nuclear extracts from cultured cells and in vitro transcription analysis. Rat embryo fibroblast (REF) cells were pas0006-291X/97 $25.00
saged in Eagle’s minimal essential medium and cells were incubated at 377C or 317C for 24 hr (5). Nuclear extracts were prepared from cultured cells and in vitro transcription assays were performed as described before (11, 12, 13). Wild-type p53 protein was purified from insect cells after infection with baculovirus (14).
RESULTS AND DISCUSSION To analyze transcriptional activation of the p21 gene promoter in vitro, a template used contains DNA sequences of 2.4 kb upstream from the p21 transcription start site. This promoter region is shown to be necessary for the p53-mediated induction in vivo (5). Nuclear extracts were prepared from cultured cells and used as a source of transcription factors. Primer extension assay was employed to measure the levels of p21 gene transcription in vitro and protein amounts in nuclear extracts, prepared from cells incubated under different conditions, were normalized to compare relative levels of transcription. To determine whether p21 gene transcription can be induced by p53 in vitro, we took advantage of rat embryo fibroblasts (REFs) containing a stably integrated murine temperature-sensitive mutant p53 (Fig. 1). Nuclear extracts were prepared from these cells after incubated at either 377C or 317C for 24 hr. The p21 transcription was almost undetectable in the extracts from cells incubated at 37 C where majority of the temperature-sensitive p53 is in the mutant conformation (lane 2). But, the transcription level was markedly increased in cell extracts prepared after cultured at 317C where the majority of p53 is present in the wild-type conformation (lane 1). Importantly, addition of purified wildtype p53 protein can restore the transcriptional activation of p21 in the nuclear extracts prepared from cells incubated at 377C (lane 3). These results indicate that p21 gene is inducible in vitro by wild-type p53, but not mutant p53, and suggest that p53-dependent transcriptional activation of p21 can be recapitulated by this in vitro transcription system. Related to the high incidence of p53 gene mutation in its DNA binding domain, next we determined that p53 must bind DNA to activate transcription from p21 gene promoter in vitro. An oligonucleotide-competition
300
Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
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BBRC 6637
/
692b$$$$81
04-22-97 11:45:04
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AP: BBRC
Vol. 234, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. The p21 promoter is transcriptionally activated in vitro by wild-type p53, but not by mutant p53. Nuclear extracts were prepared from REF cells containing temperature-sensitive mutant p53 after incubation at either 377C or 317C. Primer extension was used to detect transcripts from the p21 promoter template. The template contained a p53 responsive element (02.4 kb) and minimal p21 promoter region (50 bp including TATA box and transcription start site) (5). Lane 3 contained wild-type p53 protein purified from insect cells.
assay was performed for analyzing the role of p53 DNA binding in p21 transcription (Fig. 2). Transcription was activated by p53 in cell extracts prepared after cultured at 317C (lane 2), but not at 377C (lane 1). In nuclear
FIG. 2. In vitro transcriptional activation of p21 promoter requires DNA binding activity of p53. Nuclear extracts were prepared from REF cells containing temperature-sensitive mutant p53 after incubation at either 377C or 317C. Transcription reactions were performed in the absence or presence of increasing amounts of an oligonucleotide containing a p53 binding site. Primer extension was used to detect the transcripts from the p21 promoter template.
FIG. 3. The p53 interacts with an enhancer of p21 promoter for transcriptional activation in vitro. Nuclear extracts were prepared from REF cells containing temperature-sensitive mutant p53 after incubation at either 377C or 317C. Transcription reactions were performed with DNA templates with or without a p53 binding site. Primer extension was used to detect the transcripts from the p21 promoter templates.
extracts prepared from cells containing wild-type conformation of p53, transcriptional activation of p21 was inhibited by addition of excess oligonucleotide containing the p53 binding site (compare lane 2 with lanes 3 and 4). However, addition of comparable amounts of an oligonucleotide lacking a p53 binding site did not decrease the level of p21 transcription (data not shown). These data indicate that DNA binding of p53 is important for p21 transcriptional activation in vitro. The p53 responsive element was previously identified around 2.4 kb upstream of p21 transcription start site (5). To directly know the importance of this site for p21 transcriptional activation, we compared the transcriptional activities of p21 promoter in the presence and absence of the p53 binding site (Fig. 3). In the case of p21 promoter template containing a p53 binding site, no detectable transcription was observed in the nuclear extracts from cells incubated at 377C (lane 1) but transcription activities were markedly increased at 317C (lane 2), consistent with the data of Fig. 1. However, when the 2.4 kb upstream p53 binding site was deleted, most of the transcriptional activities was abolished in the extracts prepared after incubation at 317C (compare lanes 2 and 3). This defect was consistently observed with various concentrations of extracts. These results suggests that the 2.4 kb upstream p53 binding site is, at least some part, involved in p21 transcriptional activation by p53.
301
AID
BBRC 6637
/
692b$$$$82
04-22-97 11:45:04
bbrcg
AP: BBRC
Vol. 234, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Here wild-type p53 protein, but not mutant p53, has been shown to activate p21 transcription in vitro. This activation is dependent on the ability of p53 binding to a DNA. These in vitro data are consistent with the view that p53 induces p21, a cell cycle inhibitor, thereby arresting cell cycle and mutation of p53 cannot control this checkpoint pathway in response to DNA damage since it is defective in the transcriptional activation of specific target genes including p21 (10). This established in vitro assay systems will be very helpful to study the detailed molecular mechanisms of p21 promoter activation by p53 and to understand connections between cell cycle and transcriptional regulations during development and neoplastic transformation. ACKNOWLEDGMENT This study is supported by the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation (DRG-1329) to T. K. Kim.
REFERENCES 1. McKnight, S. L., and Yamamoto, K. R. (1992) in Transcriptional Regulation, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
2. Elledge, S. J., Winston, J., and Harper, J. W. (1996) Trends Cell Biol. 6, 388–392. 3. Hunter, T. (1997) Cell 88, 333–346. 4. Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., and Elledge, S. J. (1993) Cell 75, 805–816. 5. El-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W., and Volgelstein, B. (1993) Cell 75, 817–825. 6. Halvey, O., Novitch, B. G., Spicer, D. B., Skapek, S. X., Rhee, J., Hannon, G. J., Beach, D., and Lassar, A. B. (1995) Science 267, 1018–1021. 7. Skapek, S. X., Rhee, J., Spicer, D. B., and Lassar, A. B. (1995) Science 267, 1022–1024. 8. Parker, S. B., Eichele, G., Zhang, P., Rawls, A., Sands, A. T., Bradley, A., Olson, E. N., Harper, J. W., and Elledge, S. J. (1995) Science 267, 1024–1027. 9. Liu, M., Lee, M.-H., Cohen, M., Bommakanti, M., and Freedman, L. P. (1996) Genes & Dev. 10, 142–153. 10. Levine, A. J. (1997) Cell 88, 323–331. 11. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475–1489. 12. Kim, T. K., and Roeder, R. G. (1994) Nature 369, 252–255. 13. Kim, T. K., Zhao, Y., Hui, G., and Roeder, R. G. (1995) J. Biol. Chem. 270, 10976–10981. 14. Farmer, G., Bargonetti, J., Zhu, H., Friedman, P., Prywes, R., and Prives, C. (1992) Nature 358, 83–86.
302
AID
BBRC 6637
/
692b$$$$82
04-22-97 11:45:04
bbrcg
AP: BBRC
234, 300–302 (1997)
RC976637
In Vitro Transcriptional Activation of p21 Promoter by p53 Tae Kook Kim Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138
Received April 11, 1997
The tumor suppressor p53 can control cell growth by inducing cell cycle arrest. This is effected, at least in part, by transcriptional activation of p21 gene, a cell cycle inhibitor. Here in vitro transcription assay for p21 has been established. The assay recapitulates activation of p21 promoter mediated by p53 in vitro. Thus this in vitro assay will allow to study how transcriptional regulation of p21 can be linked to cell cycle controls during development and neoplastic transformation processes. q 1997 Academic Press
Transcriptional regulation is critical in cellular proliferation and differentiation processes (1). The development of multicellular organisms requires precise control of cell division. A lot of evidence has now demonstrated important connections between transcriptional regulation and cell cycle controls during development (2). Irregulations of this interplay can lead to the abnormal development and neoplastic transformation (3). Examination of the expression of p21 gene (4, 5), a cell cycle inhibitor, shows a nice correlation between p21 transcription and the withdrawal of cells from the division cycle during differentiation (6, 7, 8, 9). And ectopic expression of p21 induces the differentiation (7). In addition, transcription of the p21 gene is induced by a wide range of cell growth regulatory signals including DNA damage from a p53-dependent mechanism (5). The p53 protein acts as part of a checkpoint pathway which regulates cell proliferation in response to many signals including DNA damage (10). Thus appropriate regulation of p21 levels by transcription is important for normal cell division, developmental decisions and checkpoint controls. Loss of these controls can contribute to neoplastic transformation. To gain insights into detailed molecular mechanisms in p21 activation, in vitro transcription assay system has been set up in the present study. The established assay readily recapitulates p21 promoter activation by p53 in vitro. MATERIALS AND METHODS Preparation of nuclear extracts from cultured cells and in vitro transcription analysis. Rat embryo fibroblast (REF) cells were pas0006-291X/97 $25.00
saged in Eagle’s minimal essential medium and cells were incubated at 377C or 317C for 24 hr (5). Nuclear extracts were prepared from cultured cells and in vitro transcription assays were performed as described before (11, 12, 13). Wild-type p53 protein was purified from insect cells after infection with baculovirus (14).
RESULTS AND DISCUSSION To analyze transcriptional activation of the p21 gene promoter in vitro, a template used contains DNA sequences of 2.4 kb upstream from the p21 transcription start site. This promoter region is shown to be necessary for the p53-mediated induction in vivo (5). Nuclear extracts were prepared from cultured cells and used as a source of transcription factors. Primer extension assay was employed to measure the levels of p21 gene transcription in vitro and protein amounts in nuclear extracts, prepared from cells incubated under different conditions, were normalized to compare relative levels of transcription. To determine whether p21 gene transcription can be induced by p53 in vitro, we took advantage of rat embryo fibroblasts (REFs) containing a stably integrated murine temperature-sensitive mutant p53 (Fig. 1). Nuclear extracts were prepared from these cells after incubated at either 377C or 317C for 24 hr. The p21 transcription was almost undetectable in the extracts from cells incubated at 37 C where majority of the temperature-sensitive p53 is in the mutant conformation (lane 2). But, the transcription level was markedly increased in cell extracts prepared after cultured at 317C where the majority of p53 is present in the wild-type conformation (lane 1). Importantly, addition of purified wildtype p53 protein can restore the transcriptional activation of p21 in the nuclear extracts prepared from cells incubated at 377C (lane 3). These results indicate that p21 gene is inducible in vitro by wild-type p53, but not mutant p53, and suggest that p53-dependent transcriptional activation of p21 can be recapitulated by this in vitro transcription system. Related to the high incidence of p53 gene mutation in its DNA binding domain, next we determined that p53 must bind DNA to activate transcription from p21 gene promoter in vitro. An oligonucleotide-competition
300
Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.
AID
BBRC 6637
/
692b$$$$81
04-22-97 11:45:04
bbrcg
AP: BBRC
Vol. 234, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 1. The p21 promoter is transcriptionally activated in vitro by wild-type p53, but not by mutant p53. Nuclear extracts were prepared from REF cells containing temperature-sensitive mutant p53 after incubation at either 377C or 317C. Primer extension was used to detect transcripts from the p21 promoter template. The template contained a p53 responsive element (02.4 kb) and minimal p21 promoter region (50 bp including TATA box and transcription start site) (5). Lane 3 contained wild-type p53 protein purified from insect cells.
assay was performed for analyzing the role of p53 DNA binding in p21 transcription (Fig. 2). Transcription was activated by p53 in cell extracts prepared after cultured at 317C (lane 2), but not at 377C (lane 1). In nuclear
FIG. 2. In vitro transcriptional activation of p21 promoter requires DNA binding activity of p53. Nuclear extracts were prepared from REF cells containing temperature-sensitive mutant p53 after incubation at either 377C or 317C. Transcription reactions were performed in the absence or presence of increasing amounts of an oligonucleotide containing a p53 binding site. Primer extension was used to detect the transcripts from the p21 promoter template.
FIG. 3. The p53 interacts with an enhancer of p21 promoter for transcriptional activation in vitro. Nuclear extracts were prepared from REF cells containing temperature-sensitive mutant p53 after incubation at either 377C or 317C. Transcription reactions were performed with DNA templates with or without a p53 binding site. Primer extension was used to detect the transcripts from the p21 promoter templates.
extracts prepared from cells containing wild-type conformation of p53, transcriptional activation of p21 was inhibited by addition of excess oligonucleotide containing the p53 binding site (compare lane 2 with lanes 3 and 4). However, addition of comparable amounts of an oligonucleotide lacking a p53 binding site did not decrease the level of p21 transcription (data not shown). These data indicate that DNA binding of p53 is important for p21 transcriptional activation in vitro. The p53 responsive element was previously identified around 2.4 kb upstream of p21 transcription start site (5). To directly know the importance of this site for p21 transcriptional activation, we compared the transcriptional activities of p21 promoter in the presence and absence of the p53 binding site (Fig. 3). In the case of p21 promoter template containing a p53 binding site, no detectable transcription was observed in the nuclear extracts from cells incubated at 377C (lane 1) but transcription activities were markedly increased at 317C (lane 2), consistent with the data of Fig. 1. However, when the 2.4 kb upstream p53 binding site was deleted, most of the transcriptional activities was abolished in the extracts prepared after incubation at 317C (compare lanes 2 and 3). This defect was consistently observed with various concentrations of extracts. These results suggests that the 2.4 kb upstream p53 binding site is, at least some part, involved in p21 transcriptional activation by p53.
301
AID
BBRC 6637
/
692b$$$$82
04-22-97 11:45:04
bbrcg
AP: BBRC
Vol. 234, No. 2, 1997
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Here wild-type p53 protein, but not mutant p53, has been shown to activate p21 transcription in vitro. This activation is dependent on the ability of p53 binding to a DNA. These in vitro data are consistent with the view that p53 induces p21, a cell cycle inhibitor, thereby arresting cell cycle and mutation of p53 cannot control this checkpoint pathway in response to DNA damage since it is defective in the transcriptional activation of specific target genes including p21 (10). This established in vitro assay systems will be very helpful to study the detailed molecular mechanisms of p21 promoter activation by p53 and to understand connections between cell cycle and transcriptional regulations during development and neoplastic transformation. ACKNOWLEDGMENT This study is supported by the Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation (DRG-1329) to T. K. Kim.
REFERENCES 1. McKnight, S. L., and Yamamoto, K. R. (1992) in Transcriptional Regulation, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
2. Elledge, S. J., Winston, J., and Harper, J. W. (1996) Trends Cell Biol. 6, 388–392. 3. Hunter, T. (1997) Cell 88, 333–346. 4. Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., and Elledge, S. J. (1993) Cell 75, 805–816. 5. El-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W., and Volgelstein, B. (1993) Cell 75, 817–825. 6. Halvey, O., Novitch, B. G., Spicer, D. B., Skapek, S. X., Rhee, J., Hannon, G. J., Beach, D., and Lassar, A. B. (1995) Science 267, 1018–1021. 7. Skapek, S. X., Rhee, J., Spicer, D. B., and Lassar, A. B. (1995) Science 267, 1022–1024. 8. Parker, S. B., Eichele, G., Zhang, P., Rawls, A., Sands, A. T., Bradley, A., Olson, E. N., Harper, J. W., and Elledge, S. J. (1995) Science 267, 1024–1027. 9. Liu, M., Lee, M.-H., Cohen, M., Bommakanti, M., and Freedman, L. P. (1996) Genes & Dev. 10, 142–153. 10. Levine, A. J. (1997) Cell 88, 323–331. 11. Dignam, J. D., Lebovitz, R. M., and Roeder, R. G. (1983) Nucleic Acids Res. 11, 1475–1489. 12. Kim, T. K., and Roeder, R. G. (1994) Nature 369, 252–255. 13. Kim, T. K., Zhao, Y., Hui, G., and Roeder, R. G. (1995) J. Biol. Chem. 270, 10976–10981. 14. Farmer, G., Bargonetti, J., Zhu, H., Friedman, P., Prywes, R., and Prives, C. (1992) Nature 358, 83–86.
302
AID
BBRC 6637
/
692b$$$$82
04-22-97 11:45:04
bbrcg
AP: BBRC