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c-fos and Signal Transduction In Vivo
Toxicology in Vitro 12 (1998) 523±524
c-fos and Signal Transduction In Vivo T. CURRAN Department of Developmental Neurobiology, St Jude Children's Research Hospital, Memphis, TN 38105, USA
Much of our knowledge of mammalian gene regulation comes from studies carried out in cultured cells and in cell-free biochemical systems. The advent of mouse transgenic and gene-knockout technologies provides an opportunity to test the hypotheses generated from these investigations in a physiological setting. In many cases, the regulation of transgenic reporter constructs and the phenotype of speci®c gene knock-outs defy simple interpretations and challenge prevailing dogma. We analysed c-fos regulation in vivo using transgenic mice (Smeyne et al., 1992 and 1993). A cfoslacZ fusion gene was constructed that recapitulates the appropriate regulation of c-fos in vivo using X-gal staining. This construct contains a complete c-fos genomic clone, including introns, and all of the known regulatory elements (Fig. 1). We tested the role of the known regulatory elements, the SIE, SRE, FAP and CaCRE, in the c-fos promoter by introducing speci®c point mutations into this foslacZ construct (Robertson et al., 1995). The results indicate that the loss of any one regulatory element severely compromises the function of the cfos promoter even in response to simple stimuli. For example, mutation of the SIE abolished the response of ®broblasts, cultured from transgenic embryos, to serum, phorbol ester and cAMP stimulation. This suggests that the c-fos promoter functions as an integrated unit to respond to extracellular stimuli rather than as a collection of individual response elements to speci®c second messengers (Fig. 1). These results were not consistent with data obtained from transient transfection assays carried out with the same constructs. Thus, it appears that the state of DNA, after integration into the chromosome and germline transmission, aects the ability of promoters to be activated by several signalling mechanisms. We also found that simply growing primary cells from the tissues of transgenic mice deregulated the tight control of foslacZ expression seen in vivo. Thus, it is important to study the regulation of gene expression in a whole animal context to appreciate fully the mechanisms responsible for the precise control of transcription activation.
Ref-1 was ®rst identi®ed as an activity that stimulated Fos-Jun DNA binding through an unusual redox mechanism (Abate et al., 1990a,b; Xanthoudakis and Curran, 1992). Puri®ed Fos and Jun proteins exhibit only low levels of AP-1 DNA binding activity until treated with reducing agents, such as DTT or b-mercaptoethanol, or by cellular extracts. This eect is mediated by a conserved cysteine residue located in the DNA binding region that lies in close proximity to the AP-1 site. This cysteine must be reduced to allow DNA binding of AP-1 proteins. However, in the inactive state it appears to form a non-disul®de oxidation product, perhaps a sulfenic acid. Mutation of the critical cysteine to serine confers a gain-of-function phenotype; Fos and Jun no longer require reducing agents or Ref-1 in order to bind to DNA. In vivo this mutation enhances the transforming activity of fos (Okuno et al., 1991). This implies that redox regulation of AP-1 (and perhaps other transcription factors) plays a role the control of gene expression in signal transduction pathways in living cells. We puri®ed the protein present in cellular extracts that is responsible for stimulation of AP-1 DNA binding activity, sequenced the N-terminus and used this information to clone the gene (Xanthoudakis and Curran, 1992). Nucleotide sequence analysis of the ref-1 gene revealed that it was identical to a previously described abasic endonuclease that functions in base-excision DNA repair processes. Mutagenesis analysis revealed that the DNA repair and redox functions of Ref-1 are located in separate domains of the protein (Xanthoudakis et al., 1994). To investigate the function of Ref-1 in vivo, we created a targeted disruption of the gene (Xanthoudakis et al., 1996). Although we readily obtained heterozygous mice carrying the disrupted allele (Ref-1 +/-), we were unable to obtain homozygous null embryos. An analysis of early development revealed that -/embryos failed to mature past E5.5. Thus, Ref-1 is an essential gene that is required for early embryonic development. At present, we do not know whether the redox or the DNA repair functions of
0887-2333/98/$19.00+0.00 # 1998 Published by Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII: S0887-2333(98)00028-9
524
T. Curran AcknowledgementsÐThis work was supported in part by NIH Cancer Center Support CORE Grant P30 CA21765, NIH Grant 1 R01 NS 36558 and by the American Lebanese Syrian Associated Charities (ALSAC).
REFERENCES
Fig. 1. Schematic representation of the c-fos promoter. Several regulatory elements have been de®ned in transient transfection assays that are both necessary and sucient for the activation of gene expression through the indicated signaling pathways. However, the results of transgene experiments suggest that all of these elements operate in concert in the context of the natural promoter.
Ref-1, or both in concert, are required for normal development. Ref-1 is expressed broadly but its levels can be induced by hypoxia (Yao et al., 1994). Recent studies have suggested that Ref-1 may act on a broad range of transcription factors including p53 (Jayaraman et al., 1997). The activation of p53 by Ref-1 may involve more than one mechanism and it requires the C-terminal region of p53. Thus, it is possible that Ref-1 provides a general signalling role by coupling alterations in the cellular redox environment, or other signals elicited by genotoxic stress, to cellular phenotypic responses by altering the activity of transcription factors while function in DNA repair processes.
Abate C., Luk D. and Curran T. (1990a) A ubiquitous nuclear protein stimulates the DNA-binding activity of fos and jun indirectly. Cell Growth and Dierentiation 1, 455±462. Abate C., Patel L., Rauscher F. J.d. and Curran T. (1990b) Redox regulation of fos and jun DNA-binding activity in vitro. Science 249, 1157±1161. Jayaraman L., Murthy K. G., Zhu C., Curran T., Xanthoudakis S. and Prives G. (1997) Identi®cation of redox/repair protein Ref-1 as a potent activator of p53. Genes and Development 11, 558±570. Okuno H., Suzuki T., Yoshida T., Hashimoto T., Curran T. and Iba H. (1991) Inhibition of jun transformation by a mutated fos gene: design of an anti-oncogene. Oncogene 6, 1491±1497. Robertson L. M., Kerppola T. K., Vendrell M., Luk D., Smeyne R. J., Bocchiaro C., Morgan J. I. and Curran T. (1995) Regulation of c-fos expression in transgenic mice requires multiple interdependent transcription control elements. Neuron 14, 241±252. Smeyne R. J., Schilling K., Robertson L., Luk D., Oberdick J., Curran T. and Morgan J. I. (1992) fos-lacZ transgenic mice: mapping sites of gene induction in the central nervous system. Neuron 8, 13±23. Xanthoudakis S. and Curran T. (1992) Identi®cation and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA-binding activity. EMBO Journal 11, 653±665. Xanthoudakis S., Miao G. G. and Curran T. (1994) The redox and DNA-repair activities of Ref-1 are encoded by nonoverlapping domains. Proceedings of the National Academy of Sciences of the U.S.A. 91, 23±27. Xanthoudakis S., Smeyne R. J., Wallace J. D. and Curran T. (1996) The redox/DNA repair protein, Ref-1, is essential for early embryonic development in mice. Proceedings of the National Academy of Sciences of the U.S.A. 93, 8919±8923. Yao K. S., Xanthoudakis S., Curran T. and O'Dwyer P. J. (1994) Activation of AP-1 and of nuclear redox factor, Ref-1, in the response of HT29 colon cancer cells to hypoxia. Molecular and Cellular Biology 14, 5997± 6003.
c-fos and Signal Transduction In Vivo T. CURRAN Department of Developmental Neurobiology, St Jude Children's Research Hospital, Memphis, TN 38105, USA
Much of our knowledge of mammalian gene regulation comes from studies carried out in cultured cells and in cell-free biochemical systems. The advent of mouse transgenic and gene-knockout technologies provides an opportunity to test the hypotheses generated from these investigations in a physiological setting. In many cases, the regulation of transgenic reporter constructs and the phenotype of speci®c gene knock-outs defy simple interpretations and challenge prevailing dogma. We analysed c-fos regulation in vivo using transgenic mice (Smeyne et al., 1992 and 1993). A cfoslacZ fusion gene was constructed that recapitulates the appropriate regulation of c-fos in vivo using X-gal staining. This construct contains a complete c-fos genomic clone, including introns, and all of the known regulatory elements (Fig. 1). We tested the role of the known regulatory elements, the SIE, SRE, FAP and CaCRE, in the c-fos promoter by introducing speci®c point mutations into this foslacZ construct (Robertson et al., 1995). The results indicate that the loss of any one regulatory element severely compromises the function of the cfos promoter even in response to simple stimuli. For example, mutation of the SIE abolished the response of ®broblasts, cultured from transgenic embryos, to serum, phorbol ester and cAMP stimulation. This suggests that the c-fos promoter functions as an integrated unit to respond to extracellular stimuli rather than as a collection of individual response elements to speci®c second messengers (Fig. 1). These results were not consistent with data obtained from transient transfection assays carried out with the same constructs. Thus, it appears that the state of DNA, after integration into the chromosome and germline transmission, aects the ability of promoters to be activated by several signalling mechanisms. We also found that simply growing primary cells from the tissues of transgenic mice deregulated the tight control of foslacZ expression seen in vivo. Thus, it is important to study the regulation of gene expression in a whole animal context to appreciate fully the mechanisms responsible for the precise control of transcription activation.
Ref-1 was ®rst identi®ed as an activity that stimulated Fos-Jun DNA binding through an unusual redox mechanism (Abate et al., 1990a,b; Xanthoudakis and Curran, 1992). Puri®ed Fos and Jun proteins exhibit only low levels of AP-1 DNA binding activity until treated with reducing agents, such as DTT or b-mercaptoethanol, or by cellular extracts. This eect is mediated by a conserved cysteine residue located in the DNA binding region that lies in close proximity to the AP-1 site. This cysteine must be reduced to allow DNA binding of AP-1 proteins. However, in the inactive state it appears to form a non-disul®de oxidation product, perhaps a sulfenic acid. Mutation of the critical cysteine to serine confers a gain-of-function phenotype; Fos and Jun no longer require reducing agents or Ref-1 in order to bind to DNA. In vivo this mutation enhances the transforming activity of fos (Okuno et al., 1991). This implies that redox regulation of AP-1 (and perhaps other transcription factors) plays a role the control of gene expression in signal transduction pathways in living cells. We puri®ed the protein present in cellular extracts that is responsible for stimulation of AP-1 DNA binding activity, sequenced the N-terminus and used this information to clone the gene (Xanthoudakis and Curran, 1992). Nucleotide sequence analysis of the ref-1 gene revealed that it was identical to a previously described abasic endonuclease that functions in base-excision DNA repair processes. Mutagenesis analysis revealed that the DNA repair and redox functions of Ref-1 are located in separate domains of the protein (Xanthoudakis et al., 1994). To investigate the function of Ref-1 in vivo, we created a targeted disruption of the gene (Xanthoudakis et al., 1996). Although we readily obtained heterozygous mice carrying the disrupted allele (Ref-1 +/-), we were unable to obtain homozygous null embryos. An analysis of early development revealed that -/embryos failed to mature past E5.5. Thus, Ref-1 is an essential gene that is required for early embryonic development. At present, we do not know whether the redox or the DNA repair functions of
0887-2333/98/$19.00+0.00 # 1998 Published by Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII: S0887-2333(98)00028-9
524
T. Curran AcknowledgementsÐThis work was supported in part by NIH Cancer Center Support CORE Grant P30 CA21765, NIH Grant 1 R01 NS 36558 and by the American Lebanese Syrian Associated Charities (ALSAC).
REFERENCES
Fig. 1. Schematic representation of the c-fos promoter. Several regulatory elements have been de®ned in transient transfection assays that are both necessary and sucient for the activation of gene expression through the indicated signaling pathways. However, the results of transgene experiments suggest that all of these elements operate in concert in the context of the natural promoter.
Ref-1, or both in concert, are required for normal development. Ref-1 is expressed broadly but its levels can be induced by hypoxia (Yao et al., 1994). Recent studies have suggested that Ref-1 may act on a broad range of transcription factors including p53 (Jayaraman et al., 1997). The activation of p53 by Ref-1 may involve more than one mechanism and it requires the C-terminal region of p53. Thus, it is possible that Ref-1 provides a general signalling role by coupling alterations in the cellular redox environment, or other signals elicited by genotoxic stress, to cellular phenotypic responses by altering the activity of transcription factors while function in DNA repair processes.
Abate C., Luk D. and Curran T. (1990a) A ubiquitous nuclear protein stimulates the DNA-binding activity of fos and jun indirectly. Cell Growth and Dierentiation 1, 455±462. Abate C., Patel L., Rauscher F. J.d. and Curran T. (1990b) Redox regulation of fos and jun DNA-binding activity in vitro. Science 249, 1157±1161. Jayaraman L., Murthy K. G., Zhu C., Curran T., Xanthoudakis S. and Prives G. (1997) Identi®cation of redox/repair protein Ref-1 as a potent activator of p53. Genes and Development 11, 558±570. Okuno H., Suzuki T., Yoshida T., Hashimoto T., Curran T. and Iba H. (1991) Inhibition of jun transformation by a mutated fos gene: design of an anti-oncogene. Oncogene 6, 1491±1497. Robertson L. M., Kerppola T. K., Vendrell M., Luk D., Smeyne R. J., Bocchiaro C., Morgan J. I. and Curran T. (1995) Regulation of c-fos expression in transgenic mice requires multiple interdependent transcription control elements. Neuron 14, 241±252. Smeyne R. J., Schilling K., Robertson L., Luk D., Oberdick J., Curran T. and Morgan J. I. (1992) fos-lacZ transgenic mice: mapping sites of gene induction in the central nervous system. Neuron 8, 13±23. Xanthoudakis S. and Curran T. (1992) Identi®cation and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA-binding activity. EMBO Journal 11, 653±665. Xanthoudakis S., Miao G. G. and Curran T. (1994) The redox and DNA-repair activities of Ref-1 are encoded by nonoverlapping domains. Proceedings of the National Academy of Sciences of the U.S.A. 91, 23±27. Xanthoudakis S., Smeyne R. J., Wallace J. D. and Curran T. (1996) The redox/DNA repair protein, Ref-1, is essential for early embryonic development in mice. Proceedings of the National Academy of Sciences of the U.S.A. 93, 8919±8923. Yao K. S., Xanthoudakis S., Curran T. and O'Dwyer P. J. (1994) Activation of AP-1 and of nuclear redox factor, Ref-1, in the response of HT29 colon cancer cells to hypoxia. Molecular and Cellular Biology 14, 5997± 6003.