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Apoptosis: A new twist to the tale?
Dispatch
1057
Apoptosis: A new twist to the tale? Emma Warbrick
The DNA helicases XPB and XPD, components of transcription factor TFIIH, have been implicated in a p53-induced apoptotic pathway. These new findings suggest a role for the core TFIIH complex in the coordination, not only of transcription, the cell cycle and DNA repair, but also of apoptosis.
Table 1
p89 (RAD25/SSL2) XPB, ERCC3
3′–5′ DNA helicase
Address: Department of Anatomy and Physiology, Medical Sciences Institute, University of Dundee, Dundee DD1 4HN, Scotland, UK.
p80 (RAD3)
5′–3′ DNA helicase
Current Biology 1996, Vol 6 No 9:1057–1059 © Current Biology Ltd ISSN 0960-9822
The p53 protein is best known as the product of a tumour suppressor gene, but it also plays a pivotal role in the coordination of adaptive cellular responses to stress, whether due to hypoxia, metabolic disarray or DNA damage. The activation or expression of wild-type p53 in many types of cell results in cell-cycle arrest. Other types of cell, however, undergo rapid apoptotic death following the expression of wild-type p53, and there is currently intense interest in how cells choose between these two fates. These observations are not simply an artefact of in vitro cell culture systems, as in whole animals the induction of p53 protein expression and its downstream consequences are tightly regulated in distinct and tissue-specific manner [1]. Understanding these processes is of considerable practical importance, given the important role of p53 pathways in determining susceptibility to the effects of cancer radiotherapy and chemotherapy. How does p53 activate the apoptotic pathway? p53 is a nuclear phosphoprotein and a transcription factor capable of activating or repressing transcription of a wide range of target genes. It appears, however, that transcriptional regulation is only one way in which p53 can regulate growth arrest and apoptosis: several independent studies have shown that some p53 activity distinct from transcriptional activation in important for apoptosis [2–5]. Controversially, Attardi et al. [6] find no evidence for transcriptionindependent mechanisms of p53-mediated apoptosis and suggest that the interpretation of previous studies may be compromised by the presence of wild-type p53 in the cell lines used. Intriguing new results from Wang et al. [7] implicate the XPB and XPD helicases as components of a p53-mediated apoptotic pathway. These proteins, products of two of the xeroderma pigmentosum (XP) genes, are well known for their role in nucleotide excision–repair, but they are also components of the basal transcription factor TFIIH,
The constituent subunits of human ‘core’ TFIIH TFIIH component (yeast gene)
Alternative names
XPD, ERCC2
Proposed function
p62 (TFB1) p44 (SSL1)
Zinc finger (DNA binding?)
p34
Zinc finger (DNA binding?)
required for the initiation of transcription, though not for elongation, by RNA polymerase II. A ‘core’ TFIIH complex consisting of at least five polypeptides has been identified (Table 1). This core may not exist in vivo as a functionally discrete entity, but may represent a particularly stable complex of polypeptides which remain associated through fractionation steps in vitro. This core TFIIH complex participates in various functions within the cell, according to its interacting partners (Fig. 1). Figure 1
Cdk CAK
Cell-cycle regulation p53
TFIIH TFIIH CAK
Apoptosis
Coordination of DNA replication and repair
NER machinery
TFIIH p53
Regulation of transcription
DNA repair
© 1996 Current Biology
TFIIH and its partners. This diagram shows a subset of the regulatory interactions known to involve TFIIH. The core TFIIH complex is essential for the initiation of transcription, and is associated in this process with the CAK ternary complex; CAK is also required for the activity of Cdks which regulate cell-cycle progression. TFIIH is also an essential component of the nucleotide excision–repair (NER) machinery; DNA repair must be coordinated with DNA replication for the cell to maintain viability. p53-dependent transcriptional transactivation — which requires TFIIH — induces cell-cycle arrest or apoptosis. As discussed in the text, Wang et al. [7] have recently reported evidence that p53 interacts with TFIIH to induce apoptosis independently of transcriptional transactivation.
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Current Biology 1996, Vol 6 No 9
Figure 2
Transcriptional activation
Sequence-specific DNA binding
Nuclear localization sequence Carboxy-terminal domain Tetramerization
Binds TFIIH subunit p62 [8] Binds XPB/XPD [8]
The interaction of p53 with components of the core TFIIH complex. In some cases, the results may appear contradictory, but it appears that the amino terminus of p53 interacts with the p62 component of TFIIH, while the carboxyl terminus interacts with XPB and XPD. As discussed in the text, the latter interaction appears sufficient to trigger apoptosis.
Competes for binding to XPB/XPD [10] Does not induce apoptosis [7] Induces apoptosis [7] Mouse fragment induces HeLa cell apoptosis [2] © 1996 Current Biology
In its transcriptional role, TFIIH has an associated kinase activity that phosphorylates the carboxy-terminal domain of the largest subunit of RNA polymerase II. This phosphorylation is key step in the initiation of transcription in vivo, through its precise role has not yet been elucidated. The kinase activity resides in a ternary complex of MAT1, cyclin H and the cyclin-dependent kinase (Cdk) Cdk4. This ternary complex is also known Cdk-activating kinase (CAK), in which guise it is responsible for the threonine phosphorylation that is essential for Cdk activity. The observation that the same kinase complex is involved in the regulation of both transcription and the cell cycle is evidence of their co-regulation. In contrast, when TFIIH is involved in nucleotide excision–repair, it is not associated with this kinase complex, but instead forms part of a multiprotein complex — a ‘repairosome’ — with the other components of the nucleotide excision–repair pathway (Fig. 1). A new twist to the TFIIH tale has emerged with the discovery that TFIIH and p53 interact via a series of multiple protein–protein contacts (Fig. 2). The acidic transcriptional activation domains of both p53 and the viral protein VP16 bind the p62 component of TFIIH [8,9]. A carboxy-terminal region of p53 binds to XPB and XPD and this region of p53 inhibits their DNA helicase activities [7,8,10]. In its capacity as a transcriptional regulator, it is possibly not surprising that p53 interacts with the components of the transcriptional machinery. p53 is known to bind TBPassociated factors (TAFs), which are components of the transcription factor TFIID that interact with the TATAbox binding protein (TBP). It is also known to interact with several helicases — p53 was in fact first identified through its interaction with the helicase SV40 large T antigen, and it has since been found also to interact with Rad3 (the yeast XPD homologue) and with CSB (ERCC6), a helicase involved in transcription-coupled
nucleotide excision–repair [10]. Reassuringly, the recent observation that p53-mediated apoptosis is significantly reduced, though not abolished, in XPB and XPD mutant fibroblasts suggests that the postulated interaction between p53 and XPB/XPD occurs in vivo and is functionally important [7]. This effect is specifically dependent on the presence of wild-type XPB/XPD proteins, and does not appear to be due to a global defect in nucleotide excision–repair. The implication is that XPB and XPD are components of a p53-mediated apoptotic pathway. Is this form of apoptotic signalling dependent on the activation of transcription by p53? Experiments with mutant forms of human p53 showed that their apoptotic activity did not correlate with their ability to activate transcription [7]. Interestingly, loss of the carboxy-terminal 50 amino acids of p53 significantly reduced its apoptotic activity without the loss of ability to activate transcription [7]. A p53-derived carboxy-terminal peptide that includes the proposed XPB/XPD-interaction domain can induce apoptosis upon microinjection [7]. These results suggest that p53 can interact directly with XPB and XPD to induce apoptosis independently of sequence-specific transcriptional activation. In a parallel series of experiments, Haupt et al. [2] found that the amino-terminal 214 amino acids of murine p53, which lacks the ability to activate transcription, can trigger apoptosis in HeLa cells. The differences between these results and those of Wang et al. [7] may reflect differences in the experimental systems, which include the cell lines used, the state of the endogenous p53 protein — which is inactivated by binding to the human papillomavirus (HPV) E6 protein in HeLa cells — and the species of origin of the artificially expressed p53. How could the interaction of p53 with the XPB and XPD helicases act to signal apoptosis? The inhibition of XPB and XPD helicase activity by p53 in vitro suggests to the naive observer that p53 would act in the cell to inhibit
Dispatch
DNA repair and/or transcription. Although the effect of p53 activity upon rates of DNA repair is a highly controversial topic, one can conceive of a situation in which the p53–XPB/XPD interaction downregulates repair so that DNA lesions persist and are sensed by the cell, which takes them as a signal to undergo apoptosis. If this were so, then all cells with DNA-repair defects would be expected to undergo abnormally high levels of apoptosis irrespective of p53 activation, which does not appear to be the case. A second possibility is that high levels of p53 downregulate the transcriptional activity of TFIIH. The results of Wang et al. [7] indicate that the effects of mutations in XPG and XPD on p53-mediated apoptosis are unlikely to be due to a global TFIIH defect, although it is possible that certain promoters are affected differentially [7]. An alternative model has been proposed in which TFIIH binds to RNA polymerase II stalled at a site of DNA damage, and participates with the rest of the nucleotide excision–repair machinery to repair the lesion. The binding of p53 to components of TFIIH might activate p53, which would then result in a delay in cell-cycle progression or apoptosis. However, this is at odds with the observation that TFIIH acts in the initiation of transcription rather than the elongation step. In summary, it appears that a core TFIIH complex is involved in coordinating basal transcription, nucleotide excision–repair and possibly also — via its association with CAK — cell-cycle control. Recently published results suggest that TFIIH also plays a role in regulating p53mediated apoptosis, though the precise mechanism and the downstream effectors of this pathway remain to be investigated. Perhaps CAK phosphorylates p53 bound to damaged DNA and thus affects its activity. An attractive model has core TFIIH not only coordinating repair and transcription, but also integrating these processes into the cell cycle and acting in concert with p53 to signal apoptosis when cell damage is perceived as unrepairable. References 1. Midgely CA, Owens B, Briscoe CV, Brynmore Thomas D, Lane DP, Hall PA: Coupling between gamma irradiation, p53 induction and the apoptotic response depends upon cell type in vivo. J Cell Sci 1995, 108:1843–1848. 2. Haupt Y, Rowan S, Shaulian E, Vousden KH, Oren M: Induction of apoptosis in HeLa cells by trans-activation-deficient p53. Genes Dev 1995, 9:2170–2183. 3. Wagner AJ, Kokontis JM, Hay N: Myc-mediated apoptosis requires wild-type p53 in a manner independent of cell cycle arrest and the ability of p53 to induce p21waf1/cip1. Genes Dev 1994, 8:2817–2830. 4. Caelles C, Helmberg A, Karin M: p53-dependent apoptosis in the absence of transcriptional activation of p53-target genes. Nature 1994, 370:220–223. 5. Sabbatini P, Lin J, Levine AJ, White E: Essential role for p53mediated transcription in E1A-induced apoptosis. Genes Dev 1995, 9:2184–2192. 6. Attardi LD, Lowe SW, Brugarolas J, Jacks T: Transcriptional activation by p53, but not induction of the p21 gene, is essential for oncogene mediated apoptosis. EMBO J 1996, 15:3693–3701.
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7. Wang XW, Vermeulen W, Coursen JD, Gibson M, Lupold SE, Forrester K, Xu G, Elmore L, Yeh H, Hoeijmakers JHJ, Harris CC: The XPB and XPD DNA helicases are components of the p53 mediated apoptosis pathway. Genes Dev 1996, 10:1219–1232. 8. Leveillard T, Andera L, Bissonnette N, Schaeffer L, Bracco L, Egly J-M, Wasylyk B: Functional interactions between p53 and the TFIIH complex are affected by tumour associated mutations. EMBO J 1996, 15:1615–1624. 9. Xiao H, Pearson A, Coulombe B, Truant R, Zhang S, Regier JL, Triezenberg SJ, Reinberg D, Flores O, Inglis CJ, Greenblatt J: Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53. Mol Cell Biol 1994, 14:7013–7024. 10. Wang XW, Yeh H, Schaeffner L, Roy R, Moncollin V, Egly J-M, Wang Z, Friedberg EC, Evans MK, Taffe BG, et al.: p53 modulation of TFIIH-associated nucleotide excision repair activity. Nat Genet 1995, 10:188–195.
1057
Apoptosis: A new twist to the tale? Emma Warbrick
The DNA helicases XPB and XPD, components of transcription factor TFIIH, have been implicated in a p53-induced apoptotic pathway. These new findings suggest a role for the core TFIIH complex in the coordination, not only of transcription, the cell cycle and DNA repair, but also of apoptosis.
Table 1
p89 (RAD25/SSL2) XPB, ERCC3
3′–5′ DNA helicase
Address: Department of Anatomy and Physiology, Medical Sciences Institute, University of Dundee, Dundee DD1 4HN, Scotland, UK.
p80 (RAD3)
5′–3′ DNA helicase
Current Biology 1996, Vol 6 No 9:1057–1059 © Current Biology Ltd ISSN 0960-9822
The p53 protein is best known as the product of a tumour suppressor gene, but it also plays a pivotal role in the coordination of adaptive cellular responses to stress, whether due to hypoxia, metabolic disarray or DNA damage. The activation or expression of wild-type p53 in many types of cell results in cell-cycle arrest. Other types of cell, however, undergo rapid apoptotic death following the expression of wild-type p53, and there is currently intense interest in how cells choose between these two fates. These observations are not simply an artefact of in vitro cell culture systems, as in whole animals the induction of p53 protein expression and its downstream consequences are tightly regulated in distinct and tissue-specific manner [1]. Understanding these processes is of considerable practical importance, given the important role of p53 pathways in determining susceptibility to the effects of cancer radiotherapy and chemotherapy. How does p53 activate the apoptotic pathway? p53 is a nuclear phosphoprotein and a transcription factor capable of activating or repressing transcription of a wide range of target genes. It appears, however, that transcriptional regulation is only one way in which p53 can regulate growth arrest and apoptosis: several independent studies have shown that some p53 activity distinct from transcriptional activation in important for apoptosis [2–5]. Controversially, Attardi et al. [6] find no evidence for transcriptionindependent mechanisms of p53-mediated apoptosis and suggest that the interpretation of previous studies may be compromised by the presence of wild-type p53 in the cell lines used. Intriguing new results from Wang et al. [7] implicate the XPB and XPD helicases as components of a p53-mediated apoptotic pathway. These proteins, products of two of the xeroderma pigmentosum (XP) genes, are well known for their role in nucleotide excision–repair, but they are also components of the basal transcription factor TFIIH,
The constituent subunits of human ‘core’ TFIIH TFIIH component (yeast gene)
Alternative names
XPD, ERCC2
Proposed function
p62 (TFB1) p44 (SSL1)
Zinc finger (DNA binding?)
p34
Zinc finger (DNA binding?)
required for the initiation of transcription, though not for elongation, by RNA polymerase II. A ‘core’ TFIIH complex consisting of at least five polypeptides has been identified (Table 1). This core may not exist in vivo as a functionally discrete entity, but may represent a particularly stable complex of polypeptides which remain associated through fractionation steps in vitro. This core TFIIH complex participates in various functions within the cell, according to its interacting partners (Fig. 1). Figure 1
Cdk CAK
Cell-cycle regulation p53
TFIIH TFIIH CAK
Apoptosis
Coordination of DNA replication and repair
NER machinery
TFIIH p53
Regulation of transcription
DNA repair
© 1996 Current Biology
TFIIH and its partners. This diagram shows a subset of the regulatory interactions known to involve TFIIH. The core TFIIH complex is essential for the initiation of transcription, and is associated in this process with the CAK ternary complex; CAK is also required for the activity of Cdks which regulate cell-cycle progression. TFIIH is also an essential component of the nucleotide excision–repair (NER) machinery; DNA repair must be coordinated with DNA replication for the cell to maintain viability. p53-dependent transcriptional transactivation — which requires TFIIH — induces cell-cycle arrest or apoptosis. As discussed in the text, Wang et al. [7] have recently reported evidence that p53 interacts with TFIIH to induce apoptosis independently of transcriptional transactivation.
1058
Current Biology 1996, Vol 6 No 9
Figure 2
Transcriptional activation
Sequence-specific DNA binding
Nuclear localization sequence Carboxy-terminal domain Tetramerization
Binds TFIIH subunit p62 [8] Binds XPB/XPD [8]
The interaction of p53 with components of the core TFIIH complex. In some cases, the results may appear contradictory, but it appears that the amino terminus of p53 interacts with the p62 component of TFIIH, while the carboxyl terminus interacts with XPB and XPD. As discussed in the text, the latter interaction appears sufficient to trigger apoptosis.
Competes for binding to XPB/XPD [10] Does not induce apoptosis [7] Induces apoptosis [7] Mouse fragment induces HeLa cell apoptosis [2] © 1996 Current Biology
In its transcriptional role, TFIIH has an associated kinase activity that phosphorylates the carboxy-terminal domain of the largest subunit of RNA polymerase II. This phosphorylation is key step in the initiation of transcription in vivo, through its precise role has not yet been elucidated. The kinase activity resides in a ternary complex of MAT1, cyclin H and the cyclin-dependent kinase (Cdk) Cdk4. This ternary complex is also known Cdk-activating kinase (CAK), in which guise it is responsible for the threonine phosphorylation that is essential for Cdk activity. The observation that the same kinase complex is involved in the regulation of both transcription and the cell cycle is evidence of their co-regulation. In contrast, when TFIIH is involved in nucleotide excision–repair, it is not associated with this kinase complex, but instead forms part of a multiprotein complex — a ‘repairosome’ — with the other components of the nucleotide excision–repair pathway (Fig. 1). A new twist to the TFIIH tale has emerged with the discovery that TFIIH and p53 interact via a series of multiple protein–protein contacts (Fig. 2). The acidic transcriptional activation domains of both p53 and the viral protein VP16 bind the p62 component of TFIIH [8,9]. A carboxy-terminal region of p53 binds to XPB and XPD and this region of p53 inhibits their DNA helicase activities [7,8,10]. In its capacity as a transcriptional regulator, it is possibly not surprising that p53 interacts with the components of the transcriptional machinery. p53 is known to bind TBPassociated factors (TAFs), which are components of the transcription factor TFIID that interact with the TATAbox binding protein (TBP). It is also known to interact with several helicases — p53 was in fact first identified through its interaction with the helicase SV40 large T antigen, and it has since been found also to interact with Rad3 (the yeast XPD homologue) and with CSB (ERCC6), a helicase involved in transcription-coupled
nucleotide excision–repair [10]. Reassuringly, the recent observation that p53-mediated apoptosis is significantly reduced, though not abolished, in XPB and XPD mutant fibroblasts suggests that the postulated interaction between p53 and XPB/XPD occurs in vivo and is functionally important [7]. This effect is specifically dependent on the presence of wild-type XPB/XPD proteins, and does not appear to be due to a global defect in nucleotide excision–repair. The implication is that XPB and XPD are components of a p53-mediated apoptotic pathway. Is this form of apoptotic signalling dependent on the activation of transcription by p53? Experiments with mutant forms of human p53 showed that their apoptotic activity did not correlate with their ability to activate transcription [7]. Interestingly, loss of the carboxy-terminal 50 amino acids of p53 significantly reduced its apoptotic activity without the loss of ability to activate transcription [7]. A p53-derived carboxy-terminal peptide that includes the proposed XPB/XPD-interaction domain can induce apoptosis upon microinjection [7]. These results suggest that p53 can interact directly with XPB and XPD to induce apoptosis independently of sequence-specific transcriptional activation. In a parallel series of experiments, Haupt et al. [2] found that the amino-terminal 214 amino acids of murine p53, which lacks the ability to activate transcription, can trigger apoptosis in HeLa cells. The differences between these results and those of Wang et al. [7] may reflect differences in the experimental systems, which include the cell lines used, the state of the endogenous p53 protein — which is inactivated by binding to the human papillomavirus (HPV) E6 protein in HeLa cells — and the species of origin of the artificially expressed p53. How could the interaction of p53 with the XPB and XPD helicases act to signal apoptosis? The inhibition of XPB and XPD helicase activity by p53 in vitro suggests to the naive observer that p53 would act in the cell to inhibit
Dispatch
DNA repair and/or transcription. Although the effect of p53 activity upon rates of DNA repair is a highly controversial topic, one can conceive of a situation in which the p53–XPB/XPD interaction downregulates repair so that DNA lesions persist and are sensed by the cell, which takes them as a signal to undergo apoptosis. If this were so, then all cells with DNA-repair defects would be expected to undergo abnormally high levels of apoptosis irrespective of p53 activation, which does not appear to be the case. A second possibility is that high levels of p53 downregulate the transcriptional activity of TFIIH. The results of Wang et al. [7] indicate that the effects of mutations in XPG and XPD on p53-mediated apoptosis are unlikely to be due to a global TFIIH defect, although it is possible that certain promoters are affected differentially [7]. An alternative model has been proposed in which TFIIH binds to RNA polymerase II stalled at a site of DNA damage, and participates with the rest of the nucleotide excision–repair machinery to repair the lesion. The binding of p53 to components of TFIIH might activate p53, which would then result in a delay in cell-cycle progression or apoptosis. However, this is at odds with the observation that TFIIH acts in the initiation of transcription rather than the elongation step. In summary, it appears that a core TFIIH complex is involved in coordinating basal transcription, nucleotide excision–repair and possibly also — via its association with CAK — cell-cycle control. Recently published results suggest that TFIIH also plays a role in regulating p53mediated apoptosis, though the precise mechanism and the downstream effectors of this pathway remain to be investigated. Perhaps CAK phosphorylates p53 bound to damaged DNA and thus affects its activity. An attractive model has core TFIIH not only coordinating repair and transcription, but also integrating these processes into the cell cycle and acting in concert with p53 to signal apoptosis when cell damage is perceived as unrepairable. References 1. Midgely CA, Owens B, Briscoe CV, Brynmore Thomas D, Lane DP, Hall PA: Coupling between gamma irradiation, p53 induction and the apoptotic response depends upon cell type in vivo. J Cell Sci 1995, 108:1843–1848. 2. Haupt Y, Rowan S, Shaulian E, Vousden KH, Oren M: Induction of apoptosis in HeLa cells by trans-activation-deficient p53. Genes Dev 1995, 9:2170–2183. 3. Wagner AJ, Kokontis JM, Hay N: Myc-mediated apoptosis requires wild-type p53 in a manner independent of cell cycle arrest and the ability of p53 to induce p21waf1/cip1. Genes Dev 1994, 8:2817–2830. 4. Caelles C, Helmberg A, Karin M: p53-dependent apoptosis in the absence of transcriptional activation of p53-target genes. Nature 1994, 370:220–223. 5. Sabbatini P, Lin J, Levine AJ, White E: Essential role for p53mediated transcription in E1A-induced apoptosis. Genes Dev 1995, 9:2184–2192. 6. Attardi LD, Lowe SW, Brugarolas J, Jacks T: Transcriptional activation by p53, but not induction of the p21 gene, is essential for oncogene mediated apoptosis. EMBO J 1996, 15:3693–3701.
1059
7. Wang XW, Vermeulen W, Coursen JD, Gibson M, Lupold SE, Forrester K, Xu G, Elmore L, Yeh H, Hoeijmakers JHJ, Harris CC: The XPB and XPD DNA helicases are components of the p53 mediated apoptosis pathway. Genes Dev 1996, 10:1219–1232. 8. Leveillard T, Andera L, Bissonnette N, Schaeffer L, Bracco L, Egly J-M, Wasylyk B: Functional interactions between p53 and the TFIIH complex are affected by tumour associated mutations. EMBO J 1996, 15:1615–1624. 9. Xiao H, Pearson A, Coulombe B, Truant R, Zhang S, Regier JL, Triezenberg SJ, Reinberg D, Flores O, Inglis CJ, Greenblatt J: Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53. Mol Cell Biol 1994, 14:7013–7024. 10. Wang XW, Yeh H, Schaeffner L, Roy R, Moncollin V, Egly J-M, Wang Z, Friedberg EC, Evans MK, Taffe BG, et al.: p53 modulation of TFIIH-associated nucleotide excision repair activity. Nat Genet 1995, 10:188–195.