- Email: [email protected]
Stemming Danger with Golgified BAX
Molecular Cell
Previews REFERENCES Belitsky, M., Avshalom, H., Erental, A., Yelin, I., Kumar, S., London, N., Sperber, M., Schueler-Furman, O., and Engelberg-Kulka, H. (2011). Mol. Cell 41, 625–635. Carmona-Gutierrez, D., Eisenberg, T., Bu¨ttner, S., Meisinger, C., Kroemer, G., and Madeo, F. (2010). Cell Death Differ. 17, 763–773. Dwyer, D.J., Camacho, D.M., Kohanski, M.A., Callura, J.M., and Collins, J.J. (2012). Mol. Cell 46, this issue, 561–572.
Dwyer, D.J., Kohanski, M.A., Hayete, B., and Collins, J.J. (2007). Mol. Syst. Biol. 3, 91. Erental, A., Sharon, I., and Engelberg-Kulka, H. (2012). PLoS Biol. 10, e1001281. Galluzzi, L., Kepp, O., Trojel-Hansen, C., and Kroemer, G. (2012). EMBO Rep. 13, 322–330. Herker, E., Jungwirth, H., Lehmann, K.A., Maldener, C., Fro¨hlich, K.U., Wissing, S., Bu¨ttner, S., Fehr, M., Sigrist, S., and Madeo, F. (2004). J. Cell Biol. 164, 501–507.
Kohanski, M.A., Dwyer, D.J., Hayete, B., Lawrence, C.A., and Collins, J.J. (2007). Cell 130, 797–810. Kolodkin-Gal, I., and Engelberg-Kulka, H. (2006). J. Bacteriol. 188, 3420–3423. Madeo, F., Fro¨hlich, E., and Fro¨hlich, K.U. (1997). J. Cell Biol. 139, 729–734. Simon, H.U., Haj-Yehia, A., and Levi-Schaffer, F. (2000). Apoptosis 5, 415–418.
Stemming Danger with Golgified BAX Loren D. Walensky1,* 1Department of Pediatric Oncology and the Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA *Correspondence: [email protected] DOI 10.1016/j.molcel.2012.05.034
In this issue of Molecular Cell, Dumitru et al. (2012) report that hES cells localize a conformationally activated form of proapoptotic BAX to the trans Golgi network, a previously unanticipated launch pad for mitochondrial assault in response to DNA damage.
BCL-2 family proteins are charged with protecting the organism from unwanted cellular excess or demise. To achieve the proper balance, antiapoptotic BCL-2 family proteins such as BCL-XL have the capacity to protect mitochondria, the power plants of the cell, from permeabilization by proapoptotic members such as BAX, thereby preserving cell survival. Conversely, when persistence of damaged cells threatens the organism, activated proapoptotic proteins with the ability to form destructive mitochondrial pores become the saviors, eliminating renegade cells for the benefit of the whole. To render the appropriate lifedeath decision in response to a litany of cellular stressors across a broad diversity of tissues, BCL-2 family proteins are subject to exquisite regulation. In particular, pore-forming proapoptotic members, such as BAX, must be carefully controlled to avoid wanton activation and cellular destruction, yet stand ready for rapid deployment in the face of threatening external and internal stimuli. Dumitru et al. (2012) report the provocative
finding that a conformationally activated form of BAX specifically localizes to the trans Golgi network (TGN) in human embryonic stem (hES) cells, enabling rapid apoptosis of DNA-damaged stem cells to potentially avoid the developmental consequences of menacing genetic defects. To date, a variety of mechanisms have been implicated in BAX regulation. Chief among them is the autoinhibitory structure of BAX itself, which buries the hydrophobic pore-forming surfaces at the core of the protein (Suzuki et al., 2000). Only when triggered by a change in physiologic conditions, such as pH (Khaled et al., 1999) or heat (Pagliari et al., 2005), or directly activated through protein interaction (Gavathiotis et al., 2010), does a major conformational change ensue, moving BAX from cytosol to mitochondria to exert its proapoptotic effect (Figure 1, top). Thus, inherent in the BAX activation mechanism is regulation by subcellular localization (Wolter et al., 1997). An autoactivated isoform of BAX, BAX-b, eliminates this cytosolic
554 Molecular Cell 46, June 8, 2012 ª2012 Elsevier Inc.
step, existing in a tonically mobilized form that constitutively targets the mitochondria (Fu et al., 2009). Once at the mitochondria, activated BAX can be restrained, at least temporarily, through sequestration of its critical death domain by a specialized groove on the surface of antiapoptotic BCL-2 family proteins (Sattler et al., 1997). Antiapoptotic protein shuttling of BAX from mitochondria back to the cytosol or ‘‘retrotranslocation’’ has recently emerged as another BAXsuppressive mechanism (Edlich et al., 2011). In the case of BAX-b, proteosomal degradation is a key mode of negative regulation (Fu et al., 2009). Dumitru et al. (2012) find that, in the uniquely privileged context of embryonic stem cells, BAX has a previously unrecognized subcellular localization and mode of action. Motivated by elucidating the mechanistic basis for the especially rapid apoptotic response of hES cells to DNA damage, the authors employed shRNA analyses to implicate BAX, rather than BAK, as the driving executioner protein. Surprisingly, BAX was already at least
Molecular Cell
Previews
Figure 1. A Novel Localization and Mode of Regulation for Proapoptotic BAX in hES Cells (Top) In differentiated cells, BAX resides inactive in the cytosol until triggered by stress stimuli, such as DNA damage. BAX then undergoes a major conformational change, revealing an N-terminal epitope recognized by the 6A7 antibody, releasing its C-terminal helix for mitochondrial outer membrane insertion, and exposing its proapoptotic BH3 domain (Gavathiotis et al., 2010). Self-association of conformationally activated BAX porates the mitochondrion, which releases key apoptogenic factors such as cytochrome c. (Bottom) Dumitru et al. (2012) find that 6A7-positive BAX is constitutively localized to the trans Golgi network in hES cells and translocates to the mitochondrion (in a p53-dependent fashion) upon DNA damage, yielding an especially rapid apoptotic response. The observed tonic acetylation of Ku70 may contribute to the disinhibition of BAX in this context. Thus, hES cells appear to harbor a unique mode of BAX regulation that may serve to protect the organism from propagating harmful mutations at the earliest stages of embryonic development.
partially activated in untreated hES cells, as detected by the conformationspecific 6A7 anti-BAX antibody (Hsu and Youle, 1997). Subcellular organelle colocalization studies mapped the translocation of activated BAX from the TGN to the mitochondria upon apoptotic stimulation with DNA-damaging agents (Figure 1, bottom). Strikingly, this phenomenon was unique to hES cells. Upon early hES cell differentiation, exposure of the BAX activation epitope, the TGN localization, and the rapid apoptotic response to DNA damage were no longer observed. The authors propose that this novel localization of conformationally activated BAX in unstimulated hES cells enables a unique mechanism for rapid apoptosis in response to DNA damage, so that propagation of mutations at the most critical stages of embryonic development can be avoided. This unanticipated finding raises many intriguing questions. What preactivates
BAX in untreated hES cells? How is BAX preferentially (and constitutively) targeted to the TGN? What keeps BAX in check at the TGN such that membrane integrity and organeller function is not disrupted? In response to DNA damage, how is TGN-associated BAX trafficked to mitochondria? How does hES differentiation dissolve the pathway? Dumitru et al. (2012) provide several mechanistic leads that warrant further exploration and integration. First, conformationally active BAX at the TGN was exclusively associated with hES cells in S phase of the cell cycle. Second, both the rapid apoptotic response to DNA damage and BAX translocation from TGN to mitochondria was p53 dependent, as demonstrated by p53 knockdown studies. How p53 specifically regulates BAX in this context is unresolved. Whereas the rapid kinetics of the p53-dependent BAX translocation and apoptotic response seemed inconsistent with a p53 trans-
criptional effect, the activity was indeed blocked by inhibition of protein translation with cycloheximide. Finally, the disposition of BAX at the TGN was not impacted by pharmacologic BCL-2/ BCL-XL inhibition and appeared enabled by constitutive acetylation of Ku70, preventing its inhibitory interaction with BAX (Figure 1, bottom). The localization of BAX at the TGN in unstimulated hES cells provides yet another example of the complexity and multidimensionality of the BCL-2 family interaction network. With emerging apoptotic and nonapoptotic roles for BCL-2 family proteins in a diversity of subcellular localizations, including the mitochondria, endoplasmic reticulum, and nucleus, this work further raises the question of whether BAX contributes to the architecture and homeostatic function of the TGN in hES cells. Nevertheless, to stem the danger from DNA damage, embryonic stem cells appear to position
Molecular Cell 46, June 8, 2012 ª2012 Elsevier Inc. 555
Molecular Cell
Previews conformationally active BAX at a unique TGN launch pad for mitochondrial apoptosis. REFERENCES
Fu, N.Y., Sukumaran, S.K., Kerk, S.Y., and Yu, V.C. (2009). Mol. Cell 33, 15–29.
Pagliari, L.J., Kuwana, T., Bonzon, C., Newmeyer, D.D., Tu, S., Beere, H.M., and Green, D.R. (2005). Proc. Natl. Acad. Sci. USA 102, 17975–17980.
Gavathiotis, E., Reyna, D.E., Davis, M.L., Bird, G.H., and Walensky, L.D. (2010). Mol. Cell 40, 481–492.
Sattler, M., Liang, H., Nettesheim, D., Meadows, R.P., Harlan, J.E., Eberstadt, M., Yoon, H.S., Shuker, S.B., Chang, B.S., Minn, A.J., et al. (1997). Science 275, 983–986.
Dumitru, R., Gama, V., Fagan, B.M., Bower, J.J., Swahari, V., Pevny, L.H., and Deshmukh, M. (2012). Mol. Cell 46, this issue, 573–583.
Hsu, Y.T., and Youle, R.J. (1997). J. Biol. Chem. 23, 13,829–13,834.
Edlich, F., Banerjee, S., Suzuki, M., Cleland, M.M., Arnoult, D., Wang, C., Neutzner, A., Tjandra, N., and Youle, R.J. (2011). Cell 145, 104–116.
Khaled, A.R., Kim, K., Hofmeister, R., Muegge, K., and Durum, S.K. (1999). Proc. Natl. Acad. Sci. USA 96, 14476–14481.
Suzuki, M., Youle, R.J., and Tjandra, N. (2000). Cell 103, 645–654. Wolter, K.G., Hsu, Y.T., Smith, C.L., Nechushtan, A., Xi, X.G., and Youle, R.J. (1997). J. Cell Biol. 139, 1281–1292.
TGF-b Drives DNA Demethylation David Wotton1,* 1Department of Biochemistry and Molecular Genetics, and Center for Cell Signaling, University of Virginia, Charlottesville, VA 22908, USA *Correspondence: [email protected] DOI 10.1016/j.molcel.2012.05.031
In this issue of Molecular Cell, Thillainadesan et al. (2012) provide evidence that Smad proteins promote locus-specific active DNA demethylation as part of the transforming growth factor b (TGF-b) transcriptional response. DNA methylation is a well-known mechanism for gene silencing, but comparatively little is known about how DNA methylation is rapidly reversed in a locus-specific manner (Wu and Zhang, 2010). The loss of cytosine methylation is thought to occur primarily passively, as DNA is replicated. Although there are some examples of active DNA demethylation during embryonic development, these events are not gene specific, and still occur relatively slowly compared to the dynamic histone modifications associated with induction of gene expression. Direct removal of the methyl group from 5-methylcytosine (5mC) is unlikely, given the strength of the carbon-carbon bond, and until recently there has been no clear consensus for an alternative mechanism. However, some instances of active demethylation have been well documented. For example, a dynamic pattern of DNA demethylation and remethylation at the pS2/TFF1 gene correlates with cycles of estrogen receptor binding and gene expression (Metivier et al., 2008). Recruitment of the thymine DNA glycosylase (TDG) and enzymes of the break excision
repair (BER) pathway is required for pS2/ TFF1 demethylation and expression. Such evidence of dynamic gene-specific active demethylation, together with recent work on the mechanisms by which 5mC may be sequentially modified, excised, and replaced by the DNA repair machinery, has helped build an emerging consensus on the mechanism of active DNA demethylation (Wu and Zhang, 2011). The work by Torchia and colleagues adds to the small, but growing, body of evidence that rapid, genespecific active DNA demethylation can be directed by transcription factor complexes as part of the response to signaling (Thillainadesan et al., 2012). Thillainadesan et al. (2012) focus on the regulation of the p15ink4b gene, which is induced as part of the TGF-b antiproliferative response. The mechanisms of p15ink4b induction by TGF-b are complex, and include direct binding of the Smad transcription factors to the proximal promoter, and to a further upstream element, in association with forkhead cofactors (Gomis et al., 2006). The upstream Smad/forkhead binding element is close
556 Molecular Cell 46, June 8, 2012 ª2012 Elsevier Inc.
to a CpG island that can be methylated when p15ink4a expression is silenced. Thillainadesan et al. show that a corepressor complex consisting of the oncogene, ZNF217; CoREST; and the DNA methyltransferase, Dnmt3a, directs promoter methylation and gene silencing. Interestingly, following TGF-b addition there is a rapid decrease in p15ink4a methylation, which is detectable within 20 min and is independent of DNA replication, clearly pointing to an active mechanism. The CoREST complex and Dnmt3a leave the p15ink4a locus and are replaced by the TGF-b-responsive Smad transcription factors (Figure 1A). With the Smads, there is corecruitment of some of the usual coactivators, such as CBP, and also of the enzymes that can facilitate DNA demethylation. This includes the cytidine deaminase, AID; the DNA glycosylases, TDG and MBD4; and components of the BER machinery. Although the mechanisms of active DNA methylation are becoming clearer, not everything is fully resolved yet. The TET (ten eleven translocation) proteins, which are methylcytosine dioxygenases,
Previews REFERENCES Belitsky, M., Avshalom, H., Erental, A., Yelin, I., Kumar, S., London, N., Sperber, M., Schueler-Furman, O., and Engelberg-Kulka, H. (2011). Mol. Cell 41, 625–635. Carmona-Gutierrez, D., Eisenberg, T., Bu¨ttner, S., Meisinger, C., Kroemer, G., and Madeo, F. (2010). Cell Death Differ. 17, 763–773. Dwyer, D.J., Camacho, D.M., Kohanski, M.A., Callura, J.M., and Collins, J.J. (2012). Mol. Cell 46, this issue, 561–572.
Dwyer, D.J., Kohanski, M.A., Hayete, B., and Collins, J.J. (2007). Mol. Syst. Biol. 3, 91. Erental, A., Sharon, I., and Engelberg-Kulka, H. (2012). PLoS Biol. 10, e1001281. Galluzzi, L., Kepp, O., Trojel-Hansen, C., and Kroemer, G. (2012). EMBO Rep. 13, 322–330. Herker, E., Jungwirth, H., Lehmann, K.A., Maldener, C., Fro¨hlich, K.U., Wissing, S., Bu¨ttner, S., Fehr, M., Sigrist, S., and Madeo, F. (2004). J. Cell Biol. 164, 501–507.
Kohanski, M.A., Dwyer, D.J., Hayete, B., Lawrence, C.A., and Collins, J.J. (2007). Cell 130, 797–810. Kolodkin-Gal, I., and Engelberg-Kulka, H. (2006). J. Bacteriol. 188, 3420–3423. Madeo, F., Fro¨hlich, E., and Fro¨hlich, K.U. (1997). J. Cell Biol. 139, 729–734. Simon, H.U., Haj-Yehia, A., and Levi-Schaffer, F. (2000). Apoptosis 5, 415–418.
Stemming Danger with Golgified BAX Loren D. Walensky1,* 1Department of Pediatric Oncology and the Program in Cancer Chemical Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02215, USA *Correspondence: [email protected] DOI 10.1016/j.molcel.2012.05.034
In this issue of Molecular Cell, Dumitru et al. (2012) report that hES cells localize a conformationally activated form of proapoptotic BAX to the trans Golgi network, a previously unanticipated launch pad for mitochondrial assault in response to DNA damage.
BCL-2 family proteins are charged with protecting the organism from unwanted cellular excess or demise. To achieve the proper balance, antiapoptotic BCL-2 family proteins such as BCL-XL have the capacity to protect mitochondria, the power plants of the cell, from permeabilization by proapoptotic members such as BAX, thereby preserving cell survival. Conversely, when persistence of damaged cells threatens the organism, activated proapoptotic proteins with the ability to form destructive mitochondrial pores become the saviors, eliminating renegade cells for the benefit of the whole. To render the appropriate lifedeath decision in response to a litany of cellular stressors across a broad diversity of tissues, BCL-2 family proteins are subject to exquisite regulation. In particular, pore-forming proapoptotic members, such as BAX, must be carefully controlled to avoid wanton activation and cellular destruction, yet stand ready for rapid deployment in the face of threatening external and internal stimuli. Dumitru et al. (2012) report the provocative
finding that a conformationally activated form of BAX specifically localizes to the trans Golgi network (TGN) in human embryonic stem (hES) cells, enabling rapid apoptosis of DNA-damaged stem cells to potentially avoid the developmental consequences of menacing genetic defects. To date, a variety of mechanisms have been implicated in BAX regulation. Chief among them is the autoinhibitory structure of BAX itself, which buries the hydrophobic pore-forming surfaces at the core of the protein (Suzuki et al., 2000). Only when triggered by a change in physiologic conditions, such as pH (Khaled et al., 1999) or heat (Pagliari et al., 2005), or directly activated through protein interaction (Gavathiotis et al., 2010), does a major conformational change ensue, moving BAX from cytosol to mitochondria to exert its proapoptotic effect (Figure 1, top). Thus, inherent in the BAX activation mechanism is regulation by subcellular localization (Wolter et al., 1997). An autoactivated isoform of BAX, BAX-b, eliminates this cytosolic
554 Molecular Cell 46, June 8, 2012 ª2012 Elsevier Inc.
step, existing in a tonically mobilized form that constitutively targets the mitochondria (Fu et al., 2009). Once at the mitochondria, activated BAX can be restrained, at least temporarily, through sequestration of its critical death domain by a specialized groove on the surface of antiapoptotic BCL-2 family proteins (Sattler et al., 1997). Antiapoptotic protein shuttling of BAX from mitochondria back to the cytosol or ‘‘retrotranslocation’’ has recently emerged as another BAXsuppressive mechanism (Edlich et al., 2011). In the case of BAX-b, proteosomal degradation is a key mode of negative regulation (Fu et al., 2009). Dumitru et al. (2012) find that, in the uniquely privileged context of embryonic stem cells, BAX has a previously unrecognized subcellular localization and mode of action. Motivated by elucidating the mechanistic basis for the especially rapid apoptotic response of hES cells to DNA damage, the authors employed shRNA analyses to implicate BAX, rather than BAK, as the driving executioner protein. Surprisingly, BAX was already at least
Molecular Cell
Previews
Figure 1. A Novel Localization and Mode of Regulation for Proapoptotic BAX in hES Cells (Top) In differentiated cells, BAX resides inactive in the cytosol until triggered by stress stimuli, such as DNA damage. BAX then undergoes a major conformational change, revealing an N-terminal epitope recognized by the 6A7 antibody, releasing its C-terminal helix for mitochondrial outer membrane insertion, and exposing its proapoptotic BH3 domain (Gavathiotis et al., 2010). Self-association of conformationally activated BAX porates the mitochondrion, which releases key apoptogenic factors such as cytochrome c. (Bottom) Dumitru et al. (2012) find that 6A7-positive BAX is constitutively localized to the trans Golgi network in hES cells and translocates to the mitochondrion (in a p53-dependent fashion) upon DNA damage, yielding an especially rapid apoptotic response. The observed tonic acetylation of Ku70 may contribute to the disinhibition of BAX in this context. Thus, hES cells appear to harbor a unique mode of BAX regulation that may serve to protect the organism from propagating harmful mutations at the earliest stages of embryonic development.
partially activated in untreated hES cells, as detected by the conformationspecific 6A7 anti-BAX antibody (Hsu and Youle, 1997). Subcellular organelle colocalization studies mapped the translocation of activated BAX from the TGN to the mitochondria upon apoptotic stimulation with DNA-damaging agents (Figure 1, bottom). Strikingly, this phenomenon was unique to hES cells. Upon early hES cell differentiation, exposure of the BAX activation epitope, the TGN localization, and the rapid apoptotic response to DNA damage were no longer observed. The authors propose that this novel localization of conformationally activated BAX in unstimulated hES cells enables a unique mechanism for rapid apoptosis in response to DNA damage, so that propagation of mutations at the most critical stages of embryonic development can be avoided. This unanticipated finding raises many intriguing questions. What preactivates
BAX in untreated hES cells? How is BAX preferentially (and constitutively) targeted to the TGN? What keeps BAX in check at the TGN such that membrane integrity and organeller function is not disrupted? In response to DNA damage, how is TGN-associated BAX trafficked to mitochondria? How does hES differentiation dissolve the pathway? Dumitru et al. (2012) provide several mechanistic leads that warrant further exploration and integration. First, conformationally active BAX at the TGN was exclusively associated with hES cells in S phase of the cell cycle. Second, both the rapid apoptotic response to DNA damage and BAX translocation from TGN to mitochondria was p53 dependent, as demonstrated by p53 knockdown studies. How p53 specifically regulates BAX in this context is unresolved. Whereas the rapid kinetics of the p53-dependent BAX translocation and apoptotic response seemed inconsistent with a p53 trans-
criptional effect, the activity was indeed blocked by inhibition of protein translation with cycloheximide. Finally, the disposition of BAX at the TGN was not impacted by pharmacologic BCL-2/ BCL-XL inhibition and appeared enabled by constitutive acetylation of Ku70, preventing its inhibitory interaction with BAX (Figure 1, bottom). The localization of BAX at the TGN in unstimulated hES cells provides yet another example of the complexity and multidimensionality of the BCL-2 family interaction network. With emerging apoptotic and nonapoptotic roles for BCL-2 family proteins in a diversity of subcellular localizations, including the mitochondria, endoplasmic reticulum, and nucleus, this work further raises the question of whether BAX contributes to the architecture and homeostatic function of the TGN in hES cells. Nevertheless, to stem the danger from DNA damage, embryonic stem cells appear to position
Molecular Cell 46, June 8, 2012 ª2012 Elsevier Inc. 555
Molecular Cell
Previews conformationally active BAX at a unique TGN launch pad for mitochondrial apoptosis. REFERENCES
Fu, N.Y., Sukumaran, S.K., Kerk, S.Y., and Yu, V.C. (2009). Mol. Cell 33, 15–29.
Pagliari, L.J., Kuwana, T., Bonzon, C., Newmeyer, D.D., Tu, S., Beere, H.M., and Green, D.R. (2005). Proc. Natl. Acad. Sci. USA 102, 17975–17980.
Gavathiotis, E., Reyna, D.E., Davis, M.L., Bird, G.H., and Walensky, L.D. (2010). Mol. Cell 40, 481–492.
Sattler, M., Liang, H., Nettesheim, D., Meadows, R.P., Harlan, J.E., Eberstadt, M., Yoon, H.S., Shuker, S.B., Chang, B.S., Minn, A.J., et al. (1997). Science 275, 983–986.
Dumitru, R., Gama, V., Fagan, B.M., Bower, J.J., Swahari, V., Pevny, L.H., and Deshmukh, M. (2012). Mol. Cell 46, this issue, 573–583.
Hsu, Y.T., and Youle, R.J. (1997). J. Biol. Chem. 23, 13,829–13,834.
Edlich, F., Banerjee, S., Suzuki, M., Cleland, M.M., Arnoult, D., Wang, C., Neutzner, A., Tjandra, N., and Youle, R.J. (2011). Cell 145, 104–116.
Khaled, A.R., Kim, K., Hofmeister, R., Muegge, K., and Durum, S.K. (1999). Proc. Natl. Acad. Sci. USA 96, 14476–14481.
Suzuki, M., Youle, R.J., and Tjandra, N. (2000). Cell 103, 645–654. Wolter, K.G., Hsu, Y.T., Smith, C.L., Nechushtan, A., Xi, X.G., and Youle, R.J. (1997). J. Cell Biol. 139, 1281–1292.
TGF-b Drives DNA Demethylation David Wotton1,* 1Department of Biochemistry and Molecular Genetics, and Center for Cell Signaling, University of Virginia, Charlottesville, VA 22908, USA *Correspondence: [email protected] DOI 10.1016/j.molcel.2012.05.031
In this issue of Molecular Cell, Thillainadesan et al. (2012) provide evidence that Smad proteins promote locus-specific active DNA demethylation as part of the transforming growth factor b (TGF-b) transcriptional response. DNA methylation is a well-known mechanism for gene silencing, but comparatively little is known about how DNA methylation is rapidly reversed in a locus-specific manner (Wu and Zhang, 2010). The loss of cytosine methylation is thought to occur primarily passively, as DNA is replicated. Although there are some examples of active DNA demethylation during embryonic development, these events are not gene specific, and still occur relatively slowly compared to the dynamic histone modifications associated with induction of gene expression. Direct removal of the methyl group from 5-methylcytosine (5mC) is unlikely, given the strength of the carbon-carbon bond, and until recently there has been no clear consensus for an alternative mechanism. However, some instances of active demethylation have been well documented. For example, a dynamic pattern of DNA demethylation and remethylation at the pS2/TFF1 gene correlates with cycles of estrogen receptor binding and gene expression (Metivier et al., 2008). Recruitment of the thymine DNA glycosylase (TDG) and enzymes of the break excision
repair (BER) pathway is required for pS2/ TFF1 demethylation and expression. Such evidence of dynamic gene-specific active demethylation, together with recent work on the mechanisms by which 5mC may be sequentially modified, excised, and replaced by the DNA repair machinery, has helped build an emerging consensus on the mechanism of active DNA demethylation (Wu and Zhang, 2011). The work by Torchia and colleagues adds to the small, but growing, body of evidence that rapid, genespecific active DNA demethylation can be directed by transcription factor complexes as part of the response to signaling (Thillainadesan et al., 2012). Thillainadesan et al. (2012) focus on the regulation of the p15ink4b gene, which is induced as part of the TGF-b antiproliferative response. The mechanisms of p15ink4b induction by TGF-b are complex, and include direct binding of the Smad transcription factors to the proximal promoter, and to a further upstream element, in association with forkhead cofactors (Gomis et al., 2006). The upstream Smad/forkhead binding element is close
556 Molecular Cell 46, June 8, 2012 ª2012 Elsevier Inc.
to a CpG island that can be methylated when p15ink4a expression is silenced. Thillainadesan et al. show that a corepressor complex consisting of the oncogene, ZNF217; CoREST; and the DNA methyltransferase, Dnmt3a, directs promoter methylation and gene silencing. Interestingly, following TGF-b addition there is a rapid decrease in p15ink4a methylation, which is detectable within 20 min and is independent of DNA replication, clearly pointing to an active mechanism. The CoREST complex and Dnmt3a leave the p15ink4a locus and are replaced by the TGF-b-responsive Smad transcription factors (Figure 1A). With the Smads, there is corecruitment of some of the usual coactivators, such as CBP, and also of the enzymes that can facilitate DNA demethylation. This includes the cytidine deaminase, AID; the DNA glycosylases, TDG and MBD4; and components of the BER machinery. Although the mechanisms of active DNA methylation are becoming clearer, not everything is fully resolved yet. The TET (ten eleven translocation) proteins, which are methylcytosine dioxygenases,