- Email: [email protected]
All Things to All People
Chen, S.X., Cherry, A., Tari, P.K., Podgorski, K., Kwong, Y.K.K., and Haas, K. (2012). Cell 151, this issue, 41–55.
triou, E., Bear, D.M., and Greenberg, M.E. (2008). Neuron 60, 1022–1038.
Dunfield, D., and Haas, K. (2009). Neuron 64, 240–250.
Huang, Y.Y., Colino, A., Selig, D.K., and Malenka, R.C. (1992). Science 255, 730–733.
Flavell, S.W., Kim, T.K., Gray, J.M., Harmin, D.A., Hemberg, M., Hong, E.J., Markenscoff-Papadimi-
Kirkwood, A., Rioult, M.C., and Bear, M.F. (1996). Nature 381, 526–528.
Li, Z., and Sheng, M. (2012). Mol. Brain 5, 15. Niell, C.M., Meyer, M.P., and Smith, S.J. (2004). Nat. Neurosci. 7, 254–260. Schwartz, N., Schohl, A., and Ruthazer, E.S. (2009). Neuron 62, 655–669.
All Things to All People Trevor D. Littlewood,1 Peter Kreuzaler,1 and Gerard I. Evan1,* 1Department
of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cell.2012.09.006
Myc is an enigma wrapped in a mystery. Attempts to identify Myc target genes, particularly in cancer, have been fraught with dead ends and context-specific functions. Lin et al. and Nie et al. address this conundrum by showing that Myc acts to amplify the output of existing transcriptionally active genes. Originally discovered as a stowaway in some defective avian retroviruses that acutely elicit myelocytic leukemia, Myc rose to notoriety both as the prototypical cooperating oncogene that, together with Ras, can oncogenically transform fibroblasts in vitro and as one of the immediate early growth response genes that are rapidly induced in various cell types upon mitogenic stimulation. Myc is a member of a class of dimeric transcription factor— the basic-helix-loop-helix-leucine zipper proteins and, as a heterodimer with its partner protein Max, binds DNA, with a predilection for a palindromic E-box element CACGTG. Myc is widely, almost universally, present in proliferating normal somatic cells and expression of Myc protein and mRNA, both of which are very short-lived, are continuously dependent upon mitogenic signaling. By contrast, Myc expression in cancer cells is typically deregulated and elevated. Finally, ectopic expression of Myc is, alone, able to induce proliferation of many adult somatic cell types, whereas cells denuded of Myc, whether normal or neoplastic, replicate very slowly, if at all. Distill this heady brew together, and we are left with a transcription factor that exerts its biological activity through the
modulation of target genes involved in cell replication. Since then, however, our understanding of Myc has been painfully slow. Efforts to identify Myc target genes suggest that Myc modulates up to a third of the transcriptome. Whatever the latest trend in cancer biology—cell cycle, cell growth, apoptosis, metabolism, cancer stem cells, micro RNAs, angiogenesis, inflammation—Myc is in there regulating most of the key genes. Attempts to identify a unitary Myc signature have likewise been frustrated by the cell-type- and cell-context-dependent nature of Myc activity. Myc seems to be all things to all people. Myc is like the Cheshire cat in Alice: the longer you study its ‘‘function,’’ the more elusive it becomes and, in the end, all that is left is a derisive smile and a hint of leucine zipper. But now, two papers published in this issue of Cell (Lin et al., 2012 and Nie et al., 2012) may offer some relief, although their conclusions may not bring comfort because they conclude that Myc does not and never will have one transcriptional signature. Rather, they suggest that the activity of Myc is completely contextual and depends upon cell type and status. Both papers address the
question of what elevated levels of Myc do (that physiological levels may not). The stark conclusions from both papers, which are reached through genome-wide CHIP-Seq analysis and sophisticated data extraction, are that, rather than engaging its dedicated own transcriptional program, Myc serves to amplify the output of existing transcriptionally active genes. Put simply, Myc is a general amplifier of any given transcriptional state a cell finds itself in at the time of Myc activation (Figure 1). Both groups demonstrate that as Myc protein levels rise, Myc is loaded quantitatively onto active promoters (as demonstrated by co-occupancy of RNA pol II and the presence of active chromatin marks), enhancing their transcription. By contrast, Myc does not localize to the promoters of silent genes, suggesting that Myc cannot itself not instruct de novo gene activation. Hence, Myc is a contingent transcriptional amplifier. Interestingly, Myc amplification is logarithmic, disproportionally enhancing transcription of highly active genes. Of note, the loading of Myc on active genes remains dependent upon specific CACGTG E-box elements within each target gene’s promoter and proximity of such E boxes to the transcriptional start
Cell 151, September 28, 2012 ª2012 Elsevier Inc. 11
Figure 1. Myc Is an Amplifier, Not a Specifier, of Transcriptional Activation Image was designed by Rafael Casellas and Ethan Taylor.
site (TSS) appears to correlate with highly expressed genes. For years, one conundrum in Myc biology has been the role played by elevated Myc expression in cancer cells. Is elevation of Myc merely a spandrel byproduct of the various oncogenic mutations that deregulate its expression or does excess Myc do oncogenic things that less Myc doesn’t do? For example, by binding lower affinity E-box elements, preternaturally elevated levels of Myc might commandeer supernumerary genes with oncogenic activity into the Myc pantheon. Both studies indicate that this is not the case. They show that increasing Myc levels does indeed result in loading of Myc onto lower affinity noncanonical E-boxes, typically in enhancer regions more distant from the TSS; however, this occurs only in those genes at which Myc
is already bound via canonical CACGTG E-box elements. Hence, more Myc increases transcription only from those genes that are already Myc targets but not from new genes. Both groups suggest that increased Myc occupancy promotes transcriptional elongation and Lin et al. show increased occupancy of the elongation factor pTEF-b at Myc bound sites when Myc level is elevated and increased phosphorylation at a serine residue in RNA pol II associated with elongation rather than transcriptional initiation, consistent with the appearance of pol II in gene bodies (Nie et al.). This underpins previous data in which Myc was found to be important for transcriptional pause release of Pol II (Rahl et al., 2010). The elevated levels of Myc in tumor cells exacerbate existing Myc functions—they do not recruit new ones.
12 Cell 151, September 28, 2012 ª2012 Elsevier Inc.
So the idea that emerges from these data is that Myc acts as a general amplifier or augmentor of transcription, serving to lock the transcriptional program in place and buffer cells against transient perturbations and exigencies. This satisfyingly explains the elusiveness of the ‘‘Myc signature’’—in essence, the Myc signature is an entirely contextual construct: a consequence of each cell’s state rather than a cause. However, this straightforward and unifying model has some problems. First, if Myc serves to reinforce existing states, one would surely imagine that it would be induced and engaged by all types of instructional signals—for example, factors that induce differentiation and senescence. However, Myc activation by and large seems to be the exclusive purview of mitogenic signals. Indeed, persistent Myc expression typically locks cells in a continually proliferating state and renders them refractory to signals that would otherwise promote growth arrest and terminal differentiation. This private relationship between Myc and mitogenesis argues for a rather more specific role for Myc, as a coordinator of cell proliferation. Furthermore, Myc is not limited merely to reinforcing an existing cellular state—it can also instruct them: expression of Myc alone, even at physiological levels, is sufficient to drive quiescent mesenchymal, epithelial and lymphoid cells to proliferate and, concurrently, to engage all the diverse biological programs that proliferating cells need to grow and expand into the soma—for example, the switch to biosynthetic ‘‘Warburg’’ metabolism and activation of cell growth and the production of the inflammatory and angiogenic signals required to remodel surrounding stroma and blood vessels. One possible way to accommodate the apparent contradiction that Myc reinforces any pre-existing transcriptional state with the general observation that Myc activation selectively drives proliferation and dedifferentiation is to invoke the idea that differing levels of Myc are required to engage different biological outputs. Only low levels of Myc can engage proliferation, whereas higher levels are needed to lock in other phenotypes such as differentiation. However, once proliferation is engaged, it secondarily represses differentiation—hence the observed outcome that Myc induces cell
proliferation and dedifferentiation. The notion that cells respond to different Myc levels in different ways is in accord with the observations by Nie et al. (2012) that increasing levels of Myc load only onto already active genes, logarithmically but selectively increasing their output: the tacit implication of this is that cells have evolved to sense differential Myc levels. Indeed, we already know that different Myc levels do elicit different outputs— physiological levels of Myc are sufficient to drive proliferation but aberrantly elevated levels trigger apoptosis (Murphy et al., 2008). This reflects the general principle that our evolved tumor suppression mechanisms seem to discriminate between normal and oncogenic signals by sensing the aberrantly elevated flux of oncogenic signals (Feldser et al., 2010; Junttila et al., 2010; Murphy et al., 2008). However, unlike proliferation and differentiation, which are mutually contradictory, proliferation and apoptosis are not—the
earliest studies showed that both proliferation and apoptosis occur together in populations of cells harboring high levels of Myc. If proliferation negates differentiation, and the augmentation of the differentiated state only arises at Myc levels that have already engaged proliferation, when would this higher threshold ever be biologically relevant? Such questions, and countless others, are sure to keep the upcoming generation of cancer biologists busy and the current ones out of retirement. What we do have, thanks to these two seminal studies, is the glimpse of a coherent and holistic view of Myc. We have come a long way since the 1980s’ view wherein Myc exerted its pleiotropic biological effects through just a few, critical target genes, whose identification would unlock Myc’s mysteries. Now Myc regulates a third of everything—and which third depends on everything else. It’s strange the way things turn out.
REFERENCES Feldser, D., Cashman, C., Kostova, K., Ouyang, C., Bronson, R., Shin, H., Winslow, M., Hemann, M., and Jacks, T. (2010). Nature 468, 572–575. Junttila, M.R., Karnezis, A.N., Garcia, D., Madriles, F., Kortlever, R.M., Rostker, F., Brown Swigart, L., Pham, D.M., Seo, Y., Evan, G.I., and Martins, C.P. (2010). Nature 468, 567–571. Lin, C.Y., Love´n, J., Rahl, P.B., Paranal, R.M., Burge, C.B., Bradner, J.E., Lee, T.I., and Young, R.A. (2012). Cell 151, this issue, 56–67. Murphy, D.J., Junttila, M.R., Pouyet, L., Karnezis, A., Shchors, K., Bui, D.A., Brown-Swigart, L., Johnson, L., and Evan, G.I. (2008). Cancer Cell 14, 447–457. Nie, Z., Hu, G., Wei, G., Cui, K., Yamane, A., Resch, W., Wang, R., Green, D.R., Tessarollo, L., Casellas, R., et al. (2012). Cell 151, this issue, 68–79. Rahl, P.B., Lin, C.Y., Seila, A.C., Flynn, R.A., McCuine, S., Burge, C.B., Sharp, P.A., and Young, R.A. (2010). Cell 141, 432–445.
Cell 151, September 28, 2012 ª2012 Elsevier Inc. 13
triou, E., Bear, D.M., and Greenberg, M.E. (2008). Neuron 60, 1022–1038.
Dunfield, D., and Haas, K. (2009). Neuron 64, 240–250.
Huang, Y.Y., Colino, A., Selig, D.K., and Malenka, R.C. (1992). Science 255, 730–733.
Flavell, S.W., Kim, T.K., Gray, J.M., Harmin, D.A., Hemberg, M., Hong, E.J., Markenscoff-Papadimi-
Kirkwood, A., Rioult, M.C., and Bear, M.F. (1996). Nature 381, 526–528.
Li, Z., and Sheng, M. (2012). Mol. Brain 5, 15. Niell, C.M., Meyer, M.P., and Smith, S.J. (2004). Nat. Neurosci. 7, 254–260. Schwartz, N., Schohl, A., and Ruthazer, E.S. (2009). Neuron 62, 655–669.
All Things to All People Trevor D. Littlewood,1 Peter Kreuzaler,1 and Gerard I. Evan1,* 1Department
of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK *Correspondence: [email protected] http://dx.doi.org/10.1016/j.cell.2012.09.006
Myc is an enigma wrapped in a mystery. Attempts to identify Myc target genes, particularly in cancer, have been fraught with dead ends and context-specific functions. Lin et al. and Nie et al. address this conundrum by showing that Myc acts to amplify the output of existing transcriptionally active genes. Originally discovered as a stowaway in some defective avian retroviruses that acutely elicit myelocytic leukemia, Myc rose to notoriety both as the prototypical cooperating oncogene that, together with Ras, can oncogenically transform fibroblasts in vitro and as one of the immediate early growth response genes that are rapidly induced in various cell types upon mitogenic stimulation. Myc is a member of a class of dimeric transcription factor— the basic-helix-loop-helix-leucine zipper proteins and, as a heterodimer with its partner protein Max, binds DNA, with a predilection for a palindromic E-box element CACGTG. Myc is widely, almost universally, present in proliferating normal somatic cells and expression of Myc protein and mRNA, both of which are very short-lived, are continuously dependent upon mitogenic signaling. By contrast, Myc expression in cancer cells is typically deregulated and elevated. Finally, ectopic expression of Myc is, alone, able to induce proliferation of many adult somatic cell types, whereas cells denuded of Myc, whether normal or neoplastic, replicate very slowly, if at all. Distill this heady brew together, and we are left with a transcription factor that exerts its biological activity through the
modulation of target genes involved in cell replication. Since then, however, our understanding of Myc has been painfully slow. Efforts to identify Myc target genes suggest that Myc modulates up to a third of the transcriptome. Whatever the latest trend in cancer biology—cell cycle, cell growth, apoptosis, metabolism, cancer stem cells, micro RNAs, angiogenesis, inflammation—Myc is in there regulating most of the key genes. Attempts to identify a unitary Myc signature have likewise been frustrated by the cell-type- and cell-context-dependent nature of Myc activity. Myc seems to be all things to all people. Myc is like the Cheshire cat in Alice: the longer you study its ‘‘function,’’ the more elusive it becomes and, in the end, all that is left is a derisive smile and a hint of leucine zipper. But now, two papers published in this issue of Cell (Lin et al., 2012 and Nie et al., 2012) may offer some relief, although their conclusions may not bring comfort because they conclude that Myc does not and never will have one transcriptional signature. Rather, they suggest that the activity of Myc is completely contextual and depends upon cell type and status. Both papers address the
question of what elevated levels of Myc do (that physiological levels may not). The stark conclusions from both papers, which are reached through genome-wide CHIP-Seq analysis and sophisticated data extraction, are that, rather than engaging its dedicated own transcriptional program, Myc serves to amplify the output of existing transcriptionally active genes. Put simply, Myc is a general amplifier of any given transcriptional state a cell finds itself in at the time of Myc activation (Figure 1). Both groups demonstrate that as Myc protein levels rise, Myc is loaded quantitatively onto active promoters (as demonstrated by co-occupancy of RNA pol II and the presence of active chromatin marks), enhancing their transcription. By contrast, Myc does not localize to the promoters of silent genes, suggesting that Myc cannot itself not instruct de novo gene activation. Hence, Myc is a contingent transcriptional amplifier. Interestingly, Myc amplification is logarithmic, disproportionally enhancing transcription of highly active genes. Of note, the loading of Myc on active genes remains dependent upon specific CACGTG E-box elements within each target gene’s promoter and proximity of such E boxes to the transcriptional start
Cell 151, September 28, 2012 ª2012 Elsevier Inc. 11
Figure 1. Myc Is an Amplifier, Not a Specifier, of Transcriptional Activation Image was designed by Rafael Casellas and Ethan Taylor.
site (TSS) appears to correlate with highly expressed genes. For years, one conundrum in Myc biology has been the role played by elevated Myc expression in cancer cells. Is elevation of Myc merely a spandrel byproduct of the various oncogenic mutations that deregulate its expression or does excess Myc do oncogenic things that less Myc doesn’t do? For example, by binding lower affinity E-box elements, preternaturally elevated levels of Myc might commandeer supernumerary genes with oncogenic activity into the Myc pantheon. Both studies indicate that this is not the case. They show that increasing Myc levels does indeed result in loading of Myc onto lower affinity noncanonical E-boxes, typically in enhancer regions more distant from the TSS; however, this occurs only in those genes at which Myc
is already bound via canonical CACGTG E-box elements. Hence, more Myc increases transcription only from those genes that are already Myc targets but not from new genes. Both groups suggest that increased Myc occupancy promotes transcriptional elongation and Lin et al. show increased occupancy of the elongation factor pTEF-b at Myc bound sites when Myc level is elevated and increased phosphorylation at a serine residue in RNA pol II associated with elongation rather than transcriptional initiation, consistent with the appearance of pol II in gene bodies (Nie et al.). This underpins previous data in which Myc was found to be important for transcriptional pause release of Pol II (Rahl et al., 2010). The elevated levels of Myc in tumor cells exacerbate existing Myc functions—they do not recruit new ones.
12 Cell 151, September 28, 2012 ª2012 Elsevier Inc.
So the idea that emerges from these data is that Myc acts as a general amplifier or augmentor of transcription, serving to lock the transcriptional program in place and buffer cells against transient perturbations and exigencies. This satisfyingly explains the elusiveness of the ‘‘Myc signature’’—in essence, the Myc signature is an entirely contextual construct: a consequence of each cell’s state rather than a cause. However, this straightforward and unifying model has some problems. First, if Myc serves to reinforce existing states, one would surely imagine that it would be induced and engaged by all types of instructional signals—for example, factors that induce differentiation and senescence. However, Myc activation by and large seems to be the exclusive purview of mitogenic signals. Indeed, persistent Myc expression typically locks cells in a continually proliferating state and renders them refractory to signals that would otherwise promote growth arrest and terminal differentiation. This private relationship between Myc and mitogenesis argues for a rather more specific role for Myc, as a coordinator of cell proliferation. Furthermore, Myc is not limited merely to reinforcing an existing cellular state—it can also instruct them: expression of Myc alone, even at physiological levels, is sufficient to drive quiescent mesenchymal, epithelial and lymphoid cells to proliferate and, concurrently, to engage all the diverse biological programs that proliferating cells need to grow and expand into the soma—for example, the switch to biosynthetic ‘‘Warburg’’ metabolism and activation of cell growth and the production of the inflammatory and angiogenic signals required to remodel surrounding stroma and blood vessels. One possible way to accommodate the apparent contradiction that Myc reinforces any pre-existing transcriptional state with the general observation that Myc activation selectively drives proliferation and dedifferentiation is to invoke the idea that differing levels of Myc are required to engage different biological outputs. Only low levels of Myc can engage proliferation, whereas higher levels are needed to lock in other phenotypes such as differentiation. However, once proliferation is engaged, it secondarily represses differentiation—hence the observed outcome that Myc induces cell
proliferation and dedifferentiation. The notion that cells respond to different Myc levels in different ways is in accord with the observations by Nie et al. (2012) that increasing levels of Myc load only onto already active genes, logarithmically but selectively increasing their output: the tacit implication of this is that cells have evolved to sense differential Myc levels. Indeed, we already know that different Myc levels do elicit different outputs— physiological levels of Myc are sufficient to drive proliferation but aberrantly elevated levels trigger apoptosis (Murphy et al., 2008). This reflects the general principle that our evolved tumor suppression mechanisms seem to discriminate between normal and oncogenic signals by sensing the aberrantly elevated flux of oncogenic signals (Feldser et al., 2010; Junttila et al., 2010; Murphy et al., 2008). However, unlike proliferation and differentiation, which are mutually contradictory, proliferation and apoptosis are not—the
earliest studies showed that both proliferation and apoptosis occur together in populations of cells harboring high levels of Myc. If proliferation negates differentiation, and the augmentation of the differentiated state only arises at Myc levels that have already engaged proliferation, when would this higher threshold ever be biologically relevant? Such questions, and countless others, are sure to keep the upcoming generation of cancer biologists busy and the current ones out of retirement. What we do have, thanks to these two seminal studies, is the glimpse of a coherent and holistic view of Myc. We have come a long way since the 1980s’ view wherein Myc exerted its pleiotropic biological effects through just a few, critical target genes, whose identification would unlock Myc’s mysteries. Now Myc regulates a third of everything—and which third depends on everything else. It’s strange the way things turn out.
REFERENCES Feldser, D., Cashman, C., Kostova, K., Ouyang, C., Bronson, R., Shin, H., Winslow, M., Hemann, M., and Jacks, T. (2010). Nature 468, 572–575. Junttila, M.R., Karnezis, A.N., Garcia, D., Madriles, F., Kortlever, R.M., Rostker, F., Brown Swigart, L., Pham, D.M., Seo, Y., Evan, G.I., and Martins, C.P. (2010). Nature 468, 567–571. Lin, C.Y., Love´n, J., Rahl, P.B., Paranal, R.M., Burge, C.B., Bradner, J.E., Lee, T.I., and Young, R.A. (2012). Cell 151, this issue, 56–67. Murphy, D.J., Junttila, M.R., Pouyet, L., Karnezis, A., Shchors, K., Bui, D.A., Brown-Swigart, L., Johnson, L., and Evan, G.I. (2008). Cancer Cell 14, 447–457. Nie, Z., Hu, G., Wei, G., Cui, K., Yamane, A., Resch, W., Wang, R., Green, D.R., Tessarollo, L., Casellas, R., et al. (2012). Cell 151, this issue, 68–79. Rahl, P.B., Lin, C.Y., Seila, A.C., Flynn, R.A., McCuine, S., Burge, C.B., Sharp, P.A., and Young, R.A. (2010). Cell 141, 432–445.
Cell 151, September 28, 2012 ª2012 Elsevier Inc. 13