- Email: [email protected]
ATM, MacroH2A.1, and SASP: The Checks and Balances of Cellular Senescence
Molecular Cell
Preview ATM, MacroH2A.1, and SASP: The Checks and Balances of Cellular Senescence Marek Kozlowski1,2 and Andreas G. Ladurner1,2,3,4,* 1Department of Physiological Chemistry, Biomedical Center, Ludwig-Maximilians-University of Munich, Butenandtstrasse 5, 81377 Munich, Germany 2International Max Planck Research School for Molecular and Cellular Life Sciences, Am Klopferspitz 18, 82152 Martinsried, Germany 3Center for Integrated Protein Science Munich (CIPSM), 81377 Munich, Germany 4Munich Cluster for Systems Neurology (SyNergy), 80336 Munich, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2015.08.010
Oncogene activation is usually not enough to induce cancer, but causes cells to arrest proliferation, alter chromatin structure, and increase protein secretion. In this issue of Molecular Cell, Chen et al. (2015) implicate the histone variant macroH2A.1 in the regulation of senescence.
Cellular senescence is a dramatic transformation of cell fate that results in an irreversible cell cycle exit. It is triggered by a variety of factors, including shortened telomeres, oxidative stress and DNA damage, and the activation of oncogenes. The senescent cell undergoes a variety of interdependent changes in signaling networks, such as the activation of endoplasmic reticulum (ER) stress, persistent activation of the DNA damage response (DDR), and increased transcription and secretion of pro-inflammatory factors such as proteases, growth factors, extracellular remodeling proteins, and cytokines. These secreted factors together compose the senescence-associated secretory phenotype (SASP) and have the ability to induce senescence also in neighboring cells, in a paracrine fashion. An associated hallmark of cellular senescence is the large-scale re-organization of chromatin structure and epigenome. Chromatin dynamics is at the heart of controlling DNA damage repair, replication, and transcription, but there is currently relatively little insight into how SASP genes are specifically regulated at the level of transcriptional control and chromatin structure. How senescence stimuli are transduced to alter the chromatin state is therefore an open question. In this issue of Molecular Cell, Chen and colleagues find that the tumor-suppressive histone variant macroH2A.1 establishes a complex regulatory feedback loop regulating the expression of SASP genes, and that an active ATM DNA damage signaling kinase triggers the removal
of macroH2A.1 from the chromatin of SASP genes (Chen et al., 2015). Moreover, they find that the DNA damage-activated enzyme PARP-1 plays a role in this process, implicating particularly the polyADP-ribose (PAR)-binding macroH2A.1.1 histone variant isoform in mediating the chromatin control of SASP gene activity. MacroH2A.1 is known to be enriched in senescence-associated heterochromatin foci (SAHF; Zhang et al., 2005), the formation of which is a leading example of the extensive chromatin reorganization that takes place as cells become senescent (Narita et al., 2003). These foci, marked by heterochromatin features such as histone methylation (H3K9me3, H3K27me3) and the binding of heterochromatin binding 1 (HP1) proteins, are important for silencing of cell proliferation-promoting genes—while the histone variant macroH2A.1 is not critical for SAHF formation, it is thought that it may be important in maintaining this silencing (Zhang et al., 2005). Studies also show that the PAR-binding splice isoform (macroH2A.1.1) becomes enriched as human cells undergo replicative senescence (Sporn et al., 2009), while the PAR-binding deficient splice isoform (macroH2A.1.2) generally does not change in expression as cells exit the cell cycle. In addition to SAHF foci, macroH2A.1 histones are found enriched on the inactivated X chromosome and act as an epigenetic barrier to epigenetic reprogramming (Pasque et al., 2011; Gaspar-Maia et al., 2013). Thus, macroH2A.1’s chromatin functions are generally
assumed to play a role in transcriptional repression. More recent work, however, shows that the specific macroH2A.1.1 isoform also plays a role in transcription activation and is enriched across multiple euchromatic domains, especially regions marked by extensive histone acetylation (H2BK12/K15/K120Ac, H2AK5Ac, H3K4/ K14/K18Ac, H4K91Ac; Chen et al., 2014). Moreover, these macroH2A.1.1 functions depend on PARP-1, owing to the ability of this atypical histone to specifically recognize PAR through its C-terminal macrodomain module. In the current analysis, Chen and colleagues sought to dissect how macroH2A.1 regulates senescence functions by virtue of its ability to contribute to chromatin plasticity. The authors find that macroH2A.1 is enriched over SASP genes in non-senescent cells. These genes become transcriptionally active upon senescence. Importantly, siRNAmediated depletion of macroH2A.1 leads to a striking reduction of the SASP gene mRNA levels when senescence is induced by H-RasV12. Further, in the absence of macroH2A.1, cells do not accumulate high levels of phosphorylated histone H2A.X (gH2A.X) compared to control senescent cells, and activation of ATM is reduced. The authors also describe that upon senescence, macroH2A.1 undergoes genome-wide redistribution, which results in 50% of novel macroH2A.1 sites across the genome. Most of these sites are enriched in heterochromatic marks such as H3K27me3 and H3K9me3, consistent with macroH2A.1’s
Molecular Cell 59, September 3, 2015 ª2015 Elsevier Inc. 713
Molecular Cell
Preview
Figure 1. MacroH2A.1 and ATM Keep SASP in Equilibrium The histone variant macroH2A.1.1 is required for full transcriptional activation of genes composing the senescence-associated secretory phenotype (SASP), further propagating senescence via a positive feedback loop. Increases in SASP are balanced by a negative feedback loop, in which the kinase ATM is activated downstream of endoplasmic reticulum (ER) stress, elevated levels of reactive oxygen species (ROS), and DNA damage response (DDR). ATM kinase signals the DDR via the phosphorylation of the histone variant H2A.X (gH2AX), and its activity is critical in the removal of macroH2A1.1 from SASP loci. This results in SASP gene transcriptional repression. Transcriptional reprogramming during SASP is accompanied by chromatin reorganization and requires the activity of the poly-ADP-ribose polymerase (PARP) 1 enzyme activity as well as macroH2A1.1, which is capable of recognizing the ADP-ribose polymers produced by PARP-1 (Chen et al., 2015).
role in the formation of SAHF heterochromatin. Interestingly, macroH2A.1 is no longer found at the SASP genes upon oncogene-induced senescence (Chen et al., 2015). But what are the molecular mechanisms that contribute to this dynamic relocation? The fact that macroH2A.1 plays a role in SASP gene expression is important, but is macroH2A.1 repressive to SASP gene transcription in non-senescent cells, or does it help to ‘‘poise’’ SASP genes for transcriptional activation upon senescence? Chen and colleagues go some way in answering these questions and are able to bring together their results into an intricate model of checks and balances through positive and negative feedback loops involving ATM, macroH2A, and SASP genes. As SASP genes are induced upon oncogene activation, secreted cytokines can function in an autocrine fashion and induce senescence in neighboring cells. Cells lacking macroH2A.1 are not susceptible to paracrine senescence, so macroH2A.1 plays a positive role in SASP expression. At the same time, the
SASP response itself is key in auto-regulation and shutting down SASP genes via a negative feedback loop (Chen et al., 2015). Expression of SASP genes leads to enhancement of ER stress signaling and increased levels of reactive oxygen species (ROS). Consequently, DNA bases become oxidized, which activates the DDR, including the kinase ATM. The activity of ATM was found to be essential in the removal of macroH2A.1 from SASP genes and shutting down their expression (Chen et al., 2015). Upon chemical inhibition of ATM, the expression of SASP genes is increased. Thus, ATM activity is critical to the negative feedback loop triggered by SASP, ER stress, and the DDR (Figure 1; Chen et al., 2015). ATM is a sensor of DNA damage, establishing a platform gH2A.X histone variant in the vicinity of DNA damage sites. Much like the PAR-binding macroH2A.1.1 histone, gH2A.X serves as a beacon to recruit other DNA repair factors to damage sites. How ATM kinase activity can repress SASP genes is not known, but indicates an unexpected repressive
714 Molecular Cell 59, September 3, 2015 ª2015 Elsevier Inc.
role of the DDR in SASP gene activity. Further: How does ATM lead to the removal of macroH2A.1? In other stress responses, such as heat shock, macroH2A.1 is displaced from the human Hsp70 promoter (Ouararhni et al., 2006), and this process involves the ability of its macrodomain of binding poly-ADP-ribosylated PARP-1. It is therefore tempting to speculate that PAR-mediated events are critical to chromatin dynamics and the DDR across a variety of cellular stress responses. It would be interesting to compare the genome-wide association profiles of gH2A.X and macroH2A.1 histone as cells undergo senescence. Moreover, it is very likely that dedicated histone chaperones, chromatin remodelers, and other chromatin-associated proteins directly promote the incorporation or removal of macroH2A from chromatin. An interesting fact emerging from the Chen et al. study is that only one of the two macroH2A.1 histone splice isoforms mediates the events described above. Specifically, the ADP-ribose and PARbinding macroH2A1.1 isoform (Kustatscher et al., 2005; Timinszky et al., 2009) cooperate together with PARP-1 in the SASP program (Chen et al., 2015), while its sibling macroH2A1.2 appears to be inert. The senescence axis of macroH2A1.1 and PARP is particularly interesting when considering that this isoform is often downregulated in cancer (Sporn et al., 2009). Chen and colleagues suggest that this may render cancer cells resistant to SASP-mediated senescence, bypassing an important barrier to oncogenesis. Moreover, decreases in macroH2A.1.1 levels may also lower the epigenetic barrier toward pluripotency, helping cancer cells lose their differentiated cell state. The new data reported in this issue of Molecular Cell add to the emerging epigenetic barrier function of the PAR-binding macroH2A.1.1 histone and provide new and interesting insights into how chromatin dynamics and signaling are integrated to transcriptionally regulate the secretory phenotype of senescent cells.
ACKNOWLEDGMENTS We acknowledge financial support from the DFG through the Collaborative Research Centers SFB646 ‘‘Networks in genome expression and
Molecular Cell
Preview maintenance’’ dynamics.’’
and
SFB1064
‘‘Chromatin
REFERENCES Chen, H., Ruiz, P.D., Novikov, L., Casill, A.D., Park, J.W., and Gamble, M.J. (2014). Nat. Struct. Mol. Biol. 21, 981–989. Chen, H., Ruiz, P.D., McKimpson, W.M., Novikov, L., Kitsis, R.N., and Gamble, M.J. (2015). Mol. Cell 59, this issue, 719–731. Gaspar-Maia, A., Qadeer, Z.A., Hasson, D., Ratnakumar, K., Leu, N.A., Leroy, G., Liu, S., Costanzi,
C., Valle-Garcia, D., Schaniel, C., et al. (2013). Nat. Commun. 4, 1565.
Pasque, V., Gillich, A., Garrett, N., and Gurdon, J.B. (2011). EMBO J. 30, 2373–2387.
Kustatscher, G., Hothorn, M., Pugieux, C., Scheffzek, K., and Ladurner, A.G. (2005). Nat. Struct. Mol. Biol. 12, 624–625.
Sporn, J.C., Kustatscher, G., Hothorn, T., Collado, M., Serrano, M., Muley, T., Schnabel, P., and Ladurner, A.G. (2009). Oncogene 28, 3423–3428.
~nez, S., Heard, E., Narita, M., Lin, Narita, M., Nu A.W., Hearn, S.A., Spector, D.L., Hannon, G.J., and Lowe, S.W. (2003). Cell 113, 703–716.
Timinszky, G., Till, S., Hassa, P.O., Hothorn, M., Kustatscher, G., Nijmeijer, B., Colombelli, J., Altmeyer, M., Stelzer, E.H., Scheffzek, K., et al. (2009). Nat. Struct. Mol. Biol. 16, 923–929.
Ouararhni, K., Hadj-Slimane, R., Ait-Si-Ali, S., Robin, P., Mietton, F., Harel-Bellan, A., Dimitrov, S., and Hamiche, A. (2006). Genes Dev. 20, 3324–3336.
Zhang, R., Poustovoitov, M.V., Ye, X., Santos, H.A., Chen, W., Daganzo, S.M., Erzberger, J.P., Serebriiskii, I.G., Canutescu, A.A., Dunbrack, R.L., et al. (2005). Dev. Cell 8, 19–30.
Molecular Cell 59, September 3, 2015 ª2015 Elsevier Inc. 715
Preview ATM, MacroH2A.1, and SASP: The Checks and Balances of Cellular Senescence Marek Kozlowski1,2 and Andreas G. Ladurner1,2,3,4,* 1Department of Physiological Chemistry, Biomedical Center, Ludwig-Maximilians-University of Munich, Butenandtstrasse 5, 81377 Munich, Germany 2International Max Planck Research School for Molecular and Cellular Life Sciences, Am Klopferspitz 18, 82152 Martinsried, Germany 3Center for Integrated Protein Science Munich (CIPSM), 81377 Munich, Germany 4Munich Cluster for Systems Neurology (SyNergy), 80336 Munich, Germany *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2015.08.010
Oncogene activation is usually not enough to induce cancer, but causes cells to arrest proliferation, alter chromatin structure, and increase protein secretion. In this issue of Molecular Cell, Chen et al. (2015) implicate the histone variant macroH2A.1 in the regulation of senescence.
Cellular senescence is a dramatic transformation of cell fate that results in an irreversible cell cycle exit. It is triggered by a variety of factors, including shortened telomeres, oxidative stress and DNA damage, and the activation of oncogenes. The senescent cell undergoes a variety of interdependent changes in signaling networks, such as the activation of endoplasmic reticulum (ER) stress, persistent activation of the DNA damage response (DDR), and increased transcription and secretion of pro-inflammatory factors such as proteases, growth factors, extracellular remodeling proteins, and cytokines. These secreted factors together compose the senescence-associated secretory phenotype (SASP) and have the ability to induce senescence also in neighboring cells, in a paracrine fashion. An associated hallmark of cellular senescence is the large-scale re-organization of chromatin structure and epigenome. Chromatin dynamics is at the heart of controlling DNA damage repair, replication, and transcription, but there is currently relatively little insight into how SASP genes are specifically regulated at the level of transcriptional control and chromatin structure. How senescence stimuli are transduced to alter the chromatin state is therefore an open question. In this issue of Molecular Cell, Chen and colleagues find that the tumor-suppressive histone variant macroH2A.1 establishes a complex regulatory feedback loop regulating the expression of SASP genes, and that an active ATM DNA damage signaling kinase triggers the removal
of macroH2A.1 from the chromatin of SASP genes (Chen et al., 2015). Moreover, they find that the DNA damage-activated enzyme PARP-1 plays a role in this process, implicating particularly the polyADP-ribose (PAR)-binding macroH2A.1.1 histone variant isoform in mediating the chromatin control of SASP gene activity. MacroH2A.1 is known to be enriched in senescence-associated heterochromatin foci (SAHF; Zhang et al., 2005), the formation of which is a leading example of the extensive chromatin reorganization that takes place as cells become senescent (Narita et al., 2003). These foci, marked by heterochromatin features such as histone methylation (H3K9me3, H3K27me3) and the binding of heterochromatin binding 1 (HP1) proteins, are important for silencing of cell proliferation-promoting genes—while the histone variant macroH2A.1 is not critical for SAHF formation, it is thought that it may be important in maintaining this silencing (Zhang et al., 2005). Studies also show that the PAR-binding splice isoform (macroH2A.1.1) becomes enriched as human cells undergo replicative senescence (Sporn et al., 2009), while the PAR-binding deficient splice isoform (macroH2A.1.2) generally does not change in expression as cells exit the cell cycle. In addition to SAHF foci, macroH2A.1 histones are found enriched on the inactivated X chromosome and act as an epigenetic barrier to epigenetic reprogramming (Pasque et al., 2011; Gaspar-Maia et al., 2013). Thus, macroH2A.1’s chromatin functions are generally
assumed to play a role in transcriptional repression. More recent work, however, shows that the specific macroH2A.1.1 isoform also plays a role in transcription activation and is enriched across multiple euchromatic domains, especially regions marked by extensive histone acetylation (H2BK12/K15/K120Ac, H2AK5Ac, H3K4/ K14/K18Ac, H4K91Ac; Chen et al., 2014). Moreover, these macroH2A.1.1 functions depend on PARP-1, owing to the ability of this atypical histone to specifically recognize PAR through its C-terminal macrodomain module. In the current analysis, Chen and colleagues sought to dissect how macroH2A.1 regulates senescence functions by virtue of its ability to contribute to chromatin plasticity. The authors find that macroH2A.1 is enriched over SASP genes in non-senescent cells. These genes become transcriptionally active upon senescence. Importantly, siRNAmediated depletion of macroH2A.1 leads to a striking reduction of the SASP gene mRNA levels when senescence is induced by H-RasV12. Further, in the absence of macroH2A.1, cells do not accumulate high levels of phosphorylated histone H2A.X (gH2A.X) compared to control senescent cells, and activation of ATM is reduced. The authors also describe that upon senescence, macroH2A.1 undergoes genome-wide redistribution, which results in 50% of novel macroH2A.1 sites across the genome. Most of these sites are enriched in heterochromatic marks such as H3K27me3 and H3K9me3, consistent with macroH2A.1’s
Molecular Cell 59, September 3, 2015 ª2015 Elsevier Inc. 713
Molecular Cell
Preview
Figure 1. MacroH2A.1 and ATM Keep SASP in Equilibrium The histone variant macroH2A.1.1 is required for full transcriptional activation of genes composing the senescence-associated secretory phenotype (SASP), further propagating senescence via a positive feedback loop. Increases in SASP are balanced by a negative feedback loop, in which the kinase ATM is activated downstream of endoplasmic reticulum (ER) stress, elevated levels of reactive oxygen species (ROS), and DNA damage response (DDR). ATM kinase signals the DDR via the phosphorylation of the histone variant H2A.X (gH2AX), and its activity is critical in the removal of macroH2A1.1 from SASP loci. This results in SASP gene transcriptional repression. Transcriptional reprogramming during SASP is accompanied by chromatin reorganization and requires the activity of the poly-ADP-ribose polymerase (PARP) 1 enzyme activity as well as macroH2A1.1, which is capable of recognizing the ADP-ribose polymers produced by PARP-1 (Chen et al., 2015).
role in the formation of SAHF heterochromatin. Interestingly, macroH2A.1 is no longer found at the SASP genes upon oncogene-induced senescence (Chen et al., 2015). But what are the molecular mechanisms that contribute to this dynamic relocation? The fact that macroH2A.1 plays a role in SASP gene expression is important, but is macroH2A.1 repressive to SASP gene transcription in non-senescent cells, or does it help to ‘‘poise’’ SASP genes for transcriptional activation upon senescence? Chen and colleagues go some way in answering these questions and are able to bring together their results into an intricate model of checks and balances through positive and negative feedback loops involving ATM, macroH2A, and SASP genes. As SASP genes are induced upon oncogene activation, secreted cytokines can function in an autocrine fashion and induce senescence in neighboring cells. Cells lacking macroH2A.1 are not susceptible to paracrine senescence, so macroH2A.1 plays a positive role in SASP expression. At the same time, the
SASP response itself is key in auto-regulation and shutting down SASP genes via a negative feedback loop (Chen et al., 2015). Expression of SASP genes leads to enhancement of ER stress signaling and increased levels of reactive oxygen species (ROS). Consequently, DNA bases become oxidized, which activates the DDR, including the kinase ATM. The activity of ATM was found to be essential in the removal of macroH2A.1 from SASP genes and shutting down their expression (Chen et al., 2015). Upon chemical inhibition of ATM, the expression of SASP genes is increased. Thus, ATM activity is critical to the negative feedback loop triggered by SASP, ER stress, and the DDR (Figure 1; Chen et al., 2015). ATM is a sensor of DNA damage, establishing a platform gH2A.X histone variant in the vicinity of DNA damage sites. Much like the PAR-binding macroH2A.1.1 histone, gH2A.X serves as a beacon to recruit other DNA repair factors to damage sites. How ATM kinase activity can repress SASP genes is not known, but indicates an unexpected repressive
714 Molecular Cell 59, September 3, 2015 ª2015 Elsevier Inc.
role of the DDR in SASP gene activity. Further: How does ATM lead to the removal of macroH2A.1? In other stress responses, such as heat shock, macroH2A.1 is displaced from the human Hsp70 promoter (Ouararhni et al., 2006), and this process involves the ability of its macrodomain of binding poly-ADP-ribosylated PARP-1. It is therefore tempting to speculate that PAR-mediated events are critical to chromatin dynamics and the DDR across a variety of cellular stress responses. It would be interesting to compare the genome-wide association profiles of gH2A.X and macroH2A.1 histone as cells undergo senescence. Moreover, it is very likely that dedicated histone chaperones, chromatin remodelers, and other chromatin-associated proteins directly promote the incorporation or removal of macroH2A from chromatin. An interesting fact emerging from the Chen et al. study is that only one of the two macroH2A.1 histone splice isoforms mediates the events described above. Specifically, the ADP-ribose and PARbinding macroH2A1.1 isoform (Kustatscher et al., 2005; Timinszky et al., 2009) cooperate together with PARP-1 in the SASP program (Chen et al., 2015), while its sibling macroH2A1.2 appears to be inert. The senescence axis of macroH2A1.1 and PARP is particularly interesting when considering that this isoform is often downregulated in cancer (Sporn et al., 2009). Chen and colleagues suggest that this may render cancer cells resistant to SASP-mediated senescence, bypassing an important barrier to oncogenesis. Moreover, decreases in macroH2A.1.1 levels may also lower the epigenetic barrier toward pluripotency, helping cancer cells lose their differentiated cell state. The new data reported in this issue of Molecular Cell add to the emerging epigenetic barrier function of the PAR-binding macroH2A.1.1 histone and provide new and interesting insights into how chromatin dynamics and signaling are integrated to transcriptionally regulate the secretory phenotype of senescent cells.
ACKNOWLEDGMENTS We acknowledge financial support from the DFG through the Collaborative Research Centers SFB646 ‘‘Networks in genome expression and
Molecular Cell
Preview maintenance’’ dynamics.’’
and
SFB1064
‘‘Chromatin
REFERENCES Chen, H., Ruiz, P.D., Novikov, L., Casill, A.D., Park, J.W., and Gamble, M.J. (2014). Nat. Struct. Mol. Biol. 21, 981–989. Chen, H., Ruiz, P.D., McKimpson, W.M., Novikov, L., Kitsis, R.N., and Gamble, M.J. (2015). Mol. Cell 59, this issue, 719–731. Gaspar-Maia, A., Qadeer, Z.A., Hasson, D., Ratnakumar, K., Leu, N.A., Leroy, G., Liu, S., Costanzi,
C., Valle-Garcia, D., Schaniel, C., et al. (2013). Nat. Commun. 4, 1565.
Pasque, V., Gillich, A., Garrett, N., and Gurdon, J.B. (2011). EMBO J. 30, 2373–2387.
Kustatscher, G., Hothorn, M., Pugieux, C., Scheffzek, K., and Ladurner, A.G. (2005). Nat. Struct. Mol. Biol. 12, 624–625.
Sporn, J.C., Kustatscher, G., Hothorn, T., Collado, M., Serrano, M., Muley, T., Schnabel, P., and Ladurner, A.G. (2009). Oncogene 28, 3423–3428.
~nez, S., Heard, E., Narita, M., Lin, Narita, M., Nu A.W., Hearn, S.A., Spector, D.L., Hannon, G.J., and Lowe, S.W. (2003). Cell 113, 703–716.
Timinszky, G., Till, S., Hassa, P.O., Hothorn, M., Kustatscher, G., Nijmeijer, B., Colombelli, J., Altmeyer, M., Stelzer, E.H., Scheffzek, K., et al. (2009). Nat. Struct. Mol. Biol. 16, 923–929.
Ouararhni, K., Hadj-Slimane, R., Ait-Si-Ali, S., Robin, P., Mietton, F., Harel-Bellan, A., Dimitrov, S., and Hamiche, A. (2006). Genes Dev. 20, 3324–3336.
Zhang, R., Poustovoitov, M.V., Ye, X., Santos, H.A., Chen, W., Daganzo, S.M., Erzberger, J.P., Serebriiskii, I.G., Canutescu, A.A., Dunbrack, R.L., et al. (2005). Dev. Cell 8, 19–30.
Molecular Cell 59, September 3, 2015 ª2015 Elsevier Inc. 715