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news and views Communication between the brain and immune system is a two-way street and has been documented in many physiological settings4. The brain provides adrenergic input for lymphoid tissues throughout the body and can directly control many aspects of inflammation through neurotransmitter receptors expressed on cells of the immune system. For example, vagus nerve activity dampens inflammation in splenic macrophages, decreasing their expression of genes encoding inflammatory molecules through T cell–derived acetylcholine, and also decreases antibody production by B cells5. Similarly, adrenergic stimulation of the lymph nodes can restrict T cell mobility and suppress inflammation6. GABA, a major inhibitory neurotransmitter, has been shown to control autoimmune responses7. As for the widely studied communication in the opposite direction—effects of the immune system on the brain—perhaps the example best understood is in sickness behavior. Here, peripheral cytokines signal to the brain through afferents of the vagus nerve, or directly to circumventricular organs, causing stereotypical changes in behavior during illness8. Numerous other examples demonstrate beneficial effects of the immune system on the CNS in the healthy state. For example, a normal T cell compartment is needed for proper spatial learning, and mice lacking T cells display cognitive impairment9. T cells mediating such pro-cognitive effects have been found in the meningeal spaces and exert their effects, at least in part, through secreted interleukin 4 (ref. 10). Cells of the immune system have also been shown to participate in maintaining adult neurogenesis, in promoting
appropriate stress responses, and in both protective and destructive and immune responses after injury11. Just as neurotransmitter receptors have abundant expression on cells of the immune system, cytokine receptors are widely expressed in the brain, and many of the brain’s functions depend on them. For example, TNF affects synaptic scaling12, and interleukin 1β has a direct effect on long-term potentiation13. Other molecules typically associated with the immune system, such as major histocompatibility complex class I, are also expressed in the brain and participate in normal development and function of the brain14. The article by Kim et al.1 prompted a flashback to my (J.K.’s) years in graduate school, during which my colleagues and I demonstrated that CNS-derived dopamine alleviates the suppressive function of regulatory T cells, allowing a more efficient effector T cell response15. From those and other studies we began to view the brain as a master organ that controls every aspect of body function, including the immune system. A fanciful experiment that would unequivocally prove the brain’s control over the immune system would be to immunize mice lacking brains and show that this immunization is less efficient than that in a mouse with a brain. Although this experiment is clearly not possible, the system used by Kim and et al. for specific deletion and addition of TNF receptors in the hypothalamus in the context of infection with L. monocytogenes1 is a clever approximation. The brain controls tissues through direct innervation, but less is known about how it regulates immunity. Can the brain distinguish between immune responses or types of
pathogens? For decades, neuroimmune interactions were thought to be inherently associated with pathology, and the conventional understanding that the CNS is an immunologically privileged organ meant that the many functional and bidirectional interactions between the brain and the immune system were overlooked. However, the notion that the brain is completely isolated from the immune system is fading, and the work of Kim et al.1 adds an example to an ever-growing list of physiological neuroimmune communication pathways, supporting the notion that instead of being carefully guarded from the immune system, the brain actually controls it. Competing financial interests The authors declare no competing financial interests. 1. Kim, et al. Nat. Immunol. 16, 525–533 (2015). 2. Gregor, M.F. & Hotamisligil, G.S. Annu. Rev. Immunol. 29, 415–445 (2011). 3. Valdearcos, M., Robblee, M.M., Benjamin, D.I., Nomura, D.K., Xu, A.W. & Koliwad, S.K. Cell Rep. 9, 2124–2138 (2014). 4. Steinman, L. Nat. Immunol. 5, 575–581 (2004). 5. Andersson, U. & Tracey, K.J. J. Exp. Med. 209, 1057–1068 (2012). 6. Nakai, A., Hayano, Y., Furuta, F., Noda, M. & Suzuki, K. J. Exp. Med. 211, 2583–2598 (2014). 7. Bhat, R. et al. Proc. Natl. Acad. Sci. USA 107, 2580–2585 (2010). 8. Dantzer, R., O’Connor, J.C., Freund, G.G., Johnson, R.W. & Kelley, K.W. Nat. Rev. Neurosci. 9, 46–56 (2008). 9. Ziv, Y. et al. Nat. Neurosci. 9, 268–275 (2006). 10. Derecki, N.C. et al. J. Exp. Med. 207, 1067–1080 (2010). 11. Kipnis, J., Gadani, S. & Derecki, N.C. Nat. Rev. Immunol. 12, 663–669 (2012). 12. Beattie, E.C. et al. Science 295, 2282–2285 (2002). 13. Schneider, H. et al. Proc. Natl. Acad. Sci. USA 95, 7778–7783 (1998). 14. Shatz, C.J. Neuron 64, 40–45 (2009). 15. Kipnis, J. et al. J. Neurosci. 24, 6133–6143 (2004).
Cytoplasmic methylation fuels leukocyte invasion Bernhard Wehrle-Haller The methyltransferase Ezh2, an epigenetic regulator associated with tumor-cell metastasis, also methlyates the cytoplasmic integrin adaptor talin. This modification inhibits the binding of talin to F-actin, which enhances the migration and invasion of dendritic cells and neutrophils.
T
he methyltransferase Ezh2, which is part of polycomb repressive complex 2 (PRC2), regulates gene expression through trimethy-
Bernhard Wehrle-Haller is in the Department of Cellular Physiology and Metabolism, University of Geneva, Centre Médical Universitaire, Geneva, Switzerland. e-mail: [email protected]
lation of histone H3 at Lys27. These epigenetic modifications control normal stem-cell differentiation but are also associated with many tumors, where PRC2 activity stimulates epithelial-to-mesenchymal transitions, migration, invasion and metastatic spread1. PRC2-induced changes in cell adhesion and migration have so far been attributed to Ezh2-mediated transcriptional repression in the nucleus. However, Gunawan et al. now
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demonstrate that Ezh2-mediated methyltransferase activity in the cytoplasm controls integrin-mediated adhesion and migration through trimethylation of the integrin adaptor talin, blocking its binding site for the cytoskeletal protein F-actin2. The authors show that this activity reduces cell spreading but enhances the migration and tissue invasion of neutrophils and dendritic cells (DCs) in vivo and in vitro. 441
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Katie Vicari/Nature Publishing Group
Chemokine Chemokine receptor
PRC2 F-actin
a
Talin
Ezh2
b
Rho GTPase Vav1
Integrin-binding site
c
d CH3
Integrin
Extracellular matrix
Figure 1 The methylation of talin by Ezh2 regulates cell migration. Rapid cell migration requires coordinated action of multiple cellular processes. (a) At the front of a cell, chemokine signals induce polymerization of the actin cytoskeleton (F-actin) to form protrusions. (b) F-actin also forms adhesion sites when linked to the extracellular matrix. Cell-surface integrins bind to the matrix and create a mechanical connection to F-actin via talin. This leads to recruitment of Vav1 to the adhesion site. (c) Vav1 provides a docking site for Ezh2, which is part of the PRC2 complex and has methylation activity in the nucleus and cytoplasm. The binding of Ezh2 activates Vav1, which then induces polymerization of F-actin through the activity of GTPases of the Rho family8. (d) Gunawan et al. show that Ezh2 also mediates methylation (CH3) of the F-actin-binding motif in talin, which results in the loss of the ‘mechanical’ connection between talin and the actin cytoskeleton and subsequent inactivation of the integrin receptor2. This induces release of the rear of the cell from the extracellular matrix, which allows rapid migration.
Molecular machineries that confer epigenetic modification of histones and promoter regions in the nucleus can also have targets in the cytoplasm and plasma membrane. For example, a study identifying 1,750 proteins with acetylated lysine residues has found that although many of these proteins carry out nuclear functions, a large subset is linked to regulation of the cytoskeleton3. This subset includes proteins such as tubulin and the actin-remodeling protein cortactin, which regulates the adhesion and polarized migration of cells4,5. The acetylation of proteins is tightly linked to the metabolic state of cells and is altered by fuel consumption or starvation6, and nutrient levels are also associated with various types of methylation of DNA and histones, including the modification of trimethylation of histone H3 at Lys27 mediated by PRC2 (ref. 7). Such links suggest ways through which metabolism can influence cellular activities such as adhesion and migration. Cytoplasmic Ezh2-mediated methyltransferase activity is crucial for actin polymerization mediated by the T cell antigen receptor and dorsal ruffling in fibroblasts induced by platelet-derived growth factor 8. However, a cytoplasmic methylation target of Ezh2 was not identified in those studies. Now, Gunawan et al. show that this cytoplasmic methylation activity is an additional participant in the cellular control of adhesion and migration2. Similar to the situation in the nucleus, where PRC2-mediated repression is targeted to specific genes by physical 442
interaction with transcription factors such as snail2 (ref. 9), specific recruitment of Ezh2 to the scaffolding protein Vav1 is needed to alter integrin-dependent adhesion to the extracellular matrix in a talin-dependent manner, as Gunawan et al. now show2. Integrins are transmembrane receptors that are critical during certain cellular processes, such as leukocyte extravasation, but are dispensable for the ‘amoeboid’ migration exhibited by invading cancer cells and for chemokine-induced DC migration 10. Thus, depending on the specific context of the tissue or migratory task to fulfill, cells adapt their adhesion and migration abilities to the respective microenvironment. This can be achieved by changing of the concentration, priming or engagement of cell-surface integrins or by modulation of their cytoplasmic link to the actin cytoskeleton. For rapid migration, it seems that efficient protrusion and adhesion formation is necessary at the cell front and controlled release is necessary at the cell rear; this requires tight control of the assembly of extracellularmatrix adhesions and their turnover 11. Gunawan et al. find that in the absence of Ezh2 in vivo, this adhesion turnover seems to be severely perturbed, which causes the arrest of inflammation-mediated extravasation of neutrophils and DCs and blocks their subsequent invasion into the surrounding tissue2 (Fig. 1). Indeed, when the authors study Ezh2deficient DCs and neutrophils in vitro, they observe normal chemotactic responses but
deficient migration caused by reduced turnover of integrin-dependent adhesion sites, which leads to enhanced cell spreading on fibronectin-coated surfaces2. That last process correlates with enhanced tyrosine phosphorylation of focal adhesion kinase at the protein’s autophosphorylation site and at its kinaseactivation loop. Moreover, Ezh2-deficient DCs form large integrin-dependent cell-matrix adhesions and stress fibers, two features not normally detected in DCs, which are typically loosely attached. Ezh2 binds to the amino-terminal autoinhibiting calponin-homology domain of Vav1, a guanine nucleotide–exchange factor for the Rho family of GTP-binding proteins8. Partial truncation of this domain leads to activation of Vav1 and actin polymerization mediated by the members of the Rho family. A causal link between Ezh2-Vav1 binding and Vav1 activation is suggested by the finding that actin polymerization induced by crosslinking of the invariant signaling protein CD3 is inhibited in T cells that lack either Ezh2 or Vav1 (ref. 8). Because deletion of Vav1 also prevents clustering of the T cell antigen receptor together with talin12, it is likely that talin-mediated activation of the integrin LFA-1 (αLβ2) and Vav1-mediated clustering of T cell antigen receptors at sites of immunological synapses are functionally linked 13. Vav1 is recruited through Src homology 3 domain–mediated binding to proline-rich motifs present in many focal-adhesion adaptors, which suggests that the formation of integrin-dependent adhesion sites provides a scaffold for specific recruitment of Vav1 and its putative activator Ezh2. To investigate whether specific targeting of Ezh2 activity to focal adhesions would reduce the strong adhesion of Ezh2-deficient DCs, the authors introduce wild-type Ezh2, enzymatically inactive Ezh2 or Ezh2 with defective Vav1-binding activity into the cells. Although wild-type Ezh2 is able to diminish both the adhesion and the spreading phenotype, neither mutant Ezh2 has this effect, which suggests that both the enzymatic function and Vav1-targeting function of Ezh2 are required for its adhesionrelated effects. It has been reported that Vav1 directly binds to talin14, a cytoplasmic protein that is concentrated at focal adhesions and that links integrins to actin filaments 11. This prompted Gunawan et al. to investigate whether purified PRC2 is able to methylate talin in vitro2. This is indeed the case, and the authors locate an Ezh2-mediated trimethylated lysine residue in the carboxyl
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terminus of talin’s F-actin-binding domain. The authors assess the functional relevance of this methylation site through the use of mimetic talin mutants and find that demethylation-state mimetic mutants enhance its interaction with F-actin and induce cell spreading, whereas methylationstate mimetic mutants reduce its binding to F-actin and lead to reduced cell spreading in a fibronectin-dependent and thus integrindependent manner. The results noted above suggest that specific recruitment of cytoplasmic PRC2 to the scaffold protein Vav1, which in turn binds to and induces Rho family–mediated functions in integrin-dependent cell-matrix adhesions, provides a spatially and temporally controlled manner with which to inactivate the interaction of talin with F-actin. This mechanism enhances the turnover of integrin-dependent
adhesion sites that are crucial for normal function of the innate and adaptive immune systems. Cytoplasmic PRC2-mediated regulation may also be relevant to tumor-cell invasion and the formation of metastasis. Enhanced expression of Ezh2 is seen in both metastatic melanoma15 and emerging neural crest cells9, in which it causes repression of the expression of genes encoding key developmental molecules, such as E-cadherin or the tumor suppressor adenosylmethionine decarboxylase 1 (refs. 9,15). It can now be proposed that enhanced Ezh2 expression leads to targeted methylation of cytoskeletal and integrin adaptors, which weakens cell-matrix adhesions and facilitates invasive cell activity. Thus, it seems that PRC2’s regulatory functions extend beyond the epigenetic level in multiple cell types.
Competing financial interests The author declares no competing financial interests. 1. Deb, G., Singh, A.K. & Gupta, S. Mol. Cancer Res. 12, 639–653 (2014). 2. Gunawan, M. et al. Nat. Immunol. 16, 505–516 (2015). 3. Choudhary, C. et al. Science 325, 834–840 (2009). 4. Deakin, N.O. & Turner, C.E. J. Cell Biol. 206, 395–413 (2014). 5. Kaluza, D. et al. EMBO J. 30, 4142–4156 (2011). 6. Marino, G. et al. Mol. Cell (2014). 7. Martínez, J.A., Milagro, F.I., Claycombe, K.J. & Schalinske, K.L. Adv. Nutr. 5, 71–81 (2014). 8. Su, I.H. et al. Cell 121, 425–436 (2005). 9. Tien, C.L. et al. Development 142, 722–731 (2015). 10. Lämmermann, T. et al. Nature 453, 51–55 (2008). 11. Wehrle-Haller, B. Curr. Opin. Cell Biol. 24, 569–581 (2012). 12. Fischer, K.D. et al. Curr. Biol. 8, 554–562 (1998). 13. Simonson, W.T., Franco, S.J. & Huttenlocher, A.J. Immunol 177, 7707–7714 (2006). 14. Garcia-Bernal, D., Parmo-Cabanas, M., Dios-Esponera, A., Samaniego, R. & Hernán Pérez de la Ossa, D. Immunity. 31, 953–964 (2009). 15. Zingg, D. et al. Nat. Commun. 6, 6051 (2015).
GBPs take AIM at Francisella Katherine A Fitzgerald & Vijay A K Rathinam Guanylate-binding proteins (GBPs) induced by type I interferon signaling cause lysis of Francisella bacteria that have reached the host-cell cytosol. The liberated bacterial DNA is then sensed by the cytosolic AIM2 inflammasome, which activates caspase-1 and leads to pyroptotic cell death.
C
ells of the innate immune system are equipped with a plethora of sensing mechanisms that respond to microbial products and elicit protective host defenses. Caspase-1-activating inflammasomes are one such mechanism. These multiprotein complexes, composed of either nucleotidebinding domain (NBD) and leucine-rich repeat (LRR)-containing proteins or the DNA receptor AIM2, control pyroptotic cell death and the proteolytic maturation of interleukin 1β (IL-1β) and IL-18. In this issue of Nature Immunology, Man et al.1 and Meunier et al.2 independently identify members of the guanylate-binding protein (GBP) family as essential mediators of AIM2 activation during infection with the cytosolic bacterial pathogen Francisella tularensis. Both groups report that GBPs act downstream of signaling via type I interferon Katherine A. Fitzgerald is in the Program in Innate Immunity, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA. Vijay A. K. Rathinam is in the Department of Immunology, University of Connecticut Health Center, Farmington, Connecticut, USA. e-mail: [email protected]
receptors and bind F. tularensis to liberate its DNA. Thus, the mobilization of GBPs during infection facilitates the exposure of otherwise hidden bacterial ligands. AIM2 binds DNA via a HIN200 domain. It also contains a pyrin domain that allows recruitment of the adaptor ASC (‘apoptosisassociated speck-like protein containing a caspase-recruitment domain’) and caspase-1 in cells transfected with DNA or infected with DNA viruses or cytosolic bacteria3. AIM2 is part of a larger family of PYHIN proteins, all of which are encoded by interferon-inducible genes. Type I interferons induce AIM2 expression in most cell types. However, macrophages have constitutively high expression of AIM2, which allows efficient activation of the AIM2 inflammasome in response to cytosolic delivery of DNA without the need for autocrine signaling via type I interferons. Interferon-independent activation of AIM2 has also been shown in macrophages infected with the herpesvirus murine cytomegalovirus3. However, several studies have identified important roles for type I interferons in promoting activation of the AIM2 inflammasome during infection with F. tularensis3–5, although exactly how this occurs was not known until now.
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Man et al. use gene-expression profiling to identify the interferon-inducible genes that contribute to activation of the AIM2 inflammasome in F. tularensis–infected macrophages and find that type I interferons control de novo expression of the transcription factor IRF1 (‘interferonregulatory factor 1’)1. IRF1 belongs to a family of interferon-regulatory factors that regulate the expression of interferon-β (IFN-β) and interferon-stimulated genes (ISGs). Published work has identified IRF3 and IRF7 as mediators of F. tularensis– induced AIM2 activation 3, since macrophages lacking both IRF3 and IRF7 fail to activate AIM2. However, Man et al. find that macrophages lacking the receptor for type I interferons (IFNAR1) are unable to induce IRF1 expression and, in turn, that IRF1 is essential for F. tularensis–driven AIM2 activation 1. Interestingly, IRF1 is dispensable for AIM2 activation in cells transfected with DNA or infected with murine cytomegalovirus (Fig. 1). In addition, other inflammasome pathways such as NLRP3 and NLRC4 remain intact in cells lacking IRF1. These results indicate a specific role for IRF1 in activation of the AIM2 inflammasome in response to F. tularensis. 443
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news and views Communication between the brain and immune system is a two-way street and has been documented in many physiological settings4. The brain provides adrenergic input for lymphoid tissues throughout the body and can directly control many aspects of inflammation through neurotransmitter receptors expressed on cells of the immune system. For example, vagus nerve activity dampens inflammation in splenic macrophages, decreasing their expression of genes encoding inflammatory molecules through T cell–derived acetylcholine, and also decreases antibody production by B cells5. Similarly, adrenergic stimulation of the lymph nodes can restrict T cell mobility and suppress inflammation6. GABA, a major inhibitory neurotransmitter, has been shown to control autoimmune responses7. As for the widely studied communication in the opposite direction—effects of the immune system on the brain—perhaps the example best understood is in sickness behavior. Here, peripheral cytokines signal to the brain through afferents of the vagus nerve, or directly to circumventricular organs, causing stereotypical changes in behavior during illness8. Numerous other examples demonstrate beneficial effects of the immune system on the CNS in the healthy state. For example, a normal T cell compartment is needed for proper spatial learning, and mice lacking T cells display cognitive impairment9. T cells mediating such pro-cognitive effects have been found in the meningeal spaces and exert their effects, at least in part, through secreted interleukin 4 (ref. 10). Cells of the immune system have also been shown to participate in maintaining adult neurogenesis, in promoting
appropriate stress responses, and in both protective and destructive and immune responses after injury11. Just as neurotransmitter receptors have abundant expression on cells of the immune system, cytokine receptors are widely expressed in the brain, and many of the brain’s functions depend on them. For example, TNF affects synaptic scaling12, and interleukin 1β has a direct effect on long-term potentiation13. Other molecules typically associated with the immune system, such as major histocompatibility complex class I, are also expressed in the brain and participate in normal development and function of the brain14. The article by Kim et al.1 prompted a flashback to my (J.K.’s) years in graduate school, during which my colleagues and I demonstrated that CNS-derived dopamine alleviates the suppressive function of regulatory T cells, allowing a more efficient effector T cell response15. From those and other studies we began to view the brain as a master organ that controls every aspect of body function, including the immune system. A fanciful experiment that would unequivocally prove the brain’s control over the immune system would be to immunize mice lacking brains and show that this immunization is less efficient than that in a mouse with a brain. Although this experiment is clearly not possible, the system used by Kim and et al. for specific deletion and addition of TNF receptors in the hypothalamus in the context of infection with L. monocytogenes1 is a clever approximation. The brain controls tissues through direct innervation, but less is known about how it regulates immunity. Can the brain distinguish between immune responses or types of
pathogens? For decades, neuroimmune interactions were thought to be inherently associated with pathology, and the conventional understanding that the CNS is an immunologically privileged organ meant that the many functional and bidirectional interactions between the brain and the immune system were overlooked. However, the notion that the brain is completely isolated from the immune system is fading, and the work of Kim et al.1 adds an example to an ever-growing list of physiological neuroimmune communication pathways, supporting the notion that instead of being carefully guarded from the immune system, the brain actually controls it. Competing financial interests The authors declare no competing financial interests. 1. Kim, et al. Nat. Immunol. 16, 525–533 (2015). 2. Gregor, M.F. & Hotamisligil, G.S. Annu. Rev. Immunol. 29, 415–445 (2011). 3. Valdearcos, M., Robblee, M.M., Benjamin, D.I., Nomura, D.K., Xu, A.W. & Koliwad, S.K. Cell Rep. 9, 2124–2138 (2014). 4. Steinman, L. Nat. Immunol. 5, 575–581 (2004). 5. Andersson, U. & Tracey, K.J. J. Exp. Med. 209, 1057–1068 (2012). 6. Nakai, A., Hayano, Y., Furuta, F., Noda, M. & Suzuki, K. J. Exp. Med. 211, 2583–2598 (2014). 7. Bhat, R. et al. Proc. Natl. Acad. Sci. USA 107, 2580–2585 (2010). 8. Dantzer, R., O’Connor, J.C., Freund, G.G., Johnson, R.W. & Kelley, K.W. Nat. Rev. Neurosci. 9, 46–56 (2008). 9. Ziv, Y. et al. Nat. Neurosci. 9, 268–275 (2006). 10. Derecki, N.C. et al. J. Exp. Med. 207, 1067–1080 (2010). 11. Kipnis, J., Gadani, S. & Derecki, N.C. Nat. Rev. Immunol. 12, 663–669 (2012). 12. Beattie, E.C. et al. Science 295, 2282–2285 (2002). 13. Schneider, H. et al. Proc. Natl. Acad. Sci. USA 95, 7778–7783 (1998). 14. Shatz, C.J. Neuron 64, 40–45 (2009). 15. Kipnis, J. et al. J. Neurosci. 24, 6133–6143 (2004).
Cytoplasmic methylation fuels leukocyte invasion Bernhard Wehrle-Haller The methyltransferase Ezh2, an epigenetic regulator associated with tumor-cell metastasis, also methlyates the cytoplasmic integrin adaptor talin. This modification inhibits the binding of talin to F-actin, which enhances the migration and invasion of dendritic cells and neutrophils.
T
he methyltransferase Ezh2, which is part of polycomb repressive complex 2 (PRC2), regulates gene expression through trimethy-
Bernhard Wehrle-Haller is in the Department of Cellular Physiology and Metabolism, University of Geneva, Centre Médical Universitaire, Geneva, Switzerland. e-mail: [email protected]
lation of histone H3 at Lys27. These epigenetic modifications control normal stem-cell differentiation but are also associated with many tumors, where PRC2 activity stimulates epithelial-to-mesenchymal transitions, migration, invasion and metastatic spread1. PRC2-induced changes in cell adhesion and migration have so far been attributed to Ezh2-mediated transcriptional repression in the nucleus. However, Gunawan et al. now
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demonstrate that Ezh2-mediated methyltransferase activity in the cytoplasm controls integrin-mediated adhesion and migration through trimethylation of the integrin adaptor talin, blocking its binding site for the cytoskeletal protein F-actin2. The authors show that this activity reduces cell spreading but enhances the migration and tissue invasion of neutrophils and dendritic cells (DCs) in vivo and in vitro. 441
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Katie Vicari/Nature Publishing Group
Chemokine Chemokine receptor
PRC2 F-actin
a
Talin
Ezh2
b
Rho GTPase Vav1
Integrin-binding site
c
d CH3
Integrin
Extracellular matrix
Figure 1 The methylation of talin by Ezh2 regulates cell migration. Rapid cell migration requires coordinated action of multiple cellular processes. (a) At the front of a cell, chemokine signals induce polymerization of the actin cytoskeleton (F-actin) to form protrusions. (b) F-actin also forms adhesion sites when linked to the extracellular matrix. Cell-surface integrins bind to the matrix and create a mechanical connection to F-actin via talin. This leads to recruitment of Vav1 to the adhesion site. (c) Vav1 provides a docking site for Ezh2, which is part of the PRC2 complex and has methylation activity in the nucleus and cytoplasm. The binding of Ezh2 activates Vav1, which then induces polymerization of F-actin through the activity of GTPases of the Rho family8. (d) Gunawan et al. show that Ezh2 also mediates methylation (CH3) of the F-actin-binding motif in talin, which results in the loss of the ‘mechanical’ connection between talin and the actin cytoskeleton and subsequent inactivation of the integrin receptor2. This induces release of the rear of the cell from the extracellular matrix, which allows rapid migration.
Molecular machineries that confer epigenetic modification of histones and promoter regions in the nucleus can also have targets in the cytoplasm and plasma membrane. For example, a study identifying 1,750 proteins with acetylated lysine residues has found that although many of these proteins carry out nuclear functions, a large subset is linked to regulation of the cytoskeleton3. This subset includes proteins such as tubulin and the actin-remodeling protein cortactin, which regulates the adhesion and polarized migration of cells4,5. The acetylation of proteins is tightly linked to the metabolic state of cells and is altered by fuel consumption or starvation6, and nutrient levels are also associated with various types of methylation of DNA and histones, including the modification of trimethylation of histone H3 at Lys27 mediated by PRC2 (ref. 7). Such links suggest ways through which metabolism can influence cellular activities such as adhesion and migration. Cytoplasmic Ezh2-mediated methyltransferase activity is crucial for actin polymerization mediated by the T cell antigen receptor and dorsal ruffling in fibroblasts induced by platelet-derived growth factor 8. However, a cytoplasmic methylation target of Ezh2 was not identified in those studies. Now, Gunawan et al. show that this cytoplasmic methylation activity is an additional participant in the cellular control of adhesion and migration2. Similar to the situation in the nucleus, where PRC2-mediated repression is targeted to specific genes by physical 442
interaction with transcription factors such as snail2 (ref. 9), specific recruitment of Ezh2 to the scaffolding protein Vav1 is needed to alter integrin-dependent adhesion to the extracellular matrix in a talin-dependent manner, as Gunawan et al. now show2. Integrins are transmembrane receptors that are critical during certain cellular processes, such as leukocyte extravasation, but are dispensable for the ‘amoeboid’ migration exhibited by invading cancer cells and for chemokine-induced DC migration 10. Thus, depending on the specific context of the tissue or migratory task to fulfill, cells adapt their adhesion and migration abilities to the respective microenvironment. This can be achieved by changing of the concentration, priming or engagement of cell-surface integrins or by modulation of their cytoplasmic link to the actin cytoskeleton. For rapid migration, it seems that efficient protrusion and adhesion formation is necessary at the cell front and controlled release is necessary at the cell rear; this requires tight control of the assembly of extracellularmatrix adhesions and their turnover 11. Gunawan et al. find that in the absence of Ezh2 in vivo, this adhesion turnover seems to be severely perturbed, which causes the arrest of inflammation-mediated extravasation of neutrophils and DCs and blocks their subsequent invasion into the surrounding tissue2 (Fig. 1). Indeed, when the authors study Ezh2deficient DCs and neutrophils in vitro, they observe normal chemotactic responses but
deficient migration caused by reduced turnover of integrin-dependent adhesion sites, which leads to enhanced cell spreading on fibronectin-coated surfaces2. That last process correlates with enhanced tyrosine phosphorylation of focal adhesion kinase at the protein’s autophosphorylation site and at its kinaseactivation loop. Moreover, Ezh2-deficient DCs form large integrin-dependent cell-matrix adhesions and stress fibers, two features not normally detected in DCs, which are typically loosely attached. Ezh2 binds to the amino-terminal autoinhibiting calponin-homology domain of Vav1, a guanine nucleotide–exchange factor for the Rho family of GTP-binding proteins8. Partial truncation of this domain leads to activation of Vav1 and actin polymerization mediated by the members of the Rho family. A causal link between Ezh2-Vav1 binding and Vav1 activation is suggested by the finding that actin polymerization induced by crosslinking of the invariant signaling protein CD3 is inhibited in T cells that lack either Ezh2 or Vav1 (ref. 8). Because deletion of Vav1 also prevents clustering of the T cell antigen receptor together with talin12, it is likely that talin-mediated activation of the integrin LFA-1 (αLβ2) and Vav1-mediated clustering of T cell antigen receptors at sites of immunological synapses are functionally linked 13. Vav1 is recruited through Src homology 3 domain–mediated binding to proline-rich motifs present in many focal-adhesion adaptors, which suggests that the formation of integrin-dependent adhesion sites provides a scaffold for specific recruitment of Vav1 and its putative activator Ezh2. To investigate whether specific targeting of Ezh2 activity to focal adhesions would reduce the strong adhesion of Ezh2-deficient DCs, the authors introduce wild-type Ezh2, enzymatically inactive Ezh2 or Ezh2 with defective Vav1-binding activity into the cells. Although wild-type Ezh2 is able to diminish both the adhesion and the spreading phenotype, neither mutant Ezh2 has this effect, which suggests that both the enzymatic function and Vav1-targeting function of Ezh2 are required for its adhesionrelated effects. It has been reported that Vav1 directly binds to talin14, a cytoplasmic protein that is concentrated at focal adhesions and that links integrins to actin filaments 11. This prompted Gunawan et al. to investigate whether purified PRC2 is able to methylate talin in vitro2. This is indeed the case, and the authors locate an Ezh2-mediated trimethylated lysine residue in the carboxyl
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terminus of talin’s F-actin-binding domain. The authors assess the functional relevance of this methylation site through the use of mimetic talin mutants and find that demethylation-state mimetic mutants enhance its interaction with F-actin and induce cell spreading, whereas methylationstate mimetic mutants reduce its binding to F-actin and lead to reduced cell spreading in a fibronectin-dependent and thus integrindependent manner. The results noted above suggest that specific recruitment of cytoplasmic PRC2 to the scaffold protein Vav1, which in turn binds to and induces Rho family–mediated functions in integrin-dependent cell-matrix adhesions, provides a spatially and temporally controlled manner with which to inactivate the interaction of talin with F-actin. This mechanism enhances the turnover of integrin-dependent
adhesion sites that are crucial for normal function of the innate and adaptive immune systems. Cytoplasmic PRC2-mediated regulation may also be relevant to tumor-cell invasion and the formation of metastasis. Enhanced expression of Ezh2 is seen in both metastatic melanoma15 and emerging neural crest cells9, in which it causes repression of the expression of genes encoding key developmental molecules, such as E-cadherin or the tumor suppressor adenosylmethionine decarboxylase 1 (refs. 9,15). It can now be proposed that enhanced Ezh2 expression leads to targeted methylation of cytoskeletal and integrin adaptors, which weakens cell-matrix adhesions and facilitates invasive cell activity. Thus, it seems that PRC2’s regulatory functions extend beyond the epigenetic level in multiple cell types.
Competing financial interests The author declares no competing financial interests. 1. Deb, G., Singh, A.K. & Gupta, S. Mol. Cancer Res. 12, 639–653 (2014). 2. Gunawan, M. et al. Nat. Immunol. 16, 505–516 (2015). 3. Choudhary, C. et al. Science 325, 834–840 (2009). 4. Deakin, N.O. & Turner, C.E. J. Cell Biol. 206, 395–413 (2014). 5. Kaluza, D. et al. EMBO J. 30, 4142–4156 (2011). 6. Marino, G. et al. Mol. Cell (2014). 7. Martínez, J.A., Milagro, F.I., Claycombe, K.J. & Schalinske, K.L. Adv. Nutr. 5, 71–81 (2014). 8. Su, I.H. et al. Cell 121, 425–436 (2005). 9. Tien, C.L. et al. Development 142, 722–731 (2015). 10. Lämmermann, T. et al. Nature 453, 51–55 (2008). 11. Wehrle-Haller, B. Curr. Opin. Cell Biol. 24, 569–581 (2012). 12. Fischer, K.D. et al. Curr. Biol. 8, 554–562 (1998). 13. Simonson, W.T., Franco, S.J. & Huttenlocher, A.J. Immunol 177, 7707–7714 (2006). 14. Garcia-Bernal, D., Parmo-Cabanas, M., Dios-Esponera, A., Samaniego, R. & Hernán Pérez de la Ossa, D. Immunity. 31, 953–964 (2009). 15. Zingg, D. et al. Nat. Commun. 6, 6051 (2015).
GBPs take AIM at Francisella Katherine A Fitzgerald & Vijay A K Rathinam Guanylate-binding proteins (GBPs) induced by type I interferon signaling cause lysis of Francisella bacteria that have reached the host-cell cytosol. The liberated bacterial DNA is then sensed by the cytosolic AIM2 inflammasome, which activates caspase-1 and leads to pyroptotic cell death.
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ells of the innate immune system are equipped with a plethora of sensing mechanisms that respond to microbial products and elicit protective host defenses. Caspase-1-activating inflammasomes are one such mechanism. These multiprotein complexes, composed of either nucleotidebinding domain (NBD) and leucine-rich repeat (LRR)-containing proteins or the DNA receptor AIM2, control pyroptotic cell death and the proteolytic maturation of interleukin 1β (IL-1β) and IL-18. In this issue of Nature Immunology, Man et al.1 and Meunier et al.2 independently identify members of the guanylate-binding protein (GBP) family as essential mediators of AIM2 activation during infection with the cytosolic bacterial pathogen Francisella tularensis. Both groups report that GBPs act downstream of signaling via type I interferon Katherine A. Fitzgerald is in the Program in Innate Immunity, Department of Medicine, University of Massachusetts Medical School, Worcester, Massachusetts, USA. Vijay A. K. Rathinam is in the Department of Immunology, University of Connecticut Health Center, Farmington, Connecticut, USA. e-mail: [email protected]
receptors and bind F. tularensis to liberate its DNA. Thus, the mobilization of GBPs during infection facilitates the exposure of otherwise hidden bacterial ligands. AIM2 binds DNA via a HIN200 domain. It also contains a pyrin domain that allows recruitment of the adaptor ASC (‘apoptosisassociated speck-like protein containing a caspase-recruitment domain’) and caspase-1 in cells transfected with DNA or infected with DNA viruses or cytosolic bacteria3. AIM2 is part of a larger family of PYHIN proteins, all of which are encoded by interferon-inducible genes. Type I interferons induce AIM2 expression in most cell types. However, macrophages have constitutively high expression of AIM2, which allows efficient activation of the AIM2 inflammasome in response to cytosolic delivery of DNA without the need for autocrine signaling via type I interferons. Interferon-independent activation of AIM2 has also been shown in macrophages infected with the herpesvirus murine cytomegalovirus3. However, several studies have identified important roles for type I interferons in promoting activation of the AIM2 inflammasome during infection with F. tularensis3–5, although exactly how this occurs was not known until now.
nature immunology volume 16 number 5 MAy 2015
Man et al. use gene-expression profiling to identify the interferon-inducible genes that contribute to activation of the AIM2 inflammasome in F. tularensis–infected macrophages and find that type I interferons control de novo expression of the transcription factor IRF1 (‘interferonregulatory factor 1’)1. IRF1 belongs to a family of interferon-regulatory factors that regulate the expression of interferon-β (IFN-β) and interferon-stimulated genes (ISGs). Published work has identified IRF3 and IRF7 as mediators of F. tularensis– induced AIM2 activation 3, since macrophages lacking both IRF3 and IRF7 fail to activate AIM2. However, Man et al. find that macrophages lacking the receptor for type I interferons (IFNAR1) are unable to induce IRF1 expression and, in turn, that IRF1 is essential for F. tularensis–driven AIM2 activation 1. Interestingly, IRF1 is dispensable for AIM2 activation in cells transfected with DNA or infected with murine cytomegalovirus (Fig. 1). In addition, other inflammasome pathways such as NLRP3 and NLRC4 remain intact in cells lacking IRF1. These results indicate a specific role for IRF1 in activation of the AIM2 inflammasome in response to F. tularensis. 443
