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Epigenetic Control of Autophagy: Nuclear Events Gain More Attention
Molecular Cell
Minireview Epigenetic Control of Autophagy: Nuclear Events Gain More Attention Sung Hee Baek1,* and Keun Il Kim2,* 1Creative Research Initiatives Center for Chromatin Dynamics, School of Biological Sciences, Seoul National University, Seoul 08826, South Korea 2Department of Biological Sciences, Sookmyung Women’s University, Seoul 04310, South Korea *Correspondence: [email protected] (S.H.B.), [email protected] (K.I.K.) http://dx.doi.org/10.1016/j.molcel.2016.12.027
Autophagy is an evolutionarily conserved catabolic process. Although the components of autophagy in cytoplasm have been well-studied, the molecular basis for the epigenetic regulation of autophagy is poorly understood. It is becoming more important to propose a ‘‘whole-cell view’’ of autophagy embracing both cytoplasmic and nuclear events. Thus, it is great timing to summarize current status and discuss future direction. Introduction Macroautophagy/autophagy is a highly conserved self-digestion process, essential for maintaining homeostasis and viability in response to various stresses including nutrient and energy starvation (Lum et al., 2005; Mizushima et al., 2008). Dysregulated autophagy is related to many human diseases, including cancer and neurodegenerative diseases (Levine and Kroemer, 2008). Although autophagy is mainly seen as a cytoplasmic event, recent studies reveal that transcriptional and epigenetic regulation occurring in the nucleus is critical for the autophagic process €llgrabe et al., 2014). Histone modifications exerted by CARM1 (Fu H3R17 methyltransferase (Shin et al., 2016), G9a H3K9 methyltransferase (Artal-Martinez de Narvajas et al., 2013), EZH2 H3K27 methyltransferase (Wei et al., 2015), SIRT1 H4K16 deacetylase, and its counterpart hMOF H4K16 acetyltransferase €llgrabe et al., 2013) have been reported as critical nuclear (Fu events of autophagy thus far. This minireview will be focused on the epigenetic and transcriptional control of autophagy, which is mainly triggered by upstream signaling cascades and then modulated by epigenetic enzymes in the nucleus. Histone Modifications and Epigenetic Enzymes Linked to the Regulation of Autophagy Histones are subject to a variety of post-translational modifications (PTMs) including phosphorylation, methylation, acetylation, and ubiquitination by groups of specific epigenetic enzymes (Kouzarides, 2007). These diverse histone PTMs have the potential to affect the overall chromatin states for either activating or repressing transcription, mainly by modulating the accessibility of transcription factors to the chromatin (Baek, 2011; Lawrence et al., 2016). The histone PTMs can serve as specific binding sites for transcription factors and coregulators. Therefore, histone modifications form a signal platform which can accommodate specific cofactors to local chromatin region linking upstream signal to downstream gene expression. What could be roles of histone modifications during autophagy? Also, can the altered histone modifications be associated with the dysfunction of autophagic process? Accumulating evidence indicates that histone modifications contribute to the control of autophagic flux, cell fate decision, and sustained autophagy. Therefore, specific histone modifications have emerged
as an integral part of the autophagic process. Understanding altered histone modification landscape in autophagy-related diseases compared to healthy states would be an exciting area to study in the future. Here, we summarize specific histone marks directly or indirectly related to autophagy, along with the responsible epigenetic enzymes and their functional outcomes affecting various biological processes (Figure 1). Sustained Autophagy: H3R17 Dimethylation by CARM1 Arginine methylation in histones is carried out by a family of protein arginine methyltransferases (PRMTs) (Yang and Bedford, 2013). Histone H3R17 dimethylation that is solely mediated by coactivator-associated arginine methyltransferase 1 (CARM1, also called PRMT4) is linked to transcriptional activation. While acute and rapid response of autophagy occurs primarily in the cytoplasm, prolonged starvation results in the activation of transcriptional program and changes in epigenetic network. Recent study has revealed a new link between H3R17 dimethylation and autophagy (Shin et al., 2016). Glucose starvation increases genome-wide H3R17 dimethylation resulting from CARM1 induction in the nucleus, and CARM1 functions as a coactivator of transcription factor EB (TFEB) for activation of autophagy and lysosomal genes. Intriguingly, CARM1 level is tightly regulated by the ubiquitindependent degradation system. Under glucose-rich conditions, nuclear CARM1 is degraded by SKP2-SCF E3 ubiquitin ligase. When glucose starvation persists and transcription of various autophagy-related genes is needed to sustain autophagy, AMP-activated protein kinase (AMPK) accumulates in the nucleus. Among AMPK isoforms, AMPKa2 is preferentially expressed in the nucleus (Salt et al., 1998). Unlike AMPKa1, the level of AMPKa2 protein and phosphorylated AMPK, its active form, increases in the nucleus upon glucose starvation. It is noteworthy that the timeframe of nuclear AMPKa2 accumulation is concomitant with the induction of CARM1. AMPKdependent downregulation of SKP2 in the nucleus allows CARM1 to escape from SCF E3 ubiquitin ligase, resulting in its stabilization upon nutrient starvation. This stabilization of histone modifiers in the nucleus is an efficient way to regulate target gene expression and could be a prototype of protein stabilization in specific compartments of the cells during starvation-induced autophagy. Molecular Cell 65, March 2, 2017 ª 2016 Elsevier Inc. 781
Molecular Cell
Minireview Figure 1. Histone Modifications and Epigenetic Enzymes Linked to the Regulation of Autophagy Autophagic histone modifications and responsible epigenetic enzymes are depicted. CARM1dependent H3R17 dimethylation functions in sustained autophagy (A). Autophagic life-anddeath decision via the hMOF-SIRT1 control of H4K16 acetylation is identified (B). H3K9 dimethylation by G9a and H3K27 trimethylation by EZH2 are suppression marks for autophagy (C). Each function is discussed in the main text. Ac, acetylated; me, methylated.
Ellagic acid selectively blocks histone H3R17 methylation (Selvi et al., 2010). Given that inhibition of H3R17me2 mark by ellagic acid almost completely blocks autophagy occurrence both in vitro and in vivo, ellagic acid might have a potential to be developed as a therapeutic agent in autophagy-related diseases. These findings provide evidence of CARM1-mediated histone arginine methylation as a critical nuclear event in the regulation of autophagy and shed light on potential therapeutic targeting of the recently identified AMPK-SKP2-CARM1 signaling axis in autophagy-related diseases. Cell Survival versus Death Decision: H4K16 Acetylation Status by hMOF-SIRT1 Although autophagy is known to be cytoprotective, dysregulated autophagy results in cell death (Green and Levine, 2014). While uncovering how autophagy governs life and death of the cell is important, hMOF acetyltransferase-SIRT1 deacetylase, which controls H4K16 acetylation (H4K16Ac) status, turned out to be €llgrabe et al., 2014). Autophagy indeterminant in the process (Fu duction by various stimuli is associated with reduced acetylation of H4K16. The genome-wide investigation reveals that H4K16 deacetylation by rapamycin treatment is associated with transcriptional activation of autophagy-related genes. Rapamycin-induced autophagic process coincides with downregulation of hMOF and deacetylation of H4K16 without changing SIRT1 protein levels or activity. Most importantly, inhibition of H4K16Ac downregulation by overexpression of hMOF or inhibition of SIRT1 upon autophagy increases autophagic flux and results in cell death. Independent of 782 Molecular Cell 65, March 2, 2017
H4K16 deacetylation, SIRT1 has been shown to deacetylate nuclear LC3 to license its release to cytoplasm during starvation (Huang et al., 2015). SIRT1inhibitors including VPA and Ex527 are promising candidates for the treatment of cancer and autophagy-related diseases. Suppressed Autophagy: H3K9 Dimethylation by G9a and H3K27 Trimethylation by EZH2 H3K9 can be methylated by histone methyltransferases including G9a, and H3K9 methylation is associated with transcriptional repression. Recent study showed that G9a-mediated H3K9 methylation acts as a repressor of autophagy under nutrient-rich conditions (Artal-Martinez de Narvajas et al., 2013). Nutrient deprivation leads to dissociation of G9a from the promoter, consequently resulting in transcriptional activation. Pharmacological inhibition of G9a results in autophagy occurrence. Further, when naive CD4+ T cell is activated, which accompanies autophagy gene expression, dissociation of G9a and recruitment of c-Jun transcription factor at several autophagy gene promoters are involved in the progression of autophagy. G9a is therefore an epigenetic regulator of autophagy, and the inhibition of G9amediated gene repression is important during autophagy induction. Enhancer of zeste homolog 2 (EZH2) mainly catalyzes H3K27 trimethylation and represses transcription of several negative regulators of the mechanistic target of rapamycin (mTOR) pathway, including TSC2, DEPTOR, RHOA, and GPI, under nutrient-rich conditions (Wei et al., 2015). EZH2 is recruited to those target promoters via metastasis-associated 1 family member 2 (MTA2) to silence target genes exemplified by TSC2. Downregulation of TSC2 by EZH2 results in mTOR activation, leading to inhibition of autophagy. Further, knockdown of EZH2 leads to the formation of autophagosome, and inhibition of EZH2 activity induces autophagy. Given that the expression of EZH2 and MTA2 correlates negatively with expression of TSC2 in human colon cancer tissues, a potential functional link among epigenetic regulation, autophagy induction, and tumorigenesis has been suggested. Although further studies are needed, selective inhibitors for EZH2 would not only prevent silencing of tumor suppressor genes but also activate
Molecular Cell
Minireview
Figure 2. Upstream Stimuli and Known Histone Modifications in Autophagy Known histone modifications linked to enhanced autophagy or suppressed autophagy along with reported upstream stimuli are presented.
autophagy, an interesting area of research for cancer treatment. Other Histone Modifications Related to Autophagy Some other histone modifications have been reported to be potentially linked to autophagy, although responsible enzymes have not been uncovered yet. H3K56 acetylation, H4K20 methylation, and H2BK120 monoubiquitination are involved in €llgrabe suppressed autophagy (Figure 2) (Chen et al., 2016; Fu et al., 2014). Histone modifications have a genome-wide role in regulating transcription. There are obligatory histone PTMs involved in transcriptional activation such as H3K9Ac, H3K14Ac, and H3K36me, and thus some of the histone PTMs which change upon autophagic process might not be specific marks related to autophagy. However, we speculate that there are signal-induced particular histone PTMs at autophagy genes (i.e., CARM1 and H3R17me) affecting the autophagic process.
Therefore, it would be challenging to explore how upstream signaling pathways affect downstream target gene expressions along with differential regulation of histone modifications and epigenetic enzymes to confer the specificity of autophagy regulation. Further studies will reveal more autophagy-related histone marks and the specificity of autophagy regulation by histone modifications. Interplay between the Cytoplasm and the Nucleus for the Regulation of Autophagy Program In addition to the histone modifications and epigenetic enzymes that relay cytoplasmic events to the nucleus, several transcription factors play a role in signal transfer. Various transcription factors involved in the autophagic process either function in both compartments or are sequestered in the cytoplasm and translocate into the nucleus to participate in distinct nuclear Molecular Cell 65, March 2, 2017 783
Molecular Cell
Minireview regulations of autophagy. It is now widely accepted that transcriptional regulation occurs to replenish cytoplasmic material €llgrabe et al., 2014; Shin et al., and sustain autophagy (Fu 2016). The induction of autophagy leads to the activation and nuclear translocation of various transcription factors acting as crucial regulators of the autophagic process. These transcription factors might function together with histone-modifying enzymes, although the experimental evidence is limited to the CARM1-TFEB collaboration in sustained autophagy upon glucose starvation. The first example is TFEB, a member of the basic helix-loophelix leucine zipper family (Settembre et al., 2011). Under nutrient-rich conditions, TFEB is phosphorylated by mTORC1 or ERK2 and localized in the cytoplasm by binding to 14-3-3 (Raben and Puertollano, 2016). Upon autophagy induction, TFEB is dephosphorylated by calcineurin and translocates to the nucleus for transcriptional activation of autophagy genes and lysosomal genes. The cytoplasm-to-nucleus translocation of TFEB was also observed under ER stress response (Martina et al., 2016). The second example is forkhead box O (FOXO) family members of transcription factors, regulated by phosphorylationdependent nuclear-cytoplasmic shuttling (Van Der Heide et al., 2004). Among them, FOXO3 is involved in autophagy induction in multiple ways. FOXO3 is phosphorylated by AMPK in nutrient-deprived conditions and induces autophagy by activating transcription (Greer et al., 2007). On the other hand, transcriptional repression by FOXO3 also contributes autophagy induction. Under glucose-deprived conditions, FOXO3 phosphorylation by AMPK downregulates SKP2 transcription, resulting in CARM1 stabilization and autophagy induction (Shin et al., 2016). Although AMPK-mediated phosphorylation of FOXO3 does not change cellular localization of FOXO3 under nutrientdeprived conditions (Greer et al., 2007), reactive oxygen species-induced phosphorylation of FOXO3 by JNK induces nuclear translocation (Go´mez-Puerto et al., 2016), indicating a distinct regulatory mode of transcription factors depending on upstream autophagic stimuli. The third example is the GATA transcription factor Gln3, which is phosphorylated by mTOR, causing its retention in the cytoplasm under nutrient-rich conditions (Beck and Hall, 1999). mTOR inhibition, on the other hand, under nutrient-deprived conditions allows dephosphorylation and nuclear localization of Gln3 and activation of autophagy genes (Chan et al., 2001). Together, these observations indicate that transcriptional control of autophagy can be achieved by phosphorylation-dependent nuclear-cytoplasmic shuttling. As another nuclear event, an autophagy-targeting protein, LC3, is present in the nucleus interacting with lamin B1 and lamin-associated chromatin domains and participates in the changes of histone levels during senescence (Dou et al., 2015). Concluding Remark: Histone Code as a Decision Maker in Autophagy At the beginning of this minireview, we have asked two important questions regarding histone modifications related to autophagy. First, what could be the roles of histone modifications during autophagy? Alteration in specific histone marks during autophagy affects the transcriptional and epigenetic regulatory pro784 Molecular Cell 65, March 2, 2017
gram. Therefore, identification of autophagy-regulated specific histone marks offers a conceptual advance both to understand the transcriptional response to upstream signals eliciting the autophagic process and to form a potential aspect of longterm regulation of autophagy. Some of the histone marks exhibit genome-wide changes, as in the case of H4K16Ac and €llgrabe et al., 2013; Shin et al., 2016), upon autoH3R17me2 (Fu phagic occurrence. Understanding autophagic stimuli-induced histone modifications will allow us to identify specific target genes and their functions during autophagic process. Second, when the autophagic process becomes dysfunctional, is it associated with altered histone modifications? There is very likely a possible correlation between altered histone modifications and autophagic defects. As cancer progresses, histone modification patterns are changed compared to normal control (Sharma et al., 2010). It has been shown that disruption of normal patterns of histone modifications is related to cancer, and specific histone modification patterns are typically associated with the disease. Although studies on the functional relationship between dysregulated autophagic process and altered histone modifications are at the infant stage, there are many exciting aspects to be discovered. Future studies will hopefully tell us more exciting stories. REFERENCES Artal-Martinez de Narvajas, A., Gomez, T.S., Zhang, J.S., Mann, A.O., Taoda, Y., Gorman, J.A., Herreros-Villanueva, M., Gress, T.M., Ellenrieder, V., Bujanda, L., et al. (2013). Epigenetic regulation of autophagy by the methyltransferase G9a. Mol. Cell. Biol. 33, 3983–3993. Baek, S.H. (2011). When signaling kinases meet histones and histone modifiers in the nucleus. Mol. Cell 42, 274–284. Beck, T., and Hall, M.N. (1999). The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors. Nature 402, 689–692. Chan, T.F., Bertram, P.G., Ai, W., and Zheng, X.F. (2001). Regulation of APG14 expression by the GATA-type transcription factor Gln3p. J. Biol. Chem. 276, 6463–6467. Chen, S., Jing, Y., Kang, X., Yang, L., Wang, D.L., Zhang, W., Zhang, L., Chen, P., Chang, J.F., Yang, X.M., and Sun, F.L. (2016). Histone H2B monoubiquitination is a critical epigenetic switch for the regulation of autophagy. Nucleic Acids Res. http://dx.doi.org/10.1093/nar/gkw1025. Dou, Z., Xu, C., Donahue, G., Shimi, T., Pan, J.-A., Zhu, J., Ivanov, A., Capell, B.C., Drake, A.M., Shah, P.P., et al. (2015). Autophagy mediates degradation of nuclear lamina. Nature 527, 105–109. €llgrabe, J., Lynch-Day, M.A., Heldring, N., Li, W., Struijk, R.B., Ma, Q., Fu Hermanson, O., Rosenfeld, M.G., Klionsky, D.J., and Joseph, B. (2013). The histone H4 lysine 16 acetyltransferase hMOF regulates the outcome of autophagy. Nature 500, 468–471. €llgrabe, J., Klionsky, D.J., and Joseph, B. (2014). The return of the nucleus: Fu transcriptional and epigenetic control of autophagy. Nat. Rev. Mol. Cell Biol. 15, 65–74. Go´mez-Puerto, M.C., Verhagen, L.P., Braat, A.K., Lam, E.W., Coffer, P.J., and Lorenowicz, M.J. (2016). Activation of autophagy by FOXO3 regulates redox homeostasis during osteogenic differentiation. Autophagy 12, 1804–1816. Green, D.R., and Levine, B. (2014). To be or not to be? How selective autophagy and cell death govern cell fate. Cell 157, 65–75. Greer, E.L., Oskoui, P.R., Banko, M.R., Maniar, J.M., Gygi, M.P., Gygi, S.P., and Brunet, A. (2007). The energy sensor AMP-activated protein kinase directly regulates the mammalian FOXO3 transcription factor. J. Biol. Chem. 282, 30107–30119.
Molecular Cell
Minireview Huang, R., Xu, Y., Wan, W., Shou, X., Qian, J., You, Z., Liu, B., Chang, C., Zhou, T., Lippincott-Schwartz, J., and Liu, W. (2015). Deacetylation of nuclear LC3 drives autophagy initiation under starvation. Mol. Cell 57, 456–466. Kouzarides, T. (2007). Chromatin modifications and their function. Cell 128, 693–705. Lawrence, M., Daujat, S., and Schneider, R. (2016). Lateral thinking: how histone modifications regulate gene expression. Trends Genet. 32, 42–56. Levine, B., and Kroemer, G. (2008). Autophagy in the pathogenesis of disease. Cell 132, 27–42. Lum, J.J., DeBerardinis, R.J., and Thompson, C.B. (2005). Autophagy in metazoans: cell survival in the land of plenty. Nat. Rev. Mol. Cell Biol. 6, 439–448. Martina, J.A., Diab, H.I., Brady, O.A., and Puertollano, R. (2016). TFEB and TFE3 are novel components of the integrated stress response. EMBO J. 35, 479–495. Mizushima, N., Levine, B., Cuervo, A.M., and Klionsky, D.J. (2008). Autophagy fights disease through cellular self-digestion. Nature 451, 1069–1075. Raben, N., and Puertollano, R. (2016). TFEB and TFE3: linking lysosomes to cellular adaptation to stress. Annu. Rev. Cell Dev. Biol. 32, 255–278. Salt, I., Celler, J.W., Hawley, S.A., Prescott, A., Woods, A., Carling, D., and Hardie, D.G. (1998). AMP-activated protein kinase: greater AMP dependence, and preferential nuclear localization, of complexes containing the alpha2 isoform. Biochem. J. 334, 177–187.
Selvi, B.R., Batta, K., Kishore, A.H., Mantelingu, K., Varier, R.A., Balasubramanyam, K., Pradhan, S.K., Dasgupta, D., Sriram, S., Agrawal, S., and Kundu, T.K. (2010). Identification of a novel inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1)-mediated methylation of histone H3 Arg-17. J. Biol. Chem. 285, 7143–7152. Settembre, C., Di Malta, C., Polito, V.A., Garcia Arencibia, M., Vetrini, F., Erdin, S., Erdin, S.U., Huynh, T., Medina, D., Colella, P., et al. (2011). TFEB links autophagy to lysosomal biogenesis. Science 332, 1429–1433. Sharma, S., Kelly, T.K., and Jones, P.A. (2010). Epigenetics in cancer. Carcinogenesis 31, 27–36. Shin, H.J., Kim, H., Oh, S., Lee, J.-G., Kee, M., Ko, H.-J., Kweon, M.-N., Won, K.J., and Baek, S.H. (2016). AMPK-SKP2-CARM1 signalling cascade in transcriptional regulation of autophagy. Nature 534, 553–557. Van Der Heide, L.P., Hoekman, M.F.M., and Smidt, M.P. (2004). The ins and outs of FoxO shuttling: mechanisms of FoxO translocation and transcriptional regulation. Biochem. J. 380, 297–309. Wei, F.-Z., Cao, Z., Wang, X., Wang, H., Cai, M.-Y., Li, T., Hattori, N., Wang, D., Du, Y., Song, B., et al. (2015). Epigenetic regulation of autophagy by the methyltransferase EZH2 through an MTOR-dependent pathway. Autophagy 11, 2309–2322. Yang, Y., and Bedford, M.T. (2013). Protein arginine methyltransferases and cancer. Nat. Rev. Cancer 13, 37–50.
Molecular Cell 65, March 2, 2017 785
Minireview Epigenetic Control of Autophagy: Nuclear Events Gain More Attention Sung Hee Baek1,* and Keun Il Kim2,* 1Creative Research Initiatives Center for Chromatin Dynamics, School of Biological Sciences, Seoul National University, Seoul 08826, South Korea 2Department of Biological Sciences, Sookmyung Women’s University, Seoul 04310, South Korea *Correspondence: [email protected] (S.H.B.), [email protected] (K.I.K.) http://dx.doi.org/10.1016/j.molcel.2016.12.027
Autophagy is an evolutionarily conserved catabolic process. Although the components of autophagy in cytoplasm have been well-studied, the molecular basis for the epigenetic regulation of autophagy is poorly understood. It is becoming more important to propose a ‘‘whole-cell view’’ of autophagy embracing both cytoplasmic and nuclear events. Thus, it is great timing to summarize current status and discuss future direction. Introduction Macroautophagy/autophagy is a highly conserved self-digestion process, essential for maintaining homeostasis and viability in response to various stresses including nutrient and energy starvation (Lum et al., 2005; Mizushima et al., 2008). Dysregulated autophagy is related to many human diseases, including cancer and neurodegenerative diseases (Levine and Kroemer, 2008). Although autophagy is mainly seen as a cytoplasmic event, recent studies reveal that transcriptional and epigenetic regulation occurring in the nucleus is critical for the autophagic process €llgrabe et al., 2014). Histone modifications exerted by CARM1 (Fu H3R17 methyltransferase (Shin et al., 2016), G9a H3K9 methyltransferase (Artal-Martinez de Narvajas et al., 2013), EZH2 H3K27 methyltransferase (Wei et al., 2015), SIRT1 H4K16 deacetylase, and its counterpart hMOF H4K16 acetyltransferase €llgrabe et al., 2013) have been reported as critical nuclear (Fu events of autophagy thus far. This minireview will be focused on the epigenetic and transcriptional control of autophagy, which is mainly triggered by upstream signaling cascades and then modulated by epigenetic enzymes in the nucleus. Histone Modifications and Epigenetic Enzymes Linked to the Regulation of Autophagy Histones are subject to a variety of post-translational modifications (PTMs) including phosphorylation, methylation, acetylation, and ubiquitination by groups of specific epigenetic enzymes (Kouzarides, 2007). These diverse histone PTMs have the potential to affect the overall chromatin states for either activating or repressing transcription, mainly by modulating the accessibility of transcription factors to the chromatin (Baek, 2011; Lawrence et al., 2016). The histone PTMs can serve as specific binding sites for transcription factors and coregulators. Therefore, histone modifications form a signal platform which can accommodate specific cofactors to local chromatin region linking upstream signal to downstream gene expression. What could be roles of histone modifications during autophagy? Also, can the altered histone modifications be associated with the dysfunction of autophagic process? Accumulating evidence indicates that histone modifications contribute to the control of autophagic flux, cell fate decision, and sustained autophagy. Therefore, specific histone modifications have emerged
as an integral part of the autophagic process. Understanding altered histone modification landscape in autophagy-related diseases compared to healthy states would be an exciting area to study in the future. Here, we summarize specific histone marks directly or indirectly related to autophagy, along with the responsible epigenetic enzymes and their functional outcomes affecting various biological processes (Figure 1). Sustained Autophagy: H3R17 Dimethylation by CARM1 Arginine methylation in histones is carried out by a family of protein arginine methyltransferases (PRMTs) (Yang and Bedford, 2013). Histone H3R17 dimethylation that is solely mediated by coactivator-associated arginine methyltransferase 1 (CARM1, also called PRMT4) is linked to transcriptional activation. While acute and rapid response of autophagy occurs primarily in the cytoplasm, prolonged starvation results in the activation of transcriptional program and changes in epigenetic network. Recent study has revealed a new link between H3R17 dimethylation and autophagy (Shin et al., 2016). Glucose starvation increases genome-wide H3R17 dimethylation resulting from CARM1 induction in the nucleus, and CARM1 functions as a coactivator of transcription factor EB (TFEB) for activation of autophagy and lysosomal genes. Intriguingly, CARM1 level is tightly regulated by the ubiquitindependent degradation system. Under glucose-rich conditions, nuclear CARM1 is degraded by SKP2-SCF E3 ubiquitin ligase. When glucose starvation persists and transcription of various autophagy-related genes is needed to sustain autophagy, AMP-activated protein kinase (AMPK) accumulates in the nucleus. Among AMPK isoforms, AMPKa2 is preferentially expressed in the nucleus (Salt et al., 1998). Unlike AMPKa1, the level of AMPKa2 protein and phosphorylated AMPK, its active form, increases in the nucleus upon glucose starvation. It is noteworthy that the timeframe of nuclear AMPKa2 accumulation is concomitant with the induction of CARM1. AMPKdependent downregulation of SKP2 in the nucleus allows CARM1 to escape from SCF E3 ubiquitin ligase, resulting in its stabilization upon nutrient starvation. This stabilization of histone modifiers in the nucleus is an efficient way to regulate target gene expression and could be a prototype of protein stabilization in specific compartments of the cells during starvation-induced autophagy. Molecular Cell 65, March 2, 2017 ª 2016 Elsevier Inc. 781
Molecular Cell
Minireview Figure 1. Histone Modifications and Epigenetic Enzymes Linked to the Regulation of Autophagy Autophagic histone modifications and responsible epigenetic enzymes are depicted. CARM1dependent H3R17 dimethylation functions in sustained autophagy (A). Autophagic life-anddeath decision via the hMOF-SIRT1 control of H4K16 acetylation is identified (B). H3K9 dimethylation by G9a and H3K27 trimethylation by EZH2 are suppression marks for autophagy (C). Each function is discussed in the main text. Ac, acetylated; me, methylated.
Ellagic acid selectively blocks histone H3R17 methylation (Selvi et al., 2010). Given that inhibition of H3R17me2 mark by ellagic acid almost completely blocks autophagy occurrence both in vitro and in vivo, ellagic acid might have a potential to be developed as a therapeutic agent in autophagy-related diseases. These findings provide evidence of CARM1-mediated histone arginine methylation as a critical nuclear event in the regulation of autophagy and shed light on potential therapeutic targeting of the recently identified AMPK-SKP2-CARM1 signaling axis in autophagy-related diseases. Cell Survival versus Death Decision: H4K16 Acetylation Status by hMOF-SIRT1 Although autophagy is known to be cytoprotective, dysregulated autophagy results in cell death (Green and Levine, 2014). While uncovering how autophagy governs life and death of the cell is important, hMOF acetyltransferase-SIRT1 deacetylase, which controls H4K16 acetylation (H4K16Ac) status, turned out to be €llgrabe et al., 2014). Autophagy indeterminant in the process (Fu duction by various stimuli is associated with reduced acetylation of H4K16. The genome-wide investigation reveals that H4K16 deacetylation by rapamycin treatment is associated with transcriptional activation of autophagy-related genes. Rapamycin-induced autophagic process coincides with downregulation of hMOF and deacetylation of H4K16 without changing SIRT1 protein levels or activity. Most importantly, inhibition of H4K16Ac downregulation by overexpression of hMOF or inhibition of SIRT1 upon autophagy increases autophagic flux and results in cell death. Independent of 782 Molecular Cell 65, March 2, 2017
H4K16 deacetylation, SIRT1 has been shown to deacetylate nuclear LC3 to license its release to cytoplasm during starvation (Huang et al., 2015). SIRT1inhibitors including VPA and Ex527 are promising candidates for the treatment of cancer and autophagy-related diseases. Suppressed Autophagy: H3K9 Dimethylation by G9a and H3K27 Trimethylation by EZH2 H3K9 can be methylated by histone methyltransferases including G9a, and H3K9 methylation is associated with transcriptional repression. Recent study showed that G9a-mediated H3K9 methylation acts as a repressor of autophagy under nutrient-rich conditions (Artal-Martinez de Narvajas et al., 2013). Nutrient deprivation leads to dissociation of G9a from the promoter, consequently resulting in transcriptional activation. Pharmacological inhibition of G9a results in autophagy occurrence. Further, when naive CD4+ T cell is activated, which accompanies autophagy gene expression, dissociation of G9a and recruitment of c-Jun transcription factor at several autophagy gene promoters are involved in the progression of autophagy. G9a is therefore an epigenetic regulator of autophagy, and the inhibition of G9amediated gene repression is important during autophagy induction. Enhancer of zeste homolog 2 (EZH2) mainly catalyzes H3K27 trimethylation and represses transcription of several negative regulators of the mechanistic target of rapamycin (mTOR) pathway, including TSC2, DEPTOR, RHOA, and GPI, under nutrient-rich conditions (Wei et al., 2015). EZH2 is recruited to those target promoters via metastasis-associated 1 family member 2 (MTA2) to silence target genes exemplified by TSC2. Downregulation of TSC2 by EZH2 results in mTOR activation, leading to inhibition of autophagy. Further, knockdown of EZH2 leads to the formation of autophagosome, and inhibition of EZH2 activity induces autophagy. Given that the expression of EZH2 and MTA2 correlates negatively with expression of TSC2 in human colon cancer tissues, a potential functional link among epigenetic regulation, autophagy induction, and tumorigenesis has been suggested. Although further studies are needed, selective inhibitors for EZH2 would not only prevent silencing of tumor suppressor genes but also activate
Molecular Cell
Minireview
Figure 2. Upstream Stimuli and Known Histone Modifications in Autophagy Known histone modifications linked to enhanced autophagy or suppressed autophagy along with reported upstream stimuli are presented.
autophagy, an interesting area of research for cancer treatment. Other Histone Modifications Related to Autophagy Some other histone modifications have been reported to be potentially linked to autophagy, although responsible enzymes have not been uncovered yet. H3K56 acetylation, H4K20 methylation, and H2BK120 monoubiquitination are involved in €llgrabe suppressed autophagy (Figure 2) (Chen et al., 2016; Fu et al., 2014). Histone modifications have a genome-wide role in regulating transcription. There are obligatory histone PTMs involved in transcriptional activation such as H3K9Ac, H3K14Ac, and H3K36me, and thus some of the histone PTMs which change upon autophagic process might not be specific marks related to autophagy. However, we speculate that there are signal-induced particular histone PTMs at autophagy genes (i.e., CARM1 and H3R17me) affecting the autophagic process.
Therefore, it would be challenging to explore how upstream signaling pathways affect downstream target gene expressions along with differential regulation of histone modifications and epigenetic enzymes to confer the specificity of autophagy regulation. Further studies will reveal more autophagy-related histone marks and the specificity of autophagy regulation by histone modifications. Interplay between the Cytoplasm and the Nucleus for the Regulation of Autophagy Program In addition to the histone modifications and epigenetic enzymes that relay cytoplasmic events to the nucleus, several transcription factors play a role in signal transfer. Various transcription factors involved in the autophagic process either function in both compartments or are sequestered in the cytoplasm and translocate into the nucleus to participate in distinct nuclear Molecular Cell 65, March 2, 2017 783
Molecular Cell
Minireview regulations of autophagy. It is now widely accepted that transcriptional regulation occurs to replenish cytoplasmic material €llgrabe et al., 2014; Shin et al., and sustain autophagy (Fu 2016). The induction of autophagy leads to the activation and nuclear translocation of various transcription factors acting as crucial regulators of the autophagic process. These transcription factors might function together with histone-modifying enzymes, although the experimental evidence is limited to the CARM1-TFEB collaboration in sustained autophagy upon glucose starvation. The first example is TFEB, a member of the basic helix-loophelix leucine zipper family (Settembre et al., 2011). Under nutrient-rich conditions, TFEB is phosphorylated by mTORC1 or ERK2 and localized in the cytoplasm by binding to 14-3-3 (Raben and Puertollano, 2016). Upon autophagy induction, TFEB is dephosphorylated by calcineurin and translocates to the nucleus for transcriptional activation of autophagy genes and lysosomal genes. The cytoplasm-to-nucleus translocation of TFEB was also observed under ER stress response (Martina et al., 2016). The second example is forkhead box O (FOXO) family members of transcription factors, regulated by phosphorylationdependent nuclear-cytoplasmic shuttling (Van Der Heide et al., 2004). Among them, FOXO3 is involved in autophagy induction in multiple ways. FOXO3 is phosphorylated by AMPK in nutrient-deprived conditions and induces autophagy by activating transcription (Greer et al., 2007). On the other hand, transcriptional repression by FOXO3 also contributes autophagy induction. Under glucose-deprived conditions, FOXO3 phosphorylation by AMPK downregulates SKP2 transcription, resulting in CARM1 stabilization and autophagy induction (Shin et al., 2016). Although AMPK-mediated phosphorylation of FOXO3 does not change cellular localization of FOXO3 under nutrientdeprived conditions (Greer et al., 2007), reactive oxygen species-induced phosphorylation of FOXO3 by JNK induces nuclear translocation (Go´mez-Puerto et al., 2016), indicating a distinct regulatory mode of transcription factors depending on upstream autophagic stimuli. The third example is the GATA transcription factor Gln3, which is phosphorylated by mTOR, causing its retention in the cytoplasm under nutrient-rich conditions (Beck and Hall, 1999). mTOR inhibition, on the other hand, under nutrient-deprived conditions allows dephosphorylation and nuclear localization of Gln3 and activation of autophagy genes (Chan et al., 2001). Together, these observations indicate that transcriptional control of autophagy can be achieved by phosphorylation-dependent nuclear-cytoplasmic shuttling. As another nuclear event, an autophagy-targeting protein, LC3, is present in the nucleus interacting with lamin B1 and lamin-associated chromatin domains and participates in the changes of histone levels during senescence (Dou et al., 2015). Concluding Remark: Histone Code as a Decision Maker in Autophagy At the beginning of this minireview, we have asked two important questions regarding histone modifications related to autophagy. First, what could be the roles of histone modifications during autophagy? Alteration in specific histone marks during autophagy affects the transcriptional and epigenetic regulatory pro784 Molecular Cell 65, March 2, 2017
gram. Therefore, identification of autophagy-regulated specific histone marks offers a conceptual advance both to understand the transcriptional response to upstream signals eliciting the autophagic process and to form a potential aspect of longterm regulation of autophagy. Some of the histone marks exhibit genome-wide changes, as in the case of H4K16Ac and €llgrabe et al., 2013; Shin et al., 2016), upon autoH3R17me2 (Fu phagic occurrence. Understanding autophagic stimuli-induced histone modifications will allow us to identify specific target genes and their functions during autophagic process. Second, when the autophagic process becomes dysfunctional, is it associated with altered histone modifications? There is very likely a possible correlation between altered histone modifications and autophagic defects. As cancer progresses, histone modification patterns are changed compared to normal control (Sharma et al., 2010). It has been shown that disruption of normal patterns of histone modifications is related to cancer, and specific histone modification patterns are typically associated with the disease. Although studies on the functional relationship between dysregulated autophagic process and altered histone modifications are at the infant stage, there are many exciting aspects to be discovered. Future studies will hopefully tell us more exciting stories. REFERENCES Artal-Martinez de Narvajas, A., Gomez, T.S., Zhang, J.S., Mann, A.O., Taoda, Y., Gorman, J.A., Herreros-Villanueva, M., Gress, T.M., Ellenrieder, V., Bujanda, L., et al. (2013). Epigenetic regulation of autophagy by the methyltransferase G9a. Mol. Cell. Biol. 33, 3983–3993. Baek, S.H. (2011). When signaling kinases meet histones and histone modifiers in the nucleus. Mol. Cell 42, 274–284. Beck, T., and Hall, M.N. (1999). The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors. Nature 402, 689–692. Chan, T.F., Bertram, P.G., Ai, W., and Zheng, X.F. (2001). Regulation of APG14 expression by the GATA-type transcription factor Gln3p. J. Biol. Chem. 276, 6463–6467. Chen, S., Jing, Y., Kang, X., Yang, L., Wang, D.L., Zhang, W., Zhang, L., Chen, P., Chang, J.F., Yang, X.M., and Sun, F.L. (2016). Histone H2B monoubiquitination is a critical epigenetic switch for the regulation of autophagy. Nucleic Acids Res. http://dx.doi.org/10.1093/nar/gkw1025. Dou, Z., Xu, C., Donahue, G., Shimi, T., Pan, J.-A., Zhu, J., Ivanov, A., Capell, B.C., Drake, A.M., Shah, P.P., et al. (2015). Autophagy mediates degradation of nuclear lamina. Nature 527, 105–109. €llgrabe, J., Lynch-Day, M.A., Heldring, N., Li, W., Struijk, R.B., Ma, Q., Fu Hermanson, O., Rosenfeld, M.G., Klionsky, D.J., and Joseph, B. (2013). The histone H4 lysine 16 acetyltransferase hMOF regulates the outcome of autophagy. Nature 500, 468–471. €llgrabe, J., Klionsky, D.J., and Joseph, B. (2014). The return of the nucleus: Fu transcriptional and epigenetic control of autophagy. Nat. Rev. Mol. Cell Biol. 15, 65–74. Go´mez-Puerto, M.C., Verhagen, L.P., Braat, A.K., Lam, E.W., Coffer, P.J., and Lorenowicz, M.J. (2016). Activation of autophagy by FOXO3 regulates redox homeostasis during osteogenic differentiation. Autophagy 12, 1804–1816. Green, D.R., and Levine, B. (2014). To be or not to be? How selective autophagy and cell death govern cell fate. Cell 157, 65–75. Greer, E.L., Oskoui, P.R., Banko, M.R., Maniar, J.M., Gygi, M.P., Gygi, S.P., and Brunet, A. (2007). The energy sensor AMP-activated protein kinase directly regulates the mammalian FOXO3 transcription factor. J. Biol. Chem. 282, 30107–30119.
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