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SUMmOning Daxx-Mediated Repression
Molecular Cell
Previews SUMmOning Daxx-Mediated Repression Debaditya Mukhopadhyay1 and Michael J. Matunis1,* 1Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA *Correspondence: [email protected] DOI 10.1016/j.molcel.2011.03.008
An intimate relationship exists between the transcriptional coregulator Daxx, SUMO, and PML nuclear bodies. In this issue, Chang et al. (2011) provide structural insights into how phosphorylation of Daxx increases its affinity toward SUMOs and functional insights into how enhanced SUMO binding affects Daxx-PML interactions, PML nuclear body localization, and Daxx-mediated repression of genes encoding for antiapoptotic factors. The Fas death domain-associated protein (Daxx) was originally identified as a Fasinteracting protein and regulator of Fasmediated cell death (Yang et al., 1997). Since that time, Daxx has emerged as an enigmatic factor, with apparent roles in both the cytoplasm and the nucleus related to both apoptosis and cell survival (Salomoni and Khelifi, 2006). Several important themes, however, have emerged concerning Daxx function and modulation of its activities. First, Daxx associates with numerous transcription factors and modulators of chromatin structure at the promoters of a wide range of genes, repressing transcription initiation at the majority of these promoters. Second, Daxx interacts with PML and represents a constitutive component of PML nuclear bodies (PML NBs). Multiple studies have suggested that PML-NBs function as ‘‘storage depots’’ for Daxx and that its ability to act as a transcription corepressor is modulated by its sequestration or release from these depots (Lindsay et al., 2008). Related to its functions as a transcription corepressor and its association with PML NBs is the third theme concerning the effects of sumoylation on Daxx function. Daxx is covalently modified by SUMO, and it has the ability to interact noncovalently with SUMO through a C-terminal SUMO-interacting motif (SIM). Previous work by the Shih group determined that the C-terminal SIM in Daxx is critical for its association with SUMO-modified PML and localization to PML NBs, as well as for its association with SUMO-modified transcription factors and transcription corepression at gene promoters (Lin et al., 2006). This study clearly established that noncovalent SUMO binding plays a central role in
determining both the localization and function of Daxx; however, it also raised several important issues. First, although Daxx was found to bind preferentially to SUMO-1 and to be modified preferentially by SUMO-1, the molecular basis for this paralog selectivity was not fully elucidated. Second, mechanisms regulating Daxx-SUMO interactions, although anticipated to be important modulators of Daxx function, were not understood. Lastly, it remained uncertain whether interactions between Daxx and SUMO-modified PML served functions beyond simple sequestration. The new study by Chang et al. (2011) provides answers and raises new questions related to these issues. Vertebrates commonly express three SUMO paralogs (SUMO-1, -2, and -3). These paralogs are conjugated to unique subsets of proteins, suggesting that they serve distinct biological functions, presumably mediated by paralog-selective SUMO-binding proteins (Hecker et al., 2006; Zhang et al., 2008). Molecular mechanisms governing paralog-selective SUMO modification and binding, however, remain poorly understood. Daxx was previously found to be modified preferentially by SUMO-1 through a mechanism dependent on its C-terminal SIM (Lin et al., 2006). In their new study, Chang et al. discovered that SUMO-1 binding and modification is significantly and selectively enhanced by CK2-dependent phosphorylation of serines flanking this SIM (Figure 1). Nuclear magnetic resonance structural studies and isothermal calorimetry (ITC) analysis were used to investigate the molecular basis of this effect. This analysis revealed that while hydrophobic interactions are the major driving force for binding between the Daxx SIM and
4 Molecular Cell 42, April 8, 2011 ª2011 Elsevier Inc.
SUMOs in the absence of phosphorylation, phosphorylation provides sites for electrostatic interactions that preferentially enhance SUMO-1 binding over SUMO-3. However, the exact molecular basis for paralog-selective enhancement was not fully revealed, as the major residue contributing to stabilizing electrostatic interactions with the Daxx SIM was a lysine residue conserved between SUMO-1 and SUMO-3 (K39 and K34, respectively). As residues comprising the SIM-interacting surfaces of SUMO-1 and SUMO-3 are more than 50% unique, it can be anticipated that subtle but important differences exist in their interactions with SIMs. Detailed comparisons of the differences between structures of the Daxx SIM, or SIMs from other proteins, in complex with SUMO-1, -2, and -3 will be required to provide clearer insights into paralog-selective binding and, possibly, molecular predictors of selectivity. Although CK2 is a constitutively active kinase, its activity is upregulated in response to osmotic stress (Scaglioni et al., 2006). Consistent with this, Chang et al. found that osmotic stress enhanced CK2-mediated phosphorylation of the Daxx SIM and the association of Daxx with SUMO-1-modified proteins. In particular, CK2-mediated phosphorylation enhanced interactions between Daxx and SUMO-1-modified PML, and presumably the recruitment of Daxx to PML NBs (Figure 1). Contrary to a simple sequestration and inactivation of Daxx in PML NBs, as predicted from earlier studies of the glucocorticoid receptor (Lin et al., 2006), CK2-mediated phosphorylation positively affected the recruitment of Daxx to genes encoding for anti-apoptotic factors. This in turn sensitized cells to stress-induced
Molecular Cell
Previews Daxx
ATP CK2
Daxx
PP S2
S1
S2
S2 S2
S1
S1
S2
?
S1
PML
S2
Daxx
PP
Daxx
PP Paralog specific SUMOylation
S1
Daxx S2
PML S2 S1
S1 S2 S2
S2 S1
SUMO2/3 SUMO1
S2
S1
PP
S2
S1 S1 S1
Flip/Bcl2
?
Apoptotic Sensitization
S2 S1 S1 S2 S1 S1 S2 S2
S1 S2 S1 PML PML S2S2 S1
PMLS1 S2
PML
Transcription factors PML Body proteins Daxx
PP
SIM phosphorylated Daxx
PML body localization
Figure 1. CK2-Dependent Daxx-SIM Phosphorylation Controls Antiapoptotic Gene Repression CK2 phosphorylates two serine residues in the Daxx C-terminal SIM, increasing its affinity toward SUMOs, and particularly toward SUMO-1. This phosphorylation-induced enhancement of SUMO-1 binding results in preferential SUMO-1 modification and SUMO-1-dependent recruitment of Daxx to the promoters of genes coding for antiapoptotic factors. Enhanced SUMO-1 binding also stabilizes interactions between Daxx and SUMO-1-modified PML, thereby affecting Daxx localization to PML NBs. The effects of covalent SUMO-1 modification of Daxx, as well as the functional relationship between recruitment to PML NBs and gene repression, remain uncertain, as indicated by question marks.
apoptosis, as was predicted from studies correlating the association of Daxx with PML NBs and Fas-induced cell death (Torii et al., 1999). These findings raise the apparent paradox that phosphorylation of the Daxx SIM increases both its sequestration in PML NBs and its recruitment to specific gene promoters, suggesting that the simple sequestration and inactivation model of PML NBs needs to be revised. PML NBs could play a passive role in bringing about an increase in SUMO-1sensitive Daxx recruitment to specific gene promoters if the paralog profiles of the PML NBs and these promoters were inherently different. More attractively, PML NBs may play a more active role in enhancing gene repression by functioning as factories for the assembly of Daxxcontaining, SUMO-1-dependent corepressor complexes. Similar roles have been proposed for Cajal bodies and
assembly of transcription and splicing complexes (Gall, 2001). The possibility that SUMO-1-modified PML forms part of Daxx corepressor complexes recruited to promoters also remains to be investigated. The reported findings provide important insights into mechanisms affecting paralog-selective SUMO binding and modification and their downstream consequences on Daxx function that are likely to be generally applicable to the activities of a rapidly growing list of SUMO-binding proteins residing both in and out of PML NBs. Like all excellent studies, the findings also highlight challenging new questions for future research. REFERENCES Chang, C.-C., Naik, M.T., Huang, Y.-S., Jeng, J.C., Liao, P.-S., Kuo, H.-Y., Ho, C.-C., Hsieh, Y.L., Lin, C.-H., Huang, N.-J., et al. (2011). Mol. Cell 42, this issue, 62–74.
Gall, J.G. (2001). FEBS Lett. 498, 164–167. Hecker, C.M., Rabiller, M., Haglund, K., Bayer, P., and Dikic, I. (2006). J. Biol. Chem. 281, 16117– 16127. Lin, D.Y., Huang, Y.S., Jeng, J.C., Kuo, H.Y., Chang, C.C., Chao, T.T., Ho, C.C., Chen, Y.C., Lin, T.P., Fang, H.I., et al. (2006). Mol. Cell 24, 341–354. Lindsay, C.R., Morozov, V.M., and Ishov, A.M. (2008). Front. Biosci. 13, 7132–7142. Salomoni, P., and Khelifi, A.F. (2006). Trends Cell Biol. 16, 97–104. Scaglioni, P.P., Yung, T.M., Cai, L.F., ErdjumentBromage, H., Kaufman, A.J., Singh, B., TeruyaFeldstein, J., Tempst, P., and Pandolfi, P.P. (2006). Cell 126, 269–283. Torii, S., Egan, D.A., Evans, R.A., and Reed, J.C. (1999). EMBO J. 18, 6037–6049. Yang, X., Khosravi-Far, R., Chang, H.Y., and Baltimore, D. (1997). Cell 89, 1067–1076. Zhang, X.D., Goeres, J., Zhang, H., Yen, T.J., Porter, A.C., and Matunis, M.J. (2008). Mol. Cell 29, 729–741.
Molecular Cell 42, April 8, 2011 ª2011 Elsevier Inc. 5
Previews SUMmOning Daxx-Mediated Repression Debaditya Mukhopadhyay1 and Michael J. Matunis1,* 1Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205, USA *Correspondence: [email protected] DOI 10.1016/j.molcel.2011.03.008
An intimate relationship exists between the transcriptional coregulator Daxx, SUMO, and PML nuclear bodies. In this issue, Chang et al. (2011) provide structural insights into how phosphorylation of Daxx increases its affinity toward SUMOs and functional insights into how enhanced SUMO binding affects Daxx-PML interactions, PML nuclear body localization, and Daxx-mediated repression of genes encoding for antiapoptotic factors. The Fas death domain-associated protein (Daxx) was originally identified as a Fasinteracting protein and regulator of Fasmediated cell death (Yang et al., 1997). Since that time, Daxx has emerged as an enigmatic factor, with apparent roles in both the cytoplasm and the nucleus related to both apoptosis and cell survival (Salomoni and Khelifi, 2006). Several important themes, however, have emerged concerning Daxx function and modulation of its activities. First, Daxx associates with numerous transcription factors and modulators of chromatin structure at the promoters of a wide range of genes, repressing transcription initiation at the majority of these promoters. Second, Daxx interacts with PML and represents a constitutive component of PML nuclear bodies (PML NBs). Multiple studies have suggested that PML-NBs function as ‘‘storage depots’’ for Daxx and that its ability to act as a transcription corepressor is modulated by its sequestration or release from these depots (Lindsay et al., 2008). Related to its functions as a transcription corepressor and its association with PML NBs is the third theme concerning the effects of sumoylation on Daxx function. Daxx is covalently modified by SUMO, and it has the ability to interact noncovalently with SUMO through a C-terminal SUMO-interacting motif (SIM). Previous work by the Shih group determined that the C-terminal SIM in Daxx is critical for its association with SUMO-modified PML and localization to PML NBs, as well as for its association with SUMO-modified transcription factors and transcription corepression at gene promoters (Lin et al., 2006). This study clearly established that noncovalent SUMO binding plays a central role in
determining both the localization and function of Daxx; however, it also raised several important issues. First, although Daxx was found to bind preferentially to SUMO-1 and to be modified preferentially by SUMO-1, the molecular basis for this paralog selectivity was not fully elucidated. Second, mechanisms regulating Daxx-SUMO interactions, although anticipated to be important modulators of Daxx function, were not understood. Lastly, it remained uncertain whether interactions between Daxx and SUMO-modified PML served functions beyond simple sequestration. The new study by Chang et al. (2011) provides answers and raises new questions related to these issues. Vertebrates commonly express three SUMO paralogs (SUMO-1, -2, and -3). These paralogs are conjugated to unique subsets of proteins, suggesting that they serve distinct biological functions, presumably mediated by paralog-selective SUMO-binding proteins (Hecker et al., 2006; Zhang et al., 2008). Molecular mechanisms governing paralog-selective SUMO modification and binding, however, remain poorly understood. Daxx was previously found to be modified preferentially by SUMO-1 through a mechanism dependent on its C-terminal SIM (Lin et al., 2006). In their new study, Chang et al. discovered that SUMO-1 binding and modification is significantly and selectively enhanced by CK2-dependent phosphorylation of serines flanking this SIM (Figure 1). Nuclear magnetic resonance structural studies and isothermal calorimetry (ITC) analysis were used to investigate the molecular basis of this effect. This analysis revealed that while hydrophobic interactions are the major driving force for binding between the Daxx SIM and
4 Molecular Cell 42, April 8, 2011 ª2011 Elsevier Inc.
SUMOs in the absence of phosphorylation, phosphorylation provides sites for electrostatic interactions that preferentially enhance SUMO-1 binding over SUMO-3. However, the exact molecular basis for paralog-selective enhancement was not fully revealed, as the major residue contributing to stabilizing electrostatic interactions with the Daxx SIM was a lysine residue conserved between SUMO-1 and SUMO-3 (K39 and K34, respectively). As residues comprising the SIM-interacting surfaces of SUMO-1 and SUMO-3 are more than 50% unique, it can be anticipated that subtle but important differences exist in their interactions with SIMs. Detailed comparisons of the differences between structures of the Daxx SIM, or SIMs from other proteins, in complex with SUMO-1, -2, and -3 will be required to provide clearer insights into paralog-selective binding and, possibly, molecular predictors of selectivity. Although CK2 is a constitutively active kinase, its activity is upregulated in response to osmotic stress (Scaglioni et al., 2006). Consistent with this, Chang et al. found that osmotic stress enhanced CK2-mediated phosphorylation of the Daxx SIM and the association of Daxx with SUMO-1-modified proteins. In particular, CK2-mediated phosphorylation enhanced interactions between Daxx and SUMO-1-modified PML, and presumably the recruitment of Daxx to PML NBs (Figure 1). Contrary to a simple sequestration and inactivation of Daxx in PML NBs, as predicted from earlier studies of the glucocorticoid receptor (Lin et al., 2006), CK2-mediated phosphorylation positively affected the recruitment of Daxx to genes encoding for anti-apoptotic factors. This in turn sensitized cells to stress-induced
Molecular Cell
Previews Daxx
ATP CK2
Daxx
PP S2
S1
S2
S2 S2
S1
S1
S2
?
S1
PML
S2
Daxx
PP
Daxx
PP Paralog specific SUMOylation
S1
Daxx S2
PML S2 S1
S1 S2 S2
S2 S1
SUMO2/3 SUMO1
S2
S1
PP
S2
S1 S1 S1
Flip/Bcl2
?
Apoptotic Sensitization
S2 S1 S1 S2 S1 S1 S2 S2
S1 S2 S1 PML PML S2S2 S1
PMLS1 S2
PML
Transcription factors PML Body proteins Daxx
PP
SIM phosphorylated Daxx
PML body localization
Figure 1. CK2-Dependent Daxx-SIM Phosphorylation Controls Antiapoptotic Gene Repression CK2 phosphorylates two serine residues in the Daxx C-terminal SIM, increasing its affinity toward SUMOs, and particularly toward SUMO-1. This phosphorylation-induced enhancement of SUMO-1 binding results in preferential SUMO-1 modification and SUMO-1-dependent recruitment of Daxx to the promoters of genes coding for antiapoptotic factors. Enhanced SUMO-1 binding also stabilizes interactions between Daxx and SUMO-1-modified PML, thereby affecting Daxx localization to PML NBs. The effects of covalent SUMO-1 modification of Daxx, as well as the functional relationship between recruitment to PML NBs and gene repression, remain uncertain, as indicated by question marks.
apoptosis, as was predicted from studies correlating the association of Daxx with PML NBs and Fas-induced cell death (Torii et al., 1999). These findings raise the apparent paradox that phosphorylation of the Daxx SIM increases both its sequestration in PML NBs and its recruitment to specific gene promoters, suggesting that the simple sequestration and inactivation model of PML NBs needs to be revised. PML NBs could play a passive role in bringing about an increase in SUMO-1sensitive Daxx recruitment to specific gene promoters if the paralog profiles of the PML NBs and these promoters were inherently different. More attractively, PML NBs may play a more active role in enhancing gene repression by functioning as factories for the assembly of Daxxcontaining, SUMO-1-dependent corepressor complexes. Similar roles have been proposed for Cajal bodies and
assembly of transcription and splicing complexes (Gall, 2001). The possibility that SUMO-1-modified PML forms part of Daxx corepressor complexes recruited to promoters also remains to be investigated. The reported findings provide important insights into mechanisms affecting paralog-selective SUMO binding and modification and their downstream consequences on Daxx function that are likely to be generally applicable to the activities of a rapidly growing list of SUMO-binding proteins residing both in and out of PML NBs. Like all excellent studies, the findings also highlight challenging new questions for future research. REFERENCES Chang, C.-C., Naik, M.T., Huang, Y.-S., Jeng, J.C., Liao, P.-S., Kuo, H.-Y., Ho, C.-C., Hsieh, Y.L., Lin, C.-H., Huang, N.-J., et al. (2011). Mol. Cell 42, this issue, 62–74.
Gall, J.G. (2001). FEBS Lett. 498, 164–167. Hecker, C.M., Rabiller, M., Haglund, K., Bayer, P., and Dikic, I. (2006). J. Biol. Chem. 281, 16117– 16127. Lin, D.Y., Huang, Y.S., Jeng, J.C., Kuo, H.Y., Chang, C.C., Chao, T.T., Ho, C.C., Chen, Y.C., Lin, T.P., Fang, H.I., et al. (2006). Mol. Cell 24, 341–354. Lindsay, C.R., Morozov, V.M., and Ishov, A.M. (2008). Front. Biosci. 13, 7132–7142. Salomoni, P., and Khelifi, A.F. (2006). Trends Cell Biol. 16, 97–104. Scaglioni, P.P., Yung, T.M., Cai, L.F., ErdjumentBromage, H., Kaufman, A.J., Singh, B., TeruyaFeldstein, J., Tempst, P., and Pandolfi, P.P. (2006). Cell 126, 269–283. Torii, S., Egan, D.A., Evans, R.A., and Reed, J.C. (1999). EMBO J. 18, 6037–6049. Yang, X., Khosravi-Far, R., Chang, H.Y., and Baltimore, D. (1997). Cell 89, 1067–1076. Zhang, X.D., Goeres, J., Zhang, H., Yen, T.J., Porter, A.C., and Matunis, M.J. (2008). Mol. Cell 29, 729–741.
Molecular Cell 42, April 8, 2011 ª2011 Elsevier Inc. 5