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Acute Promyelocytic Leukemia is caused by expression of the oncogenic Promyelocytic Leukemia (PML)–Retinoic Acid Receptor Alpha (RARA) fusion protein. Therapy with arsenic trioxide results in degradation of PML-RARA and PML and cures the disease. Modification of PML and PML-RARA with SUMO and ubiquitin precedes ubiquitin-mediated proteolysis. To identify additional components of this pathway, we performed proteomics on PML bodies. This revealed that association of p97/VCP segregase with PML bodies is increased after arsenic treatment. Pharmacological inhibition of p97 altered the number, morphology, and size of PML bodies, accumulated SUMO and ubiquitin modified PML and blocked arsenic-induced degradation of PML-RARA and PML. p97 localized to PML bodies in response to arsenic, and siRNA-mediated depletion showed that p97 cofactors UFD1 and NPLOC4 were critical for PML degradation. Thus, the UFD1-NPLOC4-p97 segregase complex is required to extract poly-ubiquitinated, poly-SUMOylated PML from PML bodies, prior to degradation by the proteasome.

NFATc3 is the predominant member of the NFAT family of transcription factors in neurons, where it plays a pro-apoptotic role. Mechanisms controlling NFAT protein stability are poorly understood. Here we identify Trim39 as an E3 ubiquitin-ligase of NFATc3. Indeed, Trim39 binds and ubiquitinates NFATc3 in vitro and in cells where it reduces NFATc3 protein level and transcriptional activity. In contrast, silencing of endogenous Trim39 decreases NFATc3 ubiquitination and increases its activity, thereby resulting in enhanced neuronal apoptosis. We also show that Trim17 inhibits Trim39-mediated ubiquitination of NFATc3 by reducing both the E3 ubiquitin-ligase activity of Trim39 and the NFATc3/Trim39 interaction. Moreover, we identify Trim39 as a new SUMO-targeted E3 ubiquitin-ligase (STUbL). Indeed, mutation of SUMOylation sites in NFATc3 or SUMO-interacting motifs in Trim39 reduces NFATc3/Trim39 interaction and Trim39-induced ubiquitination of NFATc3. In addition, Trim39 preferentially ubiquitinates SUMOylated forms of NFATc3 in vitro. As a consequence, a SUMOylation-deficient mutant of NFATc3 exhibits increased stability and pro-apoptotic activity in neurons. Taken together, these data indicate that Trim39 modulates neuronal apoptosis by acting as a STUbL for NFATc3.

To determine the role of SUMO modification in the maintenance of pluripotent stem cells we used ML792, a potent and selective inhibitor of SUMO Activating Enzyme. Treatment of human induced pluripotent stem cells with ML792 initiated changes associated with loss of pluripotency such as reduced expression of key pluripotency markers. To identify putative effector proteins and establish sites of SUMO modification, cells were engineered to stably express either SUMO1 or SUMO2 with TGG to KGG mutations that facilitate GlyGly-K peptide immunoprecipitation and identification. A total of 976 SUMO sites were identified in 427 proteins. STRING enrichment created 3 networks of proteins with functions in regulation of gene expression, ribosome biogenesis and RNA splicing, although the latter two categories represented only 5% of the total GGK peptide intensity. The remainder have roles in transcription and chromatin structure regulation. Many of the most heavily SUMOylated proteins form a network of zinc-finger transcription factors centred on TRIM28 and associated with silencing of retroviral elements. At the level of whole proteins there was only limited evidence for SUMO paralogue-specific modification, although at the site level there appears to be a preference for SUMO2 modification over SUMO1 in acidic domains. We show that SUMO is involved in the maintenance of the pluripotent state in hiPSCs and identify many chromatin-associated proteins as bona fide SUMO substrates in human induced pluripotent stem cells.

Pluripotent stem cells represent a powerful system to identify the mechanisms governing cell fate decisions during early mammalian development. Covalent attachment of the Small Ubiquitin Like Modifier (SUMO) to proteins has emerged as an important factor in stem cell maintenance. Here we show that SUMO is required to maintain stem cells in their pluripotent state and identify many chromatin-associated proteins as bona fide SUMO substrates in human induced pluripotent stem cells (hiPSCs). Loss of SUMO increases chromatin accessibility and expression of long non-coding RNAs and human endogenous retroviral elements, indicating a role for the SUMO modification of SETDB1 and a large TRIM28 centric network of zinc finger proteins in silencing of these elements. While most protein coding genes are unaffected, the Preferentially Expressed Antigen of Melanoma (PRAME) gene locus becomes more accessible and transcription is dramatically increased after inhibition of SUMO modification. When PRAME is silent, a peak of SUMO over the transcriptional start site overlaps with ChIP-seq peaks for cohesin, RNA pol II, CTCF and ZNF143, with the latter two heavily modified by SUMO. These associations suggest that silencing of the PRAME gene is maintained by the influence of SUMO on higher order chromatin structure. Our data indicate that SUMO modification plays an important role in hiPSCs by repressing genes that disrupt pluripotency networks or drive differentiation.

NFATc3 is the predominant member of the NFAT family of transcription factor in neurons, where it plays a pro-apoptotic role. Mechanisms controlling NFAT protein stability are poorly understood. Here we identify Trim39 as an E3 ubiquitin-ligase of NFATc3. Indeed, Trim39 ubiquitinates NFATc3 in vitro and in cells, whereas silencing of endogenous Trim39 decreases NFATc3 ubiquitination. We also show that Trim17 inhibits Trim39-mediated ubiquitination of NFATc3 by reducing both the E3 ubiquitin-ligase activity of Trim39 and the NFATc3/Trim39 interaction. Moreover, mutation of SUMOylation sites in NFATc3 or SUMO-interacting motif in Trim39 reduces the NFATc3/Trim39 interaction and Trim39-induced ubiquitination of NFATc3. As a consequence, silencing of Trim39 increases the protein level and transcriptional activity of NFATc3, resulting in enhanced neuronal apoptosis. Likewise, a SUMOylation-deficient mutant of NFATc3 exhibits increased stability and pro-apoptotic activity. Taken together, these data indicate that Trim39 modulates neuronal apoptosis by acting as a SUMO-targeted E3 ubiquitin-ligase for NFATc3.

To investigate the role of SUMO modification in the maintenance of pluripotent stem cells, we used ML792, a potent and selective inhibitor of SUMO Activating Enzyme. Treatment of human induced pluripotent stem cells with ML792 resulted in the loss of key pluripotency markers. To identify putative effector proteins and establish sites of SUMO modification, cells were engineered to stably express either SUMO1 or SUMO2 with C-terminal TGG to KGG mutations that facilitate GlyGly-K peptide immunoprecipitation and identification. A total of 976 SUMO sites were identified in 427 proteins. STRING enrichment created three networks of proteins with functions in regulation of gene expression, ribosome biogenesis, and RNA splicing, although the latter two categories represented only 5% of the total GGK peptide intensity. The rest have roles in transcription and the regulation of chromatin structure. Many of the most heavily SUMOylated proteins form a network of zinc-finger transcription factors centered on TRIM28 and associated with silencing of retroviral elements. At the level of whole proteins, there was only limited evidence for SUMO paralogue-specific modification, although at the site level there appears to be a preference for SUMO2 modification over SUMO1 in acidic domains. We show that SUMO influences the pluripotent state in hiPSCs and identify many chromatin-associated proteins as bona fide SUMO substrates in human induced pluripotent stem cells., Graphical Abstract, Highlights • SUMO inhibition in human stem cells reduced pluripotency markers. • SUMO site proteomics identified 976 SUMO sites in 427 proteins. • SUMO targets functioned in gene expression, ribosome biogenesis, and RNA splicing. • There is a preference for SUMO2 over SUMO1 modification in acidic domains., In Brief The role of SUMO modification in maintenance of human induced pluripotent stem cells was investigated using an inhibitor of SUMO modification. Key markers of pluripotency were lost after inhibitor treatment. Employing SUMO site proteomics, we identified 976 sites in 427 proteins. A major network of zinc-finger transcription factors linked to TRIM28 was associated with silencing of retroviral elements. At the site level there appears to be a preference for SUMO2 modification over SUMO1 in acidic domains.

International audience; Neuronal apoptosis induced by survival factor deprivation is strongly regulated at the transcriptional level. Notably, the nuclear factor of activated T cell (NFAT) transcription factors have an important role in the control of the survival/death fate of neurons. However, the mechanisms that regulate NFAT activity in response to apoptotic stimuli and the target genes that mediate their effect on neuronal apoptosis are mostly unknown. In a previous study, we identified Trim17 as a crucial E3 ubiquitin ligase that is necessary and sufficient for neuronal apoptosis. Here, we show that Trim17 binds preferentially SUMOylated forms of NFATc3. Nonetheless, Trim17 does not promote the ubiquitination/degradation of NFATc3. NFAT transcription factors are regulated by calcium/calcineurin-dependent nuclear-cytoplasmic shuttling. Interestingly, Trim17 reduced by twofold the calcium-mediated nuclear localization of NFATc3 and, consistent with this, halved NFATc3 activity, as estimated by luciferase assays and by measurement of target gene expression. Trim17 also inhibited NFATc4 nuclear translocation and activity. NFATc4 is known to induce the expression of survival factors and, as expected, overexpression of NFATc4 protected cerebellar granule neurons from serum/KCl deprivation-induced apoptosis. Inhibition of NFATc4 by Trim17 may thus partially mediate the proapoptotic effect of Trim17. In contrast, overexpression of NFATc3 aggravated neuronal death, whereas knockdown of NFATc3 protected neurons from apoptosis. This proapoptotic effect of NFATc3 might be due to a feedback loop in which NFATc3, but not NFATc4, induces the transcription of the proapoptotic gene Trim17. Indeed, we found that overexpression or silencing of NFATc3, respectively, increased or decreased Trim17 levels, whereas NFATc4 had no significant effect on Trim17 expression. Moreover, we showed that NFATc3 binds to the promoter of the Trim17 gene together with c-Jun. Therefore, our results describe a novel mechanism regulating NFAT transcription factors beyond the calcium/calcineurin-dependent pathway and provide a possible explanation for the opposite effects of NFATc3 and NFATc4 on neuronal apoptosis.

International audience; Short-term proteasome inhibition has been shown to prevent neuronal apoptosis. However, the key pro-survival proteins that must be degraded for triggering neuronal death are mostly unknown. Here, we show that Mcl-1, an anti-apoptotic Bcl-2 family member, is degraded by the proteasome during neuronal apoptosis. Using primary cultures of cerebellar granule neurons deprived of serum and KCl, we found that ubiquitination and proteasomal degradation of Mcl-1 depended on its prior phosphorylation by GSK3, providing the first insight into post-translational regulation of Mcl-1 in neurons. In a previous study, we have reported that the E3 ubiquitin-ligase Trim17 is both necessary and sufficient for neuronal apoptosis. Here, we identified Trim17 as a novel E3 ubiquitin-ligase for Mcl-1. Indeed, Trim17 co-immunoprecipitated with Mcl-1. Trim17 ubiquitinated Mcl-1 in vitro. Overexpression of Trim17 decreased the protein level of Mcl-1 in a phosphorylation- and proteasome-dependent manner. Finally, knock down of Trim17 expression reduced both ubiquitination and degradation of Mcl-1 in neurons. Moreover, impairment of Mcl-1 phosphorylation, by kinase inhibition or point mutations, not only decreased ubiquitination and degradation of Mcl-1, but also blocked the physical interaction between Trim17 and Mcl-1. As this stabilization of Mcl-1 increased its neuroprotective effect, our data strongly suggest that Trim17-mediated ubiquitination and degradation of Mcl-1 is necessary for initiating neuronal death.