Small ubiquitin-like modifier (SUMO) modification has emerged as an important posttranslational control of protein functions. Daxx, a transcriptional corepressor, was reported to repress the transcriptional potential of several transcription factors and target to PML oncogenic domains (PODs) via SUMO-dependent interactions. The mechanism by which Daxx binds to sumoylated factors mediating transcriptional and subnuclear compartmental regulation remains unclear. Here, we define a SUMO-interacting motif (SIM) within Daxx and show it to be crucial for targeting Daxx to PODs and for transrepression of several sumoylated transcription factors, including glucocorticoid receptor (GR). In addition, the capability of Daxx SIM to bind SUMO also controls Daxx sumoylation. We further demonstrate that arsenic trioxide-induced sumoylation of PML correlates with a change of endogenous Daxx partitioning from GR-regulated gene promoter to PODs and a relief of Daxx repression on GR target gene expression. Our results provide mechanistic insights into Daxx in SUMO-dependent transcriptional control and subnuclear compartmentalization.

Small ubiquitin-like modifier (SUMO) conjugation and interaction are increasingly associated with various cellular processes. However, little is known about the cellular signaling mechanisms that regulate proteins for distinct SUMO paralog conjugation and interactions. Using the transcriptional coregulator Daxx as a model, we show that SUMO paralog-selective binding and conjugation are regulated by phosphorylation of the Daxx SUMO-interacting motif (SIM). NMR structural studies show that Daxx (732)E-I-I-V-L-S-D-S-D(740) is a bona fide SIM that binds to SUMO-1 in a parallel orientation. Daxx-SIM is phosphorylated by CK2 kinase at residues S737 and S739. Phosphorylation promotes Daxx-SIM binding affinity toward SUMO-1 over SUMO-2/3, causing Daxx preference for SUMO-1 conjugation and interaction with SUMO-1-modified factors. Furthermore, Daxx-SIM phosphorylation enhances Daxx to sensitize stress-induced cell apoptosis via antiapoptotic gene repression. Our findings provide structural insights into the Daxx-SIM:SUMO-1 complex, a model of SIM phosphorylation-enhanced SUMO paralog-selective modification and interaction, and phosphorylation-regulated Daxx function in apoptosis.

Promyelocytic leukemia protein (PML) sumoylation has been proposed to control the formation of PML nuclear bodies (NBs) and is crucial for PML-dependent cellular processes, including apoptosis and transcriptional regulation. However, the regulatory mechanisms of PML sumoylation and its specific roles in the formation of PML NBs remain largely unknown. Here, we show that histone deacetylase 7 (HDAC7) knockdown reduces the size and the number of the PML NBs in human umbilical vein endothelial cells (HUVECs). HDAC7 coexpression stimulates PML sumoylation independent of its HDAC activity. Furthermore, HDAC7 associates with the E2 SUMO ligase, Ubc9, and stimulates PML sumoylation in vitro, suggesting that it possesses a SUMO E3 ligase-like activity to promote PML sumoylation. Importantly, HDAC7 knockdown inhibits tumor necrosis factor alpha-induced PML sumoylation and the formation of PML NBs in HUVECs. These results demonstrate a novel function of HDAC7 and provide a regulatory mechanism of PML sumoylation.

While numerous small ubiquitin-like modifier (SUMO) conjugated substrates have been identified, very little is known about the cellular signalling mechanisms that differentially regulate substrate sumoylation. Here, we show that acetylation of SUMO E2 conjugase Ubc9 selectively downregulates the sumoylation of substrates with negatively charged amino acid-dependent sumoylation motif (NDSM) consisting of clustered acidic residues located downstream from the core w-K-X-E/D consensus motif, such as CBP and Elk-1, but not substrates with core w-K-X-E/D motif alone or SUMO-interacting motif. Ubc9 is acetylated at residue K65 and K65 acetylation attenuates Ubc9 binding to NDSM substrates, causing a reduction in NDSM substrate sumoylation. Furthermore, Ubc9 K65 acetylation can be downregulated by hypoxia via SIRT1, and is correlated with hypoxia-elicited modulation of sumoylation and target gene expression of CBP and Elk-1 and cell survival. Our data suggest that Ubc9 acetylation/deacetylation serves as a dynamic switch for NDSM substrate sumoylation and we report a previously undescribed SIRT1/Ubc9 regulatory axis in the modulation of protein sumoylation and the hypoxia response.

Smad4, a common-mediator of Smads, plays a central role in forming complexes with receptor-phosphorylated Smads, and then transduces transforming growth factor (TGF)-β signals into the nuclei. Although many cellular factors are involved in TGF-β induced epithelial-to-mesenchymal transition (EMT) and cell migration, very little is known with the mechanism of Smad4 regulation on pro-oncogenes response by TGF-β. Herein, we demonstrate the interaction of Sentrin-specific protease 2 (SENP2) with Smad4 through SENP2 residue 363~400. The same segment is also important for desumoylation of Smad4, and able to relieve sumoylation-mediated TGF-β repression. The SENP2363~400 segment is critical for TGF-β-induced cell migration, which is correlated with SENP2363~400 deletion mutant failed to increase matrix metalloproteinase (MMP)-9 and EMT marker gene expression. Moreover, our results suggest that the interaction and desumoylation between SENP2 and Smad4 promote cell migration in triple-negative breast cancer cells. Altogether, our data show how SENP2 regulates its substrate for desumoylation, and also the role of SENP2 in TGF-β induced cancer cell migration.