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.

Small ubiquitin-like modifier (SUMO) modification is emerging as an important control in transcription regulation. Here, we show that CREB-binding protein (CBP), a versatile transcriptional coactivator for numerous transcription factors in response to diverse signaling events, can be modified by SUMO-1 at lysine residues 999, 1034, and 1057 both in vitro and in vivo. Mutation of the SUMO acceptor lysine residues either individually or in combination enhanced CBP transcriptional activity, and expression of a SUMO protease SENP2 potentiated the transcriptional activity of CBP wild-type but not its sumoylation mutant, indicating that SUMO modification negatively regulates CBP transcriptional activity. Furthermore, we demonstrated an interaction of SUMO-1-modified CBP with the transcriptional corepressor Daxx and an essential role of Daxx in mediating SUMO-dependent transcriptional regulation of CBP through histone deacetylase 2 recruitment. Together, our findings indicate that SUMO modification and subsequent recruitment of Daxx represent a previously undescribed mechanism in modulating CBP transcriptional potential.protein-protein interaction ͉ posttranslational modification ͉ transcriptional repression ͉ SENP2 ͉ histone deacetylase 2 S umoylation, the covalent attachment of the small ubiquitinlike modifier (SUMO) peptide to lysine residues of targeted substrate, has recently emerged as an important mechanism in transcriptional control (1-3). With an increasing number of sumoylated transcription factors and cofactors being identified, SUMO modification, in most cases, appears to repress the activity of targeted transcriptional activators through altering their subcompartmentalization and͞or molecular interaction properties. For example, sumoylation silences the transcriptional activity of Sp3 by translocating it to nuclear domain 10, also named promyelocytic leukemia protein oncogenic domains (4). In addition to the regulation of the nucleo-cytoplasmic shuttling (5), Elk-1 sumoylation further recruits the histone deacetylase (HDAC) 2 to Elk-1-regulated promoters, thereby repressing their transcription (6). Sumoylation of transcriptional coactivator p300 also mediates the recruitment of HDAC6, leading to SUMO-dependent transcriptional repression (7).The CREB-binding protein (CBP), a paralogue of p300, functions as a transcriptional coactivator in multiple, signaldependent transcription events (for reviews, see refs. 8-11). The coactivator activity of CBP appears to be exerted through linking different sequence-specific transcription factors to the general transcriptional machinery and͞or through its acetyltransferase activity that can acetylate histones and͞or transcription factors, thereby activating transcription. Recent studies revealed that the activity of CBP can be dynamically regulated by posttranslational modifications such as phosphorylation (12-15) and methylation (16,17). Whether CBP can be also regulated by SUMO modification remains unknown.Daxx, initially identified as a cytoplasmic signaling molecule ...

SUMO (small ubiquitin-related modifier) modification is emerging as an important post-translational control in transcription. In general, SUMO modification is associated with transcriptional repression. Although many SUMO-modified transcription factors and co-activators have been identified, little is known about the mechanism underlying SUMOylation-elicited transcriptional repression. Here, we summarize that SUMO modification of transcription factors such as androgen receptor, glucocorticoid receptor, Smad4 and CBP [CREB (cAMP-response-element-binding protein)-binding protein] co-activator results in the recruitment of a transcriptional co-repressor Daxx, thereby causing transcriptional repression. Such a SUMO-dependent recruitment of Daxx is mediated by the interaction between the SUMO moiety of SUMOylated factors and Daxx SUMO-interacting motif. Interestingly, the transrepression effect of Daxx on these SUMOylated transcription factors can be relieved by SUMOylated PML (promyelocytic leukaemia) via altering Daxx partition from the targeted gene promoter to PML nuclear bodies. Because Daxx SUMO-interacting motif is a common binding site for SUMOylated factors, a model of competition for Daxx recruitment between SUMOylated PML and SUMOylated transcription factors was proposed. Together, our findings strongly suggest that Daxx functions as a SUMO reader in the SUMO-dependent regulation of transcription and subnuclear compartmentalization.

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.

Galectin-3 is a ubiquitous lectin exerting multiple cellular functions such as RNA splicing, protein trafficking and apoptosis. Its expression is positively correlated with the poor prognosis in lung cancer patients. Galectin-3 can promote cancer progression through its effects on cell proliferation, cell survival or cancer metastasis. However, the role of galectin-3 in the regulation of cancer stem-like cells (CSCs) is still unclear. Here, we investigated the hypothesis that galectin-3 might regulate lung CSCs via the EGF receptor (EGFR) signaling pathway. In our study, galectin-3 facilitated EGFR activation and enhanced the sphere formation activity of lung cancer cells. Furthermore, galectin-3 promoted Sox2 expression in an EGFR activation-dependent manner; importantly, forced expression of Sox2 blunted the effect of galectin-3 knockdown on lung cancer sphere formation ability. These results suggest that galectin-3 promotes EGFR activation leading to the upregulation of Sox2 expression and lung CSCs properties. Moreover, we showed that the carbohydrate-binding activity of galectin-3 was important for the regulation of EGFR activation, Sox2 expression and sphere formation. We have recently reported that c-Myc is a transcriptional activator of Sox2. We further found that galectin-3 enhanced c-Myc protein stability leading to increased c-Myc binding to the Sox2 gene promoter. We also examined the effect of the stemness factors, Oct4, Nanog and Sox2 on the expression of galectin-3. We found that Oct4 enhanced galectin-3 expression. Our results together suggest that galectin-3 enhances lung cancer stemness through the EGFR/c-Myc/Sox2 axis; Oct4, in turn, promotes galectin-3 expression, forming a positive regulatory loop in lung CSCs.

Promyelocytic leukemia protein (PML) is emerging as an important tumor suppressor. Its expression is lost during the progression of several types of cancer, including lung cancer. The EGF receptor (EGFR), a membrane-bound receptor tyrosine kinase, transduces intracellular signals responsible for cell proliferation, differentiation and migration. EGFR activity is frequently abnormally upregulated in lung adenocarcinoma (LAC) and thus is considered to be a driving oncogene for LAC. EGFR translocates into the nucleus and transcriptionally activates genes, such as CCND1, that promote cell growth. Recently, we demonstrated that PML interacted with nuclear EGFR (nEGFR) and suppressed the nEGFR-mediated transcriptional activation of CCND1 in lung cancer cells, thereby restraining cell growth. When we further investigated the interplay between PML and EGFR in lung cancer metastasis, we found that the matrix metalloprotease-2 gene (MMP2) was a novel nEGFR target gene and was repressed by PML. We provide evidence that nEGFR bound to the AT-rich sequence (ATRS) in the MMP2 promoter and enhanced its transcriptional activity. In addition, we demonstrated that PML repressed nEGFR-induced MMP2 transcription and reduced cell invasion. PML was recruited by nEGFR to the MMP2 promoter where it reduced histone acetylation, leading to the transcriptional repression of MMP2. Finally, we demonstrated that PML upregulation by interferon-b (IFNb) in lung cancer cells decreased MMP2 expression and cell invasion. Together, our results suggested that IFNb induced PML to inhibit lung cancer metastasis by repressing the nEGFR-mediated transcriptional activation of MMP2.