Small Ubiquitin-related Modifier E3 Ligase Research Articles

E3 SUMO ligase SIZ1 splicing variants localize and function according to external conditions.

SIZ1 (SAP and MIZ1) is a member of the Siz/PIAS-type RING family of E3 SUMO (small ubiquitin-related modifier) ligases that play key roles in growth, development, and stress responses in plant and animal systems. Nevertheless, splicing variants of SIZ1 have not yet been characterized. Here, we identified four splicing variants of Arabidopsis (Arabidopsis thaliana) SIZ1, which encode three different protein isoforms. The SIZ1 gene encodes an 873-amino acid (aa) protein. Among the four SIZ1 splicing variants (SSVs), SSV1 and SSV4 encode identical 885 aa proteins; SSV2 encodes an 832 aa protein; and SSV3 encodes an 884 aa protein. SSV2 mainly localized to the plasma membrane, whereas SIZ1, SSV1/SSV4, and SSV3 localized to the nucleus. Interestingly, SIZ1 and all SSVs exhibited similar E3 SUMO ligase activities and preferred SUMO1 and SUMO2 for their E3 ligase activity. Transcript levels of SSV2 were substantially increased by heat treatment, while those of SSV1, SSV3, and SSV4 transcripts were unaffected by various abiotic stresses. SSV2 directly interacted with and sumoylated cyclic nucleotide-gated ion channel 6 (CNGC6), a positive thermotolerance regulator, enhancing the stability of CNGC6. Notably, transgenic siz1-2 mutants expressing SSV2 exhibited greater heat stress tolerance than wild-type plants, whereas those expressing SIZ1 were sensitive to heat stress. Furthermore, transgenic cngc6 plants overaccumulating a mutated mCNGC6 protein (K347R, a mutation at the sumoylation site) were sensitive to heat stress, similar to the cngc6 mutants, while transgenic cngc6 plants overaccumulating CNGC6 exhibited restored heat tolerance. Together, we propose that alternative splicing is an important mechanism that regulates the function of SSVs during development or under adverse conditions, including heat stress.

A SUMO‐dependent Pathway for Cytosolic Protein Quality Control

Protein quality control (PQC) maintains proteostasis in cells, whereas PQC dysregulation is found in diseases such as cancers, cardiovascular diseases, and neurodegenerative disorders. Over the past few decades, remarkable progress has been made in understanding compartmentalized PQC pathways that operate inside the nucleus and the endoplasmic reticulum (ER). The small ubiquitin‐related modifiers (SUMOs), for example, were recently found to participate in a nuclear PQC pathway that involves selective sumoylation of misfolded proteins by the SUMO E3 ligase PML, and subsequent recognition and ubiquitylation by the SUMO‐targeted ubiquitin E3 ligase RNF4 (Guo et al., Mol Cell, 2014). Roles of SUMOs in cytoplasmic PQC, however, have not previously been reported. Using yeast mutants and human knockout cell lines, our lab has recently identified a conserved SUMO‐dependent pathway for degradation of a cytosolic, misfolded protein model substrate. The substrate consists of GFP fused to an N‐terminal nuclear export signal (NES) and a C‐terminal hydrophobic degron known as CL1 (NES‐GFP‐CL1). Using protein stability assays, we observed a decrease in the turnover rate of NES‐GFP‐CL1 in a yeast SUMO mutant strain and a human U2OS SUMO1 knockout cell line. Further exploration using nuclear and ER‐localized substrates revealed that this pathway is specific for soluble, cytosolic proteins. In addition, analysis of a U2OS SUMO2 knockout cell line revealed that the observed PQC pathway is specific for the SUMO1 paralogue. This suggests a distinct mechanism from the previously characterized nuclear PML‐SUMO‐RNF4 pathway, which targets misfolded proteins modified by SUMO2/3 polymeric chains. Using cellular fractionation studies, we observed an increase in levels of insoluble NES‐GFP‐CL1 specifically in SUMO1 knockout cells compared to wild type cells. We therefore propose a model where SUMO1 modification of misfolded cytosolic proteins promotes their turnover by enhancing solubility and preventing aggregation. Results from our studies reveal a novel cytosolic PQC regulatory network and provide insights into roles for sumoylation in PQC‐associated diseases.

A SUMO ligase OsMMS21 regulates rice development and auxin response

SUMOylation, which transfers the Small Ubiquitin-related Modifier (SUMO) polypeptides to target proteins, regulates diverse cellular processes in eukaryotes. The SUMO conjugation reaction is usually promoted by SUMO E3 ligases, but the molecular functions of this type of enzymes remain unclear in cereal crops. Here, OsMMS21, a SUMO E3 ligase, was functionally characterized in rice (Oryza sativa). Bioinformatics analysis showed that OsMMS21 harbors a conserved SP-RING domain that is essential for the activity of SUMO ligases. Biochemical data indicated that this protein is auto-SUMOylated. Besides, overexpression of OsMMS21 rescued the developmental defects of the AtMMS21 mutant, supporting that OsMMS21 is a functional homolog of the Arabidopsis SUMO ligase AtMMS21. The OsMMS21 rice T-DNA mutant displays a short-root and dwarfism phenotype. RNA-seq data revealed that the expression levels of many genes involved in signaling transduction of hormones, including auxin, are altered in the OsMMS21 mutant. Further results under the auxin treatment showed that the OsMMS21 mutant is insensitive to auxin. Collectively, our results demonstrated the molecular features of OsMMS21 and uncovered the roles of this SUMO ligase in development and auxin response, providing hints for further studies on protein SUMOylation in rice.

The SUMO E3 ligase CBX4 is identified as a poor prognostic marker of gastric cancer through multipronged OMIC analyses

Gastric cancer (GC) is one of the most common malignancies, with an ever-increasing incidence and high mortality rate. Chromobox4 (CBX4), also named hPC2, is a small ubiquitin-related modifier (SUMO) E3 ligase. Previous studies have found that high CBX4 expression is associated with tumor size, pathologic differentiation and decreased patient survival in hepatocellular carcinoma (HCC). However, the expression and prognostic value of CBX4 in GC have not been clarified. In our study, ONCOMINE, UALCAN, Kaplan-Meier Plotter, cBioPortal, DAVID 6.8 and TIMER were utilized. RT-PCR, immunohistochemistry (IHC), Western blot, CCK-8 assay, cell apoptosis assay, cell cycle assay were used to further verify in GC tissue samples or cell line. The transcriptional and protein level of CBX4 in GC tissues was found significantly elevated and a significant association between the expression of CBX4 and clinicopathological parameters was found in GC patients. Low expression of CBX4 in GC patients were correlated with a significantly improved prognosis. The functions of CBX4 are primarily related to the stem cell pluripotency signaling pathway, Hippo signaling pathway, HTLV-I infection, Notch signaling pathway, and N-glycan biosynthesis. Our results may provide novel insights for the selection of therapeutic targets and prognostic biomarkers for GC.

Protein S-glutathionylation stimulate adipogenesis by stabilizing C/EBPβ in 3T3L1 cells.

Reactive oxygen species (ROS) increase during adipogenesis and in obesity. Oxidants react with cysteine residues of proteins to form glutathione (GSH) adducts, S-glutathionylation, that are selectively removed by glutaredoxin-1 (Glrx). We have previously reported that Glrx knockout mice had increased protein S-glutathionylation and developed obesity by an unknown mechanism. In this study, we demonstrated that 3T3L1 adipocytes differentiation increased ROS and protein S-glutathionylation. Glrx ablation elevated protein S-glutathionylation and lipid content in 3T3L1 cells. Glrx replenishment decreased the lipid content of Glrx KO 3T3L1 cells. Glrx KO also increased protein expression and protein S-glutathionylation of the adipogenic transcription factor CCAAT enhancer-binding protein (C/EBP) β. Protein S-glutathionylation decreased the interaction of C/EBPβ and protein inhibitor of activated STAT (PIAS) 1, a small ubiquitin-related modifier E3 ligase that facilitates C/EBPβ degradation. Experiments with truncated mutant C/EBPβ demonstrated that PIAS1 interacted with the liver-enriched inhibitory protein (LIP) region of C/EBPβ. Furthermore, mass spectrometry analysis identified protein S-glutathionylation of Cys201 and Cys296 in the LIP region of C/EBPβ. The C201S, C296S double-mutant C/EBPβ prevented protein S-glutathionylation and preserved the interaction with PIAS1. In summary, Glrx ablation stimulated 3T3L1 cell differentiation and adipogenesis via increased protein S-glutathionylation of C/EBPβ, stabilizing and increasing C/EBPβ protein levels.

Modification of cardiac transcription factor Gata6 by SUMO

SUMOylation, covalent conjugation of small ubiquitin-related modifier (SUMO), has been emerging as a critical posttranslational modification of developmental transcription factors, as well as key regulators in the adult heart. Identifying the SUMOylated targets within cardiac transcription factors will facilitate to unravel the roles of SUMOylation in heart development and disease. Here, we show that Gata6, an essential cardiac transcription factor, can be modified by SUMO in vivo. Mutation of potential SUMOylation sites reveals that a lysine residue at amino acid position 12 of Gata6 serves as the major attachment site for SUMO. Pias1, as an E3 SUMO ligase, preferentially enhances the conjugation of SUMO1 to Gata6 through its RING finger domain. Functional analyses with SUMOylation-deficient mutant indicate that SUMOylation does not affect the subcellular localization but instead represses Gata6 transcriptional activity. Our data suggest that posttranslational modification of Gata6 by SUMO conjugation provides a novel mechanism to regulate Gata6 activity.

E3 SUMO ligase AtSIZ1 regulates the cruciferin content of Arabidopsis seeds

Arabidopsis thaliana E3 SUMO ligase SIZ1 (AtSIZ1) controls vegetative growth and development, including responses to nutrient deficiency and environmental stresses. Here, we analyzed the effect of AtSIZ1 and its E3 SUMO ligase activity on the amount of seed proteins. Proteomic analysis showed that the level of three major nutrient reservoir proteins, CRUCIFERIN1 (CRU1), CRU2, and CRU3, was reduced in the siz1-2 mutant compared with the wild type. However, quantitative real-time PCR (qRT-PCR) analysis showed that transcript levels of CRU1, CRU2, and CRU3 genes were significantly higher in the siz1-2 mutant than in the wild type. Yeast two-hybrid analysis revealed direct interaction of AtSIZ1 with CRU1, CRU2, and CRU3. The sumoylation assay revealed that CRU2, and CRU3 proteins were modified with a small ubiquitin-related modifier (SUMO) by the E3 SUMO ligase activity of AtSIZ1. Additionally, high-performance liquid chromatography (HPLC) analysis showed that the amino acid content was slightly higher in siz1-2 mutant seeds than in wild type seeds. Taken together, our data indicate that AtSIZ1 plays an important role in the accumulation and stability of seed storage proteins through its E3 ligase activity.

OsSIZ2 regulates nitrogen homeostasis and some of the reproductive traits in rice

Small ubiquitin-related modifier (SUMO) is a post-translational modification of proteins that has important roles in plant growth and development as well as nutrition study. OsSIZ1, a SUMO E3 ligase in rice (Oryza sativa), exerts regulatory influence on nitrogen (N) homeostasis. Here, we investigated the biological function of OsSIZ2, a paralog of OsSIZ1, in the responses to nitrogen, anther dehiscence, and seed length using a reverse genetics approach. The expression of OsSIZ2 was increased during N deficiency. Under -N condition, total N concentration in the root of OsSIZ2-Ri plants and ossiz2 was significantly increased compared with wild type. Further, 15N-labelled uptake assay revealed the role of OsSIZ2 in acquisition and mobilization of N. Moreover, qRT-PCR analyses revealed that several genes involved in the maintenance of N homeostasis were altered in OsSIZ2 mutants. In addition, ossiz2 indicated obvious defects in anther dehiscence, pollen fertility, and seed set percentage. Interestingly, however, the seed length was longer in the mutant compared with wild type. Overall, these results suggest pivotal roles of OsSIZ2 in regulating homeostasis of N and different agronomic traits including anther and seed development.

Nitrate Reductases Are Relocalized to the Nucleus by AtSIZ1 and Their Levels Are Negatively Regulated by COP1 and Ammonium.

Nitrate reductases (NRs) catalyze the first step in the reduction of nitrate to ammonium. NR activity is regulated by sumoylation through the E3 ligase activity of AtSIZ1. However, it is not clear how NRs interact with AtSIZ1 in the cell, or how nitrogen sources affect NR levels and their cellular localization. Here, we show that the subcellular localization of NRs is modulated by the E3 SUMO (Small ubiquitin-related modifier) ligase AtSIZ1 and that NR protein levels are regulated by nitrogen sources. Transient expression analysis of GFP fusion proteins in onion epidermal cells showed that the NRs NIA1 and NIA2 localize to the cytoplasmic membrane, and that AtSIZ1 localizes to the nucleoplasm, including nuclear bodies, when expressed separately, whereas NRs and AtSIZ1 localize to the nucleus when co-expressed. Nitrate did not affect the subcellular localization of the NRs, but it caused AtSIZ1 to move from the nucleus to the cytoplasm. NRs were not detected in ammonium-treated cells, whereas the localization of AtSIZ1 was not altered by ammonium treatment. NR protein levels increased in response to nitrate but decreased in response to ammonium. In addition, NR protein levels increased in response to a 26S proteasome inhibitor and in cop1-4 and DN-COP1-overexpressing transgenic plants. NR protein degradation occurred later in cop1-4 than in the wild-type, although the NR proteins did not interact with COP1. Therefore, AtSIZ1 controls nuclear localization of NR proteins, and ammonium negatively regulates their levels. The function and stability of NR proteins might be post-translationally modulated by ubiquitination.

Post-translational modifications of Arabidopsis E3 SUMO ligase AtSIZ1 are controlled by environmental conditions.

Sumoylation regulates numerous cellular functions in plants as well as in other eukaryotic systems. However, the regulatory mechanisms controlling E3 small ubiquitin‐related modifier (SUMO) ligase are not well understood. Here, post‐translational modification of the Arabidopsis E3 SUMO ligase AtSIZ1 was shown to be specifically controlled by abiotic stresses. AtSIZ1 ubiquitination was induced by exposure to heat stress in transgenic plants overexpressing the E3 ubiquitin ligase COP1. In addition, AtSIZ1 ubiquitination was strongly enhanced in transgenic plants overexpressing SUMO isopeptidase ESD4 under heat stress. By contrast, drought stress induced sumoylation rather than ubiquitination of AtSIZ1 and sumoylated forms of AtSIZ1 accumulated in esd4 and cop1–4 mutants. Moreover, siz1 mutants were found to be tolerant to heat and drought stresses. Taken together, these results indicate that ubiquitination and sumoylation of AtSIZ1 in response to abiotic stresses depend on the activities of COP1 and ESD4 and that the activity and stability of AtSIZ1 can be specifically controlled by different abiotic stresses.

Reversible regulation of ORC2 SUMOylation by PIAS4 and SENP2.

The small ubiquitin-related modifier (SUMO) system is essential for smooth progression of cell cycle at the G2/M phase. Many centromeric proteins are reversibly SUMOylated to ensure proper chromosome segregation at the mitosis. SUMOylation of centromeric Origin Recognition Complex subunit 2 (ORC2) at the G2/M phase is essential in maintaining genome integrity. However, how ORC2 SUMOylation is regulated remains largely unclear. Here we show that ORC2 SUMOylation is reversibly controlled by SUMO E3 ligase PIAS4 and De-SUMOylase SENP2. Either depletion of PIAS4 or overexpression of SENP2 eliminated SUMOylation of ORC2 at the G/M phase and consequently resulted in abnormal centromeric histone H3 lysine 4 methylation. Cells stably expressing SENP2 protein or small interfering RNA for PIAS4 bypassed mitosis and endoreduplicated their genome to become polyploidy. Furthermore, percentage of polyploid cells is reduced after coexpression of ORC2-SUMO2 fusion protein. Thus, the proper regulation of ORC2 SUMOylation at the G2/M phase by PIAS4 and SENP2 is critical for smooth progression of the mitotic cycle of cells.

SUMO E3 Ligases GmSIZ1a and GmSIZ1b regulate vegetative growth in soybean .

SIZ1 is a small ubiquitin‐related modifier (SUMO) E3 ligase that mediates post‐translational SUMO modification of target proteins and thereby regulates developmental processes and hormonal and environmental stress responses in Arabidopsis. However, the role of SUMO E3 ligases in crop plants is largely unknown. Here, we identified and characterized two Glycine max (soybean) SUMO E3 ligases, GmSIZ1a and GmSIZ1b. Expression of GmSIZ1a and GmSIZ1b was induced in response to salicylic acid (SA), heat, and dehydration treatment, but not in response to cold, abscisic acid (ABA), and NaCl treatment. Although GmSIZ1a was expressed at higher levels than GmSIZ1b, both genes encoded proteins with SUMO E3 ligase activity in vivo. Heterologous expression of GmSIZ1a or GmSIZ1b rescued the mutant phenotype of Arabidopsis siz1‐2, including dwarfism, constitutively activated expression of pathogen‐related genes, and ABA‐sensitive seed germination. Simultaneous downregulation of GmSIZ1a and GmSIZ1b (GmSIZ1a/b) using RNA interference (RNAi)‐mediated gene silencing decreased heat shock‐induced SUMO conjugation in soybean. Moreover, GmSIZ1RNAi plants exhibited reduced plant height and leaf size. However, unlike Arabidopsis siz1‐2 mutant plants, flowering time and SA levels were not significantly altered in GmSIZ1RNAi plants. Taken together, our results indicate that GmSIZ1a and GmSIZ1b mediate SUMO modification and positively regulate vegetative growth in soybean.

COP1 Controls Abiotic Stress Responses by Modulating AtSIZ1 Function through Its E3 Ubiquitin Ligase Activity.

Ubiquitination and sumoylation are essential post-translational modifications that regulate growth and development processes in plants, including control of hormone signaling mechanisms and responses to stress. This study showed that COP1 (Constitutive photomorphogenic 1) regulated the activity of Arabidopsis E3 SUMO (Small ubiquitin-related modifier) ligase AtSIZ1 through its E3 ubiquitin ligase activity. Yeast two hybrid analysis demonstrated that COP1 and AtSIZ1 directly interacted with one another, and subcellular localization assays indicated that COP1 and AtSIZ1 co-localized in nuclear bodies. Analysis of ubiquitination showed that AtSIZ1 was polyubiquitinated by COP1. The AtSIZ1 level was higher in cop1-4 mutants than in wild-type seedlings under light or dark conditions, and overexpression of a dominant-negative (DN)-COP1 mutant led to a substantial increase in AtSIZ1 accumulation. In addition, under drought, cold, and high salt conditions, SUMO-conjugate levels were elevated in DN-COP1-overexpressing plants and cop1-4 mutant plants compared to wild-type plants. Taken together, our results indicate that COP1 controls responses to abiotic stress by modulation of AtSIZ1 levels and activity.

Functional identification of MdSIZ1 as a SUMO E3 ligase in apple

SUMOylation, the conjugation of target proteins with SUMO (small ubiquitin-related modifier), is a type of post-translational modification in eukaryotes and involves the sequential action of activation (E1), conjugation (E2) and ligation (E3) enzymes. In Arabidopsis, the AtSIZ1 protein is a SUMO E3 ligase that promotes the conjugation of SUMO proteins to target substrates. Here, we isolated and identified a SUMO E3 ligase, MdSIZ1, in apple, which was similar to AtSIZ1. SUMOylation analysis showed that MdSIZ1 had SUMO E3 ligase activity in vitro and in vivo. SUMO conjugation was increased by high temperatures, low temperatures, and abscisic acid (ABA). The ectopic expression of MdSIZ1 in Arabidopsis siz1-2 mutant plants partially complemented the morphological mutant phenotype and enhanced the levels of SUMO conjugation. Taken together, these results suggest that MdSIZ1-mediated SUMO conjugation of target proteins is an important process that regulates the adaptation of apple plants to various environmental stresses.

PIAS3 enhances the transcriptional activity of HIF-1α by increasing its protein stability

The transcription factor hypoxia-inducible factor-1 (HIF-1) functions as a master regulator of hypoxic response by inducing the transcription of various genes responsible for cellular adaptation to hypoxia. In this study, we investigated the effects of protein inhibitor of activated STAT3 (PIAS3), a small ubiquitin-related modifier (SUMO) E3 ligase, on HIF-1-mediated transcriptional activation. We found that PIAS3 physically associated with HIF-1α. Moreover, PIAS3 overexpression enhanced the transcriptional activity of HIF-1α independently of its SUMO E3 ligase activity. Conversely, quantitative RT-PCR analysis showed that RNAi-mediated PIAS3 knockdown reduced the expression of HIF-1 target genes under hypoxia. In addition, PIAS3 knockdown induced the destabilization of HIF-1α protein, and the destabilization was reversed by the proteasome inhibitor MG132. Taken together, these results suggest that PIAS3 functions as a positive regulator of HIF-1α-mediated transcription by increasing its protein stability.

Functional characterization of DnSIZ1, a SIZ/PIAS-type SUMO E3 ligase from Dendrobium.

BackgroundSUMOylation is an important post-translational modification of eukaryotic proteins that involves the reversible conjugation of a small ubiquitin-related modifier (SUMO) polypeptide to its specific protein substrates, thereby regulating numerous complex cellular processes. The PIAS (protein inhibitor of activated signal transducers and activators of transcription [STAT]) and SIZ (scaffold attachment factor A/B/acinus/PIAS [SAP] and MIZ) proteins are SUMO E3 ligases that modulate SUMO conjugation. The characteristic features and SUMOylation mechanisms of SIZ1 protein in monocotyledon are poorly understood. Here, we examined the functions of a homolog of Arabidopsis SIZ1, a functional SIZ/PIAS-type SUMO E3 ligase from Dendrobium.ResultsIn Dendrobium, the predicted DnSIZ1 protein has domains that are highly conserved among SIZ/PIAS-type proteins. DnSIZ1 is widely expressed in Dendrobium organs and has a up-regulated trend by treatment with cold, high temperature and wounding. The DnSIZ1 protein localizes to the nucleus and shows SUMO E3 ligase activity when expressed in an Escherichia coli reconstitution system. Moreover, ectopic expression of DnSIZ1 in the Arabidopsis siz1-2 mutant partially complements several phenotypes and results in enhanced levels of SUMO conjugates in plants exposed to heat shock conditions. We observed that DnSIZ1 acts as a negative regulator of flowering transition which may be via a vernalization-induced pathway. In addition, ABA-hypersensitivity of siz1-2 seed germination can be partially suppressed by DnSIZ1.ConclusionsOur results suggest that DnSIZ1 is a functional homolog of the Arabidopsis SIZ1 with SUMO E3 ligase activity and may play an important role in the regulation of Dendrobium stress responses, flowering and development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0613-3) contains supplementary material, which is available to authorized users.

E3 SUMO ligase AtSIZ1 positively regulates SLY1-mediated GA signalling and plant development.

Gibberellins affect various plant development processes including germination, cell division and elongation, and flowering. A large number of studies have been carried out to address the molecular mechanisms that mediate gibberellin signalling effects on plant growth. However, such studies have been limited to DELLA protein degradation; the regulatory mechanisms controlling how the stability and function of SLEEPY1 (SLY1), a protein that interacts with target DELLA proteins as components of the Skp, Cullin, F-box (SCF)(SLY1) complex, are modulated at the post-translational level have not been addressed. In the present study, we show that the E3 SUMO (small ubiquitin-related modifier) ligase AtSIZ1 regulates gibberellic acid signalling in Arabidopsis species by sumoylating SLY1. SLY1 was less abundant in siz1-2 mutants than in wild-type plants, but the DELLA protein repressor of ga1-3 (RGA) was more abundant in siz1-2 mutants than in wild-type plants. SLY1 also accumulated to a high level in the SUMO protease mutant esd4. Transgenic sly1-13 mutants over-expressing SLY1 were phenotypically similar to wild-type plants; however, sly1-13 plants over-expressing a mutated mSLY1 protein (K122R, a mutation at the sumoylation site) retained the mutant dwarfing phenotype. Over-expression of SLY1in sly1-13 mutants resulted in a return of RGA levels to wild-type levels, but RGA accumulated to high levels in mutants over-expressing mSLY1. RGA was clearly detected in Arabidopsis co-expressing AtSIZ1 and mSLY1, but not in plants co-expressing AtSIZ1 and SLY1. In addition, sumoylated SLY1 interacted with RGA and SLY1 sumoylation was significantly increased by GA. Taken together, our results indicate that, in Arabidopsis, AtSIZ1 positively controls GA signalling through SLY1 sumoylation.

SUMO E3-Ligase Regulates DNA Damage-Dependent Exchange of the Histone Variant H2A.Z-2

Recently, in the regulation of DNA damage response, the importance of the exchange of histone variants as one of the important mechanisms for reorganization of damaged chromatin has been recognized. In addition, the small ubiquitin-related modifier (SUMO) system has been shown to play an important role in the posttranslational modification of DNA repair proteins. Recent studies have implicated yeast H2A.Z in the DNA repair process and reported the presence of two H2A.Z isoforms, H2A.Z-1 and H2A.Z-2, in vertebrates. However, the role of each of these vertebrate H2A.Z isoforms in the reorganization of damaged chromatin is still unclear. In this study, we examined the role of human H2A.Z isoforms in the reorganization of chromatin after the induction of DNA double strand breaks (DSBs) and the importance of SUMOylation in its mechanism of action. To examine the dynamics of H2A.Z isoforms at damaged sites, we established GM0637 cells that stably expressed either one of the GFP-H2A.Z isoforms and performed fluorescence recovery after photobleaching (FRAP) and inverse FRAP (iFRAP) analyses in combination with microirradiation. In FRAP analysis, the dynamics of H2A.Z isoform following DNA damage were quantified by determining the fluorescence recovery of the GFP signal within two independent areas – one area in the irradiated region and the other in the unirradiated region of a single nucleus – immediately following microirradiation. In iFRAP analysis, immediately following microirradiation, all fluorescence except for small regions in irradiated and unirradiated areas were bleached and the remaining GFP fluorescence was chased. Finally, to investigate the pathway that regulates H2A.Z isoform dynamics in the damaged chromatin, we performed FRAP and iFRAP analysis with cells expressing siRNA against SUMO E3-ligase, PIAS1 or PIAS4. In FRAP analysis, the fluorescence intensity of GFP-H2A.Z-2 rapidly recovered in the irradiated region, while that in the unirradiated region did not change during the observed time. In contrast, GFP-H2A.Z-1 signal intensity did not show any remarkable changes. In iFRAP analysis, slow diffusion of GFP-H2A.Z-2 into the bleached area was observed within the irradiated area, but not in the unirradiated area. Similar to FRAP analysis, GFP-H2A.Z-1 signal intensity did not show any remarkable changes. These findings indicate that vertebrate H2A.Z-2 is exchanged at damaged sites immediately after the induction of DSBs. The increase in the mobility of H2A.Z-2 after microirradiation was repressed by knockdown of PIAS4, but not PIAS1. We found that vertebrate H2A.Z-2 is involved in the regulation of DNA damage response at a very early stage via reorganization of damaged chromatin. In addition, SUMOylation may affect the regulation of H2A.Z-2 dynamics upon DSBs.

Abstract 763A: SUMOylation of ATF3 alters MC2R, NR5A1, STAR, and TP53 gene activities.

Abstract Cyclic AMP-dependent transcription factor-3 (ATF3), a stress sensor/mediator, plays an essential role in cells to maintain homeostasis and has diverse functions in cellular death and survival signal pathways. ATF3 binds to the consensus ATF/cAMP response element (CRE) to regulate its downstream target genes, such as CCNE2 (Cyclin E2), CD82 (KAI1), DDIT3 (GADD153), LDLR (LDL receptor), SNAI1, TP53, and TWIST1. A previous report has shown that the activities of ATF3 are modulated by ubiquitination; however, whether it is modified by small ubiquitin-related modifier (SUMO), an important modification in regulating a wide range of cellular processes, remains unknown. The aim of this study was to investigate whether ATF3 is post-translationally modified by SUMO proteins and also to determine the role of SUMOylation of ATF3 on MC2R, NR5A1, STAR, and TP53 gene activities. Here we demonstrate that ATF3 is clearly defined as a SUMO target protein both in vitro SUMOylation assay using recombinant proteins and at the cellular levels. Furthermore, ATF3 interacted with UBE2I (Ubc9), the only SUMO E2 enzyme found so far. In addition, PIAS3β (a SUMO E3 ligase) enhanced and SENP2 and SENP7 (two SUMOylation proteases) decreased SUMOylation of ATF3, respectively. In addition, we found that ATF3 is selectively SUMOylated at lysine residue 42 but the SUMOylation/deSUMOylation does not alter subcellular localization of ATF3. We next characterized the functional role of ATF3 SUMOylation on MC2R, NR5A1, STAR, and TP53 gene expression. We found that SUMOylation of ATF3 is required for full repression of NR5A1 and TP53 genes. While SUMOylation of ATF3 is required for full activation of MC2R gene activity, SUMOylation of ATF3 further enhances repression of STAR gene activity. Overall, we provide the first evidence that ATF3 is post-translationally modified by SUMO to regulate MC2R, NR5A1, STAR, and TP53 gene activities. Citation Format: Chiung-Min Wang, Victoria Brennan, Ninoska Gutierrez, Xirui Wang, Lizhong Wang, Wei-Hsiung Yang. SUMOylation of ATF3 alters MC2R, NR5A1, STAR, and TP53 gene activities. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 763A. doi:10.1158/1538-7445.AM2013-763A

AtSIZ1 regulates expression of nitrite reductase but not its activity

Plant nitrite reductase (NiR) catalyzes the reduction of nitrite to ammonium. We examined if NiR activity is regulated by sumoylation, and found NiR was not sumoylated by E3 small ubiquitin-related modifier ligase AtSIZ1. However, its transcript level decreased in the siz1-2 mutant, indicating that AtSIZ1 does not directly control NiR activity.