SUMO Proteases Research Articles

Charting the evolutionary path of the SUMO modification system in plants reveals molecular hardwiring of development to stress adaptation.

SUMO modification is part of the spectrum of Ubiquitin-like (UBL) systems that give rise to proteoform complexity through post-translational modifications (PTMs). Proteoforms are essential modifiers of cell signaling for plant adaptation to changing environments. Exploration of the evolutionary emergence of Ubiquitin-like (UBL) systems unveils their origin from prokaryotes, where it is linked to the mechanisms that enable sulfur uptake into biomolecules. We explore the emergence of the SUMO machinery across the plant lineage from single-cell to land plants. We reveal the evolutionary point at which plants acquired the ability to form SUMO chains through the emergence of SUMO E4 ligases, hinting at its role in facilitating multicellularity. Additionally, we explore the possible mechanism for the neofunctionalization of SUMO proteases through the fusion of conserved catalytic domains with divergent sequences. We highlight the pivotal role of SUMO proteases in plant development and adaptation, offering new insights into target specificity mechanisms of SUMO modification during plant evolution. Correlating the emergence of adaptive traits in the plant lineage with established experimental evidence for SUMO in developmental processes, we propose that SUMO modification has evolved to link developmental processes to adaptive functions in land plants.

Designing Ubiquitin-like protease 1 (Ulp1) based nano biocatalysts: A promising technology for SUMO fusion proteins

The SUMO proteases (Ulps), a group of cysteine proteases, are well known for their efficient ability to perform structure-based cleavage of SUMO tag from the protein of interest and generation of biotherapeutics with authentic N-terminus. However, the stability of Ulps has remained a challenge for the economical production of difficult-to-produce proteins in E. coli. Therefore, the present study aimed to establish the methodology for developing stable S. pombe Ulp1 preparation using different enzyme immobilization strategies. The whole-cell biocatalyst developed using the Pir1 anchor protein of Pichia cleaved the SUMO tag within 24 h of reaction incubation. The chemical immobilization using commercial epoxy and amino methacrylate beads significantly enhanced the operational reusability of SpUlp1 up to 24 cycles. Silica beads further improved the repetitive usage of the immobilized enzyme for 65 cycles. The SpUlp1 immobilization on laboratory-developed chitosan-coated iron oxide nanoparticles exhibited more than 90 % cleavage of SUMO tag from different substrates even after 100 consecutive reactions. Moreover, an effective SUMO tag removal was observed within 10 min of incubation. The operational stability of the immobilized enzyme was confirmed in a pH range of 5 to 13. The spherical nature of nanoparticles was confirmed by FESEM and TEM results. The successful chitosan coating and subsequent activation with glutaraldehyde were established via FT-IR. Furthermore, HRTEM, SAED, and XRD proved the crystalline nature of nanoparticles, while VSM confirmed the superparamagnetic behavior.

Soluble Expression of Antimicrobial Peptide BSN-37 from Escherichia coli by SUMO Fusion Technology.

Antimicrobial peptides (AMPs) are a kind of small molecular peptide that an organism produces to resist the invasion of foreign microorganisms. AMP BSN-37 is a bovine AMP that exhibits high antibacterial activity. In this paper, the optimized gene AMP BSN-37 was cloned into pCold-SUMO for fusion expression by recombinant DNA technology. The gene sequence of AMP BSN-37 was obtained by codons reverse translation, and the codons were optimized according to the codons preference of Escherichia coli (E. coli). The recombinant plasmid was constructed and identified by PCR, enzyme digestion and sequencing. Then the recombinant plasmid was transformed into BL21 E. coli to induce expression, and the IPTG concentration and time were optimized. The expressed soluble fusion protein SUMO-BSN-37 was purified by chromatography and then cleaved by SUMO proteases to release BSN-37. SDS-PAGE electrophoresis and Western blotting were used for identification. The recombinant plasmid pCold-SUMO-BSN-37 was obtained, and the fusion AMP BSN-37 was preliminarily expressed in BL21. After optimization, the optimal expression condition was 37 ℃ with 0.4 µM IPTG and 6h incubation. Under optimal conditions, a large amount of fusion AMP BSN-37 was obtained by purification. Western blotting showed that the fusion peptide was successfully expressed and had good activity. The expressed BSN-37 showed antimicrobial activity similar to that of synthesized BSN-37. In this study, soluble expression products of AMP BSN-37 were obtained, and the problem regarding the limited source of AMP BSN-37 could be effectively solved, laying a foundation for further research on AMP BSN-37.

Expression, Purification, and Enzymatic Analysis of Plant SUMO Proteases.

The conjugation of SUMO can profoundly change the behavior of substrate proteins, impacting a wide variety of cellular responses. SUMO proteases are emerging as key regulators of plant adaptation to its environment because of their instrumental role in the SUMO deconjugation process. Here we describe how to express, purify, and determine SUMO deconjugation activity of a plant SUMO protease.

Structural Basis for the SUMO2 Isoform Specificity of SENP7

SUMO proteases or deSUMOylases regulate the lifetime of SUMO-conjugated targets in the cell by cleaving off the isopetidic bond between the substrate and the SUMO modifier, thus reversing the conjugation activity of the SUMO E3 ligases. In humans the deSUMOylating activity is mainly conducted by the SENP/ULP protease family, which is constituted of six members sharing a homologous catalytic globular domain. SENP6 and SENP7 are the most divergent members of the family and they show a unique SUMO2/3 isoform preference and a particular activity for dismantling polySUMO2 chains. Here, we present the crystal structure of the catalytic domain of human SENP7 bound to SUMO2, revealing structural key elements for the SUMO2 isoform specificity of SENP7. In particular, we describe the specific contacts between SUMO2 and a unique insertion in SENP7 (named Loop1) that is responsible for the SUMO2 isoform specificity. All the other interface contacts between SENP7 and SUMO2, including the SUMO2 C-terminal tail interaction, are conserved among members of the SENP/ULP family. Our data give insight into an evolutionary adaptation to restrict the deSUMOylating activity in SENP6 and SENP7 for the SUMO2/3 isoforms.

Cancer-Associated Dysregulation of Sumo Regulators: Proteases and Ligases.

SUMOylation is a post-translational modification that has emerged in recent decades as a mechanism involved in controlling diverse physiological processes and that is essential in vertebrates. The SUMO pathway is regulated by several enzymes, proteases and ligases being the main actors involved in the control of sumoylation of specific targets. Dysregulation of the expression, localization and function of these enzymes produces physiological changes that can lead to the appearance of different types of cancer, depending on the enzymes and target proteins involved. Among the most studied proteases and ligases, those of the SENP and PIAS families stand out, respectively. While the proteases involved in this pathway have specific SUMO activity, the ligases may have additional functions unrelated to sumoylation, which makes it more difficult to study their SUMO-associated role in cancer process. In this review we update the knowledge and advances in relation to the impact of dysregulation of SUMO proteases and ligases in cancer initiation and progression.

The conjugation of SUMO to the transcription factor MYC2 functions in blue light-mediated seedling development in Arabidopsis.

A key function of photoreceptor signaling is the coordinated regulation of a large number of genes to optimize plant growth and development. The basic helix loop helix (bHLH) transcription factor MYC2 is crucial for regulating gene expression in Arabidopsis thaliana during development in blue light. Here we demonstrate that blue light induces the SUMOylation of MYC2. Non-SUMOylatable MYC2 is less effective in suppressing blue light-mediated photomorphogenesis than wild-type (WT) MYC2. MYC2 interacts physically with the SUMO proteases SUMO PROTEASE RELATED TO FERTILITY1 (SPF1) and SPF2. Blue light exposure promotes the degradation of SPF1 and SPF2 and enhances the SUMOylation of MYC2. Phenotypic analysis revealed that SPF1/SPF2 function redundantly as positive regulators of blue light-mediated photomorphogenesis. Our data demonstrate that SUMO conjugation does not affect the dimerization of MYC transcription factors but modulates the interaction of MYC2 with its cognate DNA cis-element and with the ubiquitin ligase Plant U-box 10 (PUB10). Finally, we show that non-SUMOylatable MYC2 is less stable and interacts more strongly with PUB10 than the WT. Taken together, we conclude that SUMO functions as a counterpoint to the ubiquitin-mediated degradation of MYC2, thereby enhancing its function in blue light signaling.

First person – Bin Yu

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Bin Yu is first author on ‘ SUMO proteases SENP3 and SENP5 spatiotemporally regulate the kinase activity of Aurora A’, published in JCS. Bin is a PhD graduate in the lab of Jing Yi at Shanghai Jiao Tong University School of Medicine, Shanghai, China, investigating protein post-translational modifications in cell cycle control.

SUMO proteases SENP3 and SENP5 spatiotemporally regulate the kinase activity of Aurora A.

Precise chromosome segregation is mediated by a well-assembled mitotic spindle, which requires balance of the kinase activity of Aurora A (AurA, also known as AURKA). However, how this kinase activity is regulated remains largely unclear. Here, using in vivo and in vitro assays, we report that conjugation of SUMO2 with AurA at K258 in early mitosis promotes the kinase activity of AurA and facilitates the binding with its activator Bora. Knockdown of the SUMO proteases SENP3 and SENP5 disrupts the deSUMOylation of AurA, leading to increased kinase activity and abnormalities in spindle assembly and chromosome segregation, which could be rescued by suppressing the kinase activity of AurA. Collectively, these results demonstrate that SENP3 and SENP5 deSUMOylate AurA to render spatiotemporal control on its kinase activity in mitosis. This article has an associated First Person interview with the first author of the paper.

An Insight into the Factors Influencing Specificity of the SUMO System in Plants.

Due to their sessile nature, plants are constantly subjected to various environmental stresses such as drought, salinity, and pathogen infections. Post-translational modifications (PTMs), like SUMOylation, play a vital role in the regulation of plant responses to their environment. The process of SUMOylation typically involves an enzymatic cascade containing the activation, (E1), conjugation (E2), and ligation (E3) of SUMO to a target protein. Additionally, it also requires a class of SUMO proteases that generate mature SUMO from its precursor and cleave it off the target protein, a process termed deSUMOylation. It is now clear that SUMOylation in plants is key to a plethora of adaptive responses. How this is achieved with an extremely limited set of machinery components is still unclear. One possibility is that novel SUMO components are yet to be discovered. However, current knowledge indicates that only a small set of enzymes seem to be responsible for the modification of a large number of SUMO substrates. It is yet unknown where the specificity lies within the SUMO system. Although this seems to be a crucial question in the field of SUMOylation studies, not much is known about the factors that provide specificity. In this review, we highlight the role of the localisation of SUMO components as an important factor that can play a vital role in contributing to the specificity within the process. This will introduce a new facet to our understanding of the mechanisms underlying such a dynamic process.

Phylogenetic distribution and evolutionary dynamics of nod and T3SS genes in the genus Bradyrhizobium.

Bradyrhizobium are abundant soil bacteria and the major symbiont of legumes. The recent availability of Bradyrhizobium genome sequences provides a large source of information for analysis of symbiotic traits. In this study, we investigated the evolutionary dynamics of the nodulation genes (nod) and their relationship with the genes encoding type III secretion systems (T3SS) and their effectors among bradyrhizobia. Based on the comparative analysis of 146 Bradyrhizobium genome sequences, we identified six different types of T3SS gene clusters. The two predominant cluster types are designated RhcIa and RhcIb and both belong to the RhcI-T3SS family previously described in other rhizobia. They are found in 92/146 strains, most of them also containing nod genes. RhcIa and RhcIb gene clusters differ in the genes they carry: while the translocon-encoding gene nopX is systematically found in strains containing RhcIb, the nopE and nopH genes are specifically conserved in strains containing RhcIa, suggesting that these last two genes might functionally substitute nopX and play a role related to effector translocation. Phylogenetic analysis suggests that bradyrhizobia simultaneously gained nod and RhcI-T3SS gene clusters via horizontal transfer or subsequent vertical inheritance of a symbiotic island containing both. Sequence similarity searches for known Nop effector proteins in bradyrhizobial proteomes revealed the absence of a so-called core effectome, i.e. that no effector is conserved among all Bradyrhizobium strains. However, NopM and SUMO proteases were found to be the main effector families, being represented in the majority of the genus. This study indicates that bradyrhizobial T3SSs might play a more significant symbiotic role than previously thought and provides new candidates among T3SS structural proteins and effectors for future functional investigations.

Utilizing Knockdowns, Overexpression and Tagging to Study the SUMO Proteases Ulp1 and Ulp2 in Tetrahymena thermophila

SUMO, small ubiquitin like modifier, plays a role in post‐translational modification of proteins, altering protein‐protein interactions and regulating cellular processes. Much of what is known about the SUMO pathway and SUMOylation comes from studies conducted in yeast and vertebrates. As a unicellular ciliated eukaryote, Tetrahymena thermophila exhibit complexity and diversity of cellular processes that are not present in yeast, and they possess the advantages of other single celled model organisms, such as short cell division times and ease of growth and genetic manipulation. We aimed to develop a better understanding of the function of the two SUMO proteases, Ulp1 and Ulp2, by knocking out the genes, ULP1 and ULP2, that encode these proteins. We designed knockout plasmid constructs by cloning the 5′ and 3′ flanking sequences of each gene into a vector with a neo resistance marker. The plasmid construct was then introduced into Tetrahymena, and cells were passaged into increasingly higher concentrations of paromomycin to promote phenotypic assortment. Quantitative RT‐PCR suggests that we were able to knockdown ULP2 but not ULP1, despite high paromomycin drug resistance, suggesting that ULP1 is an essential gene in Tetrahymena. We have also created plasmid constructs using a Gateway cloning method that enable us to both overexpress and YFP tag ULP1 and ULP2. Preliminary results show that Ulp1 localizes to the nuclear envelope, and Ulp2 localizes to the nucleus. Future work will focus on characterizing the overexpression phenotypes of ULP1 and ULP2 and the knockdown phenotype of ULP2 in Tetrahymena.Support or Funding InformationThis research was supported by the St. Olaf College Office of Collaborative Undergraduate Research, St. Olaf TRIO McNair Scholars Program (L.T.) and the Beta Beta Beta National Biological Honor Society (J.L.).

Dealing With Stress: A Review of Plant SUMO Proteases.

The SUMO system is a rapid dynamic post-translational mechanism employed by eukaryotic cells to respond to stress. Plant cells experience hyperSUMOylation of substrates in response to stresses such as heat, ethanol, and drought. Many SUMOylated proteins are located in the nucleus, SUMOylation altering many nuclear processes. The SUMO proteases play two key functions in the SUMO cycle by generating free SUMO; they have an important role in regulating the SUMO cycle, and by cleaving SUMO off SUMOylated proteins, they provide specificity to which proteins become SUMOylated. This review summarizes the broad literature of plant SUMO proteases describing their catalytic activity, domains and structure, evolution, localization, and response to stress and highlighting potential new areas of research in the future.

Assays of SUMO protease/isopeptidase activity and function in mammalian cells and tissues.

Covalent conjugation of the ubiquitin-related SUMO modifier to lysine residues of cellular proteins (SUMOylation) is a prevalent posttranslational modification. SUMOs are synthesized as precursor proteins that require carboxy-terminal processing prior to conjugation. Subsequently, a multistep enzymatic pathway is used for conjugation to target proteins. SUMOylation generally impacts protein-protein interactions and the assembly of multiprotein complexes. Cellular processes regulated by SUMOylation include DNA damage responses, cell cycle progression, or the control of gene expression. SUMOylation is reversible and commonly only a small fraction of a particular SUMO target is modified at a given time. Deconjugation of SUMO is catalyzed by a group of cysteine proteases termed SUMO proteases or SUMO isopeptidases. In human cells nine SUMO proteases, belonging to three separate families of cysteine proteases have been identified so far. The regulation and target specificity of individual SUMO proteases have not been dissected in detail, but the current view is that each protease controls the modification of subsets of proteins that are functionally and/or physically linked. Importantly, some SUMO proteases/isopeptidases not only function in deconjugation of SUMO from proteins, but also act in C-terminal processing of the SUMO precursors. Here we describe general methods for monitoring SUMO protease/isopeptidase activities in cell or tissue extracts.

Insights into the transcriptional and post-transcriptional regulation of the rice SUMOylation machinery and into the role of two rice SUMO proteases

BackgroundSUMOylation is an essential eukaryotic post-translation modification that, in plants, regulates numerous cellular processes, ranging from seed development to stress response. Using rice as a model crop plant, we searched for potential regulatory points that may influence the activity of the rice SUMOylation machinery genes.ResultsWe analyzed the presence of putative cis-acting regulatory elements (CREs) within the promoter regions of the rice SUMOylation machinery genes and found CREs related to different cellular processes, including hormone signaling. We confirmed that the transcript levels of genes involved in target-SUMOylation, containing ABA- and GA-related CREs, are responsive to treatments with these hormones. Transcriptional analysis in Nipponbare (spp. japonica) and LC-93-4 (spp. indica), showed that the transcript levels of all studied genes are maintained in the two subspecies, under normal growth. OsSUMO3 is an exceptional case since it is expressed at low levels or is not detectable at all in LC-93-4 roots and shoots, respectively. We revealed post-transcriptional regulation by alternative splicing (AS) for all genes studied, except for SUMO coding genes, OsSIZ2, OsOTS3, and OsELS2. Some AS forms have the potential to alter protein domains and catalytic centers. We also performed the molecular and phenotypic characterization of T-DNA insertion lines of some of the genes under study. Knockouts of OsFUG1 and OsELS1 showed increased SUMOylation levels and non-overlapping phenotypes. The fug1 line showed a dwarf phenotype, and significant defects in fertility, seed weight, and panicle architecture, while the els1 line showed early flowering and decreased plant height. We suggest that OsELS1 is an ortholog of AtEsd4, which was also supported by our phylogenetic analysis.ConclusionsOverall, we provide a comprehensive analysis of the rice SUMOylation machinery and discuss possible effects of the regulation of these genes at the transcriptional and post-transcriptional level. We also contribute to the characterization of two rice SUMO proteases, OsELS1 and OsFUG1.

SUMO-mediated regulation of NLRP3 modulates inflammasome activity

The NLRP3 inflammasome responds to infection and tissue damage, and rapidly escalates the intensity of inflammation by activating interleukin (IL)-1β, IL-18 and cell death by pyroptosis. How the NLRP3 inflammasome is negatively regulated is poorly understood. Here we show that NLRP3 inflammasome activation is suppressed by sumoylation. NLRP3 is sumoylated by the SUMO E3-ligase MAPL, and stimulation-dependent NLRP3 desumoylation by the SUMO-specific proteases SENP6 and SENP7 promotes NLRP3 activation. Defective NLRP3 sumoylation, either by NLRP3 mutation of SUMO acceptor lysines or depletion of MAPL, results in enhanced caspase-1 activation and IL-1β release. Conversely, depletion of SENP7 suppresses NLRP3-dependent ASC oligomerisation, caspase-1 activation and IL-1β release. These data indicate that sumoylation of NLRP3 restrains inflammasome activation, and identify SUMO proteases as potential drug targets for the treatment of inflammatory diseases.

Abstract LB-074: Examining SENP Localization In HEK-293 Cells Using MG-132 And Cycloheximide

Abstract Post translational modifications are changes to proteins that occur during or after synthesis by the ribosome. Often these modifications are necessary to form mature proteins. SUMOylation (Small Ubiquitin-like Modifier, SUMO) is a type of post translational modification that has a connection to a protein, Laminin Receptor, that contributes to the aggressiveness and proliferation of cancer. SUMO proteases, called SENPs, remove SUMO groups from SUMOylated proteins. Because Laminin Receptor may be a SUMOylated protein and is in the nucleus, we wanted to first explore how SENP-2 and SENP-3 localize under various cellular conditions. To understand the behavior of SENPs under cellular stress, we transfected human embryonic kidney cells, called HEK-293 cells, with SENP-2 or SENP-3 tagged to the fluorescent protein EGFP. We treated the cells with MG-132, a proteasome inhibitor, or with cycloheximide, a protein synthesis inhibitor. We used live imaging to qualitatively understand how SENP-2 and SENP-3 localize in the cell in normal conditions and under cell stress. Then, we looked for differences in cytosolic and nuclear partitioning of SENP-2 and SENP-3 by western blot. The exposure of SENPs to the two inhibitors helped us to understand the behavior of the SUMO proteases between the nucleus and cytoplasm. Citation Format: Michael Robinson, Charles S. Umbaugh, Marxa L. Figueiredo. Examining SENP Localization In HEK-293 Cells Using MG-132 And Cycloheximide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-074.

SUMO targeting of a stress-tolerant Ulp1 SUMO protease.

SUMO proteases of the SENP/Ulp family are master regulators of both sumoylation and desumoylation and regulate SUMO homeostasis in eukaryotic cells. SUMO conjugates rapidly increase in response to cellular stress, including nutrient starvation, hypoxia, osmotic stress, DNA damage, heat shock, and other proteotoxic stressors. Nevertheless, little is known about the regulation and targeting of SUMO proteases during stress. To this end we have undertaken a detailed comparison of the SUMO-binding activity of the budding yeast protein Ulp1 (ScUlp1) and its ortholog in the thermotolerant yeast Kluyveromyces marxianus, KmUlp1. We find that the catalytic UD domains of both ScUlp1 and KmUlp1 show a high degree of sequence conservation, complement a ulp1Δ mutant in vivo, and process a SUMO precursor in vitro. Next, to compare the SUMO-trapping features of both SUMO proteases we produced catalytically inactive recombinant fragments of the UD domains of ScUlp1 and KmUlp1, termed ScUTAG and KmUTAG respectively. Both ScUTAG and KmUTAG were able to efficiently bind a variety of purified SUMO isoforms and bound immobilized SUMO1 with nanomolar affinity. However, KmUTAG showed a greatly enhanced ability to bind SUMO and SUMO-modified proteins in the presence of oxidative, temperature and other stressors that induce protein misfolding. We also investigated whether a SUMO-interacting motif (SIM) in the UD domain of KmULP1 that is not conserved in ScUlp1 may contribute to the SUMO-binding properties of KmUTAG. In summary, our data reveal important details about how SUMO proteases target and bind their sumoylated substrates, especially under stress conditions. We also show that the robust pan-SUMO binding features of KmUTAG can be exploited to detect and study SUMO-modified proteins in cell culture systems.

SUMO proteases as potential targets for cancer therapy.

Sumoylation is one of the post-translational modifications of proteins, responsible for the regulation of many cellular processes, such as DNA replication and repair, transcription, signal transduction and nuclear transport. During sumoylation, SUMO proteins are covalently attached to the ε-amino group of lysine in target proteins via an enzymatic cascade that requires the sequential action of E1, E2 and E3 enzymes. An important aspect of sumoylation is its reversibility, which involves SUMO-specific proteases called SENPs. SENPs (sentrin/SUMO-specific proteases) catalyze the deconjugation of SUMO proteins using their isopeptidase activity. These enzymes participate through hydrolase activity in the reaction of SUMO protein maturation, which involves the removal of a short fragment on the C-terminus of SUMO inactive form and exposure two glycine residues. SENPs are important for maintaining the balance between sumoylated and desumoylated proteins required for normal cellular physiology. Six SENP isoforms (SENP1, SENP2, SENP3, SENP5, SENP6 and SENP7) have been identified in mammals. These SENPs can be divided into three subfamilies based on their sequence homology, substrate specificity and subcellular localization. Results of studies indicate the role of SUMO proteases in the development of human diseases including cancer, suggesting that these proteins may be attractive targets for new drugs.

The chromatin remodeling Isw1a complex is regulated by SUMOylation.

The ISWI class of proteins consists of a family of chromatin remodeling ATPases that is ubiquitous in eukaryotes and predominantly functions to slide nucleosomes laterally. The yeast Saccharomyces cerevisiae Isw1 partners with several non-essential alternative subunits - Ioc2, Ioc3, or Ioc4 - to form two distinct complexes Isw1a and Isw1b. Besides its ATPase domain, Isw1 presents a C-terminal region formed by HAND, SANT, and SLIDE domains responsible for interaction with the Ioc proteins and optimal association of Isw1 to chromatin. Despite diverse studies on the functions of the Isw1-containing complexes, molecular evidence for a regulation of this chromatin remodeling ATPase is still elusive. Results presented here indicate that Isw1 is not only ubiquitylated but also strongly SUMOylated on multiple lysine residues by the redundant Siz1/Siz2 SUMO E3 ligases. However, Isw1 is a poor substrate of the Ulp1 and Ulp2 SUMO proteases, thus resulting in a high level of modification. Extensive site-directed mutagenesis allowed us to identify the major SUMOylation sites and develop a SUMO-defective mutant of Isw1. Using this molecular tool, we show that SUMOylation of Isw1 specifically facilitates and/or stabilizes its interaction with its cofactor Ioc3 and consequently the efficient recruitment of the Isw1-Ioc3 complex onto chromatin. Together these data reveal a new regulatory mechanism for this fascinating remodeling factor.