Global analysis of SUMO chain function reveals multiple roles in chromatin regulation.

Like ubiquitin, the small ubiquitin-related modifier (SUMO) proteins can form oligomeric chains, but the biological functions of these superstructures are not well understood. Here, we created mutant yeast strains unable to synthesize SUMO chains (smt3(allR)) and subjected them to high-content microscopic screening, synthetic genetic array (SGA) analysis, and high-density transcript profiling to perform the first global analysis of SUMO chain function. This comprehensive assessment identified 144 proteins with altered localization or intensity in smt3(allR) cells, 149 synthetic genetic interactions, and 225 mRNA transcripts (primarily consisting of stress- and nutrient-response genes) that displayed a \u3e1.5-fold increase in expression levels. This information-rich resource strongly implicates SUMO chains in the regulation of chromatin. Indeed, using several different approaches, we demonstrate that SUMO chains are required for the maintenance of normal higher-order chromatin structure and transcriptional repression of environmental stress response genes in budding yeast

Physical and Functional Characterization of the SUMO System and SUMO Chains in S. cerevisiae

The ubiquitin-like proteins (Ubls) are small polypeptides that function as post-translational modifiers. Like ubiquitin, most Ubls are covalently attached to a lysine residue on target proteins. The small ubiquitin-related modifiers (SUMO) play important roles in a number of critical biological processes, such as proliferation and regulation of the cell cycle, yet their specific cellular functions have remained poorly understood. Like ubiquitin, SUMO proteins can also form oligomeric “chains”, but the functions of these structures were even less well understood. To this end, I created the first spectral library for the identification of Ub/Ubl proteins and Ub/Ubl chain linkages in mass spectrometry experiments. This tool has dramatically improved our ability to use MS to analyze the contents of biological samples for Ub and Ubls, and to identify specific types of Ub and Ubl chains in model organisms. I also used MS to conduct the first comprehensive SUMO system protein-protein interactome in any organism. In total, 452 high confidence protein-protein interactions were detected for S. cerevisiae SUMO system proteins, encompassing a total of 321 interacting partners. Yeast SUMO system components were found to interact with proteins involved in a number of different biological processes, and my mapping effort increased the number of known SUMO system interacting partners >50-fold. This study revealed that a number of transcriptional co-repressors and chromatin remodelling proteins interact physically with specific SUMO system components, with a clear division of labour between SUMO system enzymes. Finally, I conducted the first global analysis of SUMO chain function, using a combination of genetic, high-content microscopy, and high-density transcriptomics screens. Consistent with my interactomics work, this study demonstrated that inhibition of SUMO chain synthesis leads to severe chromatin condensation defects, which in-turn leads to chromosome missegregation, unscheduled transcription of stress-and nutrient-regulated genes, and aberrant intragenic transcription. Together, my work thus revealed a major role for the SUMO system in the maintenance of higher order chromatin structure and transcriptional repression.Ph

Raw data for the identification of SUMOylated proteins in S. cerevisiae subjected to two types of osmotic shock, using affinity purification coupled with mass spectrometry

The small ubiquitin-related modifier (SUMO) “stress response” (SSR) is a poorly understood evolutionarily conserved phenomenon in which steady-state SUMO conjugate levels are dramatically increased in response to environmental stresses. Here we describe the data acquired using affinity-purification coupled with mass spectrometry to identify proteins that are SUMOylated in response to two different types of osmotic stress, 1 M sorbitol and 1 M KCl. The mass spectrometry dataset described here has been uploaded to the MassIVE repository with ID: MSV000078739, and consists of 32 raw MS files acquired in data-dependent mode on a Thermo Q-Exactive instrument. iProphet-processed MS/MS search results and associated SAINT scores are also included as a reference. These data are discussed and interpreted in “The S. cerevisiae SUMO stress response is a conjugation–deconjugation cycle that targets the transcription machinery”, by Lewicki et al. in the Journal of Proteomics, 2014 [1]

Cyclic Enterobacterial Common Antigen Maintains the Outer Membrane Permeability Barrier of Escherichia coli in a Manner Controlled by YhdP.

Gram-negative bacteria have an outer membrane (OM) impermeable to many toxic compounds that can be further strengthened during stress. In Enterobacteriaceae, the envelope contains enterobacterial common antigen (ECA), a carbohydrate-derived moiety conserved throughout Enterobacteriaceae, the function of which is poorly understood. Previously, we identified several genes in Escherichia coli K-12 responsible for an RpoS-dependent decrease in envelope permeability during carbon-limited stationary phase. For one of these, yhdP, a gene of unknown function, deletion causes high levels of both vancomycin and detergent sensitivity, independent of growth phase. We isolated spontaneous suppressor mutants of yhdP with loss-of-function mutations in the ECA biosynthesis operon. ECA biosynthesis gene deletions suppressed envelope permeability from yhdP deletion independently of envelope stress responses and interactions with other biosynthesis pathways, demonstrating suppression is caused directly by removing ECA. Furthermore, yhdP deletion changed cellular ECA levels and yhdP was found to co-occur phylogenetically with the ECA biosynthesis operon. Cells make three forms of ECA: ECA lipopolysaccharide (LPS), an ECA chain linked to LPS core; ECA phosphatidylglycerol, a surface-exposed ECA chain linked to phosphatidylglycerol; and cyclic ECA, a cyclized soluble ECA molecule found in the periplasm. We determined that the suppression of envelope permeability with yhdP deletion is caused specifically by the loss of cyclic ECA, despite lowered levels of this molecule found with yhdP deletion. Furthermore, removing cyclic ECA from wild-type cells also caused changes to OM permeability. Our data demonstrate cyclic ECA acts to maintain the OM permeability barrier in a manner controlled by YhdP

A ubiquitin and ubiquitin-like protein spectral library

We have developed and validated a ubiquitin (Ub) and ubiquitin-like protein (Ubl) spectral library, consisting of 467 consensus spectra (320 unique peptides derived from autophagy-related protein 8, F-adjacent transcript 10, interferon-stimulated gene 15 kDa protein, neural precursor cell expressed developmentally down-regulated protein 8, small ubiquitin-related modifiers 1-3 and Ub, and nine of the most commonly observed Ub/Ubl chain linkages). The use of the Ub/Ubl library with a spectral matching tool (SpectraST) yields improved performance over database search engines, and can successfully identify many types of Ub/Ubl chain-derived peptides that cannot be identified by standard database search algorithms

BioID data of c-MYC interacting protein partners in cultured cells and xenograft tumors

BioID was performed using FlagBirA⁎ (the R118G biotin ligase mutant protein) and FlagBirA⁎-Myc in HEK293 T-REx cells maintained both under standard cell culture conditions and as mouse xenografts. The mass spectrometry dataset acquired in this study has been uploaded to the MassIVE repository with ID: MSV000078518, and consists of 28 ⁎.raw MS files acquired on an Orbitrap Velos instrument, collected in data-dependent mode. iProphet processed MS/MS search results are also included as a reference. This study has been published as “BioID identifies novel c-MYC interacting partners in cultured cells and xenograft tumors”, by Dingar et al. in the Journal of Proteomics, 2014 [1]

Identification of c-MYC SUMOylation by mass spectrometry.

The c-MYC transcription factor is a master regulator of many cellular processes and deregulation of this oncogene has been linked to more than 50% of all cancers. This deregulation can take many forms, including altered post-translational regulation. Here, using immunoprecipitation combined with mass spectrometry, we identified a MYC SUMOylation site (K326). Abrogation of signaling through this residue by substitution with arginine (K326R) has no obvious effects on MYC half-life, intracellular localization, transcriptional targets, nor on the biological effects of MYC overexpression in two different cell systems assessed for soft agar colony formation, proliferation, and apoptosis. While we have definitively demonstrated that MYC SUMOylation can occur on K326, future work will be needed to elucidate the mechanisms and biological significance of MYC regulation by SUMOylation

A Novel Mechanism for SUMO System Control: Regulated Ulp1 Nucleolar Sequestration ▿

The small ubiquitin-related modifiers (SUMOs) are evolutionarily conserved polypeptides that are covalently conjugated to protein targets to modulate their subcellular localization, half-life, or activity. Steady-state SUMO conjugation levels increase in response to many different types of environmental stresses, but how the SUMO system is regulated in response to these insults is not well understood. Here, we characterize a novel mode of SUMO system control: in response to elevated alcohol levels, the Saccharomyces cerevisiae SUMO protease Ulp1 is disengaged from its usual location at the nuclear pore complex (NPC) and sequestered in the nucleolus. We further show that the Ulp1 region previously demonstrated to interact with the karyopherins Kap95 and Kap60 (amino acids 150 to 340) is necessary and sufficient for nucleolar targeting and that enforced sequestration of Ulp1 in the nucleolus significantly increases steady-state SUMO conjugate levels, even in the absence of alcohol. We have thus characterized a novel mechanism of SUMO system control in which the balance between SUMO-conjugating and -deconjugating activities at the NPC is altered in response to stress via relocalization of a SUMO-deconjugating enzyme