The SUMO protease Ulp2 regulates genome stability and drug resistance in the human fungal pathogen Candida albicans

Stress-induced genome instability in microbial organisms is emerging as a critical regulatory mechanism for driving rapid and reversible adaption to drastic environmental changes. In Candida albicans, a human fungal pathogen that causes life-threatening infections, genome plasticity confers increased virulence and antifungal drug resistance. Discovering the mechanisms regulating C. albicans genome plasticity is a priority to understand how this and other microbial pathogens establish life-threatening infections and develop resistance to antifungal drugs. We identified the SUMO protease Ulp2 as a critical regulator of C. albicans genome integrity through genetic screening. Deletion of ULP2 leads to hypersensitivity to genotoxic agents and increased genome instability. This increased genome diversity causes reduced fitness under standard laboratory growth conditions but enhances adaptation to stress, making ulp2Δ/Δ cells more likely to thrive in the presence of antifungal drugs. Whole-genome sequencing indicates that ulp2Δ/Δ cells counteract antifungal drug-induced stress by developing segmental aneuploidies of chromosome R and chromosome I. We demonstrate that intrachromosomal repetitive elements drive the formation of complex novel genotypes with adaptive power.

SENP3 Promotes an Mff-Primed Bcl-xL-Drp1 Interaction Involved in Cell Death Following Ischemia

Dysregulation of the mitochondrial fission machinery has been linked to cell death following ischemia. Fission is largely dependent on recruitment of Dynamin-related protein 1 (Drp1) to the receptor Mitochondrial fission factor (Mff) located on the mitochondrial outer membrane (MOM). Drp1 is a target for SUMOylation and its deSUMOylation, mediated by the SUMO protease SENP3, enhances the Drp1-Mff interaction to promote cell death in an oxygen/glucose deprivation (OGD) model of ischemia. Another interacting partner for Drp1 is the Bcl-2 family member Bcl-xL, an important protein in cell death and survival pathways. Here we demonstrate that preventing Drp1 SUMOylation by mutating its SUMO target lysines enhances the Drp1-Bcl-xL interaction in vivo and in vitro. Moreover, SENP3-mediated deSUMOylation of Drp1 promotes the Drp1-Bcl-xL interaction. Our data suggest that Mff primes Drp1 binding to Bcl-xL at the mitochondria and that Mff and Bcl-xL can interact directly, independent of Drp1, through their transmembrane domains. Importantly, SENP3 loss in cells subjected to OGD correlates with reduced Drp1-Bcl-xL interaction, whilst recovery of SENP3 levels in cells subjected to reoxygenation following OGD correlates with increased Drp1-Bcl-xL interaction. Expressing a Bcl-xL mutant with defective Drp1 binding reduces OGD plus reoxygenation-evoked cell death. Taken together, our results indicate that SENP3-mediated deSUMOlyation promotes an Mff-primed Drp1-Bcl-xL interaction that contributes to cell death following ischemia.

The structural basis for Ulp2 recruitment to the kinetochore

ABSTRACTThe step-by-step process of chromosome segregation defines the stages of the cell division cycle. In eukaryotes, signaling pathways that control these steps converge upon the kinetochore, a multiprotein assembly that connects spindle microtubules to the centromere of each chromosome. Kinetochores control and adapt to major chromosomal transactions, including replication of centromeric DNA, biorientation of sister centromeres on the metaphase spindle, and transit of sister chromatids into daughter cells during anaphase. Although the mechanisms that ensure tight microtubule coupling at anaphase are at least partly understood, kinetochore adaptations that support other cell cycle transitions are not. We report here a mechanism that enables regulated control of kinetochore sumoylation. A conserved surface of the Ctf3/CENP-I kinetochore protein provides a binding site for the SUMO protease, Ulp2. Ctf3 mutations that disable Ulp2 recruitment cause elevated inner kinetochore sumoylation and defective chromosome segregation. The location of the site within the assembled kinetochore suggests coordination between sumoylation and other cell cycle-regulated processes.

SMC Complexes Are Guarded by the SUMO Protease Ulp2 Against SUMO-Chain-Mediated Turnover

ABSTRACTStructural maintenance of chromosomes (SMC) complexes, cohesin, condensin and Smc5/6, are essential for viability and participate in multiple processes, including sister chromatid cohesion, chromosome condensation, and DNA repair. Here we show that SUMO chains target all three SMC complexes and are antagonized by the SUMO protease Ulp2 to prevent their turnover. We uncover that the essential role of the cohesin-associated subunit Pds5 is to counteract SUMO chains jointly with Ulp2. Importantly, fusion of Ulp2 to kleisin Scc1 supports viability of PDS5 null cells and protects cohesin from proteasomal degradation mediated by the SUMO-targeted ubiquitin ligase Slx5/Slx8. The lethality of PDS5 deleted cells can also be bypassed by simultaneous loss of the PCNA unloader, Elg1, and the cohesin releaser, Wpl1, but only when Ulp2 is functional. Condensin and Smc5/6 complex are similarly guarded by Ulp2 against unscheduled SUMO-chain assembly, which we propose to time the availability of SMC complexes on chromatin.

The nuclear pore primes recombination-dependent DNA synthesis at arrested forks by promoting SUMO removal

Nuclear Pore complexes (NPCs) act as docking sites to anchor particular DNA lesions facilitating DNA repair by elusive mechanisms. Using replication fork barriers in fission yeast, we report that relocation of arrested forks to NPCs occurred after Rad51 loading and its enzymatic activity. The E3 SUMO ligase Pli1 acts at arrested forks to safeguard integrity of nascent strands and generates poly-SUMOylation which promote relocation to NPCs but impede the resumption of DNA synthesis by homologous recombination (HR). Anchorage to NPCs allows SUMO removal by the SENP SUMO protease Ulp1 and the proteasome, promoting timely resumption of DNA synthesis. Preventing Pli1-mediated SUMO chains was sufficient to bypass the need for anchorage to NPCs and the inhibitory effect of poly-SUMOylation on HR-mediated DNA synthesis. Our work establishes a novel spatial control of Recombination-Dependent Replication (RDR) at a unique sequence that is distinct from mechanisms engaged at collapsed-forks and breaks within repeated sequences.

piRNA-mediated gene regulation and adaptation to sex-specific transposon expression in D. melanogaster male germline

SUMMARYSmall non-coding piRNAs act as sequence-specific guides to repress complementary targets in Metazoa. Prior studies in Drosophila ovaries have demonstrated the function of piRNA pathway in transposon silencing and therefore genome defense. However, the ability of piRNA program to respond to different transposon landscape and the role of piRNAs in regulating host gene expression remain poorly understood. Here, we comprehensively analyzed piRNA expression and defined the repertoire of their targets in Drosophila melanogaster testes. Comparison of piRNA programs between sexes revealed sexual dimorphism in piRNA programs that parallel sex-specific transposon expression. Using a novel bioinformatic pipeline, we identified new piRNA clusters and established complex satellites as dual-strand piRNA clusters. While sharing most piRNA clusters, two sexes employ them differentially to combat sex-specific transposon landscape. We found several host genes targeted by piRNAs in testis, including CG12717/pita, a SUMO protease gene. piRNAs encoded on Y chromosome silence pita, but not its paralog, to exert sex- and paralog-specific gene regulation. Interestingly, pita is targeted by endogenous siRNAs in a sibling species, Drosophila mauritiana, suggesting distinct but related silencing strategies invented in recent evolution to regulate a conserved protein-encoding gene.

Purification and Characterization of Double-Stranded Nucleic Acid-Dependent ATPase Activities of Tagged Dicer-Related Helicase 1 and its Short Isoform in Caenorhabditis elegans

The Dicer-related helicases (DRHs) are members of a helicase subfamily, and mammalian DRHs such as retinoic acid-inducible gene-I (RIG-I), are involved in antiviral immunity. Caenorhabditis elegans DRH-1 and DRH-3 play crucial roles in antiviral function and chromosome segregation, respectively. Although intrinsic double-stranded RNA-dependent ATP-hydrolyzing activity has been observed in the recombinant DRH-3 protein prepared from Escherichia coli, there are no reports of biochemical studies of the nematode RIG-I homolog DRH-1. In this study, the secondary structure prediction by JPred4 revealed that DRH-1 and DRH-3 had distinct N-terminal regions and that a 200-amino acid N-terminal region of DRH-1 could form a structure very rich in α-helices. We investigated expressions and purifications of a codon-optimized DRH-1 with four different N-terminal tags, identifying poly-histidine (His)-small ubiquitin-like modifier (SUMO) as a suitable tag for DRH-1 preparation. Full-length (isoform a) and a N-terminal truncated (isoform b) of DRH-1 were purified as the His-SUMO-tagged fusion proteins. Finally, the nucleic acid-dependent ATPase activities were investigated for the two His-SUMO-tagged DRH-1 isoforms and His-tagged DRH-3. The tagged DRH-3 exhibited dsRNA-dependent ATPase activity. However, detectable dsRNA dependency of ATPase activities was not found in either isoform of tagged DRH-1 and a tag-free DRH-1 (isoform a) treated with SUMO protease. These observations suggest that DRH-1 and its short isoform have no or poor nucleic acid-dependent ATPase activity, unlike DRH-3 and mammalian DRHs.