Inactive mutants of the ped gene cause two phenotypes in Drosophila melanogaster: male sterility and the early translation of DHODH within spermatogenesis. Investigation of the PED amino acid sequence revealed an OTU domain and an ubiquitin interacting motif, suggesting that it is a member of the otubain sub-family of de-ubiqutinating enzymes. To test this, the putative active cysteine residue was mutated. Results show that this single cysteine residue is required for ped to confer male fertility. Purified wild type PED was also used to carry out in vitro deubiquitinating assays. These assays failed to show any ability for PED to cut ubiquitin chains of varying length or linkage type. Previously, a translational control element was identified in dhod mRNA which is required for its early translation phenotype in ped mutants. In an attempt to identify additional transcripts that have their translational timing affected by PED, the don juan-like 5′ UTR was inserted into a reporter gene and examined in a ped mutant background. No delay of this reporter gene was observed suggesting that don juan-like mRNA is not under the exact control pathway that dhod is.

Les plantes disposent de mécanismes rapides d'adaptation de leur physiologie et de leur développement à des conditions environnementales changeantes. Leur mise en œuvre dépend largement d’une capacité de reprogrammation de l'expression des gènes qui implique généralement des changements continus de l'épigénome. Chez de nombreux organismes, différentes voies d’enlèvement de la monoubiquitination de l’histone H2B (H2Bub) participent d'une part à faciliter la transcription des gènes par l'ARN polymérase II et d'autre part à éviter l'établissement d'un état permissif à la transcription par enlèvement de domaines enrichis en H2Bub sur des régions répétées telles que les séquences télomériques. Cette thèse a porté sur l’étude des voies régulant la marque chromatinienne H2Bub chez les plantes, dont la connaissance des mécanismes de contrôle dynamique est très fragmentaire. Une nouvelle ubiquitine protéase de l'espèce Arabidopsis thaliana a été identifiée comme étant un homologue fonctionnel de Ubp8, une protéine associée à l'élongation de la transcription au sein d'un module de déubiquitination d'H2Bub du complexe SAGA chez S. cerevisiae. L'identification et la caractérisation fonctionnelle de trois composants de ce module chez A. thaliana a révélé qu'il agit sur des milliers de gènes, suggérant son implication dans des mécanismes basaux de la transcription. Dans une seconde partie, il a été observé que l'abondance de deux sous-unités de ce module est régulée au cours de la photomorphogenèse par DET1, un acteur central de la signalisation de la lumière. Cette transition développementale permet l'adaptation du métabolisme et de la morphologie de la plante en réponse à la première exposition à la lumière, notamment via l'établissement de l'activité photosynthétique. La régulation post-traductionnelle du module de déubiquitination de l'histone H2B pourrait permettre d'ajuster son activité aux changements d'activité transcriptionnelle de la cellule au cours de cette transition. Une approche génétique a également permis d'identifier une redondance fonctionnelle partielle entre l'activité du module de déubiquitination et UBP26, une seconde déubiquitinase d'H2Bub connue pour son implication dans la répression des gènes PHERES1 et FLC par une activité Polycomb ainsi que de certains éléments transposables. Ces analyses ont permis de révéler une influence positive de UBP26 sur l'établissement d'un état répressif à la transcription sur des centaines de gènes et également dans certains contextes hétérochromatiniens. Collectivement, ce travail a permis de disséquer les spécificités et les redondances fonctionnelles de deux voies de déubiquitination de l'histone H2B portées par des complexes protéiques distincts. / Plants utilize rapid mechanisms to adapt their physiology and development to changing environments. Their triggering depends greatly on gene expression reprogramming leading to important changes in the epigenome. In numerous organisms, different pathways remove monoubiquitination of histone H2B (H2Bub) to facilitate gene transcription by RNA polymerase II, with H2Bub removal on repeated genomic regions, such as telomeres, prohibiting establishment of a transcription permissive state. This thesis aims to better characterize the pathways that regulate the chromatin mark H2Bub in plants. A new ubiquitin protease from Arabidopsis thaliana was identified as a potential homolog of Ubp8, a protein associated with transcription elongation within a deubiquitination module of the SAGA complex in S. cerevisiae. Identification and functional characterization of the three components of the deubiquitination module in A. thaliana reveals its action on thousands of genes, suggesting a role in basal transcription mechanism. Secondly, it was shown that the quantity of two subunits from this module is regulated during photomorphogenesis by DET1, a central protein involved in light signaling. This developmental transition allows adaptation of metabolism and morphology of the plant in response to the first light exposure, notably during photosynthesis establishment. The post-translational regulation of the histone H2Bub deubiquitination module may allow its adjustment to changes in cell transcription needs during this transition. A genetic approach identified a partial functional redundancy between the deubiquitination module activity and UBP26, a second H2Bub deubiquitinase known to repress the genes PHERES1 and FLC by a Polycomb activity and certain transposable elements. These analyses revealed a positive influence from UBP26 on establishing a repressive transcriptional state on hundreds of genes and on some heterochromatinian contexts. Collectively, this work dissected specificities and functional redundancies of two H2Bub deubiquitination pathways driven by distinct protein complexes.

Post-translational modifications are chemical changes that occur to proteins after their synthesis, which are essential to their function and regulation. Ubiquitination is a post translation modification that serves key roles in the regulation of proteins. USP7 is a deubiquitinase that has several critical substrates important for human health and disease, including the cancer relevant proteins PTEN, p53, MDM2 and DMNT1. Most of these substrates have been identified by cell biology or proteomics experiments, but a detailed biochemical and structural analysis is lacking, likely due to the challenge of generating site-specific and stoichiometrically ubiquitinated proteins. Therefore, we leveraged our expertise in protein semi-synthesis to generate these ubiquitinated substrates to study USP7’s ability to recognize and hydrolyze the ubiquitin, which will reveal key details on how USP7 selects its substrates. In our investigation, we generated several mono-ubiquitinated peptides, that could be functionalized later to install on the protein, and assayed USP7’s ability to hydrolyze the ubiquitin. / 2026-03-06T00:00:00Z

Protein ubiquitination is essential for the proper functioning of many eukaryotic cellular processes. The cleavage of ubiquitin chains from ubiquitinated proteins is performed by deubiquitinating enzymes, of which there are 16 in the Ubp (ubiquitin specific protease) group in the budding yeast Saccharomyces cerevisiae. The goal of my thesis has been to examine the biological roles and molecular functions of these enzymes using a combination of proteomic and molecular genetic approaches. As part of a large collaborative effort, interacting protein partners of the Ubps were isolated through affinity purification of tagged proteins, followed by protein identification by mass spectrometry. Purification of tagged Ubp6 led to the identification of the 19S proteasome complex, along with a novel subunit, Sem1. As the human homologue of Sem1 was previously identified as being associated with a protein involved in the repair of DNA double-strand breaks, I examined the possible role of Sem1 in DNA damage repair. A deletion of Sem1 and other 19S subunits resulted in hypersensitivity to various DNA damaging drugs, implicating the 19S complex in the process of DNA repair. iii I also found that purified Ubp2 interacted stably with the ubiquitin ligase Rsp5 and the protein Rup1. UBP2 interacts genetically with RSP5, indicating a functional relationship, while Rup1 facilitates the physical tethering of Ubp2 to Rsp5. Using the uracil permease Fur4, a Rsp5 substrate, as a model reporter, I found that ubp2Δ cells exhibited a temporal stabilization of Fur4 at the plasma membrane following the induction of endocytosis, implicating Ubp2 in protein sorting, specifically at the multivesicular body. In order to understand the role of Ubp2, I examined the effect of Ubp2 on Rsp5 function. I found that Rsp5, similar to its mammalian homologues, is auto-ubiquitinated in vivo, and that Ubp2 is able to directly deubiquitinate Rsp5 in vitro. Moreover, the presence of a substrate or Rup1 both resulted in increased autoubiquitination, implying an auto-inhibitory mechanism of Rsp5 regulation. Taken together, the data presented in this thesis implicate deubiquitinating enzymes in interesting and varied roles in the cell, and suggest a novel mechanism for the modulation of Rsp5-dependent trafficking processes.

Protein ubiquitination is essential for the proper functioning of many eukaryotic cellular processes. The cleavage of ubiquitin chains from ubiquitinated proteins is performed by deubiquitinating enzymes, of which there are 16 in the Ubp (ubiquitin specific protease) group in the budding yeast Saccharomyces cerevisiae. The goal of my thesis has been to examine the biological roles and molecular functions of these enzymes using a combination of proteomic and molecular genetic approaches. As part of a large collaborative effort, interacting protein partners of the Ubps were isolated through affinity purification of tagged proteins, followed by protein identification by mass spectrometry. Purification of tagged Ubp6 led to the identification of the 19S proteasome complex, along with a novel subunit, Sem1. As the human homologue of Sem1 was previously identified as being associated with a protein involved in the repair of DNA double-strand breaks, I examined the possible role of Sem1 in DNA damage repair. A deletion of Sem1 and other 19S subunits resulted in hypersensitivity to various DNA damaging drugs, implicating the 19S complex in the process of DNA repair. iii I also found that purified Ubp2 interacted stably with the ubiquitin ligase Rsp5 and the protein Rup1. UBP2 interacts genetically with RSP5, indicating a functional relationship, while Rup1 facilitates the physical tethering of Ubp2 to Rsp5. Using the uracil permease Fur4, a Rsp5 substrate, as a model reporter, I found that ubp2Δ cells exhibited a temporal stabilization of Fur4 at the plasma membrane following the induction of endocytosis, implicating Ubp2 in protein sorting, specifically at the multivesicular body. In order to understand the role of Ubp2, I examined the effect of Ubp2 on Rsp5 function. I found that Rsp5, similar to its mammalian homologues, is auto-ubiquitinated in vivo, and that Ubp2 is able to directly deubiquitinate Rsp5 in vitro. Moreover, the presence of a substrate or Rup1 both resulted in increased autoubiquitination, implying an auto-inhibitory mechanism of Rsp5 regulation. Taken together, the data presented in this thesis implicate deubiquitinating enzymes in interesting and varied roles in the cell, and suggest a novel mechanism for the modulation of Rsp5-dependent trafficking processes.

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Fanconi anemia (FA) is a rare genetic disease characterized by bone marrow failure and cancer predisposition. Cell lines derived from FA patient exhibit chromosomal instability and sensitivity to DNA interstand crosslinkers (ICLs) like mitomycin (MMC). The key event in Fanconi anemia pathway is the regulated ubiquitination and deubiquitination of FANCD2 and FANCI. Upon DNA damage, FANCD2 and FANCI are monoubiquitinated by FA core complex. They then move into the chromatin and serve as the landing site for downstream players, like FANCP/SLX4 and FAN1. USP1, the deubiquitinating enzyme (DUB), removes ubiquitin from FANCD-Ub/FANCI-Ub, and this step is required for the integrity of FA pathway. This dissertation addresses how USP1 is regulated in the cell. In Chapter 2, we discovered UAF1/WDR48 as a critical binding partner for USP1, by activating its enzymatic activity in vitro and in vivo. We then generated DT40 knockout cell lines of USP1 and UAF1. We showed that USP1/UAF1 complex is functionally required for homologous recombination (HR). Interestingly, PCNA-Ub is also a substrate for USP1. We discovered that hELG1, through its binding to USP1/UAF1 complex, regulates the deubiquitination of PCNA-Ub and translesion DNA synthesis (TLS). Then in Chapter 3, we discovered a tandem repeat of SUMO-like domains (SLD1 and SLD2) in the C terminus of UAF1. SLD2 binds directly to a SUMO-like domain-interacting motif (SIM) on FANCI. Deletion of the SLD2 of UAF1 or mutation of the SIM of FANCI disrupts UAF1/FANCI binding and inhibits FANCD2 deubiquitination. The SLD2 sequence of UAF1 also binds to a SIM on hELG1, and targets the USP1/UAF1 complex to its PCNA-Ub substrate. We proposed the regulated targeting of USP1/UAF1 to its DNA repair substrates, FANCD2-Ub and PCNA-Ub, by SLD-SIM interactions coordinates HR and TLS. Originating from USP1/UAF1 complex, we worked out a general mechanism of DUB regulation by WD40 proteins, which involved in two more DUBs, USP12 and USP46 (discussed in Chapter 4 and Appendix A). Lastly in Chapter 5, through bioinformatic analysis we identified a series of novel proteins containing ubiquitin-binding zinc fingers (UBZ). We then focused on SNM1A and FAAP20/C1orf86, and characterized their function in DNA crosslink repair.

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Immune cytokines tumor necrosis factor alpha and type I interferons provide front-line defense against viral infection and are regulated in part by ubiquitin (Ub) and Ub-like molecules. Ubiquitin and Ub-like molecule ISG15 share a conserved C-terminal motif where a terminal glycine residue becomes attached to cellular target proteins. Nairoviruses and arteriviruses contain an ovarian tumor domain-containing protease (OTU protease) that was found to corrupt pathways by removing Ub or ISG15 from target proteins. This broad substrate specificity is unlike mammalian deubiquitinating enzymes, which cannot recognize both substrates. To understand how viral OTU domain-containing proteases remove Ub and ISG15, the crystal structure of the Crimean-Congo Heamorhaggic Fever nairovirus (CCHFV) was determined with Ub to 2.5 Å resolution. A computational model was built of the CCHFV Otu protease bound to ISG15 as well. The CCHFV Otu protease has several structural differences from known OTU proteases, manifesting in its broad substrate recognition capability.

Immune cytokines tumor necrosis factor alpha and type I interferons provide front-line defense against viral infection and are regulated in part by ubiquitin (Ub) and Ub-like molecules. Ubiquitin and Ub-like molecule ISG15 share a conserved C-terminal motif where a terminal glycine residue becomes attached to cellular target proteins. Nairoviruses and arteriviruses contain an ovarian tumor domain-containing protease (OTU protease) that was found to corrupt pathways by removing Ub or ISG15 from target proteins. This broad substrate specificity is unlike mammalian deubiquitinating enzymes, which cannot recognize both substrates. To understand how viral OTU domain-containing proteases remove Ub and ISG15, the crystal structure of the Crimean-Congo Heamorhaggic Fever nairovirus (CCHFV) was determined with Ub to 2.5 Å resolution. A computational model was built of the CCHFV Otu protease bound to ISG15 as well. The CCHFV Otu protease has several structural differences from known OTU proteases, manifesting in its broad substrate recognition capability.