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Hyperphosphorylation Repurposes the CRL4B E3 Ligase to Coordinate Mitotic Entry and Exit

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Hyperphosphorylation Repurposes the CRL4B E3 Ligase to Coordinate Mitotic Entry and Exit Research Collection Doctoral Thesis Identification and Characterization of Novel Cullin 4-based E3 Ligases in Somatic Cell Division Author(s): da Graça Gilberto, Samuel Filipe Publication Date: 2017 Permanent Link: https://doi.org/10.3929/ethz-b-000219014 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library DISS. ETH NO. 24583 Identification and characterization of novel Cullin 4-based E3 ligases in somatic cell division A thesis submitted to attain the degree of DOCTOR OF SCIENCES of ETH ZURICH (Dr. sc. ETH Zurich) Presented by SAMUEL FILIPE DA GRAÇA GILBERTO MSc in Biochemistry, University of Lisbon Born on 21.02.1988 Citizen of Portugal Accepted on the recommendation of Prof. Dr. Matthias Peter Prof. Dr. Anton Wutz 2017 Table of contents 1. General introduction ..................................................................................................................1 1.1. The ubiquitylation machinery ....................................................................................................... 1 1.2. Principles of cell cycle regulation: a focus on CRLs and the APC/C .............................................. 3 1.3. Cullin-4 RING E3 ligases: cell-cycle regulation and beyond ........................................................ 14 1.4. Functional distinctions between CUL4A and CUL4B ................................................................... 22 1.5. Open questions and objectives ................................................................................................... 29 1.6. References .................................................................................................................................. 31 2. Article: CRL4RBBP7 is required for efficient CENP-A deposition at centromeres ............................. 46 2.1. Supplementary figures ................................................................................................................ 61 3. Manuscript in preparation: Hyperphosphorylation repurposes the CRL4B E3 ligase to coordinate mitotic entry and exit ....................................................................................................................... 66 3.1. Supplementary Figures ............................................................................................................. 100 4. Investigation of the role of CRL4WDTC1 in cell cycle progression ................................................. 117 4.1. Introduction .............................................................................................................................. 117 4.2. Results ....................................................................................................................................... 118 4.3. Discussion .................................................................................................................................. 135 4.4. Materials and Methods ............................................................................................................. 137 4.5. References ................................................................................................................................ 138 5. Extended discussion and future perspectives ........................................................................... 141 5.1. Mitotic function of CUL4B: a molecular explanation for XLID? ................................................ 142 5.2. CUL4B loss-of-function in mouse embryonic lethality, syndromic XLID and mitosis: how do they come together? .................................................................................................................................. 143 5.3. References ................................................................................................................................ 146 6. Appendices............................................................................................................................. 149 7. Publications ............................................................................................................................ 168 7.1. CRL4RBBP7 is required for efficient CENP-A deposition at centromeres ..................................... 168 7.2. Dynamic ubiquitin signaling in cell cycle regulation ................................................................. 168 8. Acknowledgements ................................................................................................................ 182 9. Curriculum vitae ..................................................................................................................... 184 i Cover: crystal structure of CUL4B (front cover) and CUL4A (back), PDB ID: 2HYE and 4A0L. ii Abstract Cell proliferation requires tightly coordinated alternating phases of DNA synthesis and separation, collectively termed the cell division cycle. The cell cycle makes use of timely regulation of protein activity to coordinate its processes and transitions. A pivotal post-translational modification central in this regulation is ubiquitylation. Specificity in ubiquitylation reactions depends on an E3 ligase, such as the Cullin-4 RING E3 ligase (CRL4) family, which use cullin-4A (CUL4A) or CUL4B as a scaffolding subunit. These cullins are regarded as acting redundantly in the regulation of multiple cellular processes, in particular DNA-related functions such as chromatin remodeling and DNA repair. CRL4 can assemble a multiplicity of different complexes that function as independent E3 ligases, by recruiting an array of substrate adaptor subunits. Evidence suggests that CRL4 regulates the cell cycle, but through mechanisms that remain mostly unexplored. It is additionally possible that CUL4A and CUL4B do not always assemble identical complexes. CUL4B loss-of-function mutations in humans result in the development of syndromic X-linked intellectual disability, and in mice the deletion of the Cul4b gene is embryonically lethal. Remarkably, CUL4A has not yet been associated with human disorders and its deletion is not lethal in mice. Therefore, the identity of CRL4 complexes which function in development, and in the cell cycle in particular, remains largely obscure. We investigated regulatory functions of CRL4 complexes on the cell cycle of HeLa cells, a cancer cell line widely used in cell cycle studies. By making use of siRNA-mediated mRNA depletion and gene deletion by CRISPR/Cas9 to modulate the activity of the respective E3 ligases, we discovered that indeed previously undescribed CRL4 complexes regulate important proliferative functions. In particular, we identified that the substrate adaptor Retinoblastoma-binding protein 7 (RBBP7) works together with CRL4 in the determination of the centromere. Specifically, CRL4RBBP7 participates in the process of loading the Histone H3 variant Centromere protein-A (CENP-A) at centromeric nucleosomes. As a consequence, cells have problems in aligning and separating their chromosomes during mitosis. While functions of CRL4 with RBBP7 are expected to depend on both CUL4A and CUL4B, we noticed that CUL4B operates in mitosis in a manner distinct from CUL4A. Indeed, knockout of CUL4B alone delays mitotic entry by approximately 2h, even in the presence of CUL4A, indicating that CRL4B is necessary for early mitotic stages. Because these cullins share extensive sequence identity that only significantly differs in an extended N-terminal sequence in CUL4B, we examined the possibility that this sequence is responsible for the functional distinction. Indeed, we found that the N-terminus of CUL4B is phosphorylated during mitosis and that this modification alters the subcellular localization of this cullin, which is no longer recruited to the chromatin. Our data supports a model where CUL4B is redirected from chromatin to ubiquitylate a yet unidentified iii Abstract substrate. Importantly, this mitotic function cannot be maintained outside of the context of mitosis and results in lethal phenotype in cells. A third independent involvement of CRL4 complexes in the regulation of the cell cycle comes from the identification of another substrate adaptor, WDTC1, which interacts with a mitotic regulator during in the late cell cycle stages, although the functional consequences of WDTC1 loss-of-function remain to be identified. Together, these studies establish newly-identified roles for CRL4 complexes in regulating cell proliferation. By placing special focus in the description of mechanisms of cell division, we clarified how CRL4-type E3 ligases participate in this extended functional network. Finally, our studies determine for the first time the mechanisms of a functional distinction between CUL4B and CUL4A, based on an alternative cullin regulatory mode. We aim at describing which novel substrates CRL4 can ubiquitylate at different cell cycle stages. Besides, we believe that our observations set the stage to uncover why CUL4A cannot compensate for CUL4B loss in human XLID patients. iv Abstract Sommario La proliferazione cellulare si basa sulla precisa coordinazione dell’alternarsi di fasi di sintesi del DNA e di divisione, collettivamente chiamate il ciclo di divisione cellulare. Il ciclo cellulare utilizza la tempestiva regolazione dell’attività delle proteine per coordinare i suoi processi e le sue transizioni. Una delle modificazioni post-traduzionali più importanti per questa
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