<> "The repository administrator has not yet configured an RDF license."^^ . <> . . "Analysis of CENP-A loading and misincorporation in Drosophila melanogaster"^^ . "Centromeric Protein-A (CENP-A, known as Centromere Identifier (CID) in Drosophila\r\nmelanogaster) is an epigenetic marker for centromere identity and propagation (Allshire &\r\nKarpen, 2008). Upon elevated expression, CENP-A mislocalizes to ectopic sites and can give\r\nrise to neocentromeres, thereby leading to genome instability (Heun et al., 2006; Shrestha\r\net al., 2017; Tomonaga, Matsushita, Yamaguchi, et al., 2003). Promiscuously-incorporated\r\nCENP-A is a prognostic and predictive marker for tumor progression (Filipescu et al., 2017).\r\nTo gain further understanding on this mechanism of mislocalization, I explored alternative\r\nCENP-A incorporation and elimination pathways, using cultured fly S2 cells and adult flies.\r\nPrevious studies suggested that component of general assembly factors RbAp48 is involved\r\nin CENP-A ectopic incorporation (Furuyama, Dalal, & Henikoff, 2006; Spiller-Becker,\r\nunpublished) and de novo deposition in multiple eukaryotic lineages (Fujita et al., 2007;\r\nHayashi et al., 2004; Lee et al., 2016). RbAp48 is found in several chromatin assembly,\r\nremodelling, and modification complexes (Doyen et al., 2013; Rai et al., 2013), but it is yet\r\nunknown whether RbAp48 carries out this function together with a multi-subunit complex.\r\nRbAp48 is a subunit of Chromatin Assembly Factor-1 (CAF-1) complex (Tyler et al., 1996).\r\nThus, I first tested the role of CAF-1 in CID mislocalization but did not find convincing\r\nevidence for an involvement of CAF-1 in CID ectopic loading. Levels of ectopically expressed\r\nand misincorporated CID were stable upon CAF-1 depletion and no physical interaction was\r\ndetected. Hence, I decided to search for the role of other potential RbAp48-containing\r\ncomplexes in CID misincorporation, using a Crosslinked Immunoprecipitation Mass Spec\r\n(Xlink-IP-MS) approach. Interestingly, I identified a dual catalytic and RbAp48-containing\r\nmulti-subunit complex so called Nucleosome Remodelling and Deacetylase (NuRD) complex.\r\nNuRD has two catalytic functions, namely nucleosome remodelling and histone\r\ndeacetylation (Denslow & Wade, 2007). I detected a physical interaction of overexpressed\r\nCID with the NuRD complex core components Mi-2, MTA1-like and RbAp48 and\r\nmisexpressed CID protein levels decreased and chromatin incorporation upon depletion of\r\nthe catalytic component Mi-2 and MTA1-like scaffold protein. Moreover, upon disruption of\r\nthe RbAp48 interaction surface with MTA1-like, the nuclear transport and misincorporation\r\nof CID were impaired. Taken together, these results suggest that RbAp48/MTA1-like binding\r\nis required for nuclear incorporation of overexpressed CID, and catalytic and scaffold\r\ncomponents of the NuRD complex are essential for the mislocalization of CID. Xlink-IP-MS\r\nalso detected hyperplastic discs (hyd), an E3 ubiquitin ligase (Moncrieff et al., 2015) as CID\r\ninteracting component. Mislocalized CENP-A is known to be targeted by E3 ligases in several\r\neukaryotes (Hewawasam et al., 2010; Moreno-Moreno et al., 2011; Ranjitkar et al., 2010).\r\nThus, I also tested the hypothesis that CID is targeted by hyd for proteasomal degradation.\r\nThe genetic and physical interaction between CID and hyd were determined. Hyd\r\noverexpression resulted in poly-ubiquitination and destabilization of CID. Endogenous and\r\nectopically expressed CID protein levels were found to increase upon hyd depletion.\r\nCollectively, these results demonstrated that hyd E3 ligase regulates CID protein levels\r\nthrough ubiquitin-mediated proteolysis. In summary, I found that the NuRD complex plays\r\nan essential role in nuclear localization and misincorporation of misexpressed CID, and its\r\nstability and incorporation are controlled by hyd E3 ligase. The findings presented in this\r\nwork may contribute to our understanding of misregulated CENP-A in cancer and new\r\nstrategies for tackling the detrimental misincorporation in many different tumor entities."^^ . "2019" . . . . . . . "Engin"^^ . "Demirdizen"^^ . "Engin Demirdizen"^^ . . . . . . "Analysis of CENP-A loading and misincorporation in Drosophila melanogaster (PDF)"^^ . . . "PhDThesis_Demirdizen.pdf"^^ . . . "Analysis of CENP-A loading and misincorporation in Drosophila melanogaster (Other)"^^ . . . . . . "indexcodes.txt"^^ . . . "Analysis of CENP-A loading and misincorporation in Drosophila melanogaster (Other)"^^ . . . . . . "lightbox.jpg"^^ . . . "Analysis of CENP-A loading and misincorporation in Drosophila melanogaster (Other)"^^ . . . . . . "preview.jpg"^^ . . . "Analysis of CENP-A loading and misincorporation in Drosophila melanogaster (Other)"^^ . . . . . . "medium.jpg"^^ . . . "Analysis of CENP-A loading and misincorporation in Drosophila melanogaster (Other)"^^ . . . . . . "small.jpg"^^ . . "HTML Summary of #25092 \n\nAnalysis of CENP-A loading and misincorporation in Drosophila melanogaster\n\n" . "text/html" . . . "570 Biowissenschaften, Biologie"@de . "570 Life sciences"@en . .