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Abstract

Centromeric Protein-A (CENP-A, known as Centromere Identifier (CID) in Drosophila melanogaster) is an epigenetic marker for centromere identity and propagation (Allshire & Karpen, 2008). Upon elevated expression, CENP-A mislocalizes to ectopic sites and can give rise to neocentromeres, thereby leading to genome instability (Heun et al., 2006; Shrestha et al., 2017; Tomonaga, Matsushita, Yamaguchi, et al., 2003). Promiscuously-incorporated CENP-A is a prognostic and predictive marker for tumor progression (Filipescu et al., 2017). To gain further understanding on this mechanism of mislocalization, I explored alternative CENP-A incorporation and elimination pathways, using cultured fly S2 cells and adult flies. Previous studies suggested that component of general assembly factors RbAp48 is involved in CENP-A ectopic incorporation (Furuyama, Dalal, & Henikoff, 2006; Spiller-Becker, unpublished) and de novo deposition in multiple eukaryotic lineages (Fujita et al., 2007; Hayashi et al., 2004; Lee et al., 2016). RbAp48 is found in several chromatin assembly, remodelling, and modification complexes (Doyen et al., 2013; Rai et al., 2013), but it is yet unknown whether RbAp48 carries out this function together with a multi-subunit complex. RbAp48 is a subunit of Chromatin Assembly Factor-1 (CAF-1) complex (Tyler et al., 1996). Thus, I first tested the role of CAF-1 in CID mislocalization but did not find convincing evidence for an involvement of CAF-1 in CID ectopic loading. Levels of ectopically expressed and misincorporated CID were stable upon CAF-1 depletion and no physical interaction was detected. Hence, I decided to search for the role of other potential RbAp48-containing complexes in CID misincorporation, using a Crosslinked Immunoprecipitation Mass Spec (Xlink-IP-MS) approach. Interestingly, I identified a dual catalytic and RbAp48-containing multi-subunit complex so called Nucleosome Remodelling and Deacetylase (NuRD) complex. NuRD has two catalytic functions, namely nucleosome remodelling and histone deacetylation (Denslow & Wade, 2007). I detected a physical interaction of overexpressed CID with the NuRD complex core components Mi-2, MTA1-like and RbAp48 and misexpressed CID protein levels decreased and chromatin incorporation upon depletion of the catalytic component Mi-2 and MTA1-like scaffold protein. Moreover, upon disruption of the RbAp48 interaction surface with MTA1-like, the nuclear transport and misincorporation of CID were impaired. Taken together, these results suggest that RbAp48/MTA1-like binding is required for nuclear incorporation of overexpressed CID, and catalytic and scaffold components of the NuRD complex are essential for the mislocalization of CID. Xlink-IP-MS also detected hyperplastic discs (hyd), an E3 ubiquitin ligase (Moncrieff et al., 2015) as CID interacting component. Mislocalized CENP-A is known to be targeted by E3 ligases in several eukaryotes (Hewawasam et al., 2010; Moreno-Moreno et al., 2011; Ranjitkar et al., 2010). Thus, I also tested the hypothesis that CID is targeted by hyd for proteasomal degradation. The genetic and physical interaction between CID and hyd were determined. Hyd overexpression resulted in poly-ubiquitination and destabilization of CID. Endogenous and ectopically expressed CID protein levels were found to increase upon hyd depletion. Collectively, these results demonstrated that hyd E3 ligase regulates CID protein levels through ubiquitin-mediated proteolysis. In summary, I found that the NuRD complex plays an essential role in nuclear localization and misincorporation of misexpressed CID, and its stability and incorporation are controlled by hyd E3 ligase. The findings presented in this work may contribute to our understanding of misregulated CENP-A in cancer and new strategies for tackling the detrimental misincorporation in many different tumor entities.