title: Analysis of CENP-A loading and misincorporation in Drosophila melanogaster
creator: Demirdizen, Engin
subject: ddc-570
subject: 570 Life sciences
description: 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factors RbAp48 is involved  in CENP-A ectopic incorporation (Furuyama, Dalal, & Henikoff, 2006; Spiller-Becker,  unpublished) and de novo deposition in multiple eukaryotic lineages (Fujita et al., 2007;  Hayashi et al., 2004; Lee et al., 2016).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.
date: 2019
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/25092/1/PhDThesis_Demirdizen.pdf
identifier: DOI:10.11588/heidok.00025092
identifier: urn:nbn:de:bsz:16-heidok-250926
identifier:   Demirdizen, Engin  (2019) Analysis of CENP-A loading and misincorporation in Drosophila melanogaster.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/25092/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng