Final Report Summary - CENHFDC (A novel role for histone chaperones in the dynamics of non-conventional substrates, the CENP-T/-W, complex at the centromere.) A novel role for histone chaperones in the dynamics of non-conventional substrates, theCENP-T/-W complex at the centromere.The centromere directs chromosome segregation during cell division by coordinating the assembly ofthe kinetochore, the principle microtubule-binding interface of mitotic chromosomes. Two essentialphases in centromere assembly and maturation have been distinguished during the cell cycle; a G1phase, in which CENP-A (also called CenH3) a centromere specific histone H3 variant, is replenishedto establish replication-competent centromeres, and a late-S/G2 phase, in which additional eventsestablish a centromeric chromatin substrate competent for kinetochore assembly. This includesassembly of the CCAN complex, which contains the histone-like CENP-T–CENP-W heterodimers andCENP-S–CENP-X heterotetramer (1). The correct and timely deposition of the CENP-T–CENP-Wprotein complex to centromeres is critical for kinetochore formation, spindle assembly and correctchromosome segregation (2-5). However, little is known how the histone-fold CCAN complex isestablished at the centromere and whether accessory factors such as chaperone proteins help thisvital assembly process.The overall aim of this project was to characterize the assembly of the CENP-T-W complex tothe centromere during S-phase with respect to the replicative state of the centromeric chromatin andidentify the mechanisms, which are involved in targeting the complex to centromeric chromatin prior tomitosis. Our project has been successfully completed and will be submitted for peer review in a highimpact scientific journal in the near future.In order to identify proteins, which were potentially involved in the deposition of the CENP-TCENP-W complex to centromeres, we employed a proteomic screening approach. We used a cellline that was expressing a green fluorescent protein (GFP) tagged version of CENP-W. This allowedus to specifically purify CENP-W-GFP and associated interacting proteins. The proteins, which copurifiedwith CENP-W, were identified by Mass spectrometry. We identified the SSRP1 subunit of theFACT complex as a CENP-W interactor. FACT (facilitates chromatin transcription) is a heterodimericcomplex comprising the evolutionarily highly conserved SSRP1 and Spt16 proteins that is required forthe chaperoning of histones with DNA (6). Co-immunoprecipitation experiments with SSRP1 and anti-GFP antibodies confirm the interaction of both CENP-W and CENP-T with both SSRP1 and Spt16.We next asked whether the interaction between the FACT complex and the CENP-T-CENP-Wcomplex was direct. An in vitro assay using recombinant SSRP1, Spt16 and CENP-T-CENP-W wasdesigned performed. We found that either SSRP1 or Spt16 could bind directly to the histone foldregion of the CENP-T-CENP-W. We then further refined the in vitro assay to determine specificallywhich regions of FACT bound to CENP-T-CENP-W. Taken together our data show that the CENP-TCENP-W complex interacts directly with SSRP1 and Spt16 in vitro and in vivo.We next asked whether the interaction between CENP-T-CENP-W and the FACT complex hada functional role. To determine this we depleted HeLa cells of FACT using siRNA. We then looked atthe levels of CENP-W and CENP-A, which remained at centromeres. We found that the levels ofCENP-W associated with centromeres were significantly reduced following depletion of FACT. Thelevels of CENP-A were not significantly reduced. This indicates that the FACT complex may play arole in the deposition of the CENP-T-CENP-W complex to centromeres.The FACT complex has roles in replication and transcription. Specifically, it travels with the RNAPolymerase II complex during transcription and facilitates the passage of the polymerase throughnucleosomes (7). WE asked whether inhibiting transcription or replication would also inhibit CENP-Wassembly at centromeres. To answer this question, we exploited the CLIP protein in vivo labelingsystem. This system has previously been used to assay the timing of assembly of CENP-A and theCENP-T/-W complex to the centromere(3, 7). Briefly, a quench-chase-pulse experiment was used,whereby the ‘old’ population of CENP-W-CLIP present in CLIP tag cell lines was quenched using aBC-block, rendering this protein population immune to future labeling by a fluorescent probe. A chasetime was used to allow synthesis of ‘new’ CLIP tagged protein, which was labeled with a fluorescentsubstrate. Cells, which had assembled CENP-W to the centromere, were assessed usingimmunofluorescence microscopy. We observed that cells in which transcription had been inhibitedusing a specific inhibitor of RNA Polymerase II, failed to show assembly of CENP-W at centromeres.This indicates that transcription may play a role in CENP-T-CENP-W assembly at centromeres.Taken together, we our data lead us to propose a model whereby the FACT complex can bind to theCENP-T-CENP-W complex in vivo and acts in a transcription dependent manner to deposit the CENPT-CENP-W complex to centromeres. Our findings identify a novel cell cycle-specific chromatinassembly pathway dependent on transcription for assembly of the CENP-T-CENP-W complex tochromatinImpact and OutlookOur project has a far-reaching potential impact, as the vital mechanisms involved in the targeting ofkinetochore components onto post-replicative CENP-A centromeric chromatin prior to mitosis have notbeen established. The observation that the essential histone chaperone FACT deposits a non-histoneprotein complex (CCAN) into chromatin is novel and promises to shed important insights into how thekinetochore is assembled. Investigating the mechanisms underlying this chaperone function atcentromeres enhances our understanding of how epigenetic components adapt the cellular machineryto perform the critical task of directing the accurate segregation of chromosomes in cell division.Bibliography1.T.Nishinoetal.,Cell148,487–501(2012).2.K.E.Gascoigne,I.M.Cheeseman,TheJournalofCellBiology201,23–32(2013).3.L.Prendergastetal.,PLoSBiol.9,e1001082(2011).4.D.R.Foltzetal.,Nat.CellBiol.8,458–469(2006).5.T.Horietal.,Cell135,1039–1052(2008).6.R.Belotserkovskaya,Science301,1090–1093(2003).7.L.E.T.Jansen,etal.,TheJournalofCellBiology176,795–805(2007). Related documents final1-cenhfd-final-report.pdf
