cntrmr is a part f th chrmsm which is crucially imprtant fr sgrgatin f chrmsms during cll divisin and chsin f sistr chrmatids. Cntrmr idntity and functin is dtrmind by th presence f H3 histn variant CENP-A (Cid in Drsphila) at specific locations in the chromatin, which serves as a nucleation site for the assembly of a host of kinetochore proteins, many of which are yet to be characterized in Drsphila.
T identify proteins that interact with Drosophila CENP-A and therefore might be involved in the generation of centromeric chromatin, we applied an affinity purification strategy for CENP-A-associated proteins. To this end, w gnratd cell lines with stably integrated transgenes allowing for inducible expression of CENP-A-GFP or CENP-A-StrepII-Flag fusion proteins. Mass spectrometry analysis after affinity purification using either anti-GFP or Strep-Tactin and anti-Flag affinity resins f nuclear extracts resulted in the identification of a number of potential partners f CENP-A which belong t different functional groups. Among the proteins that were identified with both affinity purification strategies were fly homologues of the previously identified interaction partners of mammalian CENP-A, Caf1 and the FACT complex subunit Dre4. In addition, CG2051 protein was detected. CG2051 corresponds to histn actyl transfras 1 (HAT1) whose orthologs in other species were shown to actylat newly synthesized histn H4 at lysine 5 (K5) and lysine 12 (K12) and which marks H3/H4 dimrs fr lading t chrmatin. Here we find that Drosophila CG2051 is a bona fide histone acetyltransferase and that CENP-A might also serve as a substrate for CG2051.
Using a number of biochemical approaches ranging from mutual co-immunoprecipitation experiments to gel filtration and interaction studies of recombinant proteins, we characterized different CENP-A-containing preloading complexes. Furthermore, we showed that RNAi-mediated knock-down of HAT1 in S2 cells leads to a reduction of CENP-A incorporation into chromatin. Taken together, these studies reveal several independent CENP-A-containing complexes that may constitute a pathway for the chaperoning, transport and loading of this centromere-specific histone variant in Drosophila.