Centromeres link sister chromatids together and are important for proper separation of chromosomes during cell division. Centromeres are also the assembly sites for kinetochores, highly complex protein structures involved in chromatid separation during mitosis. Recently, a research team from Germany applied mass spectrometry to analyze affinity-purified centromeric chromatin in Drosophila melanogaster.1 Although more than 80 kinetochore proteins and 16 constitutive centromere-associated network (CCAN) proteins have been identified in humans, centromere biology is still an emerging field of study with respect to D. melanogaster.
Only one centromere protein, CENP-C, has been identified in both humans and D. melanogaster to date. Barth et al. explain that another protein, CENP-ACID (CID = centromere identifier), is among the strongest candidates for identification in Drosophila. CENP-C represents an essential factor for centromeric CENP-ACID assembly and has been found to directly interact and be interdependent with CENP-ACID.
For their experiments, Barth et al. generated a stable cell line of Drosophila Schneider S2 cells that expresses a GFP-tagged centromeric histone H3 variant CENP-ACID. They used MNase to digest nuclei and prepare chromatin, performing anti-GFP affinity purification from the solubilized chromatin fraction. Following a trypsin digest, the research team performed liquid chromatography using an UltiMate 3000 HPLC system (Thermo Scientific). The research team then infused the effluent using a nano-electrospray ion source coupled to an LTQ Orbitrap hybrid ion trap-Orbitrap mass spectrometer (Thermo Scientific).
To analyze data and identify novel centromere components, the team used the Andromeda algorithm of the MaxQuant quantitative protein analysis software package (version 126.96.36.199) against the Flybase “dmel-all-translation-r5.24.fasta” database. They compared the composition of chromatin containing tagged CENP-ACID to chromatin containing tagged versions of the canonical histone H3.2 and its variant H3.3 in D. melanogaster.
The researchers identified 91 proteins specifically enriched in CENP-ACID. They further investigated the cellular localization of one-third of the centromere proteins based on iBAQ abundance, their specific enrichment in CENP-ACID-GFP chromatin purifications, and the availability of expression constructs. They identified and grouped candidate proteins with similar localization patterns together to form seven different classes.
Among the top-ranked proteins were products of CG2051, which is the Drosophila homolog of the human histone acetyltransferase 1 (HAT1) enzyme, and CG14480, which is novel and may play a role in controlling cell cycle-regulated degradation of proteins at centromeres.
The researchers also identified Hyd, a tumor suppressor that can cause defects in chromosome condensation, attachment and behavior. Additionally, they identified the protein CG11076, which may contribute to the linkage between centromeres and the nucleus.
Loss-of-function studies revealed that the selected candidates were required for proper mitotic divisions, yet the canonical pathways regulating CENP-ACID deposition or kinetochore formation were still functional. Because of this, the researchers posit that the novel centromere-associated proteins might be components of alternative pathways contributing to centromere and/or kinetochore function. For interested colleagues, all mass spectrometric data have been deposited in the ProteomeXchange, with identifier PXD000758.
1. Barth, T.K., et al. (2014, October) “Identification of novel Drosophila centromere-associated proteins,” Proteomics, 14 (pp. 2167–78), doi: 10.1002/pmic.201400052.
Post Author: Emily Humphreys. Emily has previous research experience in eye development, infectious diseases, and aging. While she enjoyed the thrill of research, She has since traded bench work for science journalism. Emily has been a regular contributor to Accelerating Science since 2012.