The CENP-H-I complex is required for the efficient incorporation of newly synthesized CENP-A into centromeres

Authors:
Okada M, Cheeseman IM, Hori T, Okawa K, McLeod IX, Yates JR, Desai A, Fukagawa T
In:
Source: Nat Cell Biol
Publication Date: (2006)
Issue: 8(5): 446-57
Research Area:
Cancer Research/Cell Biology
Cells used in publication:
DT40
Species: chicken
Tissue Origin: blood
Platform:
Nucleofector™ I/II/2b
Experiment
For the CENP-A–GFP incorporation assay, cells were nucleofected with 2 μg of CMV chicken CENP-A–GFP plasmid and 1 μg of CMV-monomeric RFP (mRFP) plasmid. Cells were cultured for 16 h and fixed with 3% paraformaldehyde. In each experiment, only the cells expressing mRFP signals were counted for scoring the efficiency of CENP-A incorporation; achieved transfection efficiencies of >80 %.
Abstract
In vertebrates, centromeres lack defined sequences and are thought to be propagated by epigenetic mechanisms involving the incorporation of specialized nucleosomes containing the histone H3 variant centromere protein (CENP)-A. However, the precise mechanisms that target CENP-A to centromeres remain poorly understood. Here, we isolated a multi-subunit complex, which includes the established inner kinetochore components CENP-H and CENP-I, and nine other proteins, from both human and chicken cells. Our analysis of these proteins demonstrates that the CENP-H-I complex can be divided into three functional sub-complexes, each of which is required for faithful chromosome segregation. Interestingly, newly expressed CENP-A is not efficiently incorporated into centromeres in knockout mutants of a subclass of CENP-H-I complex proteins, indicating that the CENP-H-I complex may function, in part, as a marker directing CENP-A deposition to centromeres.