Abstract
Centromere protein A (CENP-A) is a variant of core histone H3 that marks the centromere's location on the chromosome. The mechanisms that target the protein to the nucleus and the centromere have not been defined. In this study, we found that deletion of the first 53 but not the first 29 residues of CENP-A from the amino-terminus, resulted in its cytoplasmic localization. Two motifs, R42R43R44 and K49R52K53K56, which are reported to be required for DNA contact in the centromere nucleosome, were found to be critical for CENP-A nuclear accumulation. These two motifs potentially mediated its interaction with Importin-β but were not involved in CENP-A centromeric localization. A third novel motif, L60L61I62R63K64, was found to be essential for the centromeric accumulation of CENP-A. The nonpolar hydrophobic residues L60L61I62, but not the basic residues R63K64, were found to be the most important residues. A protein interaction assay suggested that this motif is not involved in the interaction of CENP-A with its deposition factors but potentially mediates its interaction with core histone H4 and CENP-B. Our study uncovered the role of the amino-terminus of CENP-A in localization.
Highlights
Chromatin is organized in arrays of nucleosomes in which the core histones, H2A, H2B, H3 and H4, are arranged as an octameric core around which DNA is wrapped
Compared to the exclusive nuclear localization of wild-type Centromere protein A (CENP-A) (Figure 1B and 1D, first rows), the mutant Del2-29 localized to nucleus (Figure 1B and 1D, second rows), and the mutant Del2-53 mainly localized to the cytoplasm (Figure 1B and 1D, third rows)
The results suggested that the motifs R42R43R44 and K49R52K53K56 are required for CENP-A nuclear accumulation, and the motif L60L61I62R63K64 is not involved in its cytoplasm/nuclear localization
Summary
Chromatin is organized in arrays of nucleosomes in which the core histones, H2A, H2B, H3 and H4, are arranged as an octameric core around which DNA is wrapped. The linker histones H1 bind to the linker DNA connecting adjacent nucleosomes [1]. The similarity between the major histone subtypes and the variants range from almost no amino acid differences to extremely divergent changes [2]. Histones are functionally conserved as indicated by their high degree of structural conservation [3]. Each histone contains a conserved C-terminal histone fold domain (HFD) and a less structured and unique aminoterminus, commonly referred to as an ‘N-tail’. Histones are highly basic proteins and the ‘N-tail’ provides the majority of the basic amino acids Arg (R) and Lys (K) to the protein
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