Phosphorylation of the CENP-A amino-terminus in mitotic centromeric chromatin is required for kinetochore function

The role of the mitotic phosphorylation of the amino (NH₂) terminus of Centromere Protein A (CENP-A), the histone variant epigenetic centromeric marker, remains elusive. Here, we show that the NH₂ terminus of human CENP-A is essential for mitotic progression and that localization of CENP-C, another key centromeric protein, requires only phosphorylation of the CENP-A NH₂ terminus, and is independent of the CENP-A NH₂ terminus length and amino acid sequence. Mitotic CENP-A nucleosomal complexes contain CENP-C and phosphobinding 14-3-3 proteins. In contrast, mitotic nucleosomal complexes carrying nonphosphorylatable CENP-A–S7A contained only low levels of CENP-C and no detectable 14-3-3 proteins. Direct interactions between the phosphorylated form of CENP-A and 14-3-3 proteins as well as between 14-3-3 proteins and CENP-C were demonstrated. Taken together, our results reveal that 14-3-3 proteins could act as specific mitotic “bridges,” linking phosphorylated CENP-A and CENP-C, which are necessary for the platform function of CENP-A centromeric chromatin in the assembly and maintenance of active kinetochores.Histone variants are nonallelic isoforms of conventional histones. It is widely accepted that the incorporation of histone variants generally confers novel structural and functional properties to the nucleosome (1). Centromere Protein A (CENP-A) is a histone variant, which replaces the canonical histone H3 at the centromere (2) and marks epigenetically the centromeres and the kinetochores (for reviews see refs. 3 and 4). The presence of CENP-A is required for the assembly of active kinetochores and its depletion results in numerous mitotic problems, such as chromosome misalignments and segregation defects, generation of chromosome bridges, aneuploidy, etc. (5). The resulting mitotic defects, following CENP-A depletion, were associated also with notable alterations in the composition and organization of the kinetochore, including the delocalization of the inner kinetochore proteins CENP-C, CENP-I, and CENP-H as well as the outer kinetochore components Highly Expressed in Cancer protein 1 (HEC1), Mitotic Arrest Deficient 2-like protein (Mad2), and CENP-E (5).During recent years, the studies of CENP-A were focused mainly on its histone-fold domain. The NH₂ terminus of CENP-A, which is not required for centromeric targeting (6, 7) appeared, however, to play an important role in both mitosis and meiosis. In yeast, the NH₂ tail of Chromosome Segregation Protein 4 (Cse4p) (the homolog of mammalian CENP-A) has an essential function distinct from that of the histone-fold domain in chromosome assembly and segregation (8). The reported data in Arabidopsis thaliana suggested the existence of a meiosis-specific loading pathway for CENP-A, requiring its NH₂ terminus (9). In addition, human CENP-A is phosphorylated in its NH₂ terminus at serine 7 in mitosis but the role of this phorphorylation is far from being clear (10, 11).Sequence alignments of the NH₂ termini of CENP-A from different species show very low sequence conservation in terms of amino acid composition, sequence, and length (Fig. 1A). However, expression of CENP-A with its NH₂ terminus deleted is lethal in yeast (12) and plants (13). These data create a paradox because on one hand, the NH₂ terminus of CENP-A, in certain organisms, is required for their survival and on the other hand, it appears to be nonessential because there is no evolutionary pressure to conserve at least some specific sequence elements.Open in a separate windowFig. 1.The NH₂ tail of CENP-A is required for mitosis and the H3 swapped NH₂ tail CENP-A chimera rescues the CENP-A null cell phenotype. (A) Sequence alignment of CENP-A NH₂ termini from different species. Names of species are indicated at Left. Serine residues are indicated in blue. Conservation between different species is shown (Lower). (B) Cell cycle visualization, after CENP-A suppression by siRNA treatment of naïve HeLa cells (second row) or HeLa cells stably expressing siRNA-resistant full-length GFP–CENP-A (third row) or tailless GFP–ΔN–CENP-A (fourth row) or GFP–H3–CENP-A swapped tail mutant (fifth row). First row shows naïve cells not treated with siRNA. A CREST antibody was used to visualize the centromeres in naïve cells; GFP fluorescence was used to visualize CENP-A in GFP fusion-expressing cells. An antibody against inner centromere protein (INCENP) and antilamina antibody were used to detect the midbody during cytokinesis and the nuclear envelope in interphase cells (shown in red). Blue, DNA; white arrowheads point to misaligned or lagging chromosomes and to chromatin bridges. (Scale bar, 5 µm.) (C) Detection of the GFP–CENP-A fusions and endogenous CENP-A in control (−) and siRNA treated (+) cells at 72 h posttransfection by Western blot. Cells used are indicated (Upper). Arrows indicate the positions of the GFP–CENP-A fusions or endogenous CENP-A. (D) Histograms showing the percentage of mitotic defects at 72 h posttransfection with siRNA against CENP-A in the indicated cell lines. HeLa, naïve cell line. (E) Same as D, but showing the percentage of multinucleate cells. For each experiment, at least 400 cells were counted. Data are means and SEM of five independent experiments.Here, we have analyzed the mitotic function of the NH₂ terminus of human CENP-A and its phosphorylation. We show the mere phosphorylation of CENP-A, but not its length and amino acid sequence, is required for the localization of CENP-C, a key mediator between centromeric chromatin and the outer kinetochore components. Our data reveal that in mitosis, the phosphorylated CENP-A nucleosomes are “bridged” to CENP-C via the phosphobinding 14-3-3 proteins. These 14-3-3 mitotic bridges are essential for the assembly of active kinetochores.