Adaptive evolution of foundation kinetochore proteins in primates

Abstract

Rapid evolution is a hallmark of centromeric DNA in eukaryotic genomes. Yet, the centromere itself has a conserved functional role that is mediated by the kinetochore protein complex. To broaden our understanding about both the DNA and proteins that interact at the functional centromere, we sought to gain a detailed view of the evolutionary events that have shaped the primate kinetochore. Specifically, we performed comparative mapping and sequencing of the genomic regions encompassing the genes encoding three foundation kinetochore proteins: Centromere Proteins A, B, and C (CENP-A, CENP-B, and CENP-C). A histone H3 variant, CENP-A provides the foundation of the centromere-specific nucleosome. Comparative sequence analyses of the CENP-A gene in 14 primate species revealed encoded amino-acid residues within both the histone-fold domain and the N-terminal tail that are under strong positive selection. Similar comparative analyses of CENP-C, another foundation protein essential for centromere function, identified amino-acid residues throughout the protein under positive selection in the primate lineage, including several in the centromere localization and DNA-binding regions. Perhaps surprisingly, the gene encoding CENP-B, a kinetochore protein that binds specifically to alpha-satellite DNA, was not found to be associated with signatures of positive selection. These findings point to important and distinct evolutionary forces operating on the DNA and proteins of the primate centromere.

FIG. 1.

Alignment of the deduced CENP-A protein sequences from 14 primates. (A) N-terminal tail region of CENP-A (positions 1–44; residue numbers are based on the human CENP-A sequence). Deviations from the human sequence are indicated by a different amino acid (single-letter abbreviations are depicted); a dot indicates that the amino acid is the same as in the human sequence, and a dash reflects insertion/deletion of an amino acid. Ser, Thr, and Tyr residues predicted to be phosphorylated (table 3 and supplementary table 2, Supplementary Material online) are shown in red, green, and blue, respectively. Deviations from the predicted phosphorylation status of a conserved amino acid are indicated by replacing the black dot by the single-letter amino-acid symbol of the conserved residue in black. Colored letters within the body of the alignment indicate residues predicted to be phosphorylated in that species but not in others. Black asterisks along the top indicate residues under positive selection. The green and blue boxes highlight protein kinase C motifs and cAMP- or cGMP-dependent protein kinase motifs, respectively. SPKK motifs and dipeptide motifs observed in other CENP-A homologs (Malik et al. 2002) are underlined in black or indicated by arrowheads, respectively. Red horizontal lines separate species in each of the four major branches of the primate phylogenetic tree. (B) Histone-fold domain of CENP-A (positions 45–140). Predicted protein structural features (Regnier et al. 2003) are indicated along the top; the CENP-A-targeting domain (CATD; Black et al. 2004) is also indicated. Other features of the alignment are as indicated in A.

FIG. 2.

Alignment of the deduced CENP-C-interaction domain sequences of the CENP-B protein from 16 primates. The multispecies alignment of the entire CENP-B protein sequence is provided in supplementary figure 3, Supplementary Material online, with positions 404–470 shown here. Features of the alignment are as in figure 1.

FIG. 3.

Alignment of the deduced protein sequence of the major functional domains of CENP-C from 13 primates. The major functional domains of the CENP-C protein are indicated in the model at the top. Specifically, the Instability, DNA Binding, and Dimerization domains are shown along with the two CENP-B-Interaction domains (thin black lines) and the three Mif2-homology domains (black bars). The multispecies protein sequence alignment of selected regions (A–D) is shown below the model; the labeled squares above the model show the relative positions of each of these regions. Black asterisks along the top indicate residues under positive selection with posterior probabilities of greater than 0.5; red asterisks reflect residues under positive selection with posterior probabilities of greater than 0.7. Other features of the alignment are as in figure 1. (A) N-Terminal CENP-B-interaction domain (residues 282–428; Suzuki et al. 2004), which overlaps the instability domain (residues 1–373; Lanini and McKeon 1995), contains a Mif2-homology domain (Mif2 block 1, residues 336–383; Brown 1995), and overlaps the start of the DNA-binding domain (residues 395–538; Yang et al. 1996; Sugimoto et al. 1997; Cohen et al. 2008). Black and green bars indicate overlap with the start of the DNA-binding domain (see B). (B) DNA-binding domain (residues 395–538). Highlighted below the alignment are portions of the DNA-binding domain, as determined by previous studies (black bar, residues 396–498 [Sugimoto et al. 1997]; green bar, residues 422–537 [Cohen et al. 2008]; and red bar, residues 433–520 [Yang et al. 1996]). The open red bar indicates the minimal CATD (residues 478–537; Yang et al. 1996). (C) Region containing potential PEST sequences (open black boxes), as determined in this study. (D) C-terminal CENP-B-interaction domain (residues 727–943; Suzuki et al. 2004) and dimerization domain (gray highlighted residues 820–943; Sugimoto et al. 1997), which encompass the other Mif2-homology domains (residues 736–759 and 890–943; Brown 1995), the CENP-C-signature domain (underlined in blue, residues 736–759; Meluh and Koshland 1995), and the 9 (A–I) domains of the β-jelly roll (indicated with pink lines; Dunwell et al. 2001; Cohen et al. 2008).