Recurrent evolution of DNA-binding motifs in the Drosophila centromeric histone

Abstract

All eukaryotes contain centromere-specific histone H3 variants (CenH3s), which replace H3 in centromeric chromatin. We have previously documented the adaptive evolution of the Drosophila CenH3 (Cid) in comparisons of Drosophila melanogaster and Drosophila simulans, a divergence of approximately 2.5 million years. We have proposed that rapidly changing centromeric DNA may be driving CenH3's altered DNA-binding specificity. Here, we compare Cid sequences from a phylogenetically broader group of Drosophila species to suggest that Cid has been evolving adaptively for at least 25 million years. Our analysis also reveals conserved blocks not only in the histone-fold domain but also in the N-terminal tail. In several lineages, the N-terminal tail of Cid is characterized by subgroup-specific oligopeptide expansions. These expansions resemble minor groove DNA binding motifs found in various histone tails. Remarkably, similar oligopeptides are also found in N-terminal tails of human and mouse CenH3 (Cenp-A). The recurrent evolution of these motifs in CenH3 suggests a packaging function for the N-terminal tail, which results in a unique chromatin organization at the primary constriction, the cytological marker of centromeres.

Figure 1

Phylogenetic conservation of Cid, the Drosophila CenH3, in the melanogaster group. (A) Schematic representation of Cid from different Drosophila species. The N-terminal tail is shown as a wavy blue line and the histone fold domain in red. The relative locations of three conserved sequence blocks in the tail are indicated as blue boxes (not drawn to scale). Each oligopeptide expansion is represented by red arrowheads; the amino acid repeat length is indicated by a number following the expansion and the number of iterations are indicated by the number of arrowheads. In addition, the consensus sequence of the repeats is shown in parentheses. (B) A multiple alignment of the histone fold domain of Drosophila Cids and D. melanogaster H3. Black dots represent gaps in the alignment. The alignment has been shaded to a 75% consensus by using MACBOXSHADE, with identical and similar residues shown in black and gray, respectively. Secondary structure assignments are from ref. with the exception of Loop 1, which has been redefined to encompass the flanking variable sequence (between the dotted vertical lines). For instance, because of insertions/deletions, it is unclear exactly where the α1 helix ends and the α2 helix begins within Loop 1 of Cid. H3 contacts with itself, another H3 molecule and H4 are indicated by blue diamonds, blue dots, and red dots, respectively (14). (C) Cid gene phylogeny. Bootstrap support (percentage of 1,000 trials) is shown next to each node. (D) The three conserved blocks in Cid's N-terminal tail in Logos format (Methods).

Figure 2

Molecular evolution of oligopeptide expansions in CenH3s. (A) Multiple alignment of the oligopeptide expansions in the pair of takahashii/suzukii species groups. All synonymous and replacement substitutions are shown in blue and orange relative to the first line of the alignment. Eight codon positions had substitutions that could be evaluated for natural selection by using Adaptsite; of these, seven positions had r_n < r_S (2 significant, P values shown), whereas codon 4 has r_n > r_S, although only marginally so. Also presented is a Logo of the repeat consensus, which clearly highlights the conservation of the first five codons relative to the last four. (B) Expansions in the ananassae species group. All positions have r_n < r_S (7 significant, P values shown), indicating a stringent purifying selection. This conservation is confirmed by the Logo of the ananassae consensus shown in C. (C) Match of ananassae consensus to vertebrate Cenp-As. E-values reported are from a search of about 15 putative CenH3s. The entire N-terminal tails of the human, mouse, and zebrafish Cenp-As (Danio rerio Cenp-A, accession nos. BF156113, BI877040, and BF158223; Washington Univ. St. Louis Zebrafish EST Project) are shown upstream of the histone fold domain. In addition to the ananassae consensus, we could identify matches to the SPKK motifs (blue boxes) as well as short dipeptide motifs that include a proline at every alternate site (dark arrowheads).

Figure 3

SPKK motifs in histone tails. (A) Schematics of sea urchin sperm H1 and H2B, and angiosperm H2A histones. Representative histones (–30) are shown from sea urchin (sperm): Parechinus angulosus, Echinolampas crassa, Strongylocentrotus nudus, Strongylocentrotus purpuratus, and Lytechinus pictus; and angiosperms: Oryza sativa, Triticum aestivum, Pisum sativum, and Arabidopsis thaliana. Each instance of an SPKK motif is shown with a dark arrowhead. For comparison, a histone of each type lacking these motifs is also presented. (B) Logo of a multiple alignment of all SPKK motifs shown in A. (C) A schematic representation of the nucleosome core structure (14) highlighting the tails of H2A, H2B, and (Cen)H3 where SPKK expansions have been found and their proximity to the minor groove of the DNA wrapped around the histone octamer. Note that the two molecules of H3 have different lengths of N-terminal tails in the structure. For clarity, the two molecules of histone H4 are omitted.