Simple and Complex Centromeric Satellites in Drosophila Sibling Species

Abstract

Centromeres are the chromosomal sites of assembly for kinetochores, the protein complexes that attach to spindle fibers and mediate separation of chromosomes to daughter cells in mitosis and meiosis. In most multicellular organisms, centromeres comprise a single specific family of tandem repeats-often 100-400 bp in length-found on every chromosome, typically in one location within heterochromatin. Drosophila melanogaster is unusual in that the heterochromatin contains many families of mostly short (5-12 bp) tandem repeats, none of which appear to be present at all centromeres, and none of which are found only at centromeres. Although centromere sequences from a minichromosome have been identified and candidate centromere sequences have been proposed, the DNA sequences at native Drosophila centromeres remain unknown. Here we use native chromatin immunoprecipitation to identify the centromeric sequences bound by the foundational kinetochore protein cenH3, known in vertebrates as CENP-A. In D. melanogaster, these sequences include a few families of 5- and 10-bp repeats; but in closely related D. simulans, the centromeres comprise more complex repeats. The results suggest that a recent expansion of short repeats has replaced more complex centromeric repeats in D. melanogaster.

Keywords: ChIP; Cid; prod; rotational phasing.

Figure 1

AAGAG is not necessary for centromere function on the D. melanogaster X chromosome. (A) Translocation T(1;3) e^H2 breaks on the short right arm of the X chromosome and at 93 on chromosome 3. (B) The wild-type X in the stock T(1;3) e^H2/In(3R)C lacks detectable AAGAG and CENP-A signal overlaps with AATAT signal. Hybridization signal is not found under all of the anti-CidH signal, suggesting that the kinetochore interferes with hybridization.

Figure 2

AATAG repeats in D. melanogaster. (A) A fluorescent (AATAG)₁₀ probe detects repeats left of the centromere on chromosome 2 in larval brain squashes. (B) Enrichment of varying numbers of (AATAG)_n repeats in 250-bp reads from anti-CidM ChIP of S2 cells. (C) An example of a reference sequence containing (AATAG)₂ repeats.

Figure 3

Periodicity of simcent1 repeats. Dot matrix plots of similarity for four clones with homology to simcent1 compared against themselves reveal repeats with a periodicity of ∼500 bp.

Figure 4

FISH of the simcent1 probe in D. simulans. (A) Hybridization of simcent1 to D. simulans larval neuroblasts using formaldehyde fixation. (B) simcent1 hybridization together with CENP-A detection. (C and D) Hybridization of simcent1 using formaldehyde fixation with 45% acetic acid. Arrow points to weak signal at the expected position of the X centromere.

Figure 5

Abundance of simple and complex repeats in nine Drosophila species. All species are in the melanogaster subgroup, except D. ananassae, which is in the melanogaster group, and D. pseudoobscura, which serves as an outgroup. The cladograms at the bottom depict the relationships of the species. The y-axis represents the fraction of the reads that contain the repeat, presented on log₁₀ scale. In the box and whisker plots, the box ends represent the second and third quartiles of the data separated by the median, while the whiskers represent the first and fourth quartiles. The mean is marked by X. For each centromere-enriched repeat, [mel] and [sim] indicate that in the corresponding species, the abundance of the repeat in anti-Cid IP was at least 5% of that of the most abundant centromere repeat in that species.