A Satellite-Free Centromere in Equus przewalskii Chromosome 10

Abstract

In mammals, centromeres are epigenetically specified by the histone H3 variant CENP-A and are typically associated with satellite DNA. We previously described the first example of a natural satellite-free centromere on Equus caballus chromosome 11 (ECA11) and, subsequently, on several chromosomes in other species of the genus Equus. We discovered that these satellite-free neocentromeres arose recently during evolution through centromere repositioning and/or chromosomal fusion, after inactivation of the ancestral centromere, where, in many cases, blocks of satellite sequences were maintained. Here, we investigated by FISH the chromosomal distribution of satellite DNA families in Equus przewalskii (EPR), demonstrating a good degree of conservation of the localization of the major horse satellite families 37cen and 2PI with the domestic horse. Moreover, we demonstrated, by ChIP-seq, that 37cen is the satellite bound by CENP-A and that the centromere of EPR10, the ortholog of ECA11, is devoid of satellite sequences. Our results confirm that these two species are closely related and that the event of centromere repositioning which gave rise to EPR10/ECA11 centromeres occurred in the common ancestor, before the separation of the two horse lineages.

Keywords: ChIP-seq; Equus przewalskii; centromere; karyotype evolution; satellite DNA.

Figure 1

DAPI-banded karyotype of a male Equus przewalskii. The karyotype (2n = 66) consists of 24 biarmed autosomes, 40 acrocentric autosomes, a biarmed X chromosome, and the acrocentric Y chromosome. Chromosomes are arranged and numbered according to standard nomenclature.

Figure 2

Chromosomal localization of two horse BAC clones specific for ECA5p and ECA5q. Metaphase spreads from E. caballus (A), E. caballus × E. przewalskii (B), and E. przewalskii (C) hybridized with BAC clone CHORI241-17G8 (ECA5p) on the left and BAC clone CHORI241-25H7 (ECA5q) on the right. Chromosomes were stained with DAPI (blue). The white arrows indicate BAC signals (red).

Figure 3

Hybridization of satellite DNA probes with metaphase chromosomes of E. przewalskii. Single-color FISH experiments with three satellite DNA probes: (A) 37cen (red signals), (B) 2PI (red signals), and (C) total genomic DNA (green signals). In panel (D), two-color FISH with 2PI probe (red signals) and total genomic DNA (green signals). The white arrows indicate chromosome 10, which is the only chromosome pair lacking any hybridization signal.

Figure 4

ChIP-seq characterization of centromeres in Equus przewalskii. (A) ChIP-seq identification of the EPR10 satellite-free centromere. ChIP-seq reads from primary fibroblasts of Przewalski’s horse were mapped on the EquCab2.0 horse reference genome. The CENP-A enriched domain is visualized as a peak. The y-axis reports the normalized read counts whereas the x-axis reports the coordinates on the reference genome. Under the peak, heterozygous nucleotide positions detected in the reads from immunoprecipitated DNA are indicated as rectangles. (B) Enrichment of 37cen and 2PI satellite families in the CENP-A immunoprecipitated chromatin. Values were measured as the ratio between normalized read counts (RPKM) in immunoprecipitated and input DNA. The ERE-1 retrotransposon is used as a control.