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Centromeres are essential chromosomal loci responsible for proper chromosome segregation during cell division. Centromeric chromatin is epigenetically defined by centromere-specific histone H3 variants, CENH3 proteins, that show significant heterogeneity among eukaryotic organisms. Concurrently, underlying DNA sequences, mostly composed of satellite DNAs (satDNAs) and transposons, are highly variable even between closely related species. To deepen understanding of centromere evolution, we focused our research on the three insect species from the genus Tribolium: the sibling species Tribolium castaneum and Tribolium freemani, capable of mutual mating, and phylogenetically more distant Tribolium madens. Relying on 40% similarity to the histone H3 amino acid sequence, we identified the CENH3 genes in all three species. Consistently with their phylogenetic relationships, T. castaneum cCENH3 and T. freemani fCENH3 proteins share 80% identity in amino acid sequence, while both of them are less similar to T. madens mCENH3 showing 74% identity. Based on their species-specific N- terminal sequences, we raised antibodies that detected cCENH3, fCENH3, and mCENH3 exclusively in the centromeres of T. castaneum, T. freemani, and T. madens, respectively. To disclose the composition of Tribolium centromeric DNAs, we performed ChIP-Seq experiments. Illumina sequencing revealed that the cCENH3, fCENH3, and mCENH3 nucleosomes predominantly associate with the major satDNAs of each species. IF-FISH experiments confirmed that in the three examined species their highly abundant, major satDNAs span across the functional centromeres of all chromosomes. Showing no similarity in nucleotide sequence, the major satDNAs of the three species are considered species-specific. It could be hypothesized that dramatic changes in composition of centromeric DNA sequences foster adaptive evolution of CENH3 proteins. Such premise is in accordance with the centromere paradox, which conciliates the rapidly evolving centromeric DNA and CENH3 proteins conflicting conserved centromere function. |
