Tetrameric Structure of Centromeric Nucleosomes in Interphase Drosophila Cells

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Abstract
Centromeres, the specialized chromatin structures that are responsible for equal segregation of chromosomes at mitosis, are epigenetically maintained by a centromere-specific histone H3 variant (CenH3). However, the mechanistic basis for centromere maintenance is unknown. We investigated biochemical properties of CenH3 nucleosomes from Drosophila melanogaster cells. Cross-linking of CenH3 nucleosomes identifies heterotypic tetramers containing one copy of CenH3, H2A, H2B, and H4 each. Interphase CenH3 particles display a stable association of approximately 120 DNA base pairs. Purified centromeric nucleosomal arrays have typical “beads-on-a-string” appearance by electron microscopy but appear to resist condensation under physiological conditions. Atomic force microscopy reveals that native CenH3-containing nucleosomes are only half as high as canonical octameric nucleosomes are, confirming that the tetrameric structure detected by cross-linking comprises the entire interphase nucleosome particle. This demonstration of stable half-nucleosomes in vivo provides a possible basis for the instability of centromeric nucleosomes that are deposited in euchromatic regions, which might help maintain centromere identity. The octameric structure of eukaryotic nucleosomes is universally accepted as the basic unit of chromatin. This is certainly the case for the vast bulk of nucleosomes; however, there have been no reports of the in vivo structure of nucleosomes associated with centromeres. Though centromeres make up only a minute fraction of the genomic landscape, their role in segregating chromosomes during mitosis is essential for maintaining genomic integrity. We report the characterization of centromeric chromatin from Drosophila cells, using detailed biochemical, electron microscopic, and atomic force microscopic analyses. Surprisingly, we found that, in striking contrast to bulk chromatin, centromeric nucleosomes are stable heterotypic tetramers in vivo, with one copy of CenH3 (the centromere-specific H3 variant), H2A, H2B, and H4 each, wrapping one full turn of DNA at interphase (the cell growth phase of the cell cycle). This results in nucleosome particles that are only half as high as bulk nucleosomes. These unexpected findings can help account for the dynamic behavior of CenH3-containing nucleosomes, whereby they are deposited promiscuously but are turned over in noncentromeric regions. Our demonstration of the existence of stable half-nucleosomes at centromeres suggests a novel mechanism for maintaining centromere identity.