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.
Highlights
Centromeres are specialized chromatin structures within eukaryotic chromosomes that ensure equal segregation of daughter chromosomes during mitosis [1]
It appears that centromere identity is maintained, at least in part, by the removal of centromere-specific histone H3 (CenH3) nucleosomes that are deposited in chromosome arms while retaining newly deposited CenH3 nucleosomes at centromeres, which are embedded in a quiescent heterochromatic environment [8]
A CenH3-Containing Tetramer Is Cross-Linked In Vivo We first investigated the organization of CenH3 and core histones within centromeric (CenH3) and bulk (BC) chromatin in chromatin extracts from nuclei of Drosophila S2 cells, using the primary amine cross-linking agent dimethyl
Summary
Centromeres are specialized chromatin structures within eukaryotic chromosomes that ensure equal segregation of daughter chromosomes during mitosis [1]. The overexpression of CENP-A is correlated with neocentromeres in some human cancers [3], and overexpression of Drosophila CenH3 (Cid) leads to its deposition in active euchromatin [4,5,6] and the formation of ectopic centromeres [5]. The ready formation of neocentromeres, which would lead to immediate chromosome loss, is prevented by the active removal of CenH3 deposited in chromosome arms and its degradation in the proteosome [6,7]. It appears that centromere identity is maintained, at least in part, by the removal of CenH3 nucleosomes that are deposited in chromosome arms while retaining newly deposited CenH3 nucleosomes at centromeres, which are embedded in a quiescent heterochromatic environment [8]
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