CENP-A at Human Centromeres and Neocentromeres Forms Octameric Nucleosomes with Loose Superhelical Termini

Abstract

The centromere is the chromosomal locus that serves as the site of kinetochore formation and spindle microtubule attachment in mitosis. Centromere protein A (CENP-A) is a histone H3 variant that epigenetically specifies centromere location. Conflicting evidence has emerged regarding the histone composition and stoichiometry of CENP-A nucleosomes and the predominant form of the CENP-A nucleosome throughout the cell cycle remains controversial. Indeed, several recent reports propose that CENP-A predominantly exists in half-nucleosomes (i.e. ‘hemisomes'). We have developed novel genomic analysis tools that we marry to existing massively parallel paired-end, long-read DNA sequencing technology to probe CENP-A nucleosome structure at centromeres. using this approach in patient-derived cells harboring a neocentromere (a newly arisen functional centromere) we simultaneously identify the length and complete sequence reads of nucleosome positions at base pair resolution on normal repetitive centromere DNA and the unique sequences underlying neocentromeres. Unlike bulk nucleosomes from the same preparations that predominantly protect a single peak size (∼150 bp) from nuclease digestion, CENP-A nucleosomes occupy two major peak sizes (∼130 and ∼110 bp) and a minor peak (∼150 bp). Each peak is substantially longer than what could be protected by a hemisome or any other tetramer of histones (i.e. protection of ∼60-80 bp from nuclease digestion). Further, the prominent position on centromeric DNA of the 110 bp peak is internal to the most prominent position of the 130 bp peak, indicating a stable, protected nucleosome core with loose termini. Strikingly, pure, reconstituted octameric CENP-A nucleosomes yield similarly sized terminally digested DNA fragments under conditions where their canonical H3-containing counterparts protected ∼150 bp of nucleosomal DNA. Our findings indicate that the fundamental unit of functional centromeric chromatin is an octameric nucleosome with loose termini.