Human centromeric CENP-A chromatin is a homotypic, octameric nucleosome at all cell cycle points

Abstract

In this study, the authors use new reference models for 23 human centromeres and find that at all cell cycle phases centromeric CENP-A chromatin complexes are octameric nucleosomes with two molecules of CENP-A. This finding refutes previous models that have suggested that hemisomes may briefly transition to octameric nucleosomes.

Unexpected conformational variations of the human centromeric chromatin complex

“…The sensitivity of the uncross-linked CCAN to MNase digestion can account in part for the differences in DNA protection that led to conflicting conclusions about the structure of the CENP-A nucleosome. However, by following N-ChIP with salt fractionation, we now show that CENP-A particles observed using low-salt conditions (Lacoste et al 2014;Nechemia-Arbely et al 2017) comprise only a minor fraction of the total CENP-A genome-wide. In contrast, the major N-ChIP salt fraction consists of particles that protect much larger DNA fragments, consistent with the presence of an intact CCAN complex.…”

“…We wondered whether the relative lack of large fragments obtained using N-ChIP in other published studies (Lacoste et al 2014;Henikoff et al 2015;Nechemia-Arbely et al 2017) might have resulted from the failure to solubilize most centromeric chromatin under low-salt conditions. To address this possibility, we prepared Illumina sequencing libraries from N-ChIP salt fractions and subjected them to paired-end sequencing.…”

“…Using a comparative chromatin immunoprecipitation (ChIP) with DNA sequencing (ChIP-seq) strategy that included native ChIP (N-ChIP), cross-linking ChIP (X-ChIP), and sequential ChIP (ReChIP), we showed previously that CENP-B, CENP-C, and CENP-T are physically integrated and form a coherent complex with CENP-A nucleosomes. Micrococcal nuclease (MNase) digestion of CENP-A, CENP-C, and CENP-T X-ChIP resulted in >165-bp protection over α-satellite dimers (Thakur and Henikoff 2016), whereas under native conditions, MNase digestion resulted primarily in shorter CENP-A-bound α-satellite fragments ranging from ∼100 to ∼135 bp (Hasson et al 2013;Henikoff et al 2015;Nechemia-Arbely et al 2017).…”

Remarkable Evolutionary Plasticity of Centromeric Chromatin

“…Indeed, using cross-linking and light sonication after MNase digestion, we found that CENP-T containing α-satellite particles could be recovered in robust amounts, and CENP-T particles precisely co-mapped with CENP-A and CENP-C (Thakur and Henikoff 2016). It is possible that estimated particle size differences reported in studies using native MNase ChIP-seq (Hasson et al 2013;Lacoste et al 2014;Henikoff et al 2015;Nechemia-Arbely et al 2017) can be explained by the lability of CENP-TWSX-containing particles when subjected to MNase treatment.…”

“…Most human artificial centromeres require hundreds of kilobases of an ∼170-bp α-satellite repeat array organized as higher-order repeats (HORs), including ∼17-bp consensus binding sites for CENP-B protein (Hayden et al 2013). Several studies have used ChIP-seq to show that CENP-A nucleosomes occupy α-satellite sequences, although the exact composition and conformation of the particles continue to be debated (Bui et al 2012;Hasson et al 2013;Lacoste et al 2014;Athwal et al 2015;Henikoff et al 2015;Thakur and Henikoff 2016;Nechemia-Arbely et al 2017). We have found that much of the disagreement stems from the difficulty of mapping ChIP-seq reads to tandemly repeated DNA sequences, which have proven to be intractable using standard tools for sequence assembly.…”