CENP-A octamers do not confer a reduction in nucleosome height by AFM

Walkiewicz, Marcin P.; Dimitriadis, Emilios K.; Dalal, Yamini

doi:10.1038/nsmb.2742

Cited by 23 publications

(40 citation statements)

References 21 publications

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“…In vitro, the structure of the CENP-A octameric nucleosome is almost superimposable with that of H3 but has weakened exit/entry DNA contacts due to the lack of a single Arginine in CENP-A's alpha-N helix which is present in H3's N-terminal section 2,59 . This concurrence in crystallographic and AFM data is in contrast with the previously reported rigidified CENP-A/H4 tetrameric core, which is more compact and has been interpreted to be inflexible 60,61 .…”

Section: Discussionmentioning

confidence: 99%

The supercoiling state of DNA determines the handedness of both H3 and CENP-A nucleosomes

et al. 2017

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a Nucleosomes form the unit structure of the genome in eukaryotes, thereby constituting a fundamental tenet of chromatin biology. In canonical nucleosomes, DNA wraps around the histone octamer in a left-handed toroidal ramp. Here, in singlemolecule magnetic tweezers studies of chaperone-assisted nucleosome assembly, we show that the handedness of the DNA wrapping around the nucleosome core is intrinsically ambidextrous, and depends on the pre-assembly supercoiling state of the DNA, i.e., it is not uniquely determined by the octameric histone core. Nucleosomes assembled onto negatively supercoiled DNA are found to exhibit a left-handed conformation, whereas assembly onto positively supercoiled DNA results in right-handed nucleosomes. This intrinsic flexibility to adopt both chiralities is observed both for canonical H3 nucleosomes, and for centromere-specific variant CENP-A nucleosomes. These data support recent advances suggesting an intrinsic adaptability of the nucleosome, and provide insights into how nucleosomes might rapidly reassemble after cellular processes that generate positive supercoiling in vivo. IntroductionIn eukaryotic cells, DNA wraps around histone octamers to form nucleosomes. These particles compact the genome, and mediate accessibility to the underlying DNA, thus regulating major cellular processes like transcription, replication and repair. Hence, the structure of nucleosomes has been the focus of many studies [1][2][3][4][5][6] . As the vast majority of nucleosome structural studies show, DNA wraps around the core histones in a left-handed ramp, with the histones inserting Arginines into the minor groove of DNA once every ~10.4bp. Thus, 147bp of DNA are arranged in 14 segments of a left-handed super-helical ramp around the octamer. Under specialized circumstances, there has been evidence for a minor fraction of nucleosomes existing in right-handed, partial, prenucleosomal, and unfolded forms [8][9][10] . Furthermore, similar to tetrameric nucleosomes found in the archaebacteria [11][12][13] , which can flip between right and left handed states, H3/H4 tetrasomes can adopt a left-or right-handed chirality.Flexibility in the manner in which DNA wraps about the core particle may thus arise from intrinsic adaptability of the H3/H4 interface. Indeed, recent work 18-20 supports early pioneering papers [21][22][23] showing that H3/H4 tetrasomes can inter-convert between the right-and left-handed states. While early work in the chromatin field demonstrated that octameric nucleosomes are preferentially assembled onto negatively supercoiled DNA 24,25 , absorbing the negative DNA plectonemes into the left-handed wrap found in the canonical octamer, more recent work has argued for the presence of "reversomes", transitionary forms used by nucleosomes to switch between handedness 8,26 . Adding to this topological complexity, histone variants such as CENP-A, which replace H3 in centromerespecific nucleosomes, have been correlated with positive supercoils on closed circular mini-plasmids in vivo in yeast, ...

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Section: Discussionmentioning

confidence: 99%

The supercoiling state of DNA determines the handedness of both H3 and CENP-A nucleosomes

et al. 2017

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“…Based on several earlier studies, a number of contradictory models have been proposed for the composition and stoichiometry of CENP-A nucleosomes (Furuyama and Henikoff 2009;Black and Cleveland 2011;Bui et al 2012;Miell et al 2013Miell et al , 2014Padeganeh et al 2013;Codomo et al 2014;Walkiewicz et al 2014). However, several more recent reports supported the idea that, throughout the entire cell cycle, CENP-A chromatin mainly consists of octameric nucleosomes rather than nonoctameric forms (for example, tetrasomes, hemisomes, and hexasomes) (Hasson et al 2013;Padeganeh et al 2013).…”

Section: Cenp-a Chromatin Displays a Distinct Higher-order Organizationmentioning

confidence: 96%

Structural transitions of centromeric chromatin regulate the cell cycle-dependent recruitment of CENP-N

et al. 2015

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Specific recognition of centromere-specific histone variant CENP-A-containing chromatin by CENP-N is an essential process in the assembly of the kinetochore complex at centromeres prior to mammalian cell division. However, the mechanisms of CENP-N recruitment to centromeres/kinetochores remain unknown. Here, we show that a CENP-A-specific RG loop (Arg80/Gly81) plays an essential and dual regulatory role in this process. The RG loop assists the formation of a compact "ladder-like" structure of CENP-A chromatin, concealing the loop and thus impairing its role in recruiting CENP-N. Upon G1/S-phase transition, however, centromeric chromatin switches from the compact to an open state, enabling the now exposed RG loop to recruit CENP-N prior to cell division. Our results provide the first insights into the mechanisms by which the recruitment of CENP-N is regulated by the structural transitions between compaction and relaxation of centromeric chromatin during the cell cycle.

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“…Second, provocative in vivo studies have shown that as long as key inner kinetochore proteins are directly tethered to the DNA at an exogenously tagged locus, mammalian CENP-A is dispensable for centromere function [16]. Third, crystallographic and AFM analysis [17–20] suggest that when measured in a static state, CENP-A nucleosomes are very similar to canonical H3 nucleosomes. In contrast to these reports, other experiments have however suggested that octameric CENP-A nucleosomes are more compacted and rigidified in vitro [21, 22], or possess alternative conformations in vivo [23, 24].…”

Section: Introductionmentioning

confidence: 99%

CENP-A and H3 Nucleosomes Display a Similar Stability to Force-Mediated Disassembly

et al. 2016

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Centromere-specific nucleosomes are a central feature of the kinetochore complex during mitosis, in which microtubules exert pulling and pushing forces upon the centromere. CENP-A nucleosomes have been assumed to be structurally unique, thereby providing resilience under tension relative to their H3 canonical counterparts. Here, we directly test this hypothesis by subjecting CENP-A and H3 octameric nucleosomes, assembled on random or on centromeric DNA sequences, to varying amounts of applied force by using single-molecule magnetic tweezers. We monitor individual disassembly events of CENP-A and H3 nucleosomes. Regardless of the DNA sequence, the force-mediated disassembly experiments for CENP-A and H3 nucleosomes demonstrate similar rupture forces, life time residency and disassembly steps. From these experiments, we conclude that CENP-A does not, by itself, contribute unique structural features to the nucleosome that lead to a significant resistance against force-mediated disruption. The data present insights into the mechanistic basis for how CENP-A nucleosomes might contribute to the structural foundation of the centromere in vivo.

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CENP-A octamers do not confer a reduction in nucleosome height by AFM

Cited by 23 publications

References 21 publications

The supercoiling state of DNA determines the handedness of both H3 and CENP-A nucleosomes

The supercoiling state of DNA determines the handedness of both H3 and CENP-A nucleosomes

Structural transitions of centromeric chromatin regulate the cell cycle-dependent recruitment of CENP-N

CENP-A and H3 Nucleosomes Display a Similar Stability to Force-Mediated Disassembly

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