Summary Histone variants play crucial roles in gene expression, genome integrity and chromosome segregation. However, to what extent histone variants control chromatin architecture remains largely unknown. We report genome-wide profiles of all four types of H2A variants in Arabidopsis and identify that the previously uncharacterized histone variant H2A.W specifically associates with heterochromatin. Genetic analyses show that H2A.W acts in synergy with the heterochromatic marks H3K9me2 and DNA methylation to maintain genome integrity. In vitro, H2A.W enhances chromatin condensation through a higher propensity to promote fiber-to-fiber interactions via its conserved C-terminal motif. In vivo, elimination of H2A.W causes decondensation of heterochromatin and conversely, ectopic expression of H2A.W promotes heterochromatin condensation. These results demonstrate that H2A.W plays critical roles in heterochromatin by promoting higher order chromatin condensation. Since motifs similar to the H2A.W C-terminal motif are present in other histone variants in other organisms, our findings impact our understanding of heterochromatin condensation in eukaryotes.

Centromere protein (CENP) A, a histone H3 variant, is a key epigenetic determinant of chromosome domains known as centromeres. Centromeres nucleate kinetochores, multi-subunit complexes that capture spindle microtubules to promote chromosome segregation during mitosis. Two kinetochore proteins, CENP-C and CENP-N, recognize CENP-A in the context of a rare CENP-A nucleosome. Here, we reveal the structural basis for the exquisite selectivity of CENP-N for centromeres. CENP-N uses charge and space complementarity to decode the L1 loop that is unique to CENP-A. It also engages in extensive interactions with a 15-base pair segment of the distorted nucleosomal DNA double helix, in a position predicted to exclude chromatin remodelling enzymes. Besides CENP-A, stable centromere recruitment of CENP-N requires a coincident interaction with a newly identified binding motif on nucleosome-bound CENP-C. Collectively, our studies clarify how CENP-N and CENP-C decode and stabilize the non-canonical CENP-A nucleosome to enforce epigenetic centromere specification and kinetochore assembly.

The organization of genomic DNA into nucleosomes profoundly affects all DNA-related processes in eukaryotes. The histone chaperone ‘FAcilitates Chromatin Transcription’ (FACT, consisting of subunits SPT16 and SSRP1 1 ) promotes both disassembly and reassembly of nucleosomes during gene transcription, DNA replication, and repair 2 . The mechanism by which FACT causes these opposing outcomes is unknown. Here we report two cryo-EM structures of human FACT in complex with partially assembled ‘sub-nucleosomes’, with supporting biochemical and hydrogen-deuterium exchange (HDX) data. FACT is engaged in extensive interactions with nucleosomal DNA and all histones. The large DNA-binding surface on FACT appears to be protected by the C-terminal domains of both subunits, and this inhibition is released by interaction with H2A-H2B, allowing FACT-H2A-H2B to dock onto a (H3-H4) 2 -DNA complex 3 . SPT16 binds nucleosomal DNA and tethers H2A-H2B through its C-terminal domain by acting as a placeholder for DNA. SSRP1 also contributes to DNA binding, and can assume two conformations, depending on whether a second H2A-H2B dimer is present. Our data suggest a compelling mechanism for how FACT maintains chromatin integrity during polymerase passage, by facilitating H2A-H2B dimer removal, stabilizing intermediate ‘subnucleosomal’ states, and promoting nucleosome reassembly. Our findings reconcile discrepancies regarding the many roles of FACT and underscore the dynamic interactions between histone chaperones and nucleosomes.

Since the first high-resolution structure of the nucleosome was reported in 1997, the available information on chromatin structure has increased exponentially. Here, we review insights derived from cutting-edge biophysical and structural approaches applied to the study of nucleosome dynamics and nucleosome-binding factors, with a focus on the experimental advances driving the research. In addition, we highlight emerging challenges in nucleosome structural biology.

Highlights d Marseilleviridae encode proteins that resemble fused histones H4-H3 and H2B-H2A d These histone doublets assemble into unstable nucleosomelike particles in vitro d Histone doublets localize to the viral factory and are highly abundant in the virus d They are essential for viral fitness and infectivity, a first for any virus

Chromatin accessibility is modulated by structural transitions that provide timely access to the genetic and epigenetic information during many essential nuclear processes. These transitions are orchestrated by regulatory proteins that coordinate intricate structural modifications and signalling pathways. In vitro reconstituted chromatin samples from defined components are instrumental in defining the mechanistic details of such processes. The bottleneck to appropriate in vitro analysis is the production of high quality, and quality-controlled, chromatin substrates. In this chapter we describe methods for in vitro chromatin reconstitution and quality control. We highlight the strengths and weaknesses of various approaches, and emphasize quality control steps that ensure reconstitution of a bona fide homogenous chromatin preparation. This is essential for optimal reproducibility and reliability of ensuing experiments using chromatin substrates.

Eukaryotic centromeres are defined by the presence of nucleosomes containing the histone H3 variant, centromere protein A (CENP-A). Once incorporated at centromeres, CENP-A nucleosomes are remarkably stable, exhibiting no detectable loss or exchange over many cell cycles. It is currently unclear whether this stability is an intrinsic property of CENP-A containing chromatin or whether it arises from proteins that specifically associate with CENP-A chromatin. Two proteins, CENP-C and CENP-N, are known to bind CENP-A human nucleosomes directly. Here we test the hypothesis that CENP-C or CENP-N stabilize CENP-A nucleosomes in vitro and in living cells. We show that CENP-N stabilizes CENP-A nucleosomes alone and additively with CENP-C in vitro. However, removal of CENP-C and CENP-N from cells, or mutating CENP-A so that it no longer interacts with CENP-C or CENP-N, had no effect on centromeric CENP-A stability in vivo. Thus, the stability of CENP-A nucleosomes in chromatin does not arise solely from its interactions with CENP-C or CENP-N.

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