Expansion of human centromeric arrays in cells undergoing break-induced replication.

Human centromeres are located within α-satellite arrays and evolve rapidly, which can lead to individual variation in array length. Proposed mechanisms for such alterations in length are unequal crossover between sister chromatids, gene conversion, and break-induced replication. However, the underlying molecular mechanisms responsible for the massive, complex, and homogeneous organization of centromeric arrays have not been experimentally validated. Here, we use droplet digital PCR assays to demonstrate that centromeric arrays can expand and contract within ∼20 somatic cell divisions of an alternative lengthening of telomere (ALT)-positive cell line. We find that the frequency of array variation among single-cell-derived subclones ranges from a minimum of ∼7% to a maximum of ∼100%. Further clonal evolution revealed that centromere expansion is favored over contraction. We find that the homologous recombination protein RAD52 and the helicase PIF1 are required for extensive array change, suggesting that centromere sequence evolution can occur via break-induced replication.

Expansion of human centromeric arrays in cells undergoing break-induced replication.

Human centromeres are located within α-satellite arrays and evolve rapidly, which can lead to individual variation in array lengths. Proposed mechanisms for such alterations in lengths are unequal cross-over between sister chromatids, gene conversion, and break-induced replication. However, the underlying molecular mechanisms responsible for the massive, complex, and homogeneous organization of centromeric arrays have not been experimentally validated. Here, we use droplet digital PCR assays to demonstrate that centromeric arrays can expand and contract within ~20 somatic cell divisions of a cell line. We find that the frequency of array variation among single-cell-derived subclones ranges from a minimum of ~7% to a maximum of ~100%. Further clonal evolution revealed that centromere expansion is favored over contraction. We find that the homologous recombination protein RAD52 and the helicase PIF1 are required for extensive array change, suggesting that centromere sequence evolution can occur via break-induced replication.

Giant variations in giant virus genome packaging.

Giant viruses (Nucleocytoviricota) have a largely conserved lifecycle, yet how they cram their large genomes into viral capsids is mostly unknown. The major capsid protein and the packaging ATPase (pATPase) comprise a highly conserved morphogenesis module in giant viruses, yet some giant viruses dispense with an icosahedral capsid, and others encode multiple versions of pATPases, including conjoined ATPase doublets, or encode none. Some giant viruses have acquired DNA-condensing proteins to compact their genomes, including sheath-like structures encasing folded DNA or densely packed viral nucleosomes that show a resemblance to eukaryotic nucleosomes at the telomeres. Here, we review what is known and unknown about these ATPases and condensing proteins, and place these variations in the context of viral lifecycles.

A giant virus genome is densely packaged by stable nucleosomes within virions.

The two doublet histones of Marseillevirus are distantly related to the four eukaryotic core histones and wrap 121 base pairs of DNA to form remarkably similar nucleosomes. By permeabilizing Marseillevirus virions and performing genome-wide nuclease digestion, chemical cleavage, and mass spectrometry assays, we find that the higher-order organization of Marseillevirus chromatin fundamentally differs from that of eukaryotes. Marseillevirus nucleosomes fully protect DNA within virions as closely abutted 121-bp DNA-wrapped cores without linker DNA or phasing along genes. Likewise, we observed that nucleosomes reconstituted onto multi-copy tandem repeats of a nucleosome-positioning sequence are tightly packed. Dense promiscuous packing of fully wrapped nucleosomes rather than "beads on a string" with genic punctuation represents a distinct mode of DNA packaging by histones. We suggest that doublet histones have evolved for viral genome protection and may resemble an early stage of histone differentiation leading to the eukaryotic octameric nucleosome.

A standardized nomenclature for mammalian histone genes.

Histones have a long history of research in a wide range of species, leaving a legacy of complex nomenclature in the literature. Community-led discussions at the EMBO Workshop on Histone Variants in 2011 resulted in agreement amongst experts on a revised systematic protein nomenclature for histones, which is based on a combination of phylogenetic classification and historical symbol usage. Human and mouse histone gene symbols previously followed a genome-centric system that was not applicable across all vertebrate species and did not reflect the systematic histone protein nomenclature. This prompted a collaboration between histone experts, the Human Genome Organization (HUGO) Gene Nomenclature Committee (HGNC) and Mouse Genomic Nomenclature Committee (MGNC) to revise human and mouse histone gene nomenclature aiming, where possible, to follow the new protein nomenclature whilst conforming to the guidelines for vertebrate gene naming. The updated nomenclature has also been applied to orthologous histone genes in chimpanzee, rhesus macaque, dog, cat, pig, horse and cattle, and can serve as a framework for naming other vertebrate histone genes in the future.

Viral histones: pickpocket's prize or primordial progenitor?

The common histones H2A, H2B, H3, and H4 are the characteristic components of eukaryotic nucleosomes, which function to wrap DNA and compact the genome as well as to regulate access to DNA for transcription and replication in all eukaryotes. In the past two decades, histones have also been found to be encoded in some DNA viruses, where their functions and properties are largely unknown, though recently histones from two related viruses have been shown to form nucleosome-like structures in vitro. Viral histones can be highly similar to eukaryotic histones in primary sequence, suggesting they have been recently picked up from eukaryotic hosts, or they can be radically divergent in primary sequence and may occur as conjoined histone doublets, triplets, or quadruplets, suggesting ancient origins prior to the divergence of modern eukaryotes. Here, we review what is known of viral histones and discuss their possible origins and functions. We consider how the viral life cycle may affect their properties and histories, and reflect on the possible roles of viruses in the origin of the nucleus of modern eukaryotic cells.

The genetics and epigenetics of satellite centromeres.

Centromeres, the chromosomal loci where spindle fibers attach during cell division to segregate chromosomes, are typically found within satellite arrays in plants and animals. Satellite arrays have been difficult to analyze because they comprise megabases of tandem head-to-tail highly repeated DNA sequences. Much evidence suggests that centromeres are epigenetically defined by the location of nucleosomes containing the centromere-specific histone H3 variant cenH3, independently of the DNA sequences where they are located; however, the reason that cenH3 nucleosomes are generally found on rapidly evolving satellite arrays has remained unclear. Recently, long-read sequencing technology has clarified the structures of satellite arrays and sparked rethinking of how they evolve, and new experiments and analyses have helped bring both understanding and further speculation about the role these highly repeated sequences play in centromere identification.

Histone variants at a glance.

Eukaryotic nucleosomes organize chromatin by wrapping 147 bp of DNA around a histone core particle comprising two molecules each of histone H2A, H2B, H3 and H4. The DNA entering and exiting the particle may be bound by the linker histone H1. Whereas deposition of bulk histones is confined to S-phase, paralogs of the common histones, known as histone variants, are available to carry out functions throughout the cell cycle and accumulate in post-mitotic cells. Histone variants confer different structural properties on nucleosomes by wrapping more or less DNA or by altering nucleosome stability. They carry out specialized functions in DNA repair, chromosome segregation and regulation of transcription initiation, or perform tissue-specific roles. In this Cell Science at a Glance article and the accompanying poster, we briefly examine new insights into histone origins and discuss variants from each of the histone families, focusing on how structural differences may alter their functions.

The Yin and Yang of Histone Marks in Transcription.

Nucleosomes wrap DNA and impede access for the machinery of transcription. The core histones that constitute nucleosomes are subject to a diversity of posttranslational modifications, or marks, that impact the transcription of genes. Their functions have sometimes been difficult to infer because the enzymes that write and read them are complex, multifunctional proteins. Here, we examine the evidence for the functions of marks and argue that the major marks perform a fairly small number of roles in either promoting transcription or preventing it. Acetylations and phosphorylations on the histone core disrupt histone-DNA contacts and/or destabilize nucleosomes to promote transcription. Ubiquitylations stimulate methylations that provide a scaffold for either the formation of silencing complexes or resistance to those complexes, and carry a memory of the transcriptional state. Tail phosphorylations deconstruct silencing complexes in particular contexts. We speculate that these fairly simple roles form the basis of transcriptional regulation by histone marks.

What makes a centromere?

Centromeres are the eukaryotic chromosomal sites at which the kinetochore forms and attaches to spindle microtubules to orchestrate chromosomal segregation in mitosis and meiosis. Although centromeres are essential for cell division, their sequences are not conserved and evolve rapidly. Centromeres vary dramatically in size and organization. Here we categorize their diversity and explore the evolutionary forces shaping them. Nearly all centromeres favor AT-rich DNA that is gene-free and transcribed at a very low level. Repair of frequent centromere-proximal breaks probably contributes to their rapid sequence evolution. Point centromeres are only ~125 bp and are specified by common protein-binding motifs, whereas short regional centromeres are 1-5 kb, typically have unique sequences, and may have pericentromeric repeats adapted to facilitate centromere clustering. Transposon-rich centromeres are often ~100-300 kb and are favored by RNAi machinery that silences transposons, by suppression of meiotic crossovers at centromeres, and by the ability of some transposons to target centromeres. Megabase-length satellite centromeres arise in plants and animals with asymmetric female meiosis that creates centromere competition, and favors satellite monomers one or two nucleosomes in length that position and stabilize centromeric nucleosomes. Holocentromeres encompass the length of a chromosome and may differ dramatically between mitosis and meiosis. We propose a model in which low level transcription of centromeres facilitates the formation of non-B DNA that specifies centromeres and promotes loading of centromeric nucleosomes.

Old cogs, new tricks: the evolution of gene expression in a chromatin context.

Sophisticated gene-regulatory mechanisms probably evolved in prokaryotes billions of years before the emergence of modern eukaryotes, which inherited the same basic enzymatic machineries. However, the epigenomic landscapes of eukaryotes are dominated by nucleosomes, which have acquired roles in genome packaging, mitotic condensation and silencing parasitic genomic elements. Although the molecular mechanisms by which nucleosomes are displaced and modified have been described, just how transcription factors, histone variants and modifications and chromatin regulators act on nucleosomes to regulate transcription is the subject of considerable ongoing study. We explore the extent to which these transcriptional regulatory components function in the context of the evolutionarily ancient role of chromatin as a barrier to processes acting on DNA and how chromatin proteins have diversified to carry out evolutionarily recent functions that accompanied the emergence of differentiation and development in multicellular eukaryotes.

Transcribing Centromeres: Noncoding RNAs and Kinetochore Assembly.

Chromosome segregation depends on the attachment of spindle microtubules to sites on chromosomal DNA known as centromeres, through kinetochore protein complexes. Although RNA was found in kinetochores in the 1970s, only with recent investigations has evidence emerged that loading of the centromere-specific nucleosomes that form the foundation of the kinetochore may be coupled to centromeric transcription. Centromeric transcripts are bound by several kinetochore proteins that require them for stabilization or localization. At least some centromeres have promoter activity, and many have non-B form DNA that may facilitate their transcription. Whereas other noncoding RNAs regulate gene expression or silence transposons, cotranscriptional assembly of kinetochores is a novel function for noncoding RNAs.

Simple and Complex Centromeric Satellites in Drosophila Sibling Species.

Centromeres are the chromosomal sites of assembly for kinetochores, the protein complexes that attach to spindle fibers and mediate separation of chromosomes to daughter cells in mitosis and meiosis. In most multicellular organisms, centromeres comprise a single specific family of tandem repeats-often 100-400 bp in length-found on every chromosome, typically in one location within heterochromatin. Drosophila melanogaster is unusual in that the heterochromatin contains many families of mostly short (5-12 bp) tandem repeats, none of which appear to be present at all centromeres, and none of which are found only at centromeres. Although centromere sequences from a minichromosome have been identified and candidate centromere sequences have been proposed, the DNA sequences at native Drosophila centromeres remain unknown. Here we use native chromatin immunoprecipitation to identify the centromeric sequences bound by the foundational kinetochore protein cenH3, known in vertebrates as CENP-A. In D. melanogaster, these sequences include a few families of 5- and 10-bp repeats; but in closely related D. simulans, the centromeres comprise more complex repeats. The results suggest that a recent expansion of short repeats has replaced more complex centromeric repeats in D. melanogaster.

Remarkable Evolutionary Plasticity of Centromeric Chromatin.

Centromeres were familiar to cell biologists in the late 19th century, but for most eukaryotes the basis for centromere specification has remained enigmatic. Much attention has been focused on the cenH3 (CENP-A) histone variant, which forms the foundation of the centromere. To investigate the DNA sequence requirements for centromere specification, we applied a variety of epigenomic approaches, which have revealed surprising diversity in centromeric chromatin properties. Whereas each point centromere of budding yeast is occupied by a single precisely positioned tetrameric nucleosome with one cenH3 molecule, the "regional" centromeres of fission yeast contain unphased presumably octameric nucleosomes with two cenH3s. In Caenorhabditis elegans, kinetochores assemble all along the chromosome at sites of cenH3 nucleosomes that resemble budding yeast point centromeres, whereas holocentric insects lack cenH3 entirely. The "satellite" centromeres of most animals and plants consist of cenH3-containing particles that are precisely positioned over homogeneous tandem repeats, but in humans, different α-satellite subfamilies are occupied by CENP-A nucleosomes with very different conformations. We suggest that this extraordinary evolutionary diversity of centromeric chromatin architectures can be understood in terms of the simplicity of the task of equal chromosome segregation that is continually subverted by selfish DNA sequences.

Histone variants on the move: substrates for chromatin dynamics.

Most histones are assembled into nucleosomes behind the replication fork to package newly synthesized DNA. By contrast, histone variants, which are encoded by separate genes, are typically incorporated throughout the cell cycle. Histone variants can profoundly change chromatin properties, which in turn affect DNA replication and repair, transcription, and chromosome packaging and segregation. Recent advances in the study of histone replacement have elucidated the dynamic processes by which particular histone variants become substrates of histone chaperones, ATP-dependent chromatin remodellers and histone-modifying enzymes. Here, we review histone variant dynamics and the effects of replacing DNA synthesis-coupled histones with their replication-independent variants on the chromatin landscape.

HistoneDB 2.0: a histone database with variants--an integrated resource to explore histones and their variants.

Compaction of DNA into chromatin is a characteristic feature of eukaryotic organisms. The core (H2A, H2B, H3, H4) and linker (H1) histone proteins are responsible for this compaction through the formation of nucleosomes and higher order chromatin aggregates. Moreover, histones are intricately involved in chromatin functioning and provide a means for genome dynamic regulation through specific histone variants and histone post-translational modifications. 'HistoneDB 2.0--with variants' is a comprehensive database of histone protein sequences, classified by histone types and variants. All entries in the database are supplemented by rich sequence and structural annotations with many interactive tools to explore and compare sequences of different variants from various organisms. The core of the database is a manually curated set of histone sequences grouped into 30 different variant subsets with variant-specific annotations. The curated set is supplemented by an automatically extracted set of histone sequences from the non-redundant protein database using algorithms trained on the curated set. The interactive web site supports various searching strategies in both datasets: browsing of phylogenetic trees; on-demand generation of multiple sequence alignments with feature annotations; classification of histone-like sequences and browsing of the taxonomic diversity for every histone variant. HistoneDB 2.0 is a resource for the interactive comparative analysis of histone protein sequences and their implications for chromatin function. Database URL: http://www.ncbi.nlm.nih.gov/projects/HistoneDB2.0.

Inner Kinetochore Protein Interactions with Regional Centromeres of Fission Yeast.

Centromeres of the fission yeast Schizosaccharomyces pombe lack the highly repetitive sequences that make most other "regional" centromeres refractory to analysis. To map fission yeast centromeres, we applied H4S47C-anchored cleavage mapping and native and cross-linked chromatin immunoprecipitation with paired-end sequencing. H3 nucleosomes are nearly absent from the central domain, which is occupied by centromere-specific H3 (cenH3 or CENP-A) nucleosomes with two H4s per particle that are mostly unpositioned and are more widely spaced than nucleosomes elsewhere. Inner kinetochore proteins CENP-A, CENP-C, CENP-T, CENP-I, and Scm3 are highly enriched throughout the central domain except at tRNA genes, with no evidence for preferred kinetochore assembly sites. These proteins are weakly enriched and less stably incorporated in H3-rich heterochromatin. CENP-A nucleosomes protect less DNA from nuclease digestion than H3 nucleosomes, while CENP-T protects a range of fragment sizes. Our results suggest that CENP-T particles occupy linkers between CENP-A nucleosomes and that classical regional centromeres differ from other centromeres by the absence of CENP-A nucleosome positioning.

Environmental responses mediated by histone variants.

Fluctuations in the ambient environment can trigger chromatin disruptions, involving replacement of nucleosomes or exchange of their histone subunits. Unlike canonical histones, which are available only during S-phase, replication-independent histone variants are present throughout the cell cycle and are adapted for chromatin repair. The H2A.Z variant mediates responses to environmental perturbations including fluctuations in temperature and seasonal variation. Phosphorylation of histone H2A.X rapidly marks double-strand DNA breaks for chromatin repair, which is mediated by both H2A and H3 histone variants. Other histones are used as weapons in conflicts between parasites and their hosts, which suggests broad involvement of histone variants in environmental responses beyond chromatin repair.

The CentO satellite confers translational and rotational phasing on cenH3 nucleosomes in rice centromeres.

Plant and animal centromeres comprise megabases of highly repeated satellite sequences, yet centromere function can be specified epigenetically on single-copy DNA by the presence of nucleosomes containing a centromere-specific variant of histone H3 (cenH3). We determined the positions of cenH3 nucleosomes in rice (Oryza sativa), which has centromeres composed of both the 155-bp CentO satellite repeat and single-copy non-CentO sequences. We find that cenH3 nucleosomes protect 90-100 bp of DNA from micrococcal nuclease digestion, sufficient for only a single wrap of DNA around the cenH3 nucleosome core. cenH3 nucleosomes are translationally phased with 155-bp periodicity on CentO repeats, but not on non-CentO sequences. CentO repeats have an ∼10-bp periodicity in WW dinucleotides and in micrococcal nuclease cleavage, providing evidence for rotational phasing of cenH3 nucleosomes on CentO and suggesting that satellites evolve for translational and rotational stabilization of centromeric nucleosomes.