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Many animals achieve sperm chromatin compaction and stabilisation by replacing canonical histones with sperm nuclear basic proteins (SNBPs) such as protamines during spermatogenesis. Hydrozoan cnidarians and echinoid sea urchins lack protamines and have evolved a distinctive family of sperm-specific histone H2Bs (spH2Bs) with extended N termini rich in SPK(K/R) motifs. Echinoid sperm packaging is regulated by spH2Bs. Their sperm is negatively buoyant and fertilises on the sea floor. Hydroid cnidarians undertake broadcast spawning but their sperm properties are poorly characterised. We show that Hydractinia echinata and H. symbiolongicarpus sperm chromatin possesses higher stability than somatic chromatin, with reduced accessibility to transposase Tn5 integration and to endonucleases in vitro. In contrast, nuclear dimensions are only moderately reduced in mature Hydractinia sperm. Ectopic expression of spH2B in the background of H2B.1 knockdown results in downregulation of global transcription and cell cycle arrest in embryos, without altering their nuclear density. Taken together, SPKK-containing spH2B variants act to stabilise chromatin and silence transcription in Hydractinia sperm with only limited chromatin compaction. We suggest that spH2Bs could contribute to sperm buoyancy as a reproductive adaptation.

Giardia lamblia is a pathogenic unicellular eukaryotic parasite that causes giardiasis. Its genome encodes the canonical histones H2A, H2B, H3, and H4, which share low amino acid sequence identity with their human orthologues. We determined the structure of the G. lamblia nucleosome core particle (NCP) at 3.6 Å resolution by cryo-electron microscopy. G. lamblia histones form a characteristic NCP, in which the visible 125 base-pair region of the DNA is wrapped in a left-handed supercoil. The acidic patch on the G. lamblia octamer is deeper, due to an insertion extending the H2B α1 helix and L1 loop, and thus cannot bind the LANA acidic patch binding peptide. The DNA and histone regions near the DNA entry-exit sites could not be assigned, suggesting that these regions are asymmetrically flexible in the G. lamblia NCP. Characterization by thermal unfolding in solution revealed that both the H2A–H2B and DNA association with the G. lamblia H3–H4 were weaker than those for human H3–H4. These results demonstrate the uniformity of the histone octamer as the organizing platform for eukaryotic chromatin, but also illustrate the unrecognized capability for large scale sequence variations that enable the adaptability of histone octamer surfaces and confer internal stability., Graphical Abstract Graphical AbstractG. lamblia has an equivalent nucleosome structure, despite low histone sequence identity to metazoan nucleosomes. The DNA entry-exit and acidic patch regions are unique characteristics of G. lamblia nucleosomes.

Exploratory analysis of cancer consortia data curated by the cBioPortal repository typically requires advanced programming skills and expertise to identify novel genomic prognostic markers that have the potential for both diagnostic and therapeutic exploitation. We developed GNOSIS (GeNomics explOrer using StatistIcal and Survival analysis in R), an R Shiny App incorporating a range of R packages enabling users to efficiently explore and visualise such clinical and genomic data. GNOSIS provides an intuitive graphical user interface and multiple tab panels supporting a range of functionalities, including data upload and initial exploration, data recoding and subsetting, data visualisations, statistical analysis, mutation analysis and, in particular, survival analysis to identify prognostic markers. GNOSIS also facilitates reproducible research by providing downloadable input logs and R scripts from each session, and so offers an excellent means of supporting clinician-researchers in developing their statistical computing skills.

Recent studies have highlighted the potential for missense mutations in histones to act as oncogenic drivers, leading to the term 'oncohistones'. While histone proteins are highly conserved, they are encoded by multigene families. There is heterogeneity among these genes at the level of the underlying sequence, the amino acid composition of the encoded histone isoform, and the expression levels. One question that arises, therefore, is whether all histone-encoding genes function equally as oncohistones. In this review, we consider this question and explore what this means in terms of the mechanisms by which oncohistones can exert their effects in chromatin.

Many animals achieve sperm chromatin compaction and stabilisation during spermatogenesis by replacing canonical histones with sperm nuclear basic proteins (SNBPs) such as protamines. A number of animals including hydrozoan cnidarians and echinoid sea urchins lack protamines and have instead evolved a distinctive family of sperm-specific histone H2Bs (spH2Bs) with extended N-termini rich in SPKK-related motifs. Sperm packaging in echinoids such as sea urchins is regulated by spH2Bs and their sperm is negatively buoyant for fertilization on the sea floor. Hydroid cnidarians also package sperm with spH2Bs but undertake broadcast spawning and their sperm properties are poorly characterised. We show that sperm chromatin from the hydroid Hydractinia possesses higher stability than its somatic equivalent, with reduced accessibility of sperm chromatin to transposase Tn5 integration in vivo and to endonucleases in vitro. However, nuclear dimensions are only moderately reduced in mature Hydractinia sperm compared to other cell types. Ectopic expression of spH2B in the background of H2B knockdown resulted in downregulation of global transcription and cell cycle arrest in embryos without altering their nuclear density. Taken together, spH2B variants containing SPKK-related motifs act to stabilise chromatin and silence transcription in Hydractinia sperm without significant chromatin compaction. This is consistent with a contribution of spH2B to sperm buoyancy as a reproductive adaptation.

The histone proteins defining nucleosome structure are highly conserved in common model organisms and are frequently portrayed as uniform chromatin building blocks. We surveyed over 1700 complete eukaryotic genomes and confirm that almost all encode recognisable canonical core histones. Nevertheless, divergent eukaryotes show unrecognised diversity in histone sequences and offer an opportunity to observe the potential for nucleosome variation. Recombinant histones for Plasmodium falciparum, Giardia lamblia, Encephalitozoon cuniculi and Leishmania major were prepared alongside those for human, Xenopus laevis and Saccharomyces cerevisiae. All could be assembled into nucleosomes in vitro on sequences known to direct positioning with metazoan histones. P. falciparum histones refolded into very stable nucleosomes consistent with a highly regulated transcriptional programme. In contrast, G. lamblia and E. cuniculi histones formed less stable nucleosomes and were prone to aggregation as H3-H4 tetramers. Inspection of the histone fold dimer interface residues suggested a potential to form tetrasomal arrays consistent with polymerisation. DNA binding preferences observed using systematic evolution of ligands by exponential enrichment (SELEX) for human, P. falciparum and E. cuniculi histone octamers were highly similar and reflect a shared capability to package diverse genomic sequences. This demonstrates that nucleosomal organisation is retained across eukaryotes and can accommodate genome variation, but histone protein sequences vary more than commonly recognised to provide the potential for diversity of chromatin features.Significance statementIt is widely assumed that eukaryotes package their genomes using equivalent nucleosome building blocks despite considerable variation in the composition and behaviour of cell nuclei. Our survey of available eukaryote genomes shows that histone proteins from divergent eukaryotes vary much more widely in sequence than is commonly recognised, even in histone fold dimer and DNA interaction interfaces. We demonstrate that divergent eukaryote histones nevertheless form nucleosomes on DNA sequences favoured in metazoans. These nucleosomes vary in stability but share broad DNA sequence preferences. This suggests that histone-dependent packaging does not constrain genome variation, and that chromatin behaviour can adapt by evolution of canonical core histone sequences in addition to other well-known mechanisms.

Breast cancer is the leading cause of cancer related death among women. Breast cancers are generally diagnosed and treated based on clinical and histopathological features, along with subtype classification determined by the Prosigna Breast Cancer Prognostic Gene Signature Assay (also known as PAM50). Currently the copy number alteration (CNA) landscape of the tumour is not considered. We set out to examine the role of genomic instability (GI) in breast cancer survival since CNAs reflect GI and correlate with survival in other cancers. We focused on the 70% of breast cancers classified as luminal and carried out a comprehensive survival and association analysis using Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) data to determine whether CNA Score Quartiles derived from absolute CNA counts are associated with survival. Analysis revealed that patients diagnosed with luminal A breast cancer have a CNA landscape associated with disease specific survival, suggesting that CNA Score can provide a statistically robust prognostic factor. Furthermore, stratification of patients into subtypes based on gene expression has shown that luminal A and B cases overlap, and it is in this region we largely observe luminal A cases with reduced survival outlook. Therefore, luminal A breast cancer patients with quantitatively elevated CNA counts may benefit from more aggressive therapy. This demonstrates how individual genomic landscapes can facilitate personalisation of therapeutic interventions to optimise survival outcomes.

Breast cancer is the leading cause of cancer related death among women. Breast cancers are generally diagnosed and treated based on clinical and histopathological features, along with subtype classification determined by the Prosigna Breast Cancer Prognostic Gene Signature Assay (also known as PAM50). Currently the copy number alteration (CNA) landscape of the tumour is not considered. We set out to examine the role of genomic instability (GI) in breast cancer survival since CNAs reflect GI and correlate with survival in other cancers. We focussed on the 70% of breast cancers classified as luminal and carried out a comprehensive survival and association analysis using Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) data to determine whether CNA burden quartiles derived from absolute CNA counts are associated with survival. Luminal A and B patients were stratified by PAM50 subtype and tumour grade and then tested for association with CNA burden using multiple statistical tests. Analysis revealed that patients diagnosed with luminal A grade 3 breast cancer have a CNA landscape associated with disease specific survival, suggesting that these patients could be classified as at-risk. Furthermore, luminal A grade 3 cases largely occupy a region of stratification based on gene expression at the boundary where luminal A and luminal B cases overlap. We conclude that GI reflected by absolute CNA score is a statistically robust prognostic factor for survival in luminal A grade 3 breast cancer. Therefore, luminal A grade 3 breast cancer patients in CNA burden quartiles 3 or 4 may benefit from more aggressive therapy. This demonstrates how individual genomic landscapes can facilitate personalisation of therapeutic interventions to optimise survival outcomes.

A developmental program affecting human face shape is shown by Greenberg et al. (2019) to hinge on the ability to distinguish a single methyl group between two histone variant isoforms and the action of the chromatin-remodeling enzyme SRCAP. This challenges researchers to link atomic structure to a morphological defect.

Core histone proteins H2A, H2B, H3, and H4 are encoded by a large family of genes distributed across the human genome. Canonical core histones contribute the majority of proteins to bulk chromatin packaging, and are encoded in 4 clusters by 65 coding genes comprising 17 for H2A, 18 for H2B, 15 for H3, and 15 for H4, along with at least 17 total pseudogenes. The canonical core histone genes display coding variation that gives rise to 11 H2A, 15 H2B, 4 H3, and 2 H4 unique protein isoforms. Although histone proteins are highly conserved overall, these isoforms represent a surprising and seldom recognised variation with amino acid identity as low as 77% between canonical histone proteins of the same type. The gene sequence and protein isoform diversity also exceeds commonly used subtype designations such as H2A.1 and H3.1, and exists in parallel with the well-known specialisation of variant histone proteins. RNA sequencing of histone transcripts shows evidence for differential expression of histone genes but the functional significance of this variation has not yet been investigated. To assist understanding of the implications of histone gene and protein diversity we have catalogued the entire human canonical core histone gene and protein complement. In order to organise this information in a robust, accessible, and accurate form, we applied software build automation tools to dynamically generate the canonical core histone repertoire based on current genome annotations and then to organise the information into a manuscript format. Automatically generated values are shown with a light grey background. Alongside recognition of the encoded protein diversity, this has led to multiple corrections to human histone annotations, reflecting the flux of the human genome as it is updated and enriched in reference databases. This dynamic manuscript approach is inspired by the aims of reproducible research and can be readily adapted to other gene families.

Damage-induced long non-coding RNAs (dilncRNA) synthesized at DNA double-strand breaks (DSBs) by RNA polymerase II (RNAPII) are necessary for DNA damage response (DDR) foci formation. We demonstrate that induction of DSBs results in the assembly of functional promoters that include a complete RNAPII pre-initiation complex (PIC), MED1 and CDK9. Absence or inactivation of these factors causes DDR foci reduction both in vivo and in an in vitro system that reconstitutes DDR events on nucleosomes. We also show that dilncRNAs drive molecular crowding of DDR proteins such as 53BP1 into foci that exhibit liquid-liquid phase separation (LLPS) condensate properties. We propose that the assembly of DSB-induced transcriptional promoters drives RNA synthesis which stimulates phase separation of DDR factors in the shape of foci.

Within canonical eukaryotic nuclei, DNA is packaged with highly conserved histone proteins into nucleosomes, which facilitate DNA condensation and contribute to genomic regulation. Yet the dinoflagellates, a group of unicellular algae, are a striking exception to this otherwise universal feature as they have largely abandoned histones and acquired apparently viral-derived substitutes termed DVNPs (dinoflagellate-viral-nucleoproteins). Despite the magnitude of this transition, its evolutionary drivers remain unknown. Here, using Saccharomyces cerevisiae as a model, we show that DVNP impairs growth and antagonizes chromatin by localizing to histone binding sites, displacing nucleosomes, and impairing transcription. Furthermore, DVNP toxicity can be relieved through histone depletion and cells diminish their histones in response to DVNP expression suggesting that histone reduction could have been an adaptive response to these viral proteins. These findings provide insights into eukaryotic chromatin evolution and highlight the potential for horizontal gene transfer to drive the divergence of cellular systems., Dinoflagellates are known to use dinoflagellate-viral-nucleoproteins (DVNPs) in place of histones, yet this evolutionary transition is not well understood. Here, Irwin et al. use yeast expressing DVNP to show that DVNP displaces histones and that histone reduction allows cells to cope with DVNP.

We employ targeted mass spectrometry to compare the levels of H2AX S139 phosphorylation (γH2AX) and Y142 phosphorylation. We use synthetic peptides to facilitate MS optimisation and estimate relative detection efficiencies for the different modifications. Despite phosphopeptide enrichment from large amounts of starting material, we are unable to detect endogenous H2AX Y142 phosphorylation, indicating that it is present in low abundance (

ATP-dependent chromatin remodeling enzymes play essential biological roles by mobilizing nucleosomal DNA. Yet how DNA is mobilized despite the steric constraints placed by the histone octamer remains unknown. Here, using methyl transverse-relaxation-optimized NMR spectroscopy on a 450 kDa complex, we show that the chromatin remodeler, SNF2h, distorts the histone octamer. Binding of SNF2h in an activated ATP state changes dynamics of buried histone residues. Preventing octamer distortion by site-specific disulfide linkages inhibits nucleosome sliding by SNF2h, while promoting octamer eviction by the SWI/SNF complex, RSC. Our findings indicate that the histone core of a nucleosome is more plastic than previously imagined and that octamer deformation plays different roles based on the type of chromatin remodeler. Octamer plasticity may contribute to chromatin regulation beyond ATP-dependent remodeling.

Understanding the function of chromatin has become an exciting field that interests researchers from a wide variety of disciplines. Chromatin is composed of fundamental nucleosome building blocks, and the ability to assemble defined, positioned mononucleosomes is central to experimental investigation of chromatin structure, stability and dynamics. However, the molecular principles, historical background and biochemistry of experimental techniques underlying positioned nucleosomes are sometimes unclear. This review provides an introduction from the perspective of the core histone octamer as a surface for histone–DNA interactions that directs a unique conformation for wrapped DNA. The origins and properties of the most widely used nucleosome positioning DNA sequences including the Widom 601, 5S rRNA and MMTV LTR as well as other less well-known examples are described. An overview of the main experimental methods for preparing nucleosomes and mapping their positions is also provided. These should be suitable...

Nucleosomes fulfill the apparently conflicting roles of compacting DNA within eukaryotic genomes while permitting access to regulatory factors. Central to this is their ability to stably associate with DNA while retaining the ability to undergo rearrangements that increase access to the underlying DNA. Here, we have studied different aspects of nucleosome dynamics including nucleosome sliding, histone dimer exchange, and DNA wrapping within nucleosomes. We find that alterations to histone proteins, especially the histone tails and vicinity of the histone H3 alphaN helix, can affect these processes differently, suggesting that they are mechanistically distinct. This raises the possibility that modifications to histone proteins may provide a means of fine-tuning specific aspects of the dynamic properties of nucleosomes to the context in which they are located.

Polycomb-like proteins 1–3 (PCL1–3) are substoichiometric components of the Polycomb-repressive complex 2 (PRC2) that are essential for association of the complex with chromatin. However, it remains unclear why three proteins with such apparent functional redundancy exist in mammals. Here we characterize their divergent roles in both positively and negatively regulating cellular proliferation. We show that while PCL2 and PCL3 are E2F-regulated genes expressed in proliferating cells, PCL1 is a p53 target gene predominantly expressed in quiescent cells. Ectopic expression of any PCL protein recruits PRC2 to repress the INK4A gene; however, only PCL2 and PCL3 confer an INK4A-dependent proliferative advantage. Remarkably, PCL1 has evolved a PRC2- and chromatin-independent function to negatively regulate proliferation. We show that PCL1 binds to and stabilizes p53 to induce cellular quiescence. Moreover, depletion of PCL1 phenocopies the defects in maintaining cellular quiescence associated with p53 loss. This newly evolved function is achieved by the binding of the PCL1 N-terminal PHD domain to the C-terminal domain of p53 through two unique serine residues, which were acquired during recent vertebrate evolution. This study illustrates the functional bifurcation of PCL proteins, which act in both a chromatin-dependent and a chromatin-independent manner to regulate the INK4A and p53 pathways.

The cell contains highly dynamic structures exploiting physical principles of self-organisation at the mesoscale (100 nm to 10 μ m). Examples include non-membrane bound cytoplasmic bodies, cytoskeleton-based motor networks and multi-scale chromatin organisation. The challenges of mesoscale self-organisation were discussed at a CECAM workshop in July 2014. Biologists need approaches to observe highly dynamic, low affinity, low specificity associations and to perturb single structures, while biological physicists and biomathematicians need to work closely with biologists to build and validate quantitative models. A table of terminology is included to facilitate multidisciplinary efforts to reveal the richness and diversity of mesoscale cell biology. Keywords: Workshop report, Self-organisation, Mesoscale, Cell biology, Cytoskeleton, Cytoplasmic bodies, Chromatin

Proteins with sequence similarity to the yeast Snf2 protein form a large family of ATPases that act to alter the structure of a diverse range of DNA-protein structures including chromatin. Snf2 family enzymes are related in sequence to DExx box helicases, yet they do not possess helicase activity. Recent biochemical and structural studies suggest that the mechanism by which these enzymes act involves ATP-dependent translocation on DNA. Crystal structures suggest that these enzymes travel along the minor groove, a process that can generate the torque or energy in remodelling processes. We review the recent structural and biochemical findings which suggest a common mechanistic basis underlies the action of many of both Snf2 family and DExx box helicases.

The Snf2 family of helicase-related proteins includes the catalytic subunits of ATP-dependent chromatin remodelling complexes found in all eukaryotes. These act to regulate the structure and dynamic properties of chromatin and so influence a broad range of nuclear processes. We have exploited progress in genome sequencing to assemble a comprehensive catalogue of over 1300 Snf2 family members. Multiple sequence alignment of the helicase-related regions enables 24 distinct subfamilies to be identified, a considerable expansion over earlier surveys. Where information is known, there is a good correlation between biological or biochemical function and these assignments, suggesting Snf2 family motor domains are tuned for specific tasks. Scanning of complete genomes reveals all eukaryotes contain members of multiple subfamilies, whereas they are less common and not ubiquitous in eubacteria or archaea. The large sample of Snf2 proteins enables additional distinguishing conserved sequence blocks within the helicase-like motor to be identified. The establishment of a phylogeny for Snf2 proteins provides an opportunity to make informed assignments of function, and the identification of conserved motifs provides a framework for understanding the mechanisms by which these proteins function.

Previous studies have identified sin mutations that alleviate the requirement for the yeast SWI/SNF chromatin remodelling complex, which include point changes in the yeast genes encoding core histones. Here we characterise the biochemical properties of nucleosomes bearing these mutations. We find that sin mutant nucleosomes have a high inherent thermal mobility. As the SWI/SNF complex can alter nucleosome positioning, the higher mobility of sin mutant nucleosomes provides a means by which sin mutations may substitute for SWI/SNF function. The location of sin mutations also provides a new opportunity for insights into the mechanism for nucleosome mobilisation. We find that both mutations altering histone DNA contacts at the nucleosome dyad and mutations in the dimer-tetramer interface influence nucleosome mobility. Furthermore, incorporation of H2A.Z into nucleosomes, which also alters dimer-tetramer interactions, affects nucleosome mobility. Thus, variation of histone sequence or subtype provides a means by which eukaryotes may regulate access to chromatin through alterations to nucleosome mobility.

The fundamental subunit of chromatin, the nucleosome, is not a static entity but can move along DNA via either thermal or enzyme-driven movements. Here we have monitored the movements of nucleosomes following deposition at well-defined locations on mouse mammary tumor virus promoter DNA. We found that the sites to which nucleosomes are deposited during chromatin assembly differ from those favored during thermal equilibration. Taking advantage of this, we were able to track the movement of nucleosomes over 156 bp and found that this proceeds via intermediate positions spaced between 46 and 62 bp. The remodeling enzyme ISWI was found to direct the movement of nucleosomes to sites related to those observed during thermal mobilization. In contrast, nucleosome mobilization driven by the SWI/SNF and RSC complexes were found to drive nucleosomes towards sites up to 51 bp beyond DNA ends, with little respect for the sites favored during thermal repositioning. The dynamic properties of nucleosomes we describe are likely to influence their role in gene regulation.

Nucleosomes are the ubiquitous and fundamental packaging for eukaryotic genomes, and are the substrate for many processes in the nucleus. Nucleosomes are not static entities but can readily be moved by thermal energy and ATP-dependent chromatin remodeling complexes in a process known as sliding or shifting. We summarize from a mechanical perspective the twist defect and bulge diffusion mechanisms proposed as the most likely pathway for nucleosome mobilization. We then consider the elastic properties of DNA and how this affects the potential for each mechanism, concentrating on kinetic aspects of twist diffusion and possible planar bulge sizes and summarize the experimental evidence reflecting on each. Either, or both, mechanisms could occur, and careful experimentation focusing on their uniquely distinguishing features will be required to determine their relative contributions to chromatin dynamics.

The ISWI proteins form the catalytic core of a subset of ATP-dependent chromatin-remodeling activities. Here, we studied the interaction of the ISWI protein with nucleosomal substrates. We found that the ability of nucleic acids to bind and stimulate the ATPase activity of ISWI depends on length. We also found that ISWI is able to displace triplex-forming oligonucleotides efficiently when they are introduced at sites close to a nucleosome but successively less efficiently 30 to 60 bp from its edge. The ability of ISWI to direct triplex displacement was specifically impeded by the introduction of 5- or 10-bp gaps in the 3′-5′ strand between the triplex and the nucleosome. In combination, these observations suggest that ISWI is a 3′-5′-strand-specific, ATP-dependent DNA translocase that may be capable of forcing DNA over the surface of nucleosomes.

ATP-dependent chromatin remodeling activities participate in the alteration of chromatin structure during gene regulation. All have DNA- or chromatin-stimulated ATPase activity and many can alter the structure of chromatin; however, the means by which they do this have remained unclear. Here we describe a novel activity for ATP-dependent chromatin remodeling activities, the ability to generate unconstrained negative superhelical torsion in DNA and chromatin. We find that the ability to distort DNA is shared by the yeast SWI/SNF complex, Xenopus Mi-2 complex, recombinant ISWI, and recombinant BRG1, suggesting that the generation of superhelical torsion represents a primary biomechanical activity shared by all Snf2p-related ATPase motors. The generation of superhelical torque provides a potent means by which ATP-dependent chromatin remodeling activities can manipulate chromatin structure.

Gene regulation involves the generation of a local chromatin topology that is conducive to transcription. Several classes of chromatin remodelling activity have been shown to play a role in this process. ATP-dependent chromatin-remodelling activities use energy derived from the hydrolysis of ATP to alter the structure of chromatin, making it more accessible for transcription factor binding. The yeast SWI-SWF complex is the founding member of this family of ATP-dependent chromatin-remodelling activities. We have developed a model system to study the ability of the SWI-SWF complex to alter chromatin structure. Using this system, we find that SWI-SWF is able to alter the position of nucleosomes along the DNA. This is consistent with recent reports that other ATP-dependent chromatin-remodelling activities can alter the positions of nucleosomes along DNA. This suggests that nucleosome mobilization may be a general feature of the activity of ATP-dependent chromatin-remodelling activities. Some of the mechanisms by which nucleosomes may be moved along DNA are discussed.

The generation of a local chromatin topology conducive to transcription is a key step in gene regulation. The yeast SWI/SNF complex is the founding member of a family of ATP-dependent remodelling activities capable of altering chromatin structure both in vitro and in vivo. Despite its importance, the pathway by which the SWI/SNF complex disrupts chromatin structure is unknown. Here we use a model system to demonstrate that the yeast SWI/SNF complex can reposition nucleosomes in an ATP-dependent reaction that favours attachment of the histone octamer to an acceptor site on the same molecule of DNA (in cis). We show that SWI/SNF-mediated displacement of the histone octamer is effectively blocked by a barrier introduced into the DNA, suggesting that this redistribution involves sliding or tracking of nucleosomes along DNA, and that it is achieved by a catalytic mechanism. We conclude that SWI/SNF catalyses the redistribution of nucleosomes along DNA in cis, which may represent a general mechanism by which ATP-dependent chromatin remodelling occurs.

In Xenopus somatic cells histone H1 effects the transcriptional repression of oocyte type 5 S RNA genes, without altering the transcription of the somatic type 5 S RNA genes. Using an unambiguous nucleosome mapping method we find substantial differences between the multiple in vitro nucleosome positions on the two types of genes. These nucleosome positions determine both transcription factor and H1 binding, allowing TFIIIA to bind more efficiently to nucleosomes containing the somatic 5 S RNA gene than to nucleosomes on the oocyte 5 S RNA gene. Significantly, in a binding competition between TFIIIA and H1, TFIIIA preferentially binds to the somatic nucleosome whereas H1 preferentially binds to the oocyte nucleosome, excluding TFIIIA binding. These results strongly suggest that nucleosome positioning plays a key role in the regulation of transcription of 5 S RNA genes and provide a molecular mechanism for the selective repression of the oocyte 5 S RNA genes by H1.

Modulation of eukaryotic gene expression is influenced by the organization of regulatory DNA-elements in chromatin. The mouse mammary tumor virus (MMTV) promoter exhibits regularly positioned nucleosomes that reduce the accessibility of the binding sites for sequence-specific transcription factors, in particular nuclear factor (NF1). Hormonal induction of the MMTV promoter is accompanied by remodeling of the nucleosomal structure, but the biochemical nature of these structural changes is unknown. Using recombinant histones, we have now assembled the MMTV promoter in particles containing either an octamer of the histones H3, H4, H2A and H2B or a tetramer of histones H3 and H4, and have compared the two particles in terms of structure, positioning, and exclusion of transcription factors. Using site-directed hydroxy radicals to map histone locations, two main nucleosome positions are found with dyads at position -107 and at -127. The same two main positions are found for particles containing only the H3/H4 tetramer, showing that the absence of H2A/H2B dimers does not alter positioning. The rotational orientation of the DNA double helix in both types of particles is essentially identical. However, the ends of the nucleosomal DNA as well as its central region are more accessible to cleavage reagents in the tetramer particle than in the octamer particle. In agreement with these structural features, the transcription factors NF1 and OTF1 were able to bind to their cognate sites on the tetramer particle, while they could not gain access to the same sites on the surface of the octamer particle. The DNase I digestion pattern of octamers treated with partially purified SWI/SNF complex from HeLa cells in the presence of ATP is indistinguishable from that of tetramer particles, suggesting that the SWI/SNF complex promotes ATP-dependent remodeling of the octamer particle but not of tetramer particles. These results are compatible with a hormone-induced removal of histone H2A/H2B during MMTV induction.

The four core histone proteins, H2A, H2B, H3, and H4 of Xenopus laevis have been individually expressed in milligram quantities in Escherichia coli. The full-length proteins and the "trypsin-resistant" globular domains were purified under denaturing conditions and folded into histone octamers. Both intact and truncated recombinant octamers, as well as chicken erythrocyte octamer, were assembled into nucleosome core particles using a 146 bp defined-sequence DNA fragment from a 5 S RNA gene. The three types of core particles were characterized and compared by gel electrophoresis, DNase I cleavage, and tyrosine fluorescence emission during stepwise dissociation with increasing ionic strength. Nucleosome core particles containing native and mutant histones made in bacteria have facilitated its X-ray structure determination at 2.8 A resolution.

A base-pair resolution method for determining nucleosome position in vitro has been developed to com- plement existing, less accurate methods. Cysteaminyl EDTA was tethered to a recombinant histone octamer via a mutant histone H4 with serine 47 replaced by cysteine. When assembled into nucleosome core particles, the DNA could be cut site specifically by hydroxyl radical-catalyzed chain scission by using the Fenton reaction. Strand cleavage occurs mainly at a single nucleotide close to the dyad axis of the core particle, and assignment of this location via the symmetry of the nucleosome allows base-pair resolution mapping of the histone octamer position on the DNA. The positions of the histone octamer and H3H4 tetramer were mapped on a 146-bp Lytechinus variegatus 5S rRNA sequence and a twofold-symmetric derivative. The weakness of translational determinants of nucleosome positioning relative to the overall affinity of the histone proteins for this DNA is clearly demonstrated. The predominant location of both histone octamer and H3H4 tetramer assembled on the 5S rDNA is off center. Shifting the nucleosome core particle position along DNA within a conserved rotational phase could be induced under physiologically relevant conditions. Since nucleosome shifting has important consequences for chromatin structure and gene regulation, an approach to the thermodynamic characterization of this movement is proposed. This mapping method is potentially adaptable for determining nucleosome position in chromatin in vivo.

Histone H2AX is a histone variant found in almost all eukaryotes. It makes a central contribution to genome stability through its role in the signaling of DNA damage events and by acting as a foundation for the assembly of repair foci. The H2AX protein sequence is highly similar and in some cases overlapping with replication-dependent canonical H2A, yet the H2AX gene and protein structures exhibit a number of features specific to the role of this histone in DNA repair. The most well known of these is a specific serine at the extreme C-terminus of H2AX which is phosphorylated by Phosphoinositide-3-Kinase-related protein Kinases (PIKKs) to generate the gammaH2AX mark. However, recent studies have demonstrated that phosphorylation, ubiquitylation and other post-translational modifications are also crucial for function. H2AX transcript properties suggest a capability to respond to damage events. Furthermore, the biochemical properties of H2AX protein within the nucleosome structure and its distribution within chromatin also point to features linked to its role in the DNA damage response. In particular, the theoretical inter-nucleosomal spacing of H2AX and the potential implications of amino acid residues distinguishing H2AX from canonical H2A in structure and dynamics are considered in detail. This review summarises current understanding of H2AX from a structure-function perspective.

Histone H2AX is a histone variant found in almost all eukaryotes. It makes a central contribution to genome stability through its role in the signaling of DNA damage events and by acting as a foundation for the assembly of repair foci. The H2AX protein sequence is highly similar and in some cases overlapping with replication-dependent canonical H2A, yet the H2AX gene and protein structures exhibit a number of features specific to the role of this histone in DNA repair. The most well known of these is a specific serine at the extreme C-terminus of H2AX which is phosphorylated by Phosphoinositide-3-Kinase-related protein Kinases (PIKKs) to generate the γH2AX mark. However, recent studies have demonstrated that phosphorylation, ubiquitylation and other post-translational modifications are also crucial for function. H2AX transcript properties suggest a capability to respond to damage events. Furthermore, the biochemical properties of H2AX protein within the nucleosome structure and its distribution within chromatin also point to features linked to its role in the DNA damage response. In particular, the theoretical inter-nucleosomal spacing of H2AX and the potential implications of amino acid residues distinguishing H2AX from canonical H2A in structure and dynamics are considered in detail. This review summarises current understanding of H2AX from a structure–function perspective.

Nucleosomes are the fundamental subunits of eukaryotic chromatin. They are not static entities, but can undergo a number of dynamic transitions, including spontaneous repositioning along DNA. As nucleosomes are spaced close together within genomes, it is likely that on occasion they approach each other and may even collide. Here we have used a dinucleosomal model system to show that the 147-base-pair (bp) DNA territories of two nucleosomes can overlap extensively. In the situation of an overlap by 44 bp or 54 bp, one histone dimer is lost and the resulting complex can condense to form a compact single particle. We propose a pathway in which adjacent nucleosomes promote DNA unraveling as they approach each other and that this permits their 147-bp territories to overlap, and we suggest that these events may represent early steps in a pathway for nucleosome removal via collision.

Alteration of chromatin structure by chromatin modifying and remodelling activities is a key stage in the regulation of many nuclear processes. These activities are frequently interlinked, and many chromatin remodelling enzymes contain motifs that recognise modified histones. Here we adopt a peptide ligation strategy to generate specifically modified chromatin templates and used these to study the interaction of the Chd1, Isw2 and RSC remodelling complexes with differentially acetylated nucleosomes. Specific patterns of histone acetylation are found to alter the rate of chromatin remodelling in different ways. For example, histone H3 lysine 14 acetylation acts to increase recruitment of the RSC complex to nucleosomes. However, histone H4 tetra-acetylation alters the spectrum of remodelled products generated by increasing octamer transfer in trans. In contrast, histone H4 tetra-acetylation was also found to reduce the activity of the Chd1 and Isw2 remodelling enzymes by reducing catalytic turnover without affecting recruitment. These observations illustrate a range of different means by which modifications to histones can influence the action of remodelling enzymes.

The ability to sense and respond appropriately to genetic lesions is vitally important to maintain the integrity of the genome. Emerging evidence indicates that various modulations to chromatin structure are centrally important to many aspects of the DNA damage response (DDR). Here, we discuss recently described roles for specific post-translational covalent modifications to histone proteins, as well as ATP-dependent chromatin remodelling, in DNA damage signalling and repair of DNA double strand breaks.

ISWI proteins form the catalytic core of a subset of ATP-dependent chromatin remodelling activities in eukaryotes from yeast to man. Many of these complexes have been found to reposition nucleosomes, but with different directionalities. We find that the yeast Isw1a, Isw2 and Chd1 enzymes preferentially move nucleosomes towards more central locations on short DNA fragments whereas Isw1b does not. Importantly, the inherent positioning properties of the DNA play an important role in determining where nucleosomes are relocated to by all of these enzymes. However, a key difference is that the Isw1a, Isw2 and Chd1 enzymes are unable to move nucleosomes to positions closer than 15 bp from a DNA end whereas Isw1b can. We also find that there is a correlation between the inability of enzymes to move nucleosomes close to DNA ends and the preferential binding to nucleosomes bearing linker DNA. These observations suggest that the accessibility of linker DNA together with the positioning properties of the underlying DNA play important roles in determining the outcome of remodelling by these enzymes.

ATP-dependent chromatin remodelling enzymes act to alter chromatin structure during gene regulation. Studies of the ATPase motors that drive these enzymes support the notion that they function as ATP-dependent DNA translocases with limited processivity. The action of these enzymes on nucleosomes results in the alteration of nucleosome positioning and structure. Recent studies have shown that ATP-dependent chromatin remodelling can also either remove or exchange histone dimers between nucleosomes. This provides a new means by which the incorporation of histone variants can be directed. Additional observations support roles for ATP-dependent remodelling enzymes throughout the transcription cycle.

Publisher Summary A system for the expression of recombinant histone proteins has been developed and it provides the possibility of manipulating the histone proteins by site-directed mutagenesis. This can be used to study the roles of specific regions of the histone proteins in chromatin function and to introduce amino acids at specific sites within nucleosomes for the attachment of reporter compounds. This chapter describes the use of this approach to attach compounds that facilitate the study of the dynamic properties of chromatin. Recombinant histone technology can also be used to provide a means of attaching fluorescent dyes. Techniques for the detection of fluorescent dyes have increased, enabling detection down to the level of single molecules. To date, the major use of fluorescent technology to study chromatin structure has stemmed from the expression of green or yellow fluorescent protein (GFP or YFP, respectively) histone fusions in vivo. These have provided a powerful system for the study of the dynamic properties of chromatin in vivo .

During the past year, major advances have been made towards understanding the function of ATP-dependent chromatin-remodelling activities both in vitro and in vivo. These suggest that ATP-dependent chromatin-remodelling activities are capable of both altering the structure of individual nucleosomes and acting in concert with other forms of chromatin-modifying enzymes, to regulate the formation and decondensation of chromatin fibres.

Publisher Summary This chapter discusses the base-pair resolution mapping of nucleosome positions using site-directed hydroxy radicals. The site-directed hydroxyl radical method presented in the chapter differs from free-solution hydroxyl radical methods because it localizes the chelation of the hydroxy radical-generating iron center to a specific cysteine residue, thus directing the site of attack of hydroxy radicals. Using the chelating EDTAcyst reagent, which is attached via a disulfide bond to histone H4 residue 47 located near the nucleosome pseudo dyad axis, hydroxy radicals create a single major scission per strand separated by three base pairs on complementary strands. Similar site-directed chelating reagents have also been synthesized, and there are growing numbers of reports of their use in the study of nucleic acid interactions. The chapter describes the assembly of nucleosomes containing a derivatized histone octamer.

Uniquely positioned nucleosomes were mapped in vitro on mouse mammary tumor 3' long terminal repeat (MMTV 3'LTR) DNA at base-pair resolution. Nucleosome A assembly was strongly favored over nucleosome B, and heating of each as a mononucleosome caused migration to the ends of the DNA fragment at a unique rate. Taken together with DNA sequence analysis, this suggests why MMTV 3'LTV nucleosome positions reported upstream of vector-derived sequences conflict and also how flanking genomic sequences could modulate the promoter in in vivo situations. Importantly, nucleosomes are shown to migrate for significant distances along DNA under physiologically relevant conditions, and the actual rates have been measured directly in solution. Exact positioning and shifting over greater than 60 bp has important consequences for transcription factor access to this MMTV promoter and for the role of nucleosomes in general.