Chromatin Research Research Articles

Genome-Wide Prediction of Nucleosome Occupancy in Maize Reveals Plant Chromatin Structural Features at Genes and Other Elements at Multiple Scales

The nucleosome is a fundamental structural and functional chromatin unit that affects nearly all DNA-templated events in eukaryotic genomes. It is also a biochemical substrate for higher order, cis-acting gene expression codes and the monomeric structural unit for chromatin packaging at multiple scales. To predict the nucleosome landscape of a model plant genome, we used a support vector machine computational algorithm trained on human chromatin to predict the nucleosome occupancy likelihood (NOL) across the maize (Zea mays) genome. Experimentally validated NOL plots provide a novel genomic annotation that highlights gene structures, repetitive elements, and chromosome-scale domains likely to reflect regional gene density. We established a new genome browser (http://www.genomaize.org) for viewing support vector machine-based NOL scores. This annotation provides sequence-based comprehensive coverage across the entire genome, including repetitive genomic regions typically excluded from experimental genomics data. We find that transposable elements often displayed family-specific NOL profiles that included distinct regions, especially near their termini, predicted to have strong affinities for nucleosomes. We examined transcription start site consensus NOL plots for maize gene sets and discovered that most maize genes display a typical +1 nucleosome positioning signal just downstream of the start site but not upstream. This overall lack of a -1 nucleosome positioning signal was also predicted by our method for Arabidopsis (Arabidopsis thaliana) genes and verified by additional analysis of previously published Arabidopsis MNase-Seq data, revealing a general feature of plant promoters. Our study advances plant chromatin research by defining the potential contribution of the DNA sequence to observed nucleosome positioning and provides an invariant baseline annotation against which other genomic data can be compared.

Locus-specific biochemical epigenetics/chromatin biochemistry by insertional chromatin immunoprecipitation.

Comprehensive understanding of regulation mechanisms of biological phenomena mediated by functions of genomic DNA requires identification of molecules bound to genomic regions of interest in vivo. However, nonbiased methods to identify molecules bound to specific genomic loci in vivo are limited. To perform biochemical and molecular biological analysis of specific genomic regions, we developed the insertional chromatin immunoprecipitation (iChIP) technology to purify the genomic regions of interest. We applied iChIP to direct identification of components of insulator complexes, which function as boundaries of chromatin domain, showing that it is feasible to directly identify proteins and RNA bound to a specific genomic region in vivo by using iChIP. In addition, recently, we succeeded in identifying proteins and genomic regions interacting with a single copy endogenous locus. In this paper, we will discuss the application of iChIP to epigenetics and chromatin research.

Coal mining meets chromatin research: Digging for mechanisms in epigenetic control of gene expression

AbstractEditor's suggested further reading in BioEssays Epigenetics meets mathematics: Towards a quantitative understanding of chromatin biology Abstract

Assisted reproduction treatment and epigenetic inheritance

BACKGROUNDThe subject of epigenetic risk of assisted reproduction treatment (ART), initiated by reports on an increase of children with the Beckwith–Wiedemann imprinting disorder, is very topical. Hence, there is a growing literature, including mouse studies.METHODSIn order to gain information on transgenerational epigenetic inheritance and epigenetic effects induced by ART, literature databases were searched for papers on this topic using relevant keywords.RESULTSAt the level of genomic imprinting involving CpG methylation, ART-induced epigenetic defects are convincingly observed in mice, especially for placenta, and seem more frequent than in humans. Data generally provide a warning as to the use of ovulation induction and in vitro culture. In human sperm from compromised spermatogenesis, sequence-specific DNA hypomethylation is observed repeatedly. Transmittance of sperm and oocyte DNA methylation defects is possible but, as deduced from the limited data available, largely prevented by selection of gametes for ART and/or non-viability of the resulting embryos. Some evidence indicates that subfertility itself is a risk factor for imprinting diseases. As in mouse, physiological effects from ART are observed in humans.In the human, indications for a broader target for changes in CpG methylation than imprinted DNA sequences alone have been found. In the mouse, a broader range of CpG sequences has not yet been studied. Also, a multigeneration study of systematic ART on epigenetic parameters is lacking.CONCLUSIONSThe field of epigenetic inheritance within the lifespan of an individual and between generations (via mitosis and meiosis, respectively) is growing, driven by the expansion of chromatin research. ART can induce epigenetic variation that might be transmitted to the next generation.

Bone and calcium update; bone research update. Role of steroid hormones in regulation of the epigenesis

Fat-soluble ligands like steroid hormones exert their actions through nuclear receptor-mediated transcriptional controls. Nuclear receptors are hormone-dependent transcription factors, and their ligand-dependent function is facilitated by transcriptional co-regulators, that were initially considered to bridge nuclear receptors with transcription initiation complex. However, recent progress in epigenome and chromatin research has uncovered that hormone-dependent transcriptional controls by nuclear receptors require a number of epigenetic/transcriptional co-regurators facilitating chromatin reorganization.

Antibody recognition of histone post-translational modifications: emerging issues and future prospects

Antibody recognition of histone post-translational modifications: emerging issues and future prospects

Chemical tools in chromatin research

Eukaryotes organize their DNA in the form of chromatin. This complex of DNA and packaging proteins, the histones, ensures that all genomic information fits into the limited space of the cell nucleus. In addition to compacting DNA, chromatin itself regulates the activity of encoded genes. This regulatory process involves many posttranslational modifications of histone proteins and deciphering the complex crosstalk between histone modifications and gene activity represents a central challenge for biomedical science. This task has often been supported by sophisticated chemical tools, which were crucial for many important discoveries in this field. This review provides an overview of chemical tools for chromatin research, with emphasis on classical and current examples of their applications.

Of proteins and DNA—proteomic role in the field of chromatin research

To paraphrase Robert Burns's poem To a Mouse, the best laid schemes of DNA-protein complex purification often go awry. Chromatin with its heterogeneous and dynamic protein composition remains difficult to analyze. Still critical progress has been made in recent years in characterizing the interface between DNA and proteins due, in part, to significant advances in proteomic technologies. Proteomics has progressed to a point where affinity purification of soluble complexes and protein identification by mass spectrometry are routine. The new challenge for chromatin proteomics lies in studying proteins and protein complexes in their native environment, which is on chromatin. These novel types of data represent an additional layer of information that can be used to better characterize and understand cellular processes. This review will focus on the past contributions as well as on emerging mass spectrometry-based methodologies attempting to better define the complex relationship between proteins, protein complexes and DNA.

Epigenome Microarray Platform for Proteome-Wide Dissection of Chromatin-Signaling Networks

Knowledge of protein domains that function as the biological effectors for diverse post-translational modifications of histones is critical for understanding how nuclear and epigenetic programs are established. Indeed, mutations of chromatin effector domains found within several proteins are associated with multiple human pathologies, including cancer and immunodeficiency syndromes. To date, relatively few effector domains have been identified in comparison to the number of modifications present on histone and non-histone proteins. Here we describe the generation and application of human modified peptide microarrays as a platform for high-throughput discovery of chromatin effectors and for epitope-specificity analysis of antibodies commonly utilized in chromatin research. Screening with a library containing a majority of the Royal Family domains present in the human proteome led to the discovery of TDRD7, JMJ2C, and MPP8 as three new modified histone-binding proteins. Thus, we propose that peptide microarray methodologies are a powerful new tool for elucidating molecular interactions at chromatin.

Epigenomics: A Roadmap to Chromatin

In their Letter “Epigenomics: a roadmap, but to where?” (3 October, p. 43), H. D. Madhani et al. applaud the NIH for directing funds towards chromatin research, but argue that the Epigenomics Roadmap initiative ([1][1]) is ill-conceived and diverts funds from investigator- initiated proposals. However, their criticisms are more semantic than scientific, and they ignore the role that technology development has played in driving chromatin research. As recipients of grants awarded in this program, we would like to set the record straight. ![Figure][2] CREDIT: JUPITER IMAGES We agree with Madhani et al. that epigenetic regulation is driven by transcription factor binding. However, studies of such regulatory processes have traditionally received strong NIH support, whereas the Epigenomics Roadmap aims to characterize the chromatin landscape that transcription factors act upon. Unlike transcription factors, which are diverse and often differ between eukaryotic taxa, chromatin components include histone variants and modifications that are essentially universal. Ultimately, transcription factors must act upon DNA packaged by histones, and essentially all eukaryotes use a common set of histone and DNA modifying enzymes, nucleosome remodelers, histone chaperones, and chromatin-binding proteins to facilitate transcription factor and polymerase action. We think that the NIH is justified in limiting this initiative to chromatin, and had some other term than “epigenomics” been used, there would be no basis for this complaint. A more substantive concern is that this initiative diverts funds from investigator-initiated grants, corralling individual scientists “to work together under a more rigid, directed framework.” However, 17 of the 22 grantees aim to develop novel tools and markers for chromatin research. Our three grants are high-risk, high-gain R21s; at $175,000 to $200,000 per year for 2 years, they are among the smallest NIH awards. Although we recognize that these funds might have been diverted from traditional programs of the NIH institutes that fund us (the National Institute on Drug Abuse, the National Institute of Diabetes and Digestive and Kidney Diseases, and the National Institute of Environmental Health Sciences), we believe that they deserve credit, not criticism, for investing in novel technologies for understanding chromatin. 1. 1.[↵][3]NIH Roadmap for Medical Research, Roadmap Initiatives, Epigenomics ( ). [1]: #ref-1 [2]: pending:yes [3]: #xref-ref-1-1 View reference 1. in text

Single‐molecule analysis of chromatin: Changing the view of genomes one molecule at a time

Wrapping DNA into chromatin provides a wealth of regulatory mechanisms that ensure normal growth and development in eukaryotes. Our understanding of chromatin structure, including nucleosomes and non-histone protein-DNA interactions, has benefited immensely from nuclease and chemical digestion techniques. DNA-bound proteins, such as histones or site-specific factors, protect DNA against nuclease cleavage and generate large nucleosomal or small regulatory factor footprints. Chromatin subject to distinct modes of regulation often coincides with sites of nuclease hypersensitivity or nucleosome positioning. An inherent limitation of cleavage-based analyses has been the inability to reliably analyze regions of interest when levels of digestion depart from single-hit kinetics. Moreover, cleavage-based techniques provide views that are averaged over all the molecules in a sample population. Therefore, in cases of occupancy of multiple regulatory elements by factors, one cannot define whether the factors are bound to the same or different molecules in the population. The recent development of DNA methyltransferase-based, single-molecule MAP-IT technology overcomes limitations of ensemble approaches and has opened numerous new avenues in chromatin research. Here, we review the strengths, limitations, applications and future prospects of MAP-IT ranging from structural issues to mechanistic questions in eukaryotic chromatin regulation.

Primers on chromatin

To accompany the Focus on Chromatin appearing in this issue of Nature Structural & Molecular Biology, a series of primers has been specially prepared that covers the wealth of knowledge in four areas of chromatin research. These areas include functions associated with covalent histone modifications, the enzymes that mediate these modifications, modules that recognize chromatin, and the ATP-dependent chromatin-remodeling complexes. In such a complex field, the information has inevitably been somewhat simplified. As an example, the correlation between modifications and functions are often context dependent. For instance, H3K9 methylation has been associated with transcriptional activation when present in the coding region of the gene, but has also been associated with repression. The reference list provides further reading and details, as do the Reviews and Perspective in this issue. Although there are many informative structures in this field, space constraints allowed only representative structures to be shown, followed by reference citations for related structures ('3D REF' column). The primers can be used as a stand-alone resource--feel free to tear them out of the issue or print out the PDF versions and modify or add to them yourself as new data emerge. The online versions of the primers contain hyperlinks to the Protein Data Bank as well as 3D view links that allow structural visualization.

Chromatin is wonderful stuff

Chromatin molecules have properties that set them aside from all other biomacromolecules in the cell. (i) Chromosomes, which are single chromatin molecules, are the largest macromolecules in eukaryotic cells. (ii) Chromatin molecules carry the cell's genetic and epigenetic information and all control elements that regulate and, importantly, orchestrate expression of tenths of thousands of genes. (iii) Chromatin fibers are extremely highly folded inside the cell nucleus. Moreover, chromatin folding varies considerably from cell to cell, even in otherwise identical cells. Despite this, chromatin can be accurately duplicated and faithfully separated over the two daughter cells during cell division. (iv) The highly folded chromatin fiber acts as a scaffold that spatially organizes processes inside the nucleus, such as transcription and replication. This chromatin-based compartmentalization of the nucleus is a key element in the regulation of genome function. (v) Chromatin-associated processes often are single molecule processes in which proteins that freely diffuse inside the nucleus have to find and assemble on unique sequence elements, e.g. a specific promoter, of the chromatin fiber. (vi) The structure of chromatin shows remarkable plasticity. Depending on regulatory signals its chemical structure changes continuously (e.g. due to histone modifications), its protein composition alters (e.g. incorporation of variant histones and the assembly and disassembly of transcription complexes) and folding inside the nucleus may change dramatically (e.g. depending on epigenetic changes and changes in transcriptional activity). These properties all contribute to and probably are essential for proper functioning of the eukaryotic genome. Research aiming to understand chromatin properties and unravel underlying physical principles and molecular mechanisms is hampered by the fact that we have no tools to directly visualize and analyze the chromatin fiber inside the cell. Also, chromatin structure in part depends on weak interactions that are lost upon opening the cell, making biochemical approaches difficult. To unravel chromatin-related enigmas we have to rely on combining a broad range of strategies and techniques. This special issue addresses a number of important aspects of chromatin research that are not yet encountered often in the main stream scientific literature. In the first paper Van Holde and Zlatanova [1] discuss our present understanding of what probably is a basic folding state of chromatin, the 30 nm fiber. One way to unravel principles of chromatin folding is the development of predictive computational models, which define precise questions that can be addressed experimentally. Langowski and Heermann [2] present a sample of this by modeling the nucleosomal fiber and analyzing its properties, such as flexibility as a function of linker histone H1 binding. Hancock [3] discusses an often overlooked aspect of biological organization in general and chromatin structure in particular: macromolecular crowding. It is made highly plausible that crowding phenomena are a key element in defining chromatin structure in vivo. The paper of Fakan and Van Driel [4] discusses an intriguing aspect of spatial organization of our genome. Transcription and replication occur exclusively at the surface of compact subchromosomal chromatin domains, which poses major constrains to the way the chromatin fiber is folded inside the interphase nucleus. Dorman et al. [5] E.R. Dorman, A.M. Bushey and V.G. Corces, The role of insulator elements in large-scale chromatin structure in interphase, Sem Cell Dev Biol 18 (2007), pp. 682-690. Article | PDF (1131 K) | View Record in Scopus | Cited By in Scopus (17)[5] summarize our understanding of the role of insulator elements in the genome in the spatial organization of the chromatin fiber. Insulator sequences may constitute a major class of chromatin-chromatin interactions. Sexton et al. [6] describe another important type of interchromatin interactions: transcription factories. In this type of structures transcriptionally active genes from different part of the chromatin fiber and from different chromosomes come together in the interphase nucleus, imposing further constrains on the large-scale folding pattern. Chuang and Belmont [7] address the issue of directed movement of parts of the chromatin fiber in relation to compartmentalization of the interphase nucleus. Finally, Goetze et al. [8] discuss various aspects of the large-scale organization of the chromatin fiber in mammalian cells. Together, these papers give a good overview of the different ways structure-function relationships of the chromatin fiber are addressed and summarize the main questions that we have to deal with in the forthcoming years.

Engineered apoptotic nucleases for chromatin research

We have created new genomics tools for chromatin research by genetically engineering the human and mouse major apoptotic nucleases that are responsible for internucleosomal DNA cleavage, DNA fragmentation factor (DFF). Normally, in its inactive form, DFF is a heterodimer composed of a 45-kDa chaperone inhibitor subunit (DFF45 or ICAD), and a 40-kDa latent endonuclease subunit (DFF40 or CAD). Upon caspase-3 cleavage of DFF45, DFF40 forms active endonuclease homo-oligomers. Although Saccharomyces cerevisiae lacks DFF, expression of caspase-3 is lethal in this organism, but expression of the highly sequence-specific tobacco etch virus protease (TEVP) is harmless. Therefore, we inserted TEVP cleavage sites immediately downstream of the two caspase-3 cleavage sites within DFF45, generating a novel form of DFF (DFF-T) whose nuclease activity proved to be exclusively under the control of TEVP. We demonstrate that co-expression of TEVP and DFF-T under galactose control results in nucleosomal DNA laddering and cell death in S. cerevisiae. We also created synthetic DFF genes with optimized codons for high-level expression in Eschericia coli or S. cerevisiae. We further demonstrate the excellence of the synthetic gene products for in vitro mapping of the nucleosome positions and hypersensitive sites in specific genes such as the yeast PHO5.

Deciphering the histone code using mass spectrometry

During the past decade, studies surrounding chromatin research have grown exponentially. A major focus of chromatin biology is centered on understanding of how histone modifications alter chromatin structure at the molecular and mechanistic levels. Discoveries are being made at a rapid pace due to the advent of new and innovative techniques. Mass spectrometry has emerged as a powerful tool in the field of histone research due to its speed, sensitivity, and ease of use. This has resulted in the identification of a number of novel histone modification sites. In consequence, new roles in biological processes have been discovered and hypothetical models, such as the ‘histone code’ have been reaffirmed or refined. One significant advantage to using mass spectrometric techniques is that the combinations of modifications on different sites can be determined which is crucial to deciphering the ‘histone code’. In this manuscript, the mass spectrometric approaches developed over the past decade for both qualitative and quantitative analysis of histone post-translational modifications (PTMs) are discussed.

Unique features of the apoptotic endonuclease DFF40/CAD relative to micrococcal nuclease as a structural probe for chromatinThis paper is one of a selection of papers published in this Special Issue, entitled 27th International West Coast Chromatin and Chromosome Conference, and has undergone the Journal's usual peer review process.

The gold standard for studies of nucleosomal chromatin structure for the past 30 years has been the enzyme micrococcal nuclease (MNase). During the course of our studies on the elucidation of the mechanism of action of the apoptotic nuclease DNA fragmentation factor-40 / caspase-activated deoxyribonuclease (DFF40/CAD) on naked DNA and chromatin substrates, it became clear that this enzyme is superior in certain respects to MNase for studying several aspects of chromatin structure. Here we review our published results supporting this statement. Relative to MNase, we have found that DFF40/CAD has the following properties: (i) it does not cut within nucleosomes to generate subnucleosomal DNA fragments; (ii) it is more specific for the linker regions between nucleosomes; (iii) it lacks exonuclease activity; (iv) it is specific for double-stranded DNA and makes exclusively double-stranded breaks; and (v) it attacks histone-H1-containing chromatin more efficiently. Taken together, these facts explain why DFF40/CAD generates sharper oligonucleosomal DNA ladders compared with those generated by MNase. We therefore recommend the following uses for DFF40/CAD for chromatin research: nucleosome isolation, chromatin-remodeling assays, repeat length measurements, and nucleosome-positioning assays along specific sequences. Other uses include footprinting assays of transcription factor positions, shearing chromatin for immunopreciptitation experiments (ChIP), and shearing DNA for recombinant DNA library preparation or for shotgun cloning for sequencing.

Histone variants--the structure behind the function

In recent years, the chromatin field has witnessed a renewed interest in histone variants as pertaining to their structural role, but mainly because of the functional specificity they impart to chromatin. In this review, I am going to discuss several of the most recent structural studies on core histone (H2A.Bbd, H2A.Z, H2A.X, macroH2A, H3.3, CENP-A) and linker histone variants (histone H1 microheterogeneity) focusing on their role in nucleosome stability and chromatin fibre dynamics with special emphasis on their possible functional implications. The data accumulated to date indicates that histone variability plays an important role in the histone-mediated regulation of chromatin metabolism. Understanding and deciphering the underlying structural amino acid code behind such variability remains one of the most exciting future challenges in chromatin research.

Susan Gasser

Susan Gasser

H2AX: tailoring histone H2A for chromatin-dependent genomic integrity

During the last decade, chromatin research has been focusing on the role of histone variability as a modulator of chromatin structure and function. Histone variability can be the result of either post-translational modifications or intrinsic variation at the primary structure level: histone variants. In this review, we center our attention on one of the most extensively characterized of such histone variants in recent years, histone H2AX. The molecular phylogeny of this variant seems to have run in parallel with that of the major canonical somatic H2A1 in eukaryotes. Functionally, H2AX appears to be mainly associated with maintaining the genome integrity by participating in the repair of the double-stranded DNA breaks exogenously introduced by environmental damage (ionizing radiation, chemicals) or in the process of homologous recombination during meiosis. At the structural level, these processes involve the phosphorylation of serine at the SQE motif, which is present at the very end of the C-terminal domain of H2AX, and possibly other PTMs, some of which have recently started to be defined. We discuss a model to account for how these H2AX PTMs in conjunction with chromatin remodeling complexes (such as INO80 and SWRI) can modify chromatin structure (remodeling) to support the DNA unraveling ultimately required for DNA repair.

Histone Deacetylase Inhibitors and Cancer Therapy

Cancer drug development has moved from conventional cytotoxic chemotherapeutics to a more mechanism-based targeted approach towards the common goal of tumour growth arrest. The rapid progress in chromatin research and understanding epigenetic control has supplied a plethora of potential targets for intervention in cancer. Histone deacetylases (HDACs) have been widely implicated in growth and transcriptional control, and inhibition of HDAC activity using small molecules causes apoptosis in tumour cells. Here, we review HDAC inhibitors, together with their current status of clinical development and potential utility in cancer therapy.