Doxorubicin is one of the most important anti-cancer chemotherapeutic drugs, being widely used for the treatment of solid tumors and acute leukemias. The action of doxorubicin and other anthracycline drugs has been intensively investigated during the last several decades, but the mechanisms that have been proposed for cell killing remain disparate and controversial. In this review, we examine the proposed models for doxorubicin action from the perspective of the chromatin landscape, which is altered in many types of cancer due to recurrent mutations in chromatin modifiers. We highlight recent evidence for effects of anthracyclines on DNA torsion and chromatin dynamics that may underlie basic mechanisms of doxorubicin-mediated cell death and suggest new therapeutic strategies for cancer treatment.
doxorubicin; anthracycline; cancer; DNA torsion; chromatin dynamics; chemotherapy
Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. Formaldehyde cross-linking and sonication followed by Chromatin ImmunoPrecipitation (X-ChIP) is widely used for profiling of TF binding, but is limited by low resolution and poor specificity and sensitivity. We present a simple protocol that starts with micrococcal nuclease-digested uncross-linked chromatin and is followed by affinity purification of TFs and paired-end sequencing. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of Saccharomyces cerevisiae Abf1 and Reb1 provide highly accurate base-pair resolution maps that are not biased toward accessible chromatin, and do not require input normalization. We also demonstrate the high specificity of our method when applied to larger genomes by profiling Drosophila melanogaster GAGA Factor and Pipsqueak. Our results suggest that ORGANIC profiling is a widely applicable high-resolution method for sensitive and specific profiling of direct protein-DNA interactions.
chromatin immunoprecipitation; ChIP; native; Abf1; Reb1; GAGA factor; Pipsqueak
As RNA Polymerase II (Pol II) transcribes a gene, it encounters an array of well-ordered nucleosomes. How it traverses through this array in vivo remains unresolved. One model proposes that torsional stress generated during transcription destabilizes nucleosomes ahead of Pol II. Here, we describe a method for high resolution mapping of underwound DNA using next-generation sequencing, and show that torsion is correlated with gene expression in Drosophila melanogaster cells. Accumulation of torsional stress, through topoisomerase inhibition, results in increased. Pol II at transcription start sites. Whereas Topo I inhibition results in increased nascent RNA transcripts, Topo II inhibition shows little change. Despite the different effects on Pol II elongation, topoisomerase inhibition results in increased nucleosome turnover and salt solubility within gene bodies, suggesting that the elongation-independent effects of torsional stress on nucleosome dynamics contributes to the destabilization of nucleosomes.
torsional stress; nucleosome turnover; nascent RNA; topoisomerase
Sixty years after Watson and Crick published the double helix model of DNA's structure, thirteen members of Genome Biology's Editorial Board select key advances in the field of genome biology subsequent to that discovery.
RNA polymerase II (PolII) transcribes RNA within a chromatin context, with nucleosomes acting as barriers to transcription. Despite these barriers, transcription through chromatin in vivo is highly efficient, suggesting the existence of factors that overcome this obstacle. To increase the resolution obtained by standard chromatin immunoprecipitation, we developed a novel strategy using micrococcal nuclease digestion of cross-linked chromatin. We find that the chromatin remodeler Chd1 is recruited to promoter proximal nucleosomes of genes undergoing active transcription, where Chd1 is responsible for the vast majority of PolII-directed nucleosome turnover. The expression of a dominant negative form of Chd1 results in increased stalling of PolII past the entry site of the promoter proximal nucleosomes. We find that Chd1 evicts nucleosomes downstream of the promoter in order to overcome the nucleosomal barrier and enable PolII promoter escape, thus providing mechanistic insight into the role of Chd1 in transcription and pluripotency.
DNA is tightly packaged in a material called chromatin inside the cell nucleus. To produce proteins this DNA must first be transcribed to produce a molecule of messenger RNA, which is then translated to make a protein. To assist with this process cells ‘unpack’ certain regions of the DNA so that enzymes that catalyze the different steps in this process can have access to the DNA.
A protein called Chd1 is involved in the unpacking process in yeast, but its role in more complex animals is not clear. Now, Skene et al. have shown that this protein is needed to allow the enzyme that catalyzes the transcription of DNA—an enzyme called RNA polymerase II—to do its job. Chd1 acts to unpack the tightly packaged DNA from chromatin, thus allowing the transcription of the DNA to proceed. In the absence of Chd1 activity, RNA polymerase II stalls at the gene promoter—the region of DNA that starts the transcription of a particular gene. This work highlights how the packaging of DNA in the cell is highly dynamic and controls fundamental biological processes.
Skene et al. modified a well-known genetic technique called ChIP-seq. Previous ChIP-seq protocols typically provided a blurry, low-resolution map of where proteins bound to chromatin. Skene et al. used an enzyme to ‘chew back’ the DNA to reveal the exact ‘footprints’ of the Chd1 protein and the RNA polymerase II enzyme on the chromatin in mice. It will be possible to adapt this new protocol to map the positions of other proteins, which will help to improve our understanding of the ways in which chromatin regulates access to DNA.
transcription; chromatin; RNA polymerase II; promoter escape; mouse
In budding yeast, a single cenH3 (Cse4) nucleosome occupies the ∼120-bp functional centromere, however conflicting structural models for the particle have been proposed. To resolve this controversy, we have applied H4S47C-anchored cleavage mapping, which reveals the precise position of histone H4 in every nucleosome in the genome. We find that cleavage patterns at centromeres are unique within the genome and are incompatible with symmetrical structures, including octameric nucleosomes and (Cse4/H4)2 tetrasomes. Centromere cleavage patterns are compatible with a precisely positioned core structure, one in which each of the 16 yeast centromeres is occupied by oppositely oriented Cse4/H4/H2A/H2B hemisomes in two rotational phases within the population. Centromere-specific hemisomes are also inferred from distances observed between closely-spaced H4 cleavages, as predicted from structural modeling. Our results indicate that the orientation and rotational position of the stable hemisome at each yeast centromere is not specified by the functional centromere sequence.
DNA is tightly packaged in cells for a variety of reasons—to allow it to fit inside the nucleus, to protect it from damage, and to help control the production of proteins from genes. The basic unit of packaged DNA is called a nucleosome, which consists of DNA wrapped around a structure formed by two pairs of four different proteins.
These proteins, which are called histones, have a role that extends beyond providing structural support for DNA. When cells divide, for example, pairs of ‘sister chromosomes’ are pulled apart to ensure that the two daughter cells both have the same chromosomes as the original cell. The sister chromosomes are pulled apart from a single position called a centromere, and the nucleosomes at this position contain a histone that is different from the histones found everywhere else in the cell. However, until recently it was not clear if the nucleosomes that contained these special cenH3 histones had the same structure as other nucleosomes.
Now Henikoff et al. have used a method called H4S47C-anchored cleavage mapping to study every nucleosome in the genome of the yeast S. cerevisiae. This mapping technique uses DNA sequencing to measure the precise distances between fixed points on the DNA in the nucleosome. Knowing these distances tells researchers a great deal about the number and position of the histones within each nucleosome in the genome.
Using this approach, Henikoff et al. found that nucleosomes at centromeres are different from other nucleosomes in histone number and arrangement. In particular, the nucleosome at each yeast centromere contains only one each of the four different histones in an asymmetrical orientation, in contrast to all other yeast nucleosomes, which contain two sets of four histones in a symmetrical arrangement. Furthermore, each nucleosome at a centromere can adopt one of two orientations: these orientations are mirror images of each other, and they occur with equal probability. It should also be possible to use the mapping technique developed by Henikoff et al. to study the larger and more complex centromeres found in other organisms, including humans.
centromeres; nucleosome; chemical cleavage mapping; S. cerevisiae
Centromeres vary greatly in size and sequence composition, ranging from ‘point’ centromeres with a single cenH3-containing nucleosome to ‘regional’ centromeres embedded in tandemly repeated sequences to holocentromeres that extend along the length of entire chromosomes. Point centromeres are defined by sequence, whereas regional and holocentromeres are epigenetically defined by the location of cenH3-containing nucleosomes. In this study, we show that Caenorhabditis elegans holocentromeres are organized as dispersed but discretely localized point centromeres, each forming a single cenH3-containing nucleosome. These centromeric sites co-localize with kinetochore components, and their occupancy is dependent on the cenH3 loading machinery. These sites coincide with non-specific binding sites for multiple transcription factors (‘HOT’ sites), which become occupied when cenH3 is lost. Our results show that the point centromere is the basic unit of holocentric organization in support of the classical polycentric model for holocentromeres, and provide a mechanistic basis for understanding how centromeric chromatin might be maintained.
During cell division, the chromosomes in the original cell must be replicated and these ‘sister chromosomes’ must then be divided equally between the two new daughter cells. At first, the sister chromosomes are held together near a region called the centromere, which is important because the microtubules that pull the sister chromosomes apart attach themselves to the centromere. In many cases, the centromere is a small region near the middle of the chromosomes, which produces a classic X shape. However, in some organisms centromeres span the entire length of the chromosomes. There are at least 13 plant and animal lineages with such holocentromeres.
Inside the nucleus of cells, DNA is wrapped around molecules called histones. There are five major families of histones, and histones belonging to one of these families—the H3 histones—are replaced by cenH3 variant histones at both conventional centromeres and holocentromeres. There are many unanswered questions about holocentromeres. In particular, do holocentromeres truly extend along the full length of the chromosomes, or are they found at a large number of specific sites?
Now Steiner and Henikoff have studied the distribution of cenH3 in the genome of the worm C. elegans to investigate holocentromeres in greater detail. These experiments showed that the holocentromere in C. elegans is actually made of about 700 individual centromeric sites distributed along the length of the chromosomes. Each of these sites contains just one nucleosome that contains cenH3, and these sites are likely to be the sites that microtubules attach to during cell division. Surprisingly, the same sites can also act as so-called ‘HOT–sites’: these sites are bound by many proteins that are involved in regulating the process by which genes are expressed as proteins, which suggests a link between centromeres and these regulatory proteins.
The work of Steiner and Henikoff describes how centromeric nucleosomes are distributed across the genome, but why and how cenH3 ends up at these particular 700 sites remains an open question.
CenH3; CENP-A; holocentromere; point centromere; transcription factor hotspots; C. elegans
Repeat sequences are abundant in eukaryotic genomes but many are excluded from genome assemblies. In Drosophila melanogaster classical studies of repeat content suggested variability between individuals, but they lacked the precision of modern high throughput sequencing technologies. Genome-wide profiling of chromatin features such as histone tail modifications and DNA-binding proteins relies on alignment to the reference genome and hence excludes highly repetitive sequences.
By analyzing repeat libraries, sequence complexity and k-mer counts we determined the abundances of different D. melanogaster repeat classes in flies in two public datasets, DGRP and modENCODE. We found that larval DNA was depleted of all repeat classes relative to adult and embryonic DNA, as expected from the known depletion of repeat-rich pericentromeric regions during polytenization of larval tissues. By applying a method that is independent of alignment to the genome assembly, we found that satellite repeats associate with distinct H3 tail modifications, such as H3K9me2 and H3K9me3 for short repeats and H3K9me1 for 359 bp repeats. Short AT-rich repeats however are depleted of nucleosomes and hence all histone modifications and associated chromatin proteins.
The total repeat content and association of repeat sequences with chromatin modifications can be determined despite repeats being excluded from genome assemblies, revealing unexpected distinctions in chromatin features based on sequence composition.
DNA satellites; Next-generation sequencing; ChIP-seq; Histone modification
The Swi2/Snf2 family ATPase Mot1 displaces TATA-binding protein (TBP) from DNA in vitro, but the global relationship between Mot1 and TBP in vivo is unclear. In particular, how Mot1 activates transcription is poorly understood. To address these issues, we mapped the distribution of Mot1 and TBP on native chromatin at base pair resolution. Mot1 and TBP binding sites coincide throughout the genome, and depletion of TBP results in a global decrease in Mot1 binding. We find evidence that Mot1 approaches TBP from the upstream direction, consistent with its in vitro mode of action. Strikingly, inactivation of Mot1 leads to both increases and decreases in TBP-genome association. Sites of TBP gain tend to contain robust TATA boxes, while sites of TBP loss contain poly(dA-dT) tracts that may contribute to nucleosome exclusion. Sites of TBP gain are associated with increased gene expression, while decreased TBP binding is associated with reduced gene expression. We propose that the action of Mot1 is required to clear TBP from intrinsically preferred (TATA-containing) binding sites, ensuring sufficient soluble TBP to bind intrinsically disfavored (TATA-less) sites.
Doxorubicin is an anthracycline DNA intercalator that is among the most commonly used anti-cancer drugs . Doxorubicin causes DNA double-strand breaks in rapidly dividing cells, although whether it also affects general chromatin properties is unknown. Here, we use a metabolic labeling strategy to directly measure nucleosome turnover  to examine the effect of doxorubicin on chromatin dynamics in squamous cell carcinoma cell lines derived from genetically defined mice. We find that doxorubicin enhances nucleosome turnover around gene promoters, and turnover correlates with gene expression level. Consistent with a direct action of doxorubicin, enhancement of nucleosome turnover around promoters gradually increases with time of exposure to the drug. Interestingly, enhancement occurs both in wild-type cells and in cells lacking either the p53 tumor suppressor gene or the master regulator of the DNA damage response, Atm, suggesting that doxorubicin action on nucleosome dynamics is independent of the DNA damage checkpoint. In addition, another anthracycline drug, aclarubicin, shows similar effects on enhancing nucleosome turnover around promoters. Our results suggest that anthracycline intercalation promotes nucleosome turnover around promoters by its effect on DNA topology, with possible implications for mechanisms of cell killing during cancer chemotherapy.
CATCH-IT; Squamous cell carcinoma; p53; Atm
Epigenomics, the determination of epigenetic landscapes on a genome-wide scale, has progressed at an astounding rate over the past decade. Recent technological developments have enabled base-pair resolution of various epigenomic features, leading to new insights into epigenetic regulation.
epigenomics; chromatin immunoprecipitation; ChIP-exo; micrococcal nuclease mapping; deoxyribonuclease I mapping
The structure of nucleosomes that contain the cenH3 histone variant has been controversial. In budding yeast, a single right-handed cenH3/H4/H2A/H2B tetramer wraps the ∼80-bp Centromere DNA Element II (CDE II) sequence of each centromere into a ‘hemisome’. However, attempts to reconstitute cenH3 particles in vitro have yielded exclusively ‘octasomes’, which are observed in vivo on chromosome arms only when Cse4 (yeast cenH3) is overproduced. Here, we show that Cse4 octamers remain intact under conditions of low salt and urea that dissociate H3 octamers. However, particles consisting of two DNA duplexes wrapped around a Cse4 octamer and separated by a gap efficiently split into hemisomes. Hemisome dimensions were confirmed using a calibrated gel-shift assay and atomic force microscopy, and their identity as tightly wrapped particles was demonstrated by gelFRET. Surprisingly, Cse4 hemisomes were stable in 4 M urea. Stable Cse4 hemisomes could be reconstituted using either full-length or tailless histones and with a 78-bp CDEII segment, which is predicted to be exceptionally stiff. We propose that CDEII DNA stiffness evolved to favor Cse4 hemisome over octasome formation. The precise correspondence between Cse4 hemisomes resident on CDEII in vivo and reconstituted on CDEII in vitro without any other factors implies that CDEII is sufficient for hemisome assembly.
ATP-dependent nucleosome remodelers influence genetic processes by altering nucleosome occupancy, positioning, and composition. In vitro, Saccharomyces cerevisiae ISWI and CHD remodelers require ∼30–85 bp of extranucleosomal DNA to reposition nucleosomes, but linker DNA in S. cerevisiae averages <20 bp. To address this discrepancy between in vitro and in vivo observations, we have mapped the genomic distributions of the yeast Isw1, Isw2, and Chd1 remodelers at base-pair resolution on native chromatin. Although these remodelers act in gene bodies, we find that they are also highly enriched at nucleosome-depleted regions (NDRs), where they bind to extended regions of DNA adjacent to particular transcription factors. Surprisingly, catalytically inactive remodelers show similar binding patterns. We find that remodeler occupancy at NDRs and gene bodies is associated with nucleosome turnover and transcriptional elongation rate, suggesting that remodelers act on regions of transient nucleosome unwrapping or depletion within gene bodies subsequent to transcriptional elongation.
Eukaryotic genomes are compacted into chromatin, which restricts access to DNA. In order for cells to transcribe, replicate, and repair DNA, chromatin structure must be altered. Eukaryotes have evolved chromatin remodeling enzymes that use energy derived from ATP hydrolysis to modulate chromatin structure. In vitro, yeast ISWI and CHD remodelers require 30–85 bp of extranucleosomal DNA in order to efficiently remodel chromatin, but in vivo, yeast linker DNA is, on average, <20 bp. By mapping yeast Isw1, Isw2, and Chd1 on native chromatin, we find that these remodelers bind to extended regions of linker DNA adjacent to transcription factor binding sites within nucleosome depleted regions. Remodeler binding is associated with nucleosome turnover and transcription rate, suggesting that ISWI and CHD remodelers help to reestablish proper chromatin structure following transcriptional elongation.
On 11 to 13 March 2013, BioMed Central will be hosting its inaugural conference, Epigenetics & Chromatin: Interactions and Processes, at Harvard Medical School, Cambridge, MA, USA. Epigenetics & Chromatin has now launched a special article series based on the general themes of the conference.
A half century after John Gurdon demonstrated nuclear reprogramming, for which he was awarded the 2012 Nobel Prize in Physiology or Medicine, his group provides insights into the molecular mechanisms whereby chromatin remodeling is required for nuclear reprogramming. Among the issues addressed in Gurdon's latest work are the chromatin impediments to artificially induced reprogramming, discovered by Shinya Yamanaka, who shared the award with Gurdon.
See research article: http://www.epigeneticsandchromatin.com/content/5/1/17
Histone variants are non-allelic protein isoforms that play key roles in diversifying chromatin structure. The known number of such variants has greatly increased in recent years, but the lack of naming conventions for them has led to a variety of naming styles, multiple synonyms and misleading homographs that obscure variant relationships and complicate database searches. We propose here a unified nomenclature for variants of all five classes of histones that uses consistent but flexible naming conventions to produce names that are informative and readily searchable. The nomenclature builds on historical usage and incorporates phylogenetic relationships, which are strong predictors of structure and function. A key feature is the consistent use of punctuation to represent phylogenetic divergence, making explicit the relationships among variant subtypes that have previously been implicit or unclear. We recommend that by default new histone variants be named with organism-specific paralog-number suffixes that lack phylogenetic implication, while letter suffixes be reserved for structurally distinct clades of variants. For clarity and searchability, we encourage the use of descriptors that are separate from the phylogeny-based variant name to indicate developmental and other properties of variants that may be independent of structure.
The Sorting Intolerant from Tolerant (SIFT) algorithm predicts the effect of coding variants on protein function. It was first introduced in 2001, with a corresponding website that provides users with predictions on their variants. Since its release, SIFT has become one of the standard tools for characterizing missense variation. We have updated SIFT’s genome-wide prediction tool since our last publication in 2009, and added new features to the insertion/deletion (indel) tool. We also show accuracy metrics on independent data sets. The original developers have hosted the SIFT web server at FHCRC, JCVI and the web server is currently located at BII. The URL is http://sift-dna.org (24 May 2012, date last accessed).
The centromere is a defining feature of the eukaryotic chromosome, required for attachment to spindle microtubules and segregation to the poles at both mitosis and meiosis. The fundamental unit of centromere identity is the centromere-specific nucleosome, in which the centromeric histone 3 (cenH3) variant takes the place of H3. The structure of the cenH3 nucleosome has been the subject of controversy, as mutually exclusive models have been proposed, including conventional and unconventional left-handed octamers (octasomes), hexamers with non-histone protein constituents, and right-handed heterotypic tetramers (hemisomes). Hemisomes have been isolated from native centromeric chromatin, but traditional nucleosome assembly protocols have generally yielded partially unwrapped left-handed octameric nucleosomes. In budding yeast, topology analysis and high-resolution mapping has revealed that a single right-handed cenH3 hemisome occupies the ~80-bp Centromere DNA Element II (CDEII) of each chromosome. Overproduction of cenH3 leads to promiscuous low-level incorporation of octasome-sized particles throughout the yeast genome. We propose that the right-handed cenH3 hemisome is the universal unit of centromeric chromatin, and that the inherent instability of partially unwrapped left-handed cenH3 octamers is an adaptation to prevent formation of neocentromeres on chromosome arms.
The “point” centromere of budding yeast is genetically defined by an ∼125-bp sequence. Recent fluorescence measurements of kinetochore clusters have suggested that this sequence specifies multiple centromere histone 3 (CenH3) nucleosomes. However, high-resolution mapping demonstrates that there is only one CenH3 nucleosome per centromere, providing biochemical confirmation of the point centromere model.
To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.
Genomes are packaged by complexing DNA with histone proteins, which provides an opportunity to regulate gene expression by dynamically impeding access of transcriptional regulatory proteins and RNA polymerases to DNA. The incorporation of histone variants into nucleosomes and addition of post-translational modifications to histones can alter the physical properties of nucleosomes and thereby serve as a mechanism for regulating DNA exposure. Chromatin-based gene regulation has profound effects on developmental processes including regulation of the vegetative to reproductive transition, as well as responses to pathogens and abiotic factors. Incorporation of the histone variant H2A.Z and methylation of histone H3 lysine residues 4 and 27 have emerged as key elements in the regulation of genes involved in each of these processes.
Differential expression of maternally and paternally inherited alleles of a gene is referred to as gene imprinting, a form of epigenetic gene regulation common to flowering plants and mammals. In plants, imprinting primarily occurs in the endosperm, a seed tissue that supports the embryo during its growth and development. Previously, we demonstrated that widespread DNA demethylation at remnants of transposable elements accompanies endosperm development and that a subset of these methylation changes are associated with gene imprinting. Here we assay imprinted gene expression genome-wide by performing high-throughput sequencing of RNA derived from seeds of reciprocal intraspecific crosses. We identify more than 200 loci that exhibit parent-of-origin effects on gene expression in the endosperm, including a large number of transcription factors, hormone biosynthesis and response genes, and genes that encode regulators of epigenetic information, such as methylcytosine binding proteins, histone methyltransferases, and chromatin remodelers. The majority of these genes are partially, rather than completely, imprinted, suggesting that gene dosage regulation is an important aspect of imprinted gene expression.
We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor–binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription factor–binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers, with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions to most of the conserved genome.