Genome-wide approaches to studying chromatin modifications

Over two metres of DNA is packaged into each nucleus in the human body in a manner that still allows for gene regulation. This remarkable feat is accomplished by the wrapping of DNA around histone proteins in repeating units of nucleosomes to form a structure known as chromatin. This chromatin structure is subject to various modifications that have profound influences on gene expression. Recently developed techniques to study chromatin modifications at a genome-wide scale are now allowing researchers to probe the complex components that make up epigenomes. Here we review genome-wide approaches to studying epigenomic structure and the exciting findings that have been obtained using these technologies.     

Epigenomic modification and epigenetic regulation in rice

Epigenomes including genome-wide histone modification and DNA methylation profiles are important for genome activity and for defining gene expression patterns of plant development and responses to various environmental conditions.Rice is the most important crop plant and serves as a model for cereal genomics.Rice epigenomic landscape is emerging and the function of chromatin modification regulators in gene expression,transposon repression and plant development is being characterized.Epigenomic variation that gives rise to stable or transgenerational heritable epialleles related to variation of important agronomical traits or stress responses is being characterized in rice.Implication of epigenomic variation in rice heterosis is being exploited.     

The coupling of epigenome replication with DNA replication

In multicellular organisms, each cell contains the same DNA sequence, but with different epigenetic information that determines the cell specificity. Semi-conservative DNA replication faithfully copies the parental nucleotide sequence into two DNA daughter strands during each cell cycle. At the same time, epigenetic marks such as DNA methylation and histone modifications are either precisely transmitted to the daughter cells or dynamically changed during S-phase. Recent studies indicate that in each cell cycle, many DNA replication related proteins are involved in not only genomic but also epigenomic replication. Histone modification proteins, chromatin remodeling proteins, histone variants, and RNAs participate in the epigenomic replication during S-phase. As a consequence, epigenome replication is closely linked with DNA replication during S-phase.     

Integrative epigenomic mapping defines four main chromatin states in Arabidopsis

Post-translational modification of histones and DNA methylation are important components of chromatin-level control of genome activity in eukaryotes. However, principles governing the combinatorial association of chromatin marks along the genome remain poorly understood. Here, we have generated epigenomic maps for eight histone modifications (H3K4me2 and 3, H3K27me1 and 2, H3K36me3, H3K56ac, H4K20me1 and H2Bub) in the model plant Arabidopsis and we have combined these maps with others, produced under identical conditions, for H3K9me2, H3K9me3, H3K27me3 and DNA methylation. Integrative analysis indicates that these 12 chromatin marks, which collectively cover ～90% of the genome, are present at any given position in a very limited number of combinations. Moreover, we show that the distribution of the 12 marks along the genomic sequence defines four main chromatin states, which preferentially index active genes, repressed genes, silent repeat elements and intergenic regions. Given the compact nature of the Arabidopsis genome, these four indexing states typically translate into short chromatin domains interspersed with each other. This first combinatorial view of the Arabidopsis epigenome points to simple principles of organization as in metazoans and provides a framework for further studies of chromatin-based regulatory mechanisms in plants.     

Epigenetic regulation in plant abiotic stress responses

In eukaryotic cells, gene expression is greatly influenced by the dynamic chromatin environment. Epigenetic mechanisms, including covalent modifications to DNA and histone tails and the accessibility of chromatin, create various chromatin states for stress‐responsive gene expression that is important for adaptation to harsh environmental conditions. Recent studies have revealed that many epigenetic factors participate in abiotic stress responses, and various chromatin modifications are changed when plants are exposed to stressful environments. In this review, we summarize recent progress on the cross‐talk between abiotic stress response pathways and epigenetic regulatory pathways in plants. Our review focuses on epigenetic regulation of plant responses to extreme temperatures, drought, salinity, the stress hormone abscisic acid, nutrient limitations and ultraviolet stress, and on epigenetic mechanisms of stress memory.     

Chromatin techniques for plant cells

A large number of recent studies have demonstrated that many important aspects of plant development are regulated by heritable changes in gene expression that do not involve changes in DNA sequence. Rather, these regulatory mechanisms involve modifications of chromatin structure that affect the accessibility of target genes to regulatory factors that can control their expression. The central component of chromatin is the nucleosome, containing the highly conserved histone proteins that are known to be subject to a wide range of post-translational modifications, which act as recognition codes for the binding of chromatin-associated factors. In addition to these histone modifications, DNA methylation can also have a dramatic influence on gene expression. To accommodate the burgeoning interest of the plant science community in the epigenetic control of plant development, a series of methods used routinely in our laboratories have been compiled that can facilitate the characterization of putative chromatin-binding factors at the biochemical, molecular and cellular levels.     

Engineering chromatin states: Chemical and synthetic biology approaches to investigate histone modification function

Patterns of histone post-translational modifications (PTMs) and DNA modifications establish a landscape of chromatin states with regulatory impact on gene expression, cell differentiation and development. These diverse modifications are read out by effector protein complexes, which ultimately determine their functional outcome by modulating the activity state of underlying genes. From genome-wide studies employing high-throughput ChIP-Seq methods as well as proteomic mass spectrometry studies, a large number of PTMs are known and their coexistence patterns and associations with genomic regions have been mapped in a large number of different cell types. Conversely, the molecular interplay between chromatin effector proteins and modified chromatin regions as well as their resulting biological output is less well understood on a molecular level. Within the last decade a host of chemical approaches has been developed with the goal to produce synthetic chromatin with a defined arrangement of PTMs. These methods now permit systematic functional studies of individual histone and DNA modifications, and additionally provide a discovery platform to identify further interacting nuclear proteins. Complementary chemical- and synthetic-biology methods have emerged to directly observe and modulate the modification landscape in living cells and to readily probe the effect of altered PTM patterns on biological processes. Herein, we review current methodologies allowing chemical and synthetic biological engineering of distinct chromatin states in vitro and in vivo with the aim of obtaining a molecular understanding of histone and DNA modification function. This article is part of a Special Issue entitled: Molecular mechanisms of histone modification function.     

The interplay between DNA and histone methylation: molecular mechanisms and disease implications

Methylation of cytosine in CpG dinucleotides and histone lysine and arginine residues is a chromatin modification that critically contributes to the regulation of genome integrity, replication, and accessibility. A strong correlation exists between the genome‐wide distribution of DNA and histone methylation, suggesting an intimate relationship between these epigenetic marks. Indeed, accumulating literature reveals complex mechanisms underlying the molecular crosstalk between DNA and histone methylation. These in vitro and in vivo discoveries are further supported by the finding that genes encoding DNA‐ and histone‐modifying enzymes are often mutated in overlapping human diseases. Here, we summarize recent advances in understanding how DNA and histone methylation cooperate to maintain the cellular epigenomic landscape. We will also discuss the potential implication of these insights for understanding the etiology of, and developing biomarkers and therapies for, human congenital disorders and cancers that are driven by chromatin abnormalities.     

Prime, repair, restore: the active role of chromatin in the DNA damage response

The view of DNA packaging into chromatin as a mere obstacle to DNA repair is evolving. In this review, we focus on histone variants and heterochromatin proteins as chromatin components involved in distinct levels of chromatin organization to integrate them as real players in the DNA damage response (DDR). Based on recent data, we highlight how some of these chromatin components play active roles in the DDR and contribute to the fine-tuning of damage signaling, DNA and chromatin repair. To take into account this integrated view, we revisit the existing access-repair-restore model and propose a new working model involving priming chromatin for repair and restoration as a concerted process. We discuss how this impacts on both genomic and epigenomic stability and plasticity.     

A barcode screen for epigenetic regulators reveals a role for the NuB4/HAT-B histone acetyltransferase complex in histone turnover

Dynamic modification of histone proteins plays a key role in regulating gene expression. However, histones themselves can also be dynamic, which potentially affects the stability of histone modifications. To determine the molecular mechanisms of histone turnover, we developed a parallel screening method for epigenetic regulators by analyzing chromatin states on DNA barcodes. Histone turnover was quantified by employing a genetic pulse-chase technique called RITE, which was combined with chromatin immunoprecipitation and high-throughput sequencing. In this screen, the NuB4/HAT-B complex, containing the conserved type B histone acetyltransferase Hat1, was found to promote histone turnover. Unexpectedly, the three members of this complex could be functionally separated from each other as well as from the known interacting factor and histone chaperone Asf1. Thus, systematic and direct interrogation of chromatin structure on DNA barcodes can lead to the discovery of genes and pathways involved in chromatin modification and dynamics.     

Chromatin affinity purification and quantitative mass spectrometry defining the interactome of histone modification patterns

DNA and histone modifications direct the functional state of chromatin and thereby the readout of the genome. Candidate approaches and histone peptide affinity purification experiments have identified several proteins that bind to chromatin marks. However, the complement of factors that is recruited by individual and combinations of DNA and histone modifications has not yet been defined. Here, we present a strategy based on recombinant, uniformly modified chromatin templates used in affinity purification experiments in conjunction with SILAC-based quantitative mass spectrometry for this purpose. On the prototypic H3K4me3 and H3K9me3 histone modification marks we compare our method with a histone N-terminal peptide affinity purification approach. Our analysis shows that only some factors associate with both, chromatin and peptide matrices but that a surprisingly large number of proteins differ in their association with these templates. Global analysis of the proteins identified implies specific domains mediating recruitment to the chromatin marks. Our proof-of-principle studies show that chromatin templates with defined modification patterns can be used to decipher how the histone code is read and translated.