Establishment of Histone Modifications after Chromatin Assembly

Every cell has to duplicate its entire genome during S-phase of the cell cycle. After replication, the newly synthesized DNA is rapidly assembled into chromatin. The newly assembled chromatin ‘matures’ and adopts a variety of different conformations. This differential packaging of DNA plays an important role for the maintenance of gene expression patterns and has to be reliably copied in each cell division. Posttranslational histone modifications are prime candidates for the regulation of the chromatin structure. In order to understand the maintenance of chromatin structures, it is crucial to understand the replication of histone modification patterns. To study the kinetics of histone modifications in vivo, we have pulse-labeled synchronized cells with an isotopically labeled arginine (¹⁵N₄) that is 4 Da heavier than the naturally occurring ¹⁴N₄ isoform. As most of the histone synthesis is coupled with replication, the cells were arrested at the G1/S boundary, released into S-phase and simultaneously incubated in the medium containing heavy arginine, thus labeling all newly synthesized proteins. This method allows a comparison of modification patterns on parental versus newly deposited histones. Experiments using various pulse/chase times show that particular modifications have considerably different kinetics until they have acquired a modification pattern indistinguishable from the parental histones.     

Split decision: why it matters?

The establishment and faithful maintenance of epigenetic information in the context of chromatin are crucial for a great number of biologic phenomena, including position effect variegation, Polycomb silencing, X-chromosome inactivation and genomic imprinting. However, mechanisms by which that the correct histone modification patterns propagate into daughter cells during mitotic divisions remain to be elucidated. The partitioning pattern of parental histone H3–H4 tetramers is a critical question toward our understanding of the epigenetic inheritance. In this review, we discuss why the histone H3–H4 tetramer split decision matters.     

Combinatorial chromatin modification patterns in the human genome revealed by subspace clustering

Chromatin modifications, such as post-translational modification of histone proteins and incorporation of histone variants, play an important role in regulating gene expression. Joint analyses of multiple histone modification maps are starting to reveal combinatorial patterns of modifications that are associated with functional DNA elements, providing support to the 'histone code' hypothesis. However, due to the lack of analytical methods, only a small number of chromatin modification patterns have been discovered so far. Here, we introduce a scalable subspace clustering algorithm, coherent and shifted bicluster identification (CoSBI), to exhaustively identify the set of combinatorial modification patterns across a given epigenome. Performance comparisons demonstrate that CoSBI can generate biclusters with higher intra-cluster coherency and biological relevance. We apply our algorithm to a compendium of 39 genome-wide chromatin modification maps in human CD4(+) T cells. We identify 843 combinatorial patterns that recur at >0.1% of the genome. A total of 19 chromatin modifications are observed in the combinatorial patterns, 10 of which occur in more than half of the patterns. We also identify combinatorial modification signatures for eight classes of functional DNA elements. Application of CoSBI to epigenome maps of different cells and developmental stages will aid in understanding how chromatin structure helps regulate gene expression.     

Cooperative interactions between epigenetic modifications and their function in the regulation of chromosome architecture

Epigenetic information is encoded by DNA methylation and by covalent modifications of histone tails. While defined epigenetic modification patterns have been frequently correlated with particular states of gene activity, very little is known about the integration level of epigenetic signals. Recent experiments have resulted in the characterization of several epigenetic adaptors that mediate interactions between distinct modifications. These adaptors include methyl-DNA binding proteins, chromatin remodelling enzymes and siRNAs. Complex interactions between epigenetic modifiers and adaptors provide the foundation for the stability of epigenetic inheritance. In addition, they also provide an explanation for the long-range effects of epigenetic mechanisms. We propose that a major aspect of epigenetic regulation lies in the modification of chromosome architecture and that local changes in gene expression would be secondary consequences. This view is consistent with many results from recent genomic analyses.     

Discovering common combinatorial histone modification patterns in the human genome

Histone modifications play a crucial role in regulating gene expression and cell lineage determination and maintenance at the epigenetic level. To systematically investigate this phenomenon, this paper presented a statistical hybrid clustering algorithm to identify common combinatorial histone modification patterns. We applied the algorithm to 39 histone modification marks in human CD4 + T cells and detected 854 common combinatorial histone modification patterns. Our results could cover 211 (76.17%) patterns among 277 patterns identified by the tandem mass spectrometry experiments. Based on the frequency statistical analysis, it was found that the co-occurrence frequencies of 20 backbone modifications are greater than or close to 0.2 in the 854 patterns. we also found that 15 modifications (H2BK120ac, H4K91ac, H2BK20ac, etc.), three histone acetylations (H2AK9ac, H4K16ac, and H4K12ac) and five histone methylations (H3K79me1, H3K79me2, 3K79me3, H4K20me1, and H2BK5me1) were most likely prone to coexist respectively in these patterns. In addition, we found that DNA methylation tends to combine with histone acetylation rather than histone methylation.     

Comparative Analysis of Genome-Wide Chromosomal Histone Modification Patterns in Maize Cultivars and Their Wild Relatives

Recent advances demonstrate that epigenome changes can also cause phenotypic diversity and can be heritable across generations, indicating that they may play an important role in evolutionary processes. In this study, we analyzed the chromosomal distribution of several histone modifications in five elite maize cultivars (B73, Mo17, Chang7-2, Zheng58, ZD958) and their two wild relatives (Zea mays L. ssp. parviglumis and Zea nicaraguensis) using a three-dimensional (3D) epigenome karyotyping approach by combining immunostaining and 3D reconstruction with deconvolution techniques. The distribution of these histone modifications along chromosomes demonstrated that the histone modification patterns are conserved at the chromosomal level and have not changed significantly following domestication. The comparison of histone modification patterns between metaphase chromosomes and interphase nuclei showed that some of the histone modifications were retained as the cell progressed from interphase into metaphase, although remodelling existed. This study will increase comprehension of the function of epigenetic modifications in the structure and evolution of the maize genome.     

Epigenetic regulation of nuclear lamina-associated heterochromatin by HAT1 and the acetylation of newly synthesized histones

A central component of the epigenome is the pattern of histone post-translational modifications that play a critical role in the formation of specific chromatin states. Following DNA replication, nascent chromatin is a 1:1 mixture of parental and newly synthesized histones and the transfer of modification patterns from parental histones to new histones is a fundamental step in epigenetic inheritance. Here we report that loss of HAT1, which acetylates lysines 5 and 12 of newly synthesized histone H4 during replication-coupled chromatin assembly, results in the loss of accessibility of large domains of heterochromatin, termed HAT1-dependent Accessibility Domains (HADs). HADs are mega base-scale domains that comprise ∼10% of the mouse genome. HAT1 globally represses H3 K9 me3 levels and HADs correspond to the regions of the genome that display HAT1-dependent increases in H3 K9me3 peak density. HADs display a high degree of overlap with a subset of Lamin-Associated Domains (LADs). HAT1 is required to maintain nuclear structure and integrity. These results indicate that HAT1 and the acetylation of newly synthesized histones may be critical regulators of the epigenetic inheritance of heterochromatin and suggest a new mechanism for the epigenetic regulation of nuclear lamina-heterochromatin interactions.     

The Regulation and Function of Histone Methylation

Histones are wrapped around by genomic DNA to form nucleosomes which are the basic units of chromatin. In eukaryotes histones undergo various covalent modifications such as methylation, phosphorylation, acetylation, ubiquitination and ribosylation. Histone modifications play a fundamental role in the epigenetic regulation of gene expression in multicellular eukaryotes. Histone methylation is one of the most important modifications occurring on Lysine (K) and Arginine (R) residues of histones, dynamically regulated by histone methyltransferases and demethylases. Identifications of such histone modification enzymes and to study how they work are the most fundamental questions needs to be answered. Uncovering the regulation and functions of the various histone methylation enzymes will help us to better understand the epigenetic code. This review summarizes the regulation of histone methyltransferases activity, the recruitment of methyltransferases and the distribution patterns and function of histone methylations.     

The inheritance of histone modifications depends upon the location in the chromosome in Saccharomyces cerevisiae

Histone modifications are important epigenetic features of chromatin that must be replicated faithfully. However, the molecular mechanisms required to duplicate and maintain histone modification patterns in chromatin remain to be determined. Here, we show that the introduction of histone modifications into newly deposited nucleosomes depends upon their location in the chromosome. In Saccharomyces cerevisiae, newly deposited nucleosomes consisting of newly synthesized histone H3-H4 tetramers are distributed throughout the entire chromosome. Methylation of lysine 4 on histone H3 (H3-K4), a hallmark of euchromatin, is introduced into these newly deposited nucleosomes, regardless of whether the neighboring preexisting nucleosomes harbor the K4 mutation in histone H3. Furthermore, if the heterochromatin-binding protein Sir3 is unavailable during DNA replication, histone H3-K4 methylation is introduced onto newly deposited nucleosomes in telomeric heterochromatin. Thus, a conservative distribution model most accurately explains the inheritance of histone modifications because the location of histones within euchromatin or heterochromatin determines which histone modifications are introduced.     

LinkNMF: Identification of histone modification modules in the human genome using nonnegative matrix factorization

Histone modifications are ubiquitous processes involved in various cellular mechanisms. Systemic analysis of multiple chromatin modifications has been used to characterize various chromatin states associated with functional DNA elements, gene expression, and specific biological functions. However, identification of modular modification patterns is still required to understand the functional associations between histone modification patterns and specific chromatin/DNA binding factors. To recognize modular modification patterns, we developed a novel algorithm that combines nonnegative matrix factorization (NMF) and a clique-detection algorithm. We applied it, called LinkNMF, to generate a comprehensive modification map in human CD4 + T cell promoter regions. Initially, we identified 11 modules not recognized by conventional approaches. The modules were grouped into two major classes: gene activation and repression. We found that genes targeted by each module were enriched with distinguishable biological functions, suggesting that each modular pattern plays a unique functional role. To explain the formation of modular patterns, we investigated the module-specific binding patterns of chromatin regulators. Application of LinkNMF to histone modification maps of diverse cells and developmental stages will be helpful for understanding how histone modifications regulate gene expression. The algorithm is available on our website at biodb.kaist.ac.kr/LinkNMF.     

A simple histone code opens many paths to epigenetics

Nucleosomes can be covalently modified by addition of various chemical groups on several of their exposed histone amino acids. These modifications are added and removed by enzymes (writers) and can be recognized by nucleosome-binding proteins (readers). Linking a reader domain and a writer domain that recognize and create the same modification state should allow nucleosomes in a particular modification state to recruit enzymes that create that modification state on nearby nucleosomes. This positive feedback has the potential to provide the alternative stable and heritable states required for epigenetic memory. However, analysis of simple histone codes involving interconversions between only two or three types of modified nucleosomes has revealed only a few circuit designs that allow heritable bistability. Here we show by computer simulations that a histone code involving alternative modifications at two histone positions, producing four modification states, combined with reader-writer proteins able to distinguish these states, allows for hundreds of different circuits capable of heritable bistability. These expanded possibilities result from multiple ways of generating two-step cooperativity in the positive feedback - through alternative pathways and an additional, novel cooperativity motif. Our analysis reveals other properties of such epigenetic circuits. They are most robust when the dominant nucleosome types are different at both modification positions and are not the type inserted after DNA replication. The dominant nucleosome types often recruit enzymes that create their own type or destroy the opposing type, but never catalyze their own destruction. The circuits appear to be evolutionary accessible; most circuits can be changed stepwise into almost any other circuit without losing heritable bistability. Thus, our analysis indicates that systems that utilize an expanded histone code have huge potential for generating stable and heritable nucleosome modification states and identifies the critical features of such systems.     

Additive inheritance of histone modifications in Arabidopsis thaliana intra-specific hybrids

Plant genomes are earmarked with defined patterns of chromatin marks. Little is known about the stability of these epigenomes when related, but distinct genomes are brought together by intra‐species hybridization. Arabidopsis thaliana accessions and their reciprocal hybrids were used as a model system to investigate the dynamics of histone modification patterns. The genome‐wide distribution of histone modifications H3K4me2 and H3K27me3 in the inbred parental accessions Col‐0, C24 and Cvi and their hybrid offspring was compared by chromatin immunoprecipitation in combination with genome tiling array hybridization. The analysis revealed that, in addition to DNA sequence polymorphisms, chromatin modification variations exist among accessions of A. thaliana. The range of these variations was higher for H3K27me3 (typically a repressive mark) than for H3K4me2 (typically an active mark). H3K4me2 and H3K27me3 were rather stable in response to intra‐species hybridization, with mainly additive inheritance in hybrid offspring. In conclusion, intra‐species hybridization does not result in gross changes to chromatin modifications.     

Epigenetic regulation by histone methylation and histone variants

Epigenetics is the study of heritable changes in gene expression that are not mediated at the DNA sequence level. Molecular mechanisms that mediate epigenetic regulation include DNA methylation and chromatin/histone modifications. With the identification of key histone-modifying enzymes, the biological functions of many histone posttranslational modifications are now beginning to be elucidated. Histone methylation, in particular, plays critical roles in many epigenetic phenomena. In this review, we provide an overview of recent findings that shape the current paradigms regarding the roles of histone methylation and histone variants in heterochromatin assembly and the maintenance of the boundaries between heterochromatin and euchromatin. We also highlight some of the enzymes that mediate histone methylation and discuss the stability and inheritance of this modification.     

10th NTES Conference: Nickel and arsenic compounds alter the epigenome of peripheral blood mononuclear cells

The mechanisms that underlie metal carcinogenesis are the subject of intense investigation; however, data from in vitro and in vivo studies are starting to piece together a story that implicates epigenetics as a key player. Data from our lab has shown that nickel compounds inhibit dioxygenase enzymes by displacing iron in the active site. Arsenic is hypothesized to inhibit these enzymes by diminishing ascorbate levels – an important co-factor for dioxygenases. Inhibition of histone demethylase dioxygenases can increase histone methylation levels, which also may affect gene expression. Recently, our lab conducted a series of investigations in human subjects exposed to high levels of nickel or arsenic compounds. Global levels of histone modifications in peripheral blood mononuclear cells (PBMCs) from exposed subjects were compared to low environmentally exposed controls. Results showed that nickel increased H3K4me3 and decreased H3K9me2 globally. Arsenic increased H3K9me2 and decreased H3K9ac globally. Other histone modifications affected by arsenic were sex-dependent. Nickel affected the expression of 2756 genes in human PBMCs and many of the genes were involved in immune and carcinogenic pathways. This review will describe data from our lab that demonstrates for the first time that nickel and arsenic compounds affect global levels of histone modifications and gene expression in exposed human populations.     

Variations on the topic of the "histone code"

Histones are the fundamental structural proteins intimately associated with eukaryotic DNA to form a highly ordered and condensed nucleoproteic complex termed chromatin. They are the targets of various posttranslational modifications including acetylation, methylation, phosphorylation and ubiquitination that modulate the structure/function of chromatin. The combinatorial nature of histone modifications is hypothesized to define a "histone code" that considerably extends the information potential of the genetic code, giving rise to epigenetic information. Moreover, most core histones consist of several nonallelic variants that can mark specific loci and could play an important role in establishment and maintenance of epigenetic memory. Here we will briefly present our current knowledge about histone posttranslational modifications and their implications in the regulation of epigenetic information. We will next describe core histone variants, insisting on their mode of incorporation into chromatin to discuss their epigenetic function and inheritance.