Maintenance of chromatin states: an open-and-shut case

The traditional view of chromatin envisions two states: one is 'active' and accessible to nucleases, whereas the other is 'silent' and relatively inaccessible. Recent evidence that combinations of diverse histone tail modifications represent a spectrum of chromatin states challenges this simple view. Here, we examine inter-relationships between chromatin remodeling, histone modification, DNA methylation, RNA interference, and nucleosome assembly activities. We find that the two-state view can accommodate these new findings, and that nucleosome assembly pathways may ultimately maintain euchromatic and heterochromatic states.     

Histone transfer among chaperones

The eukaryotic processes of nucleosome assembly and disassembly govern chromatin dynamics, in which histones exchange in a highly regulated manner to promote genome accessibility for all DNA-dependent processes. This regulation is partly carried out by histone chaperones, which serve multifaceted roles in co-ordinating the interactions of histone proteins with modification enzymes, nucleosome remodellers, other histone chaperones and nucleosomal DNA. The molecular details of the processes by which histone chaperones promote delivery of histones among their many functional partners are still largely undefined, but promise to offer insights into epigenome maintenance. In the present paper, we review recent findings on the histone chaperone interactions that guide the assembly of histones H3 and H4 into chromatin. This evidence supports the concepts of histone post-translational modifications and specific histone chaperone interactions as guiding principles for histone H3/H4 transactions during chromatin assembly.     

How does the histone code work?

Patterns of histone post-translational modifications correlate with distinct chromosomal states that regulate access to DNA, leading to the histone-code hypothesis. However, it is not clear how modification of flexible histone tails leads to changes in nucleosome dynamics and, thus, chromatin structure. The recent discovery that, like the flexible histone tails, the structured globular domain of the nucleosome core particle is also extensively modified adds a new and exciting dimension to the histone-code hypothesis, and calls for the re-examination of current models for the epigenetic regulation of chromatin structure. Here, we review these findings and other recent studies that suggest the structured globular domain of the nucleosome core particle plays a key role regulating chromatin dynamics.     

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.     

Effects of histone acetylation and CpG methylation on the structure of nucleosomes

Nucleosomes are the fundamental packing units of the eukaryotic genome. A nucleosome core particle comprises an octameric histone core wrapped around by ~147bp DNA. Histones and DNA are targets for covalent modifications mediated by various chromatin modification enzymes. These modifications play crucial roles in various gene regulation activities. A group of common hypotheses for the mechanisms of gene regulation involves changes in the structure and structural dynamics of chromatin induced by chromatin modifications. We employed single molecule fluorescence methods to test these hypotheses by monitoring the structure and structural dynamics of nucleosomes before and after histone acetylation and DNA methylation, two of the best-conserved chromatin modifications throughout eukaryotes. Our studies revealed that these modifications induce changes in the structure and structural dynamics of nucleosomes that may contribute directly to the formation of open or repressive chromatin conformation.     

The effect of epigenetic modifications on the secondary structures and possible binding positions of the N-terminal tail of histone H3 in the nucleosome: a computational study

The roles of histone tails as substrates for reversible chemical modifications and dynamic cognate surfaces for the binding of regulatory proteins are well established. Despite these crucial roles, experimentally derived knowledge of the structure and possible binding sites of histone tails in chromatin is limited. In this study, we utilized molecular dynamics of isolated histone H3 N-terminal peptides to investigate its structure as a function of post-translational modifications that are known to be associated with defined chromatin states. We observed a structural preference for α-helices in isoforms associated with an inactive chromatin state, while isoforms associated with active chromatin states lacked α-helical content. The physicochemical effect of the post-translational modifications was highlighted by the interaction of arginine side-chains with the phosphorylated serine residues in the inactive isoform. We also showed that the isoforms exhibit different tail lengths, and, using molecular docking of the first 15 N-terminal residues of an H3 isoform, identified potential binding sites between the superhelical gyres on the octamer surface, close to the site of DNA entry/exit in the nucleosome. We discuss the possible functional role of the binding of the H3 tail within the nucleosome on both nucleosome and chromatin structure and stability.     

Chromatin assembly: a basic recipe with various flavours

Packaging of eukaryotic genomes into chromatin is a hierarchical mechanism, starting with histone deposition onto DNA to produce nucleosome arrays, which then further fold and ultimately form functional domains. Recent studies provide interesting insight into how nucleosome assembly is coordinated with histone and DNA metabolism and underline the combined contribution of histone chaperones and chromatin remodelers. How these factors operate at a molecular level is a matter of current investigation. New data highlight the importance of histone dimers as deposition entities for de novo nucleosome assembly and identify dedicated machineries involved in histone variant deposition.     

Intra- and inter-nucleosomal protein-DNA interactions of the core histone tail domains in a model system

The core histone tail domains are key regulators of eukaryotic chromatin structure and function and alterations in the tail-directed folding of chromatin fibers and higher order structures are the probable outcome of much of the post-translational modifications occurring in these domains. The functions of the tail domains are likely to involve complex intra- and inter-nucleosomal histone-DNA interactions, yet little is known about either the structures or interactions of these domains. Here we introduce a method for examining inter-nucleosome interactions of the tail domains in a model dinucleosome and determine the propensity of each of the four N-terminal tail domains to mediate such interactions in this system. Using a strong nucleosome "positioning" sequence, we reconstituted a nucleosome containing a single histone site specifically modified with a photoinducible cross-linker within the histone tail domain, and a second nucleosome containing a radiolabeled DNA template. These two nucleosomes were then ligated together and cross-linking induced by brief UV irradiation under various solution conditions. After cross-linking, the two templates were again separated so that cross-linking representing inter-nucleosomal histone-DNA interactions could be unambiguously distinguished from intra-nucleosomal cross-links. Our results show that the N-terminal tails of H2A and H2B, but not of H3 and H4, make internucleosomal histone-DNA interactions within the dinucleosome. The relative extent of intra- to inter-nucleosome interactions was not strongly dependent on ionic strength. Additionally, we find that binding of a linker histone to the dinucleosome increased the association of the H3 and H4 tails with the linker DNA region.     

A quantitative model of nucleosome dynamics

The expression, replication and repair of eukaryotic genomes require the fundamental organizing unit of chromatin, the nucleosome, to be unwrapped and disassembled. We have developed a quantitative model of nucleosome dynamics which provides a fundamental understanding of these DNA processes. We calibrated this model using results from high precision single molecule nucleosome unzipping experiments, and then tested its predictions for experiments in which nucleosomes are disassembled by the DNA mismatch recognition complex hMSH2-hMSH6. We found that this calibrated model quantitatively describes hMSH2-hMSH6 induced disassembly rates of nucleosomes with two separate DNA sequences and four distinct histone modification states. In addition, this model provides mechanistic insight into nucleosome disassembly by hMSH2-hMSH6 and the influence of histone modifications on this disassembly reaction. This model''s precise agreement with current experiments suggests that it can be applied more generally to provide important mechanistic understanding of the numerous nucleosome alterations that occur during DNA processing.