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.     

Barriers and silencers: a theoretical toolkit for control and containment of nucleosome-based epigenetic states

Positive feedback in nucleosome modification has been proposed to allow large chromatin regions to exist stably and heritably in distinct expression states. However, modeling has shown that such epigenetic bistability requires that modifying enzymes recruited by nucleosomes are active on distant nucleosomes, potentially allowing uncontrollable spreading of modification. By modeling the silencing of mating-type loci in Saccharomyces cerevisiae, we show that a modification reaction that combines a long-range component and a locally acting component can provide bistability and can be blocked by simple barriers that interrupt the nucleosome chain. We find that robust containment of the silenced region could be achieved by the presence of a number of weak simple barriers in the surrounding chromatin and a limited capacity of the positive feedback reaction. In addition, we show that the state of the silenced region can be regulated by silencer elements acting only on neighboring nucleosomes. Thus, a relatively simple set of nucleosome-modifying enzymes and recognition domains is all that is needed to make chromatin-based epigenetics useful and safe.     

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.     

Is histone loss a common feature of DNA metabolism regulation?

Chromatin modifications play a crucial role in regulating DNA metabolism. Chromatin structures can be remodeled by covalently modifying histones, by shifting nucleosomes along the DNA, and by changing the histone composition of nucleosomes. Lately, nucleosome displacement has been extensively described within transcribed genes and DNA breaks. This review focuses on recently published work that describes the relationships between histone modification/exchange and nucleosome displacement.     

Nucleosome acetylation sequencing to study the establishment of chromatin acetylation

The establishment of posttranslational chromatin modifications is a major mechanism for regulating how genomic DNA is utilized. However, current in vitro chromatin assays do not monitor histone modifications at individual nucleosomes. Here we describe a strategy, nucleosome acetylation sequencing, that allows us to read the amount of modification at each nucleosome. In this approach, a bead-bound trinucleosome substrate is enzymatically acetylated with radiolabeled acetyl CoA by the SAGA complex from Saccharomyces cerevisae. The product is digested by restriction enzymes that cut at unique sites between the nucleosomes and then counted to quantify the extent of acetylation at each nucleosomal site. We find that we can sensitively, specifically, and reproducibly follow enzyme-mediated nucleosome acetylation. Applying this strategy, when acetylation proceeds extensively, its distribution across nucleosomes is relatively uniform. However, when substrates are used that contain nucleosomes mutated at the major sites of SAGA-mediated acetylation, or that are studied under initial rate conditions, changes in the acetylation distribution can be observed. Nucleosome acetylation sequencing should be applicable to analyzing a wide range of modifications. Additionally, because our trinucleosomes synthesis strategy is highly modular and efficient, it can be used to generate nucleosomal systems in which nucleosome composition differs across the array.     

Chromatin dynamics controls epigenetic domain formation

In multicellular organisms, nucleosomes carry epigenetic information that defines distinct patterns of gene expression, which are inherited over multiple generations. The enhanced capacity for information storage arises by nucleosome modifications, which are triggered by enzymes. Modified nucleosomes can transfer the mark to others that are in proximity by a positive-feedback (modification begets modification) mechanism. We created a generic polymer model, referred to as 3DSpreader, in which each bead, representing a nucleosome, stochastically switches between unmodified (U) and modified (M) states depending on the states of the neighbors. Modification begins at a specific nucleation site (NS) that is permanently in the M state, and could spread to other loci that is dictated by chromatin dynamics. Transfer of marks among the non-nucleation loci occurs stochastically as chromatin evolves in time. If the spreading rate is slower than the chromatin relaxation rate, which is biologically pertinent, then finite-sized domains form, driven by contacts between nucleosomes through a three-dimensional looping mechanism. Surprisingly, simulations based on the 3DSpreader model result in finite bounded domains that arise without the need for any boundary elements. Maintenance of spatially and temporally stable domains requires the presence of the NS, whose removal eliminates finite-sized modified domains. The theoretical predictions are in excellent agreement with experimental data for H3K9me3 spreading in mouse embryonic stem cells.     

Modified curved DNA that could allow local DNA underwinding at the nucleosomal pseudodyad fails to position a nucleosome in vivo.

In competitive in vitro reconstitution experiments synthetic DNA composed of tandem repeats of the repetitive sequence (A/T)3NN(G/C)3NN, specifically the 20 bp 'TG sequence' (5'-TCGGTGTTAGAGCCTGTAAC-3'), was reported to associate with the histone octamer with an affinity higher than that of nucleosomally derived DNA. However, at least two groups have independently shown that tandem repeats of the TG sequence do not accommodate a stably positioned nucleosome in vivo. It was suggested that the anisotropic flexibility of the TG sequence, governed by a 10 bp sequence periodicity, is incompatible with the required underwinding of the DNA helix at the nucleosome pseudodyad while maintaining a bending preference that can be accommodated in the remainder of the nucleosome. Here we test this hypothesis directly by studying the in vivo nucleosomal structure of modified TG sequences designed to accommodate underwinding at the pseudodyad. We show that these modifications are not sufficient to allow stable incorporation of the TG sequence repeat into a nucleosome in vivo, but do note invasion from one end of the TG heptamer of a translationally random but rotationally constrained nucleosome. We discuss possible reasons for the absence of nucleosomes from the TG sequence in vivo.     

Nucleosome-interacting proteins regulated by DNA and histone methylation

Modifications on histones or on DNA recruit proteins that regulate chromatin function. Here, we use nucleosomes methylated on DNA and on histone H3 in an affinity assay, in conjunction with a SILAC-based proteomic analysis, to identify "crosstalk" between these two distinct classes of modification. Our analysis reveals proteins whose binding to nucleosomes is regulated by methylation of CpGs, H3K4, H3K9, and H3K27 or a combination thereof. We identify the origin recognition complex (ORC), including LRWD1 as a subunit, to be a methylation-sensitive nucleosome interactor that is recruited cooperatively by DNA and histone methylation. Other interactors, such as the lysine demethylase Fbxl11/KDM2A, recognize nucleosomes methylated on histones, but their recruitment is disrupted by DNA methylation. These data establish SILAC nucleosome affinity purifications (SNAP) as a tool for studying the dynamics between different chromatin modifications and provide a modification binding "profile" for proteins regulated by DNA and histone methylation.     

Nucleosome destabilization in the epigenetic regulation of gene expression

Assembly, mobilization and disassembly of nucleosomes can influence the regulation of gene expression and other processes that act on eukaryotic DNA. Distinct nucleosome-assembly pathways deposit dimeric subunits behind the replication fork or at sites of active processes that mobilize pre-existing nucleosomes. Replication-coupled nucleosome assembly appears to be the default process that maintains silent chromatin, counteracted by active processes that destabilize nucleosomes. Nucleosome stability is regulated by the combined effects of nucleosome-positioning sequences, histone chaperones, ATP-dependent nucleosome remodellers, post-translational modifications and histone variants. Recent studies suggest that histone turnover helps to maintain continuous access to sequence-specific DNA-binding proteins that regulate epigenetic inheritance, providing a dynamic alternative to histone-marking models for the propagation of active chromatin.     

Repair of UV lesions in nucleosomes--intrinsic properties and remodeling

Nucleotide excision repair and reversal of pyrimidine dimers by photolyase (photoreactivation) are two major pathways to remove UV-lesions from DNA. Here, it is discussed how lesions are recognized and removed when the DNA is condensed into nucleosomes. During the recent years it was shown that nucleosomes inhibit photolyase and excision repair in vitro and slow down repair in vivo. The correlation of DNA-repair rates with nucleosome positions in yeast suggests that intrinsic properties of nucleosomes such as mobility and transient unwrapping of nucleosomal DNA facilitate damage recognition. Moreover, it was shown that nucleosome remodeling activities can act on UV-damaged DNA in vitro and facilitate repair suggesting that random remodeling of chromatin might contribute to damage recognition in vivo. Recent work on nucleosome structure and mobility is included to evaluate how nucleosomes accommodate DNA lesions and how nucleosome mobility and remodeling can take place on damaged DNA.     

Effects of DNA Superhelical Stress on the Stability of H2B-Ubiquitylated Nucleosomes

On the nucleosome level, histone posttranslational modifications function mainly as the regulatory signals; in addition, some posttranslational modifications can enhance nucleosome stochastic folding, which is restricted in “canonic” nucleosomes. Recently, it has been shown in vitro that symmetric or asymmetric nucleosome ubiquitylation at H2BK34 (and H2BK120, to a lesser extent) can destabilize one of the nucleosomal H2A–H2B dimers and promote nucleosome conversion to a hexasome particle [Krajewski et al. (2018). Nucleic Acids Res., 46, 7631–7642]. Such lability of H2Bub nucleosomes raises a question of whether they could accommodate transient changes in DNA torsional tensions, which are generated by virtually any process that manipulates DNA strands. Using positively or negatively supercoiled DNA minicircles and homogeneously-modified H2Bub histones, we have found that DNA topology could strongly and selectively affect nucleosome stability depending on its ubiquitylation state (here the term “nucleosome stability” means the nucleosome property to maintain its structural integrity and dynamics characteristic to “canonic” nucleosomes). The results point to a role for H2B ubiquitylation in amplifying or mitigating the effects of a DNA torque on the nucleosome stability and dynamics.     

Histone H2A ubiquitination does not preclude histone H1 binding, but it facilitates its association with the nucleosome

Histone H2A ubiquitination is a bulky posttranslational modification that occurs at the vicinity of the binding site for linker histones in the nucleosome. Therefore, we took several experimental approaches to investigate the role of ubiquitinated H2A (uH2A) in the binding of linker histones. Our results showed that uH2A was present in situ in histone H1-containing nucleosomes. Notably in vitro experiments using nucleosomes reconstituted onto 167-bp random sequence and 208-bp (5 S rRNA gene) DNA fragments showed that ubiquitination of H2A did not prevent binding of histone H1 but it rather enhanced the binding of this histone to the nucleosome. We also showed that ubiquitination of H2A did not affect the positioning of the histone octamer in the nucleosome in either the absence or the presence of linker histones.     

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.     

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.