Rapid Purification of Recombinant Histones

The development of methods to assemble nucleosomes from recombinant histones decades ago has transformed chromatin research. Nevertheless, nucleosome reconstitution remains time consuming to this day, not least because the four individual histones must be purified first. Here, we present a streamlined purification protocol of recombinant histones from bacteria. We termed this method “rapid histone purification” (RHP) as it circumvents isolation of inclusion bodies and thereby cuts out the most time-consuming step of traditional purification protocols. Instead of inclusion body isolation, whole cell extracts are prepared under strongly denaturing conditions that directly solubilize inclusion bodies. By ion exchange chromatography, the histones are purified from the extracts. The protocol has been successfully applied to all four canonical Drosophila and human histones. RHP histones and histones that were purified from isolated inclusion bodies had similar purities. The different purification strategies also did not impact the quality of octamers reconstituted from these histones. We expect that the RHP protocol can be readily applied to the purification of canonical histones from other species as well as the numerous histone variants.     

Cracking the survival code: Autophagy-related histone modifications

Modifications of histones, the chief protein components of the chromatin, have emerged as critical regulators of life and death. While the “apoptotic histone code” came to light a few years ago, accumulating evidence indicates that autophagy, a cell survival pathway, is also heavily regulated by histone-modifying proteins. In this review we describe the emerging “autophagic histone code” and the role of histone modifications in the cellular life vs. death decision.     

A Phylogenetic Study of SPBP and RAI1: Evolutionary Conservation of Chromatin Binding Modules

Our genome is assembled into and array of highly dynamic nucleosome structures allowing spatial and temporal access to DNA. The nucleosomes are subject to a wide array of post-translational modifications, altering the DNA-histone interaction and serving as docking sites for proteins exhibiting effector or “reader” modules. The nuclear proteins SPBP and RAI1 are composed of several putative “reader” modules which may have ability to recognise a set of histone modification marks. Here we have performed a phylogenetic study of their putative reader modules, the C-terminal ePHD/ADD like domain, a novel nucleosome binding region and an AT-hook motif. Interactions studies in vitro and in yeast cells suggested that despite the extraordinary long loop region in their ePHD/ADD-like chromatin binding domains, the C-terminal region of both proteins seem to adopt a cross-braced topology of zinc finger interactions similar to other structurally determined ePHD/ADD structures. Both their ePHD/ADD-like domain and their novel nucleosome binding domain are highly conserved in vertebrate evolution, and construction of a phylogenetic tree displayed two well supported clusters representing SPBP and RAI1, respectively. Their genome and domain organisation suggest that SPBP and RAI1 have occurred from a gene duplication event. The phylogenetic tree suggests that this duplication has happened early in vertebrate evolution, since only one gene was identified in insects and lancelet. Finally, experimental data confirm that the conserved novel nucleosome binding region of RAI1 has the ability to bind the nucleosome core and histones. However, an adjacent conserved AT-hook motif as identified in SPBP is not present in RAI1, and deletion of the novel nucleosome binding region of RAI1 did not significantly affect its nuclear localisation.     

Novel nucleosomal particles containing core histones and linker DNA but no histone H1

Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ∼147 bp of DNA wrapped ∼1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ∼160 bp, and then converts it to a core particle, containing ∼147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these ‘proto-chromatosomes’ are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1.     

Hyperacetylated histones facilitate chromatin assembly in vitro.

We have examined the effect of histone acetylation on the in vitro assembly of nucleosomes with DNA and purified histones at physiological ionic strength in the presence of polyglutamic acid. We have found that hyperacetylated histones assemble nucleosomes with greater efficiency, and to a greater extent, than either control or hypoacetylated histones. Assembly reactions were performed over a range of histone to DNA ratios (0.25 to 3.0, w/w) and polyglutamic acid to histone ratios (0 to 1.6, w/w). Although polyglutamic acid may act as a sink to prevent nonspecific histone-DNA interactions, our data suggest that the polyanion primarily facilitates the assembly of nucleosomes by organizing histones into a form that is amenable to deposition.     

Tetrameric structure of centromeric nucleosomes in interphase Drosophila cells

Centromeres, the specialized chromatin structures that are responsible for equal segregation of chromosomes at mitosis, are epigenetically maintained by a centromere-specific histone H3 variant (CenH3). However, the mechanistic basis for centromere maintenance is unknown. We investigated biochemical properties of CenH3 nucleosomes from Drosophila melanogaster cells. Cross-linking of CenH3 nucleosomes identifies heterotypic tetramers containing one copy of CenH3, H2A, H2B, and H4 each. Interphase CenH3 particles display a stable association of approximately 120 DNA base pairs. Purified centromeric nucleosomal arrays have typical “beads-on-a-string” appearance by electron microscopy but appear to resist condensation under physiological conditions. Atomic force microscopy reveals that native CenH3-containing nucleosomes are only half as high as canonical octameric nucleosomes are, confirming that the tetrameric structure detected by cross-linking comprises the entire interphase nucleosome particle. This demonstration of stable half-nucleosomes in vivo provides a possible basis for the instability of centromeric nucleosomes that are deposited in euchromatic regions, which might help maintain centromere identity.     

Driving proteins off DNA using applied tension.

Proteins that bind DNA so as to reduce its end-to-end length can be dissociated by application of force. The thermodynamics of this process are discussed, with special attention to the case of histones bound to DNA (i.e., a string of nucleosomes, or chromatin fiber). The histone octamer is predicted to be driven off chromatin fiber for tensions >2 piconewtons.     

Separation of nucleosomes containing histones H1 and H5

Hen erythrocyte chromatin was digested with staphylococcal nuclease and fractionated by electrophoresis in polyacrylamide gels. Instead of the three bands described for mouse carcinoma chromatin, four main discrete components (MN1, MN2, MN2E and MN3) were resolved in the mononucleosome fraction of erythrocyte chromatin. MN2 contained all five histones and a DNA fragment of 165–180 base pairs. MN2E comprised four nucleosomal histones plus histone H5 (but not H1) and a DNA fragment of 170–190 base pairs. The relatively nuclease resistant MN3 fraction of erythrocyte nucleosomes contained H1 but no H5 histone. A more accurate analysis of the MN2 fraction in mouse carcinoma nucleosomes revealed some additional microheterogeneity depending on the presence of two different subfractions of H1.     

Genetic interaction of RAD53 protein kinase with histones is important for DNA replication

Studies in budding yeast suggest the protein kinase Rad53 plays novel roles in controlling initiation of DNA replication and in maintaining cellular histone levels, and these roles are independent of Rad53-mediated regulation of the checkpoint and of nucleotide levels. In order to elucidate the role of Rad53 in replication initiation, we isolated a novel allele of RAD53, rad53-rep, that separates the checkpoint function of RAD53 from the DNA replication function. rad53-rep mutants display a chromosome loss phenotype that is suppressed by increased origin dosage, providing further evidence that Rad53 plays a role in the initiation of DNA replication. Deletion of the major histone H3–H4 pair suppresses rad53-rep-cdc7-1 synthetic lethality, suggesting Rad53''s functions in degradation of excess cellular histone and in replication initiation are related. Rad53-rep is active as a protein kinase yet fails to interact with origins of replication and like the rad53Δ mutant, the rad53-rep mutant accumulates excess soluble histones, and it is sensitive to histone dosage. In contrast, a checkpoint defective allele of RAD53 with mutations in both FHA domains, binds origins and growth of this mutant is unaffected by histone dosage. Based on these observations, we hypothesize that the origin binding and the histone degradation activities of Rad53 are central to its function in DNA replication and are independent of its checkpoint functions. We propose a model in which Rad53 acts as a “nucleosome buffer”, interacting with origins of replication to prevent the binding of excess histones to origin DNA and to maintain proper chromatin configuration.Key words: DNA replication, Rad53, histones, checkpoint, origins of replication     

Structural insight into how the human helicase subunit MCM2 may act as a histone chaperone together with ASF1 at the replication fork

MCM2 is a subunit of the replicative helicase machinery shown to interact with histones H3 and H4 during the replication process through its N-terminal domain. During replication, this interaction has been proposed to assist disassembly and assembly of nucleosomes on DNA. However, how this interaction participates in crosstalk with histone chaperones at the replication fork remains to be elucidated. Here, we solved the crystal structure of the ternary complex between the histone-binding domain of Mcm2 and the histones H3-H4 at 2.9 Å resolution. Histones H3 and H4 assemble as a tetramer in the crystal structure, but MCM2 interacts only with a single molecule of H3-H4. The latter interaction exploits binding surfaces that contact either DNA or H2B when H3-H4 dimers are incorporated in the nucleosome core particle. Upon binding of the ternary complex with the histone chaperone ASF1, the histone tetramer dissociates and both MCM2 and ASF1 interact simultaneously with the histones forming a 1:1:1:1 heteromeric complex. Thermodynamic analysis of the quaternary complex together with structural modeling support that ASF1 and MCM2 could form a chaperoning module for histones H3 and H4 protecting them from promiscuous interactions. This suggests an additional function for MCM2 outside its helicase function as a proper histone chaperone connected to the replication pathway.     

Cooperating elephants mitigate competition until the stakes get too high

Cooperation is ubiquitous in the animal kingdom as it aims to maximize benefits through joint action. Selection, however, may also favor competitive behaviors that could violate cooperation. How animals mitigate competition is hotly debated, with particular interest in primates and little attention paid thus far to nonprimates. Using a loose-string pulling apparatus, we explored cooperative and competitive behavior, as well as mitigation of the latter, in semi-wild Asian elephants (Elephas maximus). Our results showed that elephants first maintained a very high cooperation rate (average = 80.8% across 45 sessions). Elephants applied “block,” “fight back,” “leave,” “move side,” and “submission” as mitigation strategies and adjusted these strategies according to their affiliation and rank difference with competition initiators. They usually applied a “fight back” mitigation strategy as a sanction when competition initiators were low ranking or when they had a close affiliation, but were submissive if the initiators were high ranking or when they were not closely affiliated. However, when the food reward was limited, the costly competitive behaviors (“monopoly” and “fight”) increased significantly, leading to a rapid breakdown in cooperation. The instability of elephant cooperation as a result of benefit reduction mirrors that of human society, suggesting that similar fundamental principles may underlie the evolution of cooperation across species.

This study shows that in a task requiring coordinated pulling, elephants compete for access to food but work to mitigate competition in order to maintain cooperation. If the cost of competition becomes too high, however, cooperation breaks down entirely. This behavior mirrors that seen in humans and other great apes, suggesting that certain cooperative mechanisms are not unique to primates.     

A Novel Bio-Sensor Based on DNA Strand Displacement

DNA strand displacement technology performs well in sensing and programming DNA segments. In this work, we construct DNA molecular systems based on DNA strand displacement performing computation of logic gates. Specifically, a class of so-called “DNA neurons” are achieved, in which a “smart” way inspired by biological neurons encoding information is developed to encode and deliver information using DNA molecules. The “DNA neuron” is bistable, that is, it can sense DNA molecules as input signals, and release “negative” or “positive” signals DNA molecules. We design intelligent DNA molecular systems that are constructed by cascading some particularly organized “DNA neurons”, which could perform logic computation, including AND, OR, XOR logic gates, automatically. Both simulation results using visual DSD (DNA strand displacement) software and experimental results are obtained, which shows that the proposed systems can detect DNA signals with high sensitivity and accretion; moreover, the systems can process input signals automatically with complex nonlinear logic. The method proposed in this work may provide a new way to construct a sensitive molecular signal detection system with neurons spiking behavior in vitro, and can be used to develop intelligent molecular processing systems in vivo.     

Erratum to: Singh R.K. and Gunjan A. Histone tyrosine phosphorylation comes of age. Epigenetics 2011; 6:153-60.

We recently noticed that there is a major error in Figure 1 of our review published in Epignetics 2010, Volume 6, Issue 2. During the preparation of the figure, the human and yeast H2B tyrosines were numbered the same, making the human numbering incorrect. The correct Figure 1 with proper numbering of human tyrosines is below.Erratum to:Singh R.K. and Gunjan A. Histone tyrosine phosphorylation comes of age.Epigenetics 2011; 6:153-60.We recently noticed that there is a major error in Figure 1 of our review published in Epignetics 2010, Volume 6, Issue 2. During the preparation of the figure, the human and yeast H2B tyrosines were numbered the same, making the human numbering incorrect. The correct Figure 1 with proper numbering of human tyrosines is below.Open in a separate windowFigure 1. Tyrosine residues are highly conserved between budding yeast and mammalian core histones. The four canonical core histone proteins from the budding yeast Saccharomyces cerevisiae are indicated by the prefix “Sc” and denoted in blue. The mammalian core histones and the mammalian variant histone H2A.X are shown in black. The number of amino acid (aa) residues in each core histone is indicated on the right. The location of the a-helices in the secondary structure of the histone proteins is indicated by cylinders. Tyrosine residues are shown as balloons and the tyrosine residues essential for viability in budding yeast histones are indicated by red balloons. Tyrosines in mammalian histones have not yet been evaluated to determine the residues essential for viability. Note the high degree of conservation of the location as well as the spacing of all but one tyrosine residue between budding yeast and mammalian core histones (H3 Y54 being the exception). Tyrosine residues that have recently been shown to be phosphorylated in vivo are marked by yellow “explosion” signs and the letter “P.” Additional tyrosine residues that are predicted to be reasonably accessible in the nucleosomal context under certain conditions and can be potentially phosphorylated in vivo are indicated by a yellow halo only on the mammalian histones for clarity, but are likely to be just as applicable to the yeast histones. Solid yellow halo indicates higher probability of phosphorylation, while a dashed yellow halo indicates lower probability of phosphorylation.     

Effect of salt on the binding of the linker histone H1 to DNA and nucleosomes

The linker histones are involved in the salt-dependent folding of the nucleosomes into higher-order chromatin structures. To better understand the mechanism of action of these histones in chromatin, we studied the interactions of the linker histone H1 with DNA at various histone/DNA ratios and at different ionic strengths. In direct competition experiments, we have confirmed the binding of H1 to superhelical DNA in preference to linear or nicked circular DNA forms. We show that the electrophoretic mobility of the H1/supercoiled DNA complex decreases with increasing H1 concentrations and increases with ionic strengths. These results indicate that the interaction of the linker histone H1 with supercoiled DNA results in a soluble binding of H1 with DNA at low H1 or salt concentrations and aggregation at higher H1 concentrations. Moreover, we show that H1 dissociates from the DNA or nucleosomes at high salt concentrations. By the immobilized template pull-down assay, we confirm these data using the physiologically relevant nucleosome array template.