Histone Tail DNA Research Articles

Quantification of Global Histone Post Translational Modifications Using Intranuclear Flow Cytometry in Isolated Mouse Brain Microglia.

Gene expression control occurs partially by modifications in chromatin structure, including the addition and removal of posttranslational modifications to histone tails. Histone post-translational modifications (HPTMs) can either facilitate gene expression or repression. For example, acetylation of histone tail lysine residues neutralizes the positive charge and reduces interactions between the tail and negatively charged DNA. The decrease in histone tail-DNA interactions results in increased accessibility of the underlying DNA, allowing for increased transcription factor access. The acetylation mark also serves as a recognition site for bromodomain-containing transcriptional activators, together resulting in enhanced gene expression. Histone marks can be dynamically regulated during cell differentiation and in response to different cellular environments and stimuli. While next-generation sequencing approaches have begun to characterize genomic locations for individual histone modifications, only one modification can be examined concurrently. Given that there are hundreds of different HPTMs, we have developed a high throughput, quantitative measure of global HPTMs that can be used to screen histone modifications prior to conducting more extensive genome sequencing approaches. This protocol describes a flow cytometry-based method to detect global HPTMs and can be conducted using cells in culture or isolated cells from in vivo tissues. We present example data from isolated mouse brain microglia to demonstrate the sensitivity of the assay to detect global shifts in HPTMs in response to a bacteria-derived immune stimulus (lipopolysaccharide). This protocol allows for the rapid and quantitative assessment of HPTMs and can be applied to any transcriptional or epigenetic regulator that can be detected by an antibody.

Binding of regulatory proteins to nucleosomes is modulated by dynamic histone tails

Little is known about the roles of histone tails in modulating nucleosomal DNA accessibility and its recognition by other macromolecules. Here we generate extensive atomic level conformational ensembles of histone tails in the context of the full nucleosome, totaling 65 microseconds of molecular dynamics simulations. We observe rapid conformational transitions between tail bound and unbound states, and characterize kinetic and thermodynamic properties of histone tail-DNA interactions. Different histone types exhibit distinct binding modes to specific DNA regions. Using a comprehensive set of experimental nucleosome complexes, we find that the majority of them target mutually exclusive regions with histone tails on nucleosomal/linker DNA around the super-helical locations ± 1, ± 2, and ± 7, and histone tails H3 and H4 contribute most to this process. These findings are explained within competitive binding and tail displacement models. Finally, we demonstrate the crosstalk between different histone tail post-translational modifications and mutations; those which change charge, suppress tail-DNA interactions and enhance histone tail dynamics and DNA accessibility.

Sequence-Modulated Electrostatics of Poly-Peptides-DNA Interactions

Unstructured poly-peptides are abundant in biology, either alone as intrinsically disordered peptides or as flexible domains of functional proteins. One example is the family of histone tails which are poly-cationic, unstructured terminal domains protruding from the nucleosome. Capable of acting as counter-ions to negatively charged DNA, histone tails are known to interact with nucleosomal DNA, linker DNA, as well as the nucleosome core. With the goal to gain first insights into the nature of peptide-nucleic acids interactions in a biological context, here we quantify and codify the interactions between histone tails and DNA, by employing the osmotic stress method to determine the force-spacing relations of ordered DNA arrays. Remarkably, such DNA force-spacing curves sensitively depend on both total peptide charge and the biochemical sequence of peptides with the same total charge, evidencing both charge regulated and sequence/structure dependent interactions between histone tails and DNA. We speculate on the physics of histone tail-DNA interactions in terms of charge patterns and structural compliance, and further discuss the biological implications of our observations and the general functional roles of poly-peptides in nucleic acids structure, interaction, and assembly.

Rouse model with transient intramolecular contacts on a timescale of seconds recapitulates folding and fluctuation of yeast chromosomes.

DNA folding and dynamics along with major nuclear functions are determined by chromosome structural properties, which remain, thus far, elusive in vivo. Here, we combine polymer modeling and single particle tracking experiments to determine the physico-chemical parameters of chromatin in vitro and in living yeast. We find that the motion of reconstituted chromatin fibers can be recapitulated by the Rouse model using mechanical parameters of nucleosome arrays deduced from structural simulations. Conversely, we report that the Rouse model shows some inconsistencies to analyze the motion and structural properties inferred from yeast chromosomes determined with chromosome conformation capture techniques (specifically, Hi-C). We hence introduce the Rouse model with Transient Internal Contacts (RouseTIC), in which random association and dissociation occurs along the chromosome contour. The parametrization of this model by fitting motion and Hi-C data allows us to measure the kinetic parameters of the contact formation reaction. Chromosome contacts appear to be transient; associated to a lifetime of seconds and characterized by an attractive energy of –0.3 to –0.5 kBT. We suggest attributing this energy to the occurrence of histone tail-DNA contacts and notice that its amplitude sets chromosomes in ‘theta’ conditions, in which they are poised for compartmentalization and phase separation.

Histone Tail-DNA Interactions: Charge Regulation and Sequence Specificity

Histone Tail-DNA Interactions: Charge Regulation and Sequence Specificity

DNA Nanomechanics in the Nucleosome

The sequence-dependent mechanics of DNA-histone binding are essential for nucleosome positioning and mobility. Here we reanalyze nucleosome crystal structures in terms of the well-characterized base-pair scale DNA elasticity, extracting the forces and torques acting on all bound DNA base pairs. We find that the strongest forces follow a characteristic repeating pattern that recovers the 12 known DNA backbone-histone contact sites. DNA twist defects and histone point mutations modify this pattern in interpretable ways. Additional, irregular forces between contact sites reveal histone tail-DNA interactions, whereas requiring the absence of external forces leads to a structural refinement of linker DNA. Based on these observations, we propose a simple, structure-based mechanical model of the nucleosome that is able to explain the placement of DNA twist defects in 146 base-pair nucleosomes and allows an estimate of the elastic energy spectrum of nucleosome twist defect states.

Site-specific Binding Affinities within the H2B Tail Domain Indicate Specific Effects of Lysine Acetylation

Acetylation of specific lysines within the core histone tail domains plays a critical role in regulating chromatin-based activities. However, the structures and interactions of the tail domains and the molecular mechanisms by which acetylation directly alters chromatin structures are not well understood. To address these issues we developed a chemical method to quantitatively determine binding affinities of specific regions within the individual tail domains in model chromatin complexes. Examinations of specific sites within the H2B tail domain indicate that this tail contains distinct structural elements and binds within nucleosomes with affinities that would reduce the activity of tail-binding proteins 10-50-fold from that deduced from peptide binding studies. Moreover, we find that mutations mimicking lysine acetylation do not cause a global weakening of tail-DNA interactions but rather the results suggest that acetylation leads to a much more subtle and specific alteration in tail interactions than has been assumed. In addition, we provide evidence that acetylation at specific sites in the tail is not additive with several events resulting in similar, localized changes in tail binding.

The Core Histone Tail Domains Contribute to Sequence-dependent Nucleosome Positioning

The precise positioning of nucleosomes plays a critical role in the regulation of gene expression by modulating the DNA binding activity of trans-acting factors. However, molecular determinants responsible for positioning are not well understood. We examined whether the removal of the core histone tail domains from nucleosomes reconstituted with specific DNA fragments led to alteration of translational positions. Remarkably, we find that removal of tail domains from a nucleosome assembled on a DNA fragment containing a Xenopus borealis somatic-type 5S RNA gene results in repositioning of nucleosomes along the DNA, including two related major translational positions that move about 20 bp further upstream with respect to the 5S gene. In a nucleosome reconstituted with a DNA fragment containing the promoter of a Drosophila alcohol dehydrogenase gene, several translational positions shifted by about 10 bp along the DNA upon tail removal. However, the positions of nucleosomes assembled with a DNA fragment known to have one of the highest binding affinities for core histone proteins in the mouse genome were not altered by removal of core histone tail domains. Our data support the notion that the basic tail domains bind to nucleosomal DNA and influence the selection of the translational position of nucleosomes and that once tails are removed movement between translational positions occurs in a facile manner on some sequences. However, the effect of the N-terminal tails on the positioning and movement of a nucleosome appears to be dependent on the DNA sequence such that the contribution of the tails can be masked by very high affinity DNA sequences. Our results suggest a mechanism whereby sequence-dependent nucleosome positioning can be specifically altered by regulated changes in histone tail-DNA interactions in chromatin.

Computer Modeling Demonstrates that Electrostatic Attraction of Nucleosomal DNA is Mediated by Histone Tails

We conducted molecular dynamics computer simulations of charged histone tail-DNA interactions in systems mimicking nucleosome core particles (NCP). In a coarse-grained model, the NCP is modeled as a negatively charged spherical particle with flexible polycationic histone tails attached to it in a dielectric continuum with explicit mobile counterions and added salt. The size, charge, and distribution of the tails relative to the core were built to mimick real NCP. In this way, we incorporate attractive ion-ion correlation effects due to fluctuations in the ion cloud and the attractive entropic and energetic tail-bridging effects. In agreement with experimental data, increase of monovalent salt content from salt-free to physiological concentration leads to the formation of NCP aggregates; likewise, in the presence of MgCl 2, the NCPs form condensed systems via histone-tail bridging and accumulation of counterions. More detailed mechanisms of the histone tail-DNA interactions and dynamics have been obtained from all-atom molecular dynamics simulations (including water), comprising three DNA 22-mers and 14 short fragments of the H4 histone tail (amino acids 5–12) carrying three positive charges on lysine + interacting with DNA. We found correlation of the DNA-DNA distance with the presence and association of the histone tail between the DNA molecules.

Probing Core Histone Tail–DNA Interactions in a Model Dinucleosome System

Publisher Summary The chapter highlights that the H4 N-terminal tail plays a pivotal role in compaction of the array. While electrostatic interactions between the histone tails and DNA clearly contribute to tail-directed folding of the chromatin fiber, the special role of the H4 tail may be due to H4 interactions with an H2A–H2B dimer within adjacent nucleosomes. This chapter presents a technique to study internucleosome protein–DNA interactions in a model dinucleosome system. Model dinucleosomes are generated in which one nucleosome typically contains a radiolabeled DNA template and native histones, whereas the other contains a single histone site specifically modified with a UV-activatable cross-linking agent. Each of the nucleosomes is reconstituted independently and then ligated together, and cross-linking is induced by UV irradiation under various conditions. After cross-linking, the two DNA templates are separated so that internucleosomal histone–DNA cross-links can be distinguished from intranucleosomal cross-links. In addition, the chapter describes a novel method for the preparation of H3-H4 tetramers.