Nikolay Korolev

The effects of histone H4 tail acetylations on cation-induced chromatin folding and self-association

Understanding the molecular mechanisms behind regulation of chromatin folding through covalent modifications of the histone N-terminal tails is hampered by a lack of accessible chromatin containing precisely modified histones. We study the internal folding and intermolecular self-association of a chromatin system consisting of saturated 12-mer nucleosome arrays containing various combinations of completely acetylated lysines at positions 5, 8, 12 and 16 of histone H4, induced by the cations Na+, K+, Mg2+, Ca2+, cobalt-hexammine3+, spermidine3+ and spermine4+. Histones were prepared using a novel semi-synthetic approach with native chemical ligation. Acetylation of H4-K16, but not its glutamine mutation, drastically reduces cation-induced folding of the array. Neither acetylations nor mutations of all the sites K5, K8 and K12 can induce a similar degree of array unfolding. The ubiquitous K+, (as well as Rb+ and Cs+) showed an unfolding effect on unmodified arrays almost similar to that of H4-K16 acetylation. We propose that K+ (and Rb+/Cs+) binding to a site on the H2B histone (R96-L99) disrupts H4K16 ε-amino group binding to this specific site, thereby deranging H4 tail-mediated nucleosome–nucleosome stacking and that a similar mechanism operates in the case of H4-K16 acetylation. Inter-array self-association follows electrostatic behavior and is largely insensitive to the position or nature of the H4 tail charge modification.

On the Competition between Water, Sodium Ions, and Spermine in Binding to DNA: A Molecular Dynamics Computer Simulation Study

The interaction of DNA with the polyamine spermine(4+) (Spm(4+)), sodium ions, and water molecules has been studied using molecular dynamics computer simulations in a system modeling a DNA crystal. The simulation model consisted of three B-DNA decamers in a periodic hexagonal cell, containing 1200 water molecules, 8 Spm(4+), 32 Na(+), and 4 Cl(-) ions. The present paper gives a more detailed account of a recently published report of this system and compares results on this mixed Spm(4+)/Na(+)-cation system with an molecular dynamics simulation carried out for the same DNA decamer under similar conditions with only sodium counterions (Korolev et al., J. Mol. Biol. 308:907). The presence of Spm(4+) makes significant influence on the DNA hydration and on the interaction of the sodium ions with DNA. Spermine pushes water molecules out of the minor groove, whereas Na(+) attracts and organizes water around DNA. The major binding site of the Spm(4+) amino groups and the Na(+) ions is the phosphate group of DNA. The flexible polyamine spermine displays a high presence in the minor groove but does not form long-lived and structurally defined complexes. Sodium ions compete with Spm(4+) for binding to the DNA bases in the minor groove. Sodium ions also have several strong binding sites in the major groove. The ability of water molecules, Spm(4+), and Na(+) to modulate the local structure of the DNA double helix is discussed.

Competitive binding of Mg2+, Ca2+, Na+, and K+ ions to DNA in oriented DNA fibers: experimental and Monte Carlo simulation results.

Competitive binding of the most common cations of the cytoplasm (K(+), Na(+), Ca(2+), and Mg(2+)) with DNA was studied by equilibrating oriented DNA fibers with ethanol/water solutions (65 and 52% v/v EtOH) containing different combinations and concentrations of the counterions. The affinity of DNA for the cations decreases in the order Ca > Mg >> Na approximately K. The degree of Ca(2+) and/or Mg(2+) binding to DNA displays maximum changes just at physiological concentrations of salts (60-200 mM) and does not depend significantly on the ethanol concentration or on the kind of univalent cation (Na(+) or K(+)). Ca(2+) is more tightly bound to DNA and is replaced by the monovalent cations to a lesser extent than is Mg(2+). Similarly, Ca(2+) is a better competitor for binding to DNA than Mg(2+): the ion exchange equilibrium constant for a 1:1 mixture of Ca(2+) and Mg(2+) ions, K(c)(Ca)(Mg), changes from K(c)(Ca)(Mg) approximately 2 in 65% EtOH (in 3-30 mM NaCl and/or KCl) to K(c)(Ca)(Mg) approximately 1.2-1.4 in 52% EtOH (in 300 mM NaCl and/or KCl). DNA does not exhibit selectivity for Na(+) or K(+) in ethanol/water solutions either in the absence or in the presence of Ca(2+) and/or Mg(2+). The ion exchange experimental data are compared with results of grand canonical Monte Carlo (GCMC) simulations of systems of parallel and hexagonally ordered, uniformly and discretely charged polyions with the density and spatial distribution of the charged groups modeling B DNA. A quantitative agreement with experimental data on divalent-monovalent competition has been obtained for discretely charged models of the DNA polyion (for the uniformly charged cylinder model, coincidence with experiment is qualitative). The GCMC method gives also a qualitative description of experimental results for DNA binding competitions of counterions of the same charge (Ca(2+) with Mg(2+) or K(+) with Na(+)).

Similarities and differences in interaction of K+ and Na+ with condensed ordered DNA. A molecular dynamics computer simulation study.

Four 20 ns molecular dynamics simulations have been performed with two counterions, K+ or Na+, at two water contents, 15 or 20 H2O per nucleotide. A hexagonal simulation cell comprised of three identical DNA decamers [d(5′-ATGCAGTCAG) × d(5′-TGACTGCATC)] with periodic boundary condition along the DNA helix was used. The simulation setup mimics the DNA state in oriented DNA fibers or in crystals of DNA oligomers. Variation of counterion nature and water content do not alter averaged DNA structure. K+ and Na+ binding to DNA are different. K+ binds to the electronegative sites of DNA bases in the major and the minor grooves, while Na+ interacts preferentially with the phosphate groups. Increase of water causes a shift of both K+ and Na+ from the first hydration shell of O1P/O2P and of the DNA bases in the minor groove with lesser influence for the cation binding to the bases in the major groove. Mobility of both water and cations in the K–DNA systems is faster than in the Na–DNA systems: Na+ organizes and immobilizes water structure around itself and near DNA while for K+ water is less organized and more dynamic.

A universal description for the experimental behavior of salt-(in)dependent oligocation-induced DNA condensation.

We report a systematic study of the condensation of plasmid DNA by oligocations with variation of the charge, Z, from +3 to +31. The oligocations include a series of synthetic linear ε-oligo(l-lysines), (denoted εKn, n = 3–10, 31; n is the number of lysines equal to the ligand charge) and branched α-substituted homologues of εK10: εYK10, εLK10 (Z = +10); εRK10, εYRK10 and εLYRK10 (Z = +20). Data were obtained by light scattering, UV absorption monitored precipitation assay and isothermal titration calorimetry in a wide range concentrations of DNA and monovalent salt (KCl, CKCl). The dependence of EC50 (ligand concentration at the midpoint of DNA condensation) on CKCl shows the existence of a salt-independent regime at low CKCl and a salt-dependent regime with a steep rise of EC50 with increase of CKCl. Increase of the ligand charge shifts the transition from the salt-independent to salt-dependent regime to higher CKCl. A novel and simple relationship describing the EC50 dependence on DNA concentration, charge of the ligand and the salt-dependent dissociation constant of the ligand–DNA complex is derived. For the ε-oligolysines εK3–εK10, the experimental dependencies of EC50 on CKCl and Z are well-described by an equation with a common set of parameters. Implications from our findings for understanding DNA condensation in chromatin are discussed.

Influence of Histone Tails and H4 Tail Acetylations on Nucleosome–Nucleosome Interactions

Nucleosome-nucleosome interaction plays a fundamental role in chromatin folding and self-association. The cation-induced condensation of nucleosome core particles (NCPs) displays properties similar to those of chromatin fibers, with important contributions from the N-terminal histone tails. We study the self-association induced by addition of cations [Mg(2+), Ca(2+), cobalt(III)hexammine(3+), spermidine(3+) and spermine(4)(+)] for NCPs reconstituted with wild-type unmodified histones and with globular tailless histones and for NCPs with the H4 histone tail having lysine (K) acetylations or lysine-to-glutamine mutations at positions K5, K8, K12 and K16. In addition, the histone construct with the single H4K16 acetylation was investigated. Acetylated histones were prepared by a semisynthetic native chemical ligation method. The aggregation behavior of NCPs shows a general cation-dependent behavior similar to that of the self-association of nucleosome arrays. Unlike nucleosome array self-association, NCP aggregation is sensitive to position and nature of the H4 tail modification. The tetra-acetylation in the H4 tail significantly weakens the nucleosome-nucleosome interaction, while the H4 K→Q tetra-mutation displays a more modest effect. The single H4K16 acetylation also weakens the self-association of NCPs, which reflects the specific role of H4K16 in the nucleosome-nucleosome stacking. Tailless NCPs can aggregate in the presence of oligocations, which indicates that attraction also occurs by tail-independent nucleosome-nucleosome stacking and DNA-DNA attraction in the presence of cations. The experimental data were compared with the results of coarse-grained computer modeling for NCP solutions with explicit presence of mobile ions.

Application of Polyelectrolyte Theories for Analysis of DNA Melting in the Presence of Na+ and Mg2+ Ions

Numerical calculations, using Poisson-Boltzmann (PB) and counterion condensation (CC) polyelectrolyte theories, of the electrostatic free energy difference, DeltaGel, between single-stranded (coil) and double-helical DNA have been performed for solutions of NaDNA + NaCl with and without added MgCl2. Calculations have been made for conditions relevant to systems where experimental values of helix coil transition temperature (Tm) and other thermodynamic quantities have been measured. Comparison with experimental data has been possible by invoking values of Tm for solutions containing NaCl salt only. Resulting theoretical values of enthalpy, entropy, and heat capacity (for NaCl salt-containing solutions) and of Tm as a function of NaCl concentration in NaCl + MgCl2 solutions have thus been obtained. Qualitative and, to a large extent, quantitative reproduction of the experimental Tm, DeltaHm, DeltaSm, and DeltaCp values have been found from the results of polyelectrolyte theories. However, the quantitative resemblance of experimental data is considerably better for PB theory as compared to the CC model. Furthermore, some rather implausible qualitative conclusions are obtained within the CC results for DNA melting in NaCl + MgCl2 solutions. Our results argue in favor of the Poisson-Boltzmann theory, as compared to the counterion condensation theory.

Electrostatic Origin of Salt-Induced Nucleosome Array Compaction

The physical mechanism of the folding and unfolding of chromatin is fundamentally related to transcription but is incompletely characterized and not fully understood. We experimentally and theoretically studied chromatin compaction by investigating the salt-mediated folding of an array made of 12 positioning nucleosomes with 177 bp repeat length. Sedimentation velocity measurements were performed to monitor the folding provoked by addition of cations Na(+), K(+), Mg(2+), Ca(2+), spermidine(3+), Co(NH(3))(6)(3+), and spermine(4+). We found typical polyelectrolyte behavior, with the critical concentration of cation needed to bring about maximal folding covering a range of almost five orders of magnitude (from 2 μM for spermine(4+) to 100 mM for Na(+)). A coarse-grained model of the nucleosome array based on a continuum dielectric description and including the explicit presence of mobile ions and charged flexible histone tails was used in computer simulations to investigate the cation-mediated compaction. The results of the simulations with explicit ions are in general agreement with the experimental data, whereas simple Debye-Hückel models are intrinsically incapable of describing chromatin array folding by multivalent cations. We conclude that the theoretical description of the salt-induced chromatin folding must incorporate explicit mobile ions that include ion correlation and ion competition effects.

Modelling chromatin structure and dynamics: status and prospects

The packaging of genomic DNA into chromatin in the eukaryotic cell nucleus demands extensive compaction. This requires attractive nucleosome-nucleosome interactions to overcome repulsion between the negatively charged DNA segments as well as other constraints. At the same time, DNA must be dynamically accessible to the cellular machinery that operates on it. Recent progress in the experimental characterisation of the higher order structure and dynamics of well-defined chromatin fibres has stimulated the attempts at theoretical description of chromatin and the nucleosome. Here we review the present status of chromatin modelling, with particular emphasis on coarse-grained computer simulation models, the role of electrostatic interactions, and discuss future perspectives in the field.

Cation-induced polyelectrolyte–polyelectrolyte attraction in solutions of DNA and nucleosome core particles

The paper reviews our current studies on the experimentally induced cation compaction and aggregation in solutions of DNA and nucleosome core particles and the theoretical modelling of these processes using coarse-grained continuum models with explicit mobile ions and with all-atom molecular dynamics (MD) simulations. Recent experimental results on DNA condensation by cationic oligopeptides and the effects of added salt are presented. The results of MD simulations modelling DNA-DNA attraction due to the presence of multivalent ions including the polyamine spermidine and fragments of histone tails, which exhibit bridging between adjacent DNA molecules, are discussed. Experimental data on NCP aggregation, using recombinantly prepared systems are summarized. Literature data and our results of studying of the NCP solutions are compared with predictions of coarse-grained MD simulations, including the important ion correlation as well as bridging mechanisms. The importance of the results to chromatin folding and aggregation is discussed.