Abdollah Allahverdi

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.

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.

Chromatin compaction under mixed salt conditions: Opposite effects of sodium and potassium ions on nucleosome array folding

It is well known that chromatin structure is highly sensitive to the ionic environment. However, the combined effects of a physiologically relevant mixed ionic environment of K+, Mg2+ and Na+, which are the main cations of the cell cytoplasm, has not been systematically investigated. We studied folding and self-association (aggregation) of recombinant 12-mer nucleosome arrays with 177 bp DNA repeat length in solutions of mixtures of K+ and Mg2+ or Na+ and Mg2+. In the presence of Mg2+, the addition of sodium ions promotes folding of array into 30-nm fibres, whereas in mixtures of K+ and Mg2+, potassium ions abrogate folding. We found that self-association of nucleosome arrays in mixed salt solutions is synergistically promoted by Mg2+ and monovalent ions, with sodium being slightly more efficient than potassium in amplifying the self-association. The results highlight the importance of a mixed ionic environment for the compaction of chromatin under physiological conditions and demonstrate the complicated nature of the various factors that determine and regulate chromatin compaction in vivo.

The effect of salt on oligocation-induced chromatin condensation

Condensation of model chromatin in the form of fully saturated 12-mer nucleosome arrays, induced by addition of cationic ligands (ε-oligolysines with charge varied from +4 to +11), was studied in a range of KCl concentrations (10-500mM) using light scattering and precipitation assay titrations. The dependence of EC(50) (ligand concentration at the midpoint of the array condensation) on C(KCl) displays two regimes, a salt-independent at low C(KCl) and a salt-dependent at higher salt concentrations. In the salt-dependent regime EC(50) rises sharply with increase of C(KCl). Increase of ligand charge shifts the transition from the salt-independent to salt-dependent regime to higher salt. In the nucleosome array system, due to the partial neutralization of the DNA charge by histones, a lower oligocation concentration is needed to provoke condensation in the salt-independent regime compared to the related case of DNA condensation by the same cation. In the physiological range of salt concentrations (C(KCl)=50-300mM), K(+) ions assist array condensation by shifting EC(50) of the ε-oligolysines to lower values. At higher C(KCl), K(+) competes with the cationic ligands, which leads to increase of EC(50). Values of salt-dependent dissociation constant for the ε-oligolysine-nucleosome array interaction were obtained, by fitting to a general equation developed earlier for DNA, describing the dependence of EC(50) on dissociation constant, salt and polyelectrolyte concentrations.

The Influence of Ionic Environment and Histone Tails on Columnar Order of Nucleosome Core Particles

The nucleosome core particle (NCP) is the basic building block of chromatin. Nucleosome-nucleosome interactions are instrumental in chromatin compaction, and understanding NCP self-assembly is important for understanding chromatin structure and dynamics. Recombinant NCPs aggregated by multivalent cations form various ordered phases that can be studied by x-ray diffraction (small-angle x-ray scattering). In this work, the effects on the supramolecular structure of aggregated NCPs due to lysine histone H4 tail acetylations, histone H2A mutations (neutralizing the acidic patch of the histone octamer), and the removal of histone tails were investigated. The formation of ordered mainly hexagonal columnar NCP phases is in agreement with earlier studies; however, the highly homogeneous recombinant NCP systems used in this work display a more compact packing. The long-range order of the NCP columnar phase was found to be abolished or reduced by acetylation of the H4 tails, acidic patch neutralization, and removal of the H3 and H2B tails. Loss of nucleosome stacking upon removal of the H3 tails in combination with other tails was observed. In the absence of the H2A tails, the formation of an unknown highly ordered phase was observed.

Study of nickel and copper biosorption on brown algae Sargassum angustifolium: application of response surface methodology (RSM)

This study has been focused on the batch culture removal of Cu2+ and Ni2+ ions from the aqueous solution using marine brown algae Sargassum angustifolium. Influences of parameters like pH, initial metal ions concentration and biosorbent dosage on nickel and copper adsorption were also examined using the Box–Behnken design matrix. For biosorption of Cu2+ the optimum pH value was determined as 5.0, optimum biosorbent concentration to 1.0 g/L and optimum initial concentration 0.15 mmol/L. For the biosorption of Ni2+, the optimal condition was the same but the optimum pH value was determined as 6.0. Desorption experiments indicated that CH3COOH and EDTA were efficient desorbents for recovery from Cu2+ and Ni2+. The Langmuir isotherm model was applied to describe the biosorption of the Cu2+ and Ni2+ into S. angustifolium. The maximum uptake of Cu2+ and Ni2+ ions by the S. angustifolium biomass under the optimal conditions was approximately 0.94 and 0.78 mmol/g dry alga, respectively. Response surface models showed that the data were adequately fitted to a second-order polynomial model. Analysis of variance showed a high coefficient of determination value (R2=0.993 for Cu2+ and 0.991 for Ni2+) and a satisfactory second-order regression model was derived. In addition, results reported in this research demonstrated the feasibility of employing S. angustifolium as biosorbent for Ni2+ and Cu2+ removal.

Single-molecule force spectroscopy on histone H4 tail-cross-linked chromatin reveals fiber folding

The eukaryotic genome is highly compacted into a protein-DNA complex called chromatin. The cell controls access of transcriptional regulators to chromosomal DNA via several mechanisms that act on chromatin-associated proteins and provide a rich spectrum of epigenetic regulation. Elucidating the mechanisms that fold chromatin fibers into higher-order structures is therefore key to understanding the epigenetic regulation of DNA accessibility. Here, using histone H4-V21C and histone H2A-E64C mutations, we employed single-molecule force spectroscopy to measure the unfolding of individual chromatin fibers that are reversibly cross-linked through the histone H4 tail. Fibers with covalently linked nucleosomes featured the same folding characteristics as fibers containing wild-type histones but exhibited increased stability against stretching forces. By stabilizing the secondary structure of chromatin, we confirmed a nucleosome repeat length (NRL)-dependent folding. Consistent with previous crystallographic and cryo-EM studies, the obtained force-extension curves on arrays with 167-bp NRLs best supported an underlying structure consisting of zig-zag, two-start fibers. For arrays with 197-bp NRLs, we previously inferred solenoidal folding, which was further corroborated by force-extension curves of the cross-linked fibers. The different unfolding pathways exhibited by these two types of arrays and reported here extend our understanding of chromatin structure and its potential roles in gene regulation. Importantly, these findings imply that chromatin compaction by nucleosome stacking protects nucleosomal DNA from external forces up to 4 piconewtons.

Gelation of the genome by topoisomerase II targeting anticancer agents

Topoisomerase II (TOP2) regulates the topology of DNA by catalysis of a double strand passage reaction. Inhibition of this reaction prevents cell replication, and, thus, is a pathway targeted by anticancer drugs. Some details regarding the cell-killing mechanism are unknown and assays to screen for anticancer drugs are not well established. Here, we study the gelation of linear and circular DNA using microrheology assays. Gelation of the DNA–enzyme mixture was examined by tracking of multiple colloidal probe particles. The mean square displacements of the probe particles were analyzed by the time-cure superposition procedure as well as the classical derivation of the dynamic moduli. First, the passage reaction was inhibited by AMP-PNP, a non-hydrolyzable analog of ATP. The results showed gelation due to the formation of a self-catenated network of circular DNA molecules. Next, when TOP2 was inhibited by the anti-cancer drug ICRF-193, we observed a similar change in rheology. Based on these findings, we propose a cell-killing mechanism by gelation of the genome through TOP2-mediated interlocking of looped DNA segments of the replicated, intertwined chromosomes.

EZH2 promotes neoplastic transformation through VAV interaction-dependent extranuclear mechanisms

Recently, we reported that the histone methyltransferase, EZH2, controls leukocyte migration through interaction with the cytoskeleton remodeling effector, VAV, and direct methylation of the cytoskeletal regulatory protein, Talin. However, it is unclear whether this extranuclear, epigenetic-independent function of EZH2 has a profound impact on the initiation of cellular transformation and metastasis. Here, we show that EZH2 increases Talin1 methylation and cleavage, thereby enhancing adhesion turnover and promoting accelerated tumorigenesis. This transforming capacity is abolished by targeted disruption of EZH2 interaction with VAV. Furthermore, our studies demonstrate that EZH2 in the cytoplasm is closely associated with cancer stem cell properties, and that overexpression of EZH2, a mutant EZH2 lacking its nuclear localization signal (EZH2ΔNLS), or a methyl-mimicking Talin1 mutant substantially promotes JAK2-dependent STAT3 activation and cellular transformation. Taken together, our results suggest a critical role for the VAV interaction-dependent, extranuclear action of EZH2 in neoplastic transformation.