Chicken Erythrocyte Chromatin Research Articles

Transcriptionally Active Chromatin-Lessons Learned from the Chicken Erythrocyte Chromatin Fractionation.

The chicken erythrocyte model system has been valuable to the study of chromatin structure and function, specifically for genes involved in oxygen transport and the innate immune response. Several seminal features of transcriptionally active chromatin were discovered in this system. Davie and colleagues capitalized on the unique features of the chicken erythrocyte to separate and isolate transcriptionally active chromatin and silenced chromatin, using a powerful native fractionation procedure. Histone modifications, histone variants, atypical nucleosomes (U-shaped nucleosomes) and other chromatin structural features (open chromatin) were identified in these studies. More recently, the transcriptionally active chromosomal domains in the chicken erythrocyte genome were mapped by combining this chromatin fractionation method with next-generation DNA and RNA sequencing. The landscape of histone modifications relative to chromatin structural features in the chicken erythrocyte genome was reported in detail, including the first ever mapping of histone H4 asymmetrically dimethylated at Arg 3 (H4R3me2a) and histone H3 symmetrically dimethylated at Arg 2 (H3R2me2s), which are products of protein arginine methyltransferases (PRMTs) 1 and 5, respectively. PRMT1 is important in the establishment and maintenance of chicken erythrocyte transcriptionally active chromatin.

Histone N-terminal Tails Interfere with Nucleosome Traversal by RNA Polymerase II

We determined the effect of the N-terminal histone tails on nucleosome traversal by yeast and human RNA polymerase II (pol II). Removal of H2A/H2B tails, H3/H4 tails, or all tails increased complete traversal of the nucleosome by human pol II, although the increase varied considerably depending on the template and on which tails were removed. Human pol II achieved >80% traversal of one nucleosomal template lacking the H2A/H2B tails, but even in those reactions, the transcript elongation rate was lower than the rate on pure DNA templates. For yeast pol II, transcription proceeded much farther into the nucleosome in the absence of tails, but complete read-through was not substantially increased by tail removal. Transcription factor IIS provided roughly the same level of read-through stimulation for transcript elongation in the presence or absence of tails. FACT also stimulated elongation on nucleosomal templates, and this effect was similar regardless of the presence of tails. For both polymerases, removal of the H2A/H2B tails reduced pausing throughout the nucleosome, suggesting that histone tails affect a common step at most points during nucleosome traversal. We conclude that histone tails provide a significant part of the nucleosomal barrier to pol II transcript elongation.

The Effect of Internucleosomal Interaction on Folding of the Chromatin Fiber

The folding of the nucleosome chain into a chromatin fiber modulates DNA accessibility and is therefore an important factor for the control of gene expression. The fiber conformation depends crucially on the interaction between individual nucleosomes. However, this parameter has not been accurately determined experimentally, and it is affected by posttranslational histone modifications and binding of chromosomal proteins. Here, the effect of different internucleosomal interaction strengths on the fiber conformation was investigated by Monte Carlo computer simulations. The fiber geometry was modeled to fit that of chicken erythrocyte chromatin, which has been examined in numerous experimental studies. In the Monte Carlo simulation, the nucleosome shape was described as an oblate spherocylinder, and a replica exchange protocol was developed to reach thermal equilibrium for a broad range of internucleosomal interaction energies. The simulations revealed the large impact of the nucleosome geometry and the nucleosome repeat length on the compaction of the chromatin fiber. At high internucleosomal interaction energies, a lateral self-association of distant fiber parts and an interdigitation of nucleosomes were apparent. These results identify key factors for the control of the compaction and higher order folding of the chromatin fiber.

Phosphorylated serine 28 of histone H3 is associated with destabilized nucleosomes in transcribed chromatin

Histone modifications and variants have key roles in the activation and silencing of genes. Phosphorylation of histone H3 at serine 10 and serine 28 is involved in transcriptional activation of genes responding to stress or mitogen-stimulated signaling pathways. The distribution of H3-modified isoforms in G0 phase chicken erythrocyte chromatin was investigated. H3 phosphorylated at serine 28 was found highly enriched in the active/competent gene fractions, as was H3 di- and trimethylated at lysine 4. The H3 variant H3.3 in this chromatin fraction was preferentially phosphorylated at serine 28. Conversely, H3 phosphorylated at serine 10 was present in all chromatin fractions, while H3 dimethylated at lysine 9 was associated with the chromatin-containing repressed genes. H3 phosphorylated at serine 28 was located at the promoter region of the transcriptionally active, but not competent, histone H5 and β-globin genes. We provide evidence that H3.3 phosphorylated at serine 28 was present in labile nucleosomes. We propose that destabilized nucleosomes containing H3.3 phosphorylated at serine 28 aid in the dynamic disassembly–assembly of nucleosomes in active promoters.

Highly compact folding of chromatin induced by cellular cation concentrations. Evidence from atomic force microscopy studies in aqueous solution

We have performed a very extensive investigation of chromatin folding in different buffers over a wide range of ionic conditions similar to those found in eukaryotic cells. Our results show that in the presence of physiological concentrations of monovalent cations and/or low concentrations of divalent cations, small chicken erythrocyte chromatin fragments and chromatin from HeLa cells observed by transmission electron microscopy (TEM) show a compact folding, forming circular bodies of approximately 35 nm in diameter that were found previously in our laboratory in studies performed under very limited conditions. Since TEM images are obtained with dehydrated samples, we have performed atomic force microscopy (AFM) experiments to analyze chromatin structure in the presence of solutions containing different cation concentrations. The highly compact circular structures (in which individual nucleosomes are not visible as separated units) produced by small chromatin fragments in interphase ionic conditions observed by AFM are equivalent to the structures observed by TEM with chromatin samples prepared under the same ionic conditions. We have also carried out experiments of sedimentation and trypsin digestion of chromatin fragments; the results obtained confirm our AFM observations. Our results suggest that the compaction of bulk interphase chromatin in solution at room temperature is considerably higher than that generally considered in current literature. The dense chromatin folding observed in this study is consistent with the requirement of compact chromatin structures as starting elements for the building of metaphase chromosomes, but poses a difficult physical problem for gene expression during interphase.

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.

Binding of Antitumor Antibiotic Daunomycin to Histones in Chromatin and in Solution

Daunomycin is an anticancer drug that is well-known to interact with DNA in chromatin. Using a compositionally defined chicken erythrocyte chromatin fraction, we have obtained conclusive evidence that the drug is also able to interact with chromatin-bound linker histones without any noticeable binding to core histones. The drug can interact in an equal fashion with both histone H1 and H5 and to a greater extent with core histones H3/H4 and H2A/H2B as free proteins in solution. Thus, the binding of daunomycin to linker histones in the chromatin fiber is most likely due to the well-known higher accessibility of these histones to the surrounding environment of the fiber. Binding of daunomycin to linker histones appears to primarily involve the trypsin-resistant (winged-helix) domain of these proteins. The studies described here reveal the occurrence of a previously undisclosed mechanism for the antitumor activity of anthracycline drugs at the chromatin level.

Computational Modeling Predicts the Structure and Dynamics of Chromatin Fiber

Background: The compact form of the chromatin fiber is a critical regulator of fundamental processes such as transcription and replication. These reactions can occur only when the fiber is unraveled and the DNA strands contained within are exposed to interact with nuclear proteins. While progress on identifying the biochemical mechanisms that control localized folding and hence govern access to genetic information continues, the internal structure of the chromatin fiber, let alone the structural pathways for folding and unfolding, remain unknown. Results: To offer structural insights into how this nucleoprotein complex might be organized, we present a macroscopic computer model describing the mechanics of the chromatin fiber on the polymer level. We treat the core particles as electrostatically charged disks linked via charged elastic DNA segments and surrounded by a microionic hydrodynamic solution. Each nucleosome unit is represented by several hundred charges optimized so that the effective Debye-Hückel electrostatic field matches the field predicted by the nonlinear Poisson-Boltzmann equation. On the basis of Brownian dynamics simulations, we show that oligonucleosomes condense and unfold in a salt-dependent manner analogous to the chromatin fiber Conclusions: Our predicted chromatin model shows good agreement with experimental diffusion coefficients and small-angle X-ray scattering data. A fiber of width 30 nm, organized in a compact helical zigzag pattern with about 4 nucleosomes per 10 nm, naturally emerges from a repeating nucleosome folding motif. This fiber has a cross-sectional radius of gyration of R c = 8.66 nm, in close agreement with corresponding values for rat thymus and chicken erythrocyte chromatin (8.82 and 8.5 nm, respectively).

Multiple Histone Acetyltransferases Are Associated with a Chicken Erythrocyte Chromatin Fraction Enriched in Active Genes

We have examined salt-soluble chromatin released by micrococcal nuclease from a 15-day-old chicken embryo erythrocyte nuclei for histone acetyltransferase (HAT) activities. This chromatin is enriched in transcriptionally active sequences from within the active beta-globin locus and contains elevated levels of acetylated core histones. HAT activities present in this fraction target histones H4, H3, and H2A when the chromatin itself is used as the substrate. In gel HAT activity assay demonstrates that the salt-soluble chromatin fraction contains four acetyltransferase molecules distinguished by their different molecular masses (47, 33, 32, and 28 kDa). Further separation of the chromatin by centrifugation through sucrose gradients shows that the acetyltransferases segregate into chromatin-bound and chromatin-free populations. The 32- and 28-kDa HATs are associated with chromatin, whereas the 47- and 33-kDa HAT molecules are not. The chromatin-bound HAT activities predominantly target H4 to give the diacetyl and triacetyl species; some acetylation of H2A can also be seen. Our results suggest that the chromatin-associated acetyltransferases have a role in gene regulation.

Salt-Dependent Compaction of Di- and Trinucleosomes Studied by Small-Angle Neutron Scattering

Using small-angle neutron scattering (SANS), we have measured the salt-dependent static structure factor of di- and trinucleosomes from chicken erythrocytes and from COS-7 cells. We also determined the sedimentation coefficients of these dinucleosomes and dinucleosomes reconstituted on a 416-bp DNA containing two nucleosome positioning sequences of the 5S rDNA of Lytechinus variegatus at low and high salt concentrations. The internucleosomal distance d was calculated by simulation as well as Fourier back-transformation of the SANS curves and by hydrodynamic simulation of sedimentation coefficients. Nucleosome dimers from chicken erythrocyte chromatin show a decrease in d from ∼220 Å at 5 mM NaCl to 150 Å at 100 mM NaCl. For dinucleosomes from COS-7 chromatin, d decreases from 180 Å at 5 mM to 140 Å at 100 mM NaCl concentration. Our measurements on trinucleosomes are compatible with a compaction through two different mechanisms, depending on the salt concentration. Between 0 and 20 mM NaCl, the internucleosomal distance between adjacent nucleosomes remains constant, whereas the angle of the DNA strands entering and leaving the central nucleosome decreases. Above 20 mM NaCl, the adjacent nucleosomes approach each other, similar to the compaction of dinucleosomes. The internucleosomal distance of 140–150 Å at 100 mM NaCl is in agreement with distances measured by scanning force microscopy and electron microscopy on long chromatin filaments.

Pulling a single chromatin fiber reveals the forces that maintain its higher-order structure.

Single chicken erythrocyte chromatin fibers were stretched and released at room temperature with force-measuring laser tweezers. In low ionic strength, the stretch-release curves reveal a process of continuous deformation with little or no internucleosomal attraction. A persistence length of 30 nm and a stretch modulus of approximately 5 pN is determined for the fibers. At forces of 20 pN and higher, the fibers are modified irreversibly, probably through the mechanical removal of the histone cores from native chromatin. In 40-150 mM NaCl, a distinctive condensation-decondensation transition appears between 5 and 6 pN, corresponding to an internucleosomal attraction energy of approximately 2.0 kcal/mol per nucleosome. Thus, in physiological ionic strength the fibers possess a dynamic structure in which the fiber locally interconverting between "open" and "closed" states because of thermal fluctuations.

Daunomycin-induced Unfolding and Aggregation of Chromatin

Using equilibrium dialysis and sedimentation velocity analysis, we have characterized the binding of the anti-tumor drug daunomycin to chicken erythrocyte chromatin before and after depletion of linker histones and to its constitutive DNA under several ionic strengths (5, 25, and 75 mM NaCl). The equilibrium dialysis experiments reveal that the drug binds cooperatively to both the chromatin fractions and to the DNA counterpart within the range of ionic strength used in this study. A significant decrease in the binding affinity was observed at 75 mM NaCl. At any given salt concentration, daunomycin exhibits higher binding affinity for DNA than for linker histone-depleted chromatin or chromatin (in decreasing order). Binding of daunomycin to DNA does not significantly affect the sedimentation coefficient of the molecule. This is in contrast to binding to chromatin and to its linker histone-depleted counterpart. In these instances, preferential binding of the drug to the linker DNA regions induces an unfolding of the chromatin fiber that is followed by aggregation, presumably because of histone-DNA interfiber interactions.

Critical Electrolyte Concentration of Chicken Erythrocyte Chromatin.

This study examined whether chicken erythrocyte chromatin, whose great physicochemical stability is promoted by histone H5, has a critical electrolyte concentration (CEC) value comparable to that of other chromatin types with special condensed packing states but devoid of H5. The affinity for toluidine blue molecules under Mg2+ competitive binding action was thus investigated. The CEC value was found to be 0.12M, which is close to that of heterochromatin from mouse liver cells and to sperm chromatin with histone H1 variants. Acetic ethanol fixative was found to affect the CEC, possibly by influencing the chromatin packing state and DNA phosphate availability through dissociation of proteins from the DNA. The phenomenon of programmed cell death was considered to contribute to high CEC values by inducing special states of chromatin condensation.

Isolation and separation of HMG proteins and histones H1 and H5 and core histones by column chromatography on phosphocellulose.

A single-step method to dissociate histones as well as nonhistone chromosomal proteins from chicken erythrocyte chromatin and their separation into histones H1, H5, core histones, and high mobility group proteins by column chromatography on phosphocellulose is presented. NaCl at 2.0 M is effective in dissociating both histones and nonhistone proteins. The core histones elute as a complex. The pH is a critical factor in separating H5 from the core histones.

Linker histones stabilize the intrinsic salt-dependent folding of nucleosomal arrays: mechanistic ramifications for higher-order chromatin folding.

Defined nucleosomal arrays reconstituted from core histone octamers and twelve 208 bp tandem repeats of Lytechinus 5S rDNA (208-12 nucleosomal arrays) possess the ability to form an unstable folded species in MgCl2 whose extent of compaction equals that of canonical higher-order 30 nm diameter chromatin structures [Schwarz, P. M., and Hansen, J. C. (1994) J. Biol. Chem. 269, 16284-16289]. To address the mechanistic functions of linker histones in chromatin condensation, purified histone H5 has been assembled with 208-12 nucleosomal arrays in 50 mM NaCl. Novel purification procedures subsequently were developed that yielded preparations of 208-12 chromatin model systems in which a majority of the sample contained both one histone octamer per 5S rDNA repeat and one molecule of histone H5 per histone octamer. The integrity of the purified 208-12 chromatin has been extensively characterized under low-salt conditions using analytical ultracentrifugation, quantitative agarose gel electrophoresis, electron cryomicroscopy, and nuclease digestion. Results indicate that histone H5 binding to 208-12 nucleosomal arrays constrains the entering and exiting linker DNA in a way that produces structures that are indistinguishable from native chicken erythrocyte chromatin. Folding experiments performed in NaC1 and MgC12 have shown that H5 binding markedly stabilizes both the intermediate and extensively folded states of nucleosomal arrays without fundamentally altering the intrinsic nucleosomal array folding pathway. These results provide new insight into the mechanism of chromatin folding by demonstrating for the first time that distinctly different macromolecular determinants are required for formation and stabilization of higher-order chromatin structures.

Calorimetric investigation of ethidium and netropsin binding to chicken erythrocyte chromatin.

We have investigated the thermodynamic aspects of the ligand binding to chromatin, using isothermal titration calorimetry. Two classical DNA ligands were used: an intercalator, ethidium bromide, and a minor groove binder, netropsin. Stoichiometry, affinity constant, and thermodynamic parameters were determined at various salt concentrations and different temperatures. The effect of ionic strength was analyzed according to the Record theory applied to chromatin. We also compared the binding parameters on naked DNA, H1/H5-depleted chromatin, and chromatin. We demonstrated that the presence of histones on DNA still allows the ligand binding that takes place according to a simple one single-site model. For both ligand types, the thermodynamic driving force is enthalpic and the association is characterized by a somewhat weaker affinity and more scattered ligand distribution than on naked DNA. The ligand affinity is weakly altered by the salt-induced compaction of the chromatin and the binding is accompanied by a release of one counterion per ligand molecule. The temperature-dependent studies revealed the existence of a small heat capacity change associated with ligand binding to chromatin, together with an enthalpy-entropy compensation that maintains the free energy constant over the investigated temperature range.

Polyelectrolyte counterion condensation theory explains differential scanning calorimetry studies of salt-induced condensation of chicken erythrocyte chromatin.

The salt-induced chromatin condensation in chicken erythrocyte nuclei is studied by differential scanning calorimetry (DSC). The degree of chromatin condensation is measured for condensation induced by monovalent, divalent, trivalent, or tetravalent cations and by a mixture of sodium and magnesium. These last two cations show an evident competition effect. Salt-induced chromatin condensation is shown to be an entropy-driven process. A simple model of chromatin based on the polyelectrolyte counterion condensation theory is used in order to compute the charge neutralized by the cations in each chromatin domain. The degree of chromatin condensation is shown to be related to the weighed sum of the square of the phosphate charge of each domain. The model predicts the salt and the chromatin concentration dependence of the condensation and the effect of H1 removal.

Electrophoresis of Chromatin on Nondenaturing Agarose Gels Containing Mg2+: SELF-ASSEMBLY OF SMALL CHROMATIN FRAGMENTS AND FOLDING OF THE 30-nm FIBER

We show that nondenaturing agarose gels can be used for the study of the structure and dynamic properties of native (uncross-linked) chromatin. In gels containing 1.7 mM Mg2+, chicken erythrocyte chromatin fragments having from about 6 to 50 nucleosomes produce well defined bands. These bands have an electrophoretic mobility that decreases only slightly with molecular weight. This surprising behavior is not observed in low ionic strength gels. Fragments with less than 6 nucleosomes and low content of histones H1-H5 give rise to broad bands in gels with Mg2+. In contrast, fragments containing only 3-4 nucleosomes but with the normal H1-H5 content are able to form associated structures with a mobility similar to that observed for high molecular weight chromatin. Electron microscopy results indicate that the associated fragments and the fragments of higher molecular weight show similar electrophoretic properties because they become very compact in the presence of Mg2+ and form cylindrical structures with a diameter of approximately 33 nm. Our results suggest that the interactions involved in the self-assembly of small fragments are the same that direct the folding of larger fragments; in both cases, the resulting compact chromatin structure is formed from a basic element containing 5-7 nucleosomes.

Study of DNA accessibility in the condensed chromatin structures by resonance energy transfer

The linker DNA accessibility of chicken erythrocyte chromatin was studied by diffusion-enhanced resonance energy transfer (DERET). The 4″-{9‴-[((4-carboxy-3-hydroxyphenyl)-acetatamido)-3‴,6‴,9‴-(triacetyl)-3″,6‴,9‴-triazanonamido]-2″,6″-diazanonyl}-4,5',8-trimethyl psoralen-terbium complex was photocovalently bound to linker DNA and transferred its energy to fluorescein free in solution or bound on proteins of different sizes. We observed a diminution of linker DNA accessibility in chromatin as the protein size increased. Free fluorescein and proteins (up to a molecular weight of 24,000) labeled with fluorescein isothiocyanate (FITC) showed no variation in linker accessibility as chromatin condensation from 10- to 30-nm fibers was induced by an increase in ionic strength. We can conclude from these observations that linker DNA is located on the outside of the condensed chromatin fiber or, alternatively, that small proteins are free to diffuse toward an inside-located linker DNA, even in the condensed state of chromatin, possibly through the central cavity of the solenoïd model.

Three-dimensional structure of extended chromatin fibers as revealed by tapping-mode scanning force microscopy.

Unfixed chicken erythrocyte chromatin fibers in very low salt have been imaged with a scanning force microscope operating in the tapping mode in air at ambient humidity. These images reveal a three-dimensional organization of the fibers. The planar "zig-zag" conformation is rare, and extended "beads-on-a-string" fibers are seen only in chromatin depleted of histones H1 and H5. Glutaraldehyde fixation reveals very similar structures. Fibers fixed in 10 mM salt appear somewhat more compacted. These results, when compared with modeling studies, suggest that chromatin fibers may exist as irregular three-dimensional arrays of nucleosomes even at low ionic strength.