Characteristics of the structural state of DNA in chromatin with short linker

Various fragments of pigeon brain neuron chromatin with very short linker DNA have been studied by circular dichroism (CD). The DNA structure in core particles of the brain and thymus chromatins is very similar. Linker DNA and a part of core DNA in the mononucleosomes of brain chromatin is extended. This conclusion follows from increasing CD amplitude of the brain mononucleosomes as compared with the corresponding value for thymus mononucleosomes. The internucleosome interactions stabilized the compactness of core DNA in the brain oligonucleosomes. The whole linker DNA of the brain chromatin unlike thymus chromatin is extended at low ionic strength. This fact can explain the brain chromatin ability to form a compact structure with increasing ionic strength.     

Electrostatic interactions with histone tails may bend linker DNA in chromatin

Is linker DNA bent in the 30‐nm chromatin fiber at physiological conditions? We show here that electrostatic interactions between linker DNA and histone tails including salt condensation and release may bend linker DNA, thus affecting the higher order organization of chromatin. © 2005 Wiley Periodicals, Inc. Biopolymers 81: 20–28, 2006 This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

A critical role for linker DNA in higher-order folding of chromatin fibers

Nucleosome-nucleosome interactions drive the folding of nucleosomal arrays into dense chromatin fibers. A better physical account of the folding of chromatin fibers is necessary to understand the role of chromatin in regulating DNA transactions. Here, we studied the unfolding pathway of regular chromatin fibers as a function of single base pair increments in linker length, using both rigid base-pair Monte Carlo simulations and single-molecule force spectroscopy. Both computational and experimental results reveal a periodic variation of the folding energies due to the limited flexibility of the linker DNA. We show that twist is more restrictive for nucleosome stacking than bend, and find the most stable stacking interactions for linker lengths of multiples of 10 bp. We analyzed nucleosomes stacking in both 1- and 2-start topologies and show that stacking preferences are determined by the length of the linker DNA. Moreover, we present evidence that the sequence of the linker DNA also modulates nucleosome stacking and that the effect of the deletion of the H4 tail depends on the linker length. Importantly, these results imply that nucleosome positioning in vivo not only affects the phasing of nucleosomes relative to DNA but also directs the higher-order structure of chromatin.     

Binding of androgen receptor to prostatic chromatin requires intact linker DNA.

Receptor-chromatin complexes were recovered from prostatic chromatin digested with micrococcal nuclease. The fragments of chromatin were separated on linear 7.6 to 76% (v/v) glycerol density gradients. With extensive digestion of DNA, receptor labeled with [1,2-3H]dihydrotestosterone was released from the chromatin. After 5% digestion of DNA to acid-soluble products, only a trace amount of labeled receptor was detected in the unbound form. In the latter instance, most of the labeled receptor was recovered from the gradients in association with five A260 peaks representing oligomeric and monomeric nucleosomes with a repeat length of 182 +/- 14 (mean +/- S.D.) base pairs. The concentration of receptors was highest in the A260 peaks, which contained large oligomers of nucleosomes, and lowest in fractions containing primarily monomer structures. Hence, the extent to which receptors remained bound to chromatin was dependent on the relative amount of intact, linker DNA present.     

The diameters of frozen-hydrated chromatin fibers increase with DNA linker length: evidence in support of variable diameter models for chromatin

The diameters of chromatin fibers from Thyone briareus (sea cucumber) sperm (DNA linker length, n = 87 bp) and Necturus maculosus (mudpuppy) erythrocytes (n = 48 bp) were investigated. Soluble fibers were frozen into vitrified aqueous solutions of physiological ionic strength (124 mM), imaged by cryo-EM, and measured interactively using quantitative computer image-processing techniques. Frozen-hydrated Thyone and Necturus fibers had significantly different mean diameters of 43.5 nm (SD = 4.2 nm; SEM = 0.61 nm) and 32.0 nm (SD = 3.0 nm; SEM = 0.36 nm), respectively. Evaluation of previously published EM data shows that the diameters of chromatin from a large number of sources are proportional to linker length. In addition, the inherent variability in fiber diameter suggests a relationship between fiber structure and the heterogeneity of linker length. The cryo-EM data were in quantitative agreement with space-filling double-helical crossed-linker models of Thyone and Necturus chromatin. The data, however, do not support solenoid or twisted-ribbon models for chromatin that specify a constant 30 nm diameter. To reconcile the concept of solenoidal packing with the data, we propose a variable-diameter solid-solenoid model with a fiber diameter that increases with linker length. In principle, each of the variable diameter models for chromatin can be reconciled with local variations in linker length.     

Removal of histone H1 exposes linker DNA in chromatin to DNAse I

After removal of histone H1 about 40% of DNA in chromatin acquires the sensitivity of naked DNA to DNAse I. Digestion of H1-depleted chromatin with DNAse I leads to a qualitative change in the digestion pattern, generating DNA fragments of approx. 200 b.p. and multiples, similar to those obtained with micrococcal nuclease. Both effects are reversed upon reconstitution of purified H1 to H1-depleted chromatin.     

Erythroid-specific gene chromatin has an altered association with linker histones.

The chromatin of several genes was assayed for sensitivity to DNAase I and for solubility as polynucleosomes in 0.15 M NaCl. The degree of solubility of chromatin fragments as polynucleosomes in 0.15 M NaCl correlates well with the sensitivity to DNAase I for several genes. Chromatin of repressed, housekeeping and erythroid-specific genes can be distinguished as distinct groups by the degree to which they display these properties. NaCl precipitation of chromatin fragments stripped and then reconstituted with varying quantities of H1 and H5 (linker) histones indicate that the polynucleosomes of erythroid-specific genes have altered interaction with these histones. Linker histones interacted with bulk chromatin and in the chromatin of the repressed ovalbumin and vitellogenin genes to form salt precipitable structures. Chromatin of erythroid-specific genes (histone H5 and beta-globin) as well as that of the histone H2A.F gene was resistant to linker histone induced precipitation.     

DNA microarrays with PAMAM dendritic linker systems

The DNA microarray-based analysis of single nucleotide polymorphisms (SNPs) is important for the correlation of genetic variations and individual phenotypes, and for locating disease-causing genes. To facilitate the development of surfaces suitable for immobilization of oligonucleotides, we report here a novel method for the surface immobilization of DNA using pre-fabricated polyamidoamine (PAMAM) starburst dendrimers as mediator moieties. Dendrimers containing 64 primary amino groups in their outer sphere are covalently attached to silylated glass supports and, subsequently, the dendritic macromolecules are modified with glutaric anhydride and activated with N-hydroxysuccinimide. As a result of the dendritic PAMAM linker system the surfaces reveal both a very high immobilization efficiency for amino-modified DNA-oligomers, and also a remarkable high stability during repeated regeneration and re-using cycles. The performance of dendrimer-based DNA microarrays in the discrimination of SNPs is demonstrated.     

The diameter of chromatin fibres depends on linker length

We have studied the diameter of chromatin fibres embedded in epoxy resins for three different materials: mouse thymus, chicken erythrocytes and sea cucumber spermatozoa. We confirm that the diameter of chromatin fibres increases with linker length, both values being influenced by the protein composition of chromatin.     

Structure of nucleosomes and organization of inter-nucleosomal DNA in chromatin

We have compared mononucleosomes that were obtained by hydrolysis of chromatin micrococcal nuclease from a number of sources with the length of a nucleosomal repeat 185--245 b. p. long. For hydrolysis of chromatin isolated from nuclei, a series of nucleosomes was formed: MN145 (core particle), MN165, MN175...MN205, MN215, the lengths of their DNAs differing (by approximately 10.n b.p. where n = 1, 2, 3...) by a factor of 10. A feature of hydrolysis of chromatin in nuclei was the appearance of an additional H1-depleted MN155 particle. It is suggested that upon isolation of chromatin from nuclei, its partial decompactization takes place. This decompactization changes the character of nuclease splitting and seems to be connected with rearrangement of histone H1. These observations demonstrate that besides core particles MN145 and chromatosomes MN165, the major particles of digest of nuclei appear to be MN155, and for isolated chromatin--MN175. Unlike this standard picture, mainly MN145, MN155, MN235 and MN245 are formed upon hydrolysis of sea urchin sperm nuclei.     

Structure of nucleosomes and organization of internucleosomal DNA in chromatin

We have compared the mononucleosomal pattern produced by micrococcal nuclease digestion of condensed and unfolded chromatin and chromatin in nuclei from various sources with the repeat length varying from 165 to 240 base-pairs (bp). Upon digestion of isolated H1-containing chromatin of every tested type in a low ionic strength solution (unfolded chromatin), a standard series of mononucleosomes (MN) was formed: the core particle, MN145, and H1-containing, MN165, MN175, MN185, MN195, MN205 and MN215 (the indexes give an approximate length of the nucleosomal DNA that differs in these particles by an integral number of 10 bp). In addition to the pattern of unfolded chromatin, digestion of whole nuclei or condensed chromatin (high ionic strength of Ca2+) gave rise to nuclei-specific, H1-lacking MN155. Digestion of H1-lacking chromatin produced only MN145, MN155 and MN165 particles, indicating that the histone octamer can organize up to 165 bp of nucleosomal DNA. Although digestion of isolated sea urchin sperm chromatin (repeat length of about 240 bp) at a low ionic strength gave a typical "unfolded chromatin pattern", digests of spermal nuclei contained primarily MN145, MN155, MN235 and MN245 particles. A linear arrangement of histones along DNA (primary organization) of the core particle was found to be preserved in the mononucleosomes, with the spacer DNA length from 10 to 90 bp on one (in MN155) or both sides of core DNA being a multiple of about 10 bp. In MN235, the core particle occupies preferentially a central position with the length of the spacer DNA on both sides of the core DNA being usually about 30 + 60 or 40 + 50 bp. Histone H1 is localized at the ends of these particles, i.e. close to the centre of the spacer DNA. The finding that globular part of histones H3 and sea urchin sperm H2B can covalently bind to spacer DNA suggests their involvement in the organization of chromatin superstructure. Our data indicate that decondensation of chromatin is accompanied by rearrangement of histone H1 on the spacer DNA sites adjacent to the core particle and thus support a solenoid model for the chromatin superstructure in nuclei in which the core DNA together with the spacer DNA form a continuous superhelix.     

Dynamic condensation of linker histone C-terminal domain regulates chromatin structure

The basic and intrinsically disordered C-terminal domain (CTD) of the linker histone (LH) is essential for chromatin compaction. However, its conformation upon nucleosome binding and its impact on chromatin organization remain unknown. Our mesoscale chromatin model with a flexible LH CTD captures a dynamic, salt-dependent condensation mechanism driven by charge neutralization between the LH and linker DNA. Namely, at low salt concentration, CTD condenses, but LH only interacts with the nucleosome and one linker DNA, resulting in a semi-open nucleosome configuration; at higher salt, LH interacts with the nucleosome and two linker DNAs, promoting stem formation and chromatin compaction. CTD charge reduction unfolds the domain and decondenses chromatin, a mechanism in consonance with reduced counterion screening in vitro and phosphorylated LH in vivo. Divalent ions counteract this decondensation effect by maintaining nucleosome stems and expelling the CTDs to the fiber exterior. Additionally, we explain that the CTD folding depends on the chromatin fiber size, and we show that the asymmetric structure of the LH globular head is responsible for the uneven interaction observed between the LH and the linker DNAs. All these mechanisms may impact epigenetic regulation and higher levels of chromatin folding.     

Linking two DNA duplexes with a rigid linker for DNA nanotechnology

DNA has recently emerged as a promising material for the construction of nanosized architectures. Chemically modified DNA has been suggested to be an important component of such architectural building blocks. We have designed and synthesized a novel H-shaped DNA oligonucleotide dimer that is cross-linked with a structurally rigid linker composed of phenylene and ethynylene groups. A rotatable DNA unit was constructed through the self-assembly of this H-shaped DNA component and two complementary DNA oligonucleotides. In addition to the rotatable unit, a locked DNA unit containing two H-shaped DNA components was also constructed. As an example of an extended locked structure, a hexagonal DNA origami dimer and oligomer were constructed by using H-shaped DNA as linkers.