Chromatin structure studied by linear dichroism at different salt concentrations

Abstract

SynopsisWe have studied the linear dichroism (LD) of rat liver chromatin oriented by flow. Soluble chromatin, prepared by brief nuclease digestion, is found to exhibit a positive LD at low ionic strength (1 mM NaCl), with a constant LDIA over the absorption band centered at 260 nm (A, isotropic absorbance). Several previous dichroism studies on soluble chromatin have been performed on sonicated materials and have given negative LD, probably due to the presence of uncoiled DNA. The positive dichroism can be interpreted in terms of a supercoil of DNA in chromatin with a pitch angle larger than 55", and is, for example, consistent with a model where the cylindrical nucleosome core particles are stacked face to face in the chromatin filament. In contrast to the nuclease-digested chromatin, sonicated chromatin was confirmed to exhibit negative LD. This difference can be attributed to a partial uncoiling of the linker regions between the nucleosomes due to the shearing. The structural transition of chromatin to a compact form can be observed as a reduction of the positive LD of the nuclease-digested chromatin to almost zero in 0.1M NaCl or in 0.1 mM MgC12. This transition is due to a decreased electrostatic repulsion between negative phosphate groups on the DNA chain. In the case of Na+, this can be explained as a screening effect due to the bulk concentration of Na+. With Mg2+ a considerably stronger effect may indicate a more localized binding to the phosphates. At ionic strengths higher than 0.5M NaC1, the dissociation of the histones from DNA leads to uncoiling of chromatin. The change in LD during this process shows that histone H1 contributes only to a small degree to the coiling of the DNA chain, whereas histones H3 and H4 play the major role in the coiling.

Salt‐Induced Conformational Transitions in Chromatin

“…Recently Tjerneld et al [15] reported a flow linear dichroisni study of rat liver chromatin at different salt concentrations. They found that the L D is positive at NaCl concentrations between 1 mM and IOOmM, while at concentrations higher than 0.5 M the LD signal was negative.…”

“…The latter two methods have been extensively used to study the structure of both isolated nucleosomes and high-molecular-weight chromatin fragments. These methods, however, suffer from two serious disadvantages : (a) limited application to high-ionic-strength solutions, due to the thermal and electrochemical processes in the Kerr's cell, and (b) the electric field used for orientation of the molecules probably induces structural distortions in the chromatin fibrils [15,27]. These limitations can be overcome by flow orientation of the chromatin fibrils [15,16].…”

“…These methods, however, suffer from two serious disadvantages : (a) limited application to high-ionic-strength solutions, due to the thermal and electrochemical processes in the Kerr's cell, and (b) the electric field used for orientation of the molecules probably induces structural distortions in the chromatin fibrils [15,27]. These limitations can be overcome by flow orientation of the chromatin fibrils [15,16]. This paper reports the results of flow linear dichroism measurements of calf thymus chromatin preparations in a wide range of NaCl concentrations.…”

Reinterpretation of Linear Dichroism of Chromatin Supports a Perpendicular Linker Orientation in the Folded State

“…LD reflects the average orientation of the light absorbing transition dipoles in a macroscopically oriented sample, and is expected to depend sensitively on the DNA arrangement in chromatin. Despite intense study, however, the interpretation of LD results on chromatin has been subject to a remaining ambiguity, since electrically oriented and flow oriented chromatin, in an intermediate range of ionic strengths, persistently show different features: flow LD (FLD) being positive (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) and electric LD (ELD) generally negative (11,(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). This incongruity has been suggested to reside in different preparation procedures, buffer conditions or in experimental artifacts (28).…”

Orientation of DNA in agarose gels

“…In solution linear dichroism has been applied to cases wherein DNA has been aligned by an electric field (Fredericq and Houssier, 1973;Mitra et al, 1984;McGhee et al, 1980) and flow (Tjerneld et al, 1982). The introduction of fluorescent labels which bind to DNA was an important improvement, because it increased the sensitivity of detection (Mitra et al, 1984;Kubista et al, 1985;Yoshida et al, 1987).…”