Stephan I. Dimitrov

A triple helix model for the structure of chromatin fiber

A model of chromatin fiber structure is presented in which a repeating unit of a trinucleosome forms a 3‐dimensional zigzag. Twisting and compression of the zigzag result in a triple helix structure. The model is built mainly on the flow linear dichroism data showing that (a) nucleosomal disc faces are tilted relative to the fiber axis, (b) the orientation of nucleosomes does not change upon folding and unfolding of chromatin, and (c) the orientation of nucleosomes is maintained by the globular domain of histone HI.

Polyamine-DNA Interactions. Condensation of Chromatin and Naked DNA

We have used flow linear dichroism (LD) and light scattering at 90 degrees to study the condensation of both DNA and calf thymus chromatin by polyamines, such as spermine, spermidine and its analogs designated by formula NH3+(CH2)iNH2+(CH2)jNH3+, where i = 2,3 and j = 2,3, putrescine, cadaverine and MgCl2. It has been found that the different polyamines affect DNA and chromatin in a similar way. The level of compaction of the chromatin fibers induced by spermine, spermidine and the triamines NH3+(CH2)3NH2+(CH2)3NH3+ and NH3+(CH2)3NH2+(CH2)2NH3+ and MgCl2 is found to be identical. The triamine NH3+(CH2)3NH2+(CH2)2NH3+ and the diamines studied condense neither chromatin nor DNA. This drastic difference in the action of the triamines indicates that not only the charge, but also the structure of the polycations might play essential roles in their interactions with DNA and chromatin. It is shown that a mixture of mono- and multivalent cations affect DNA and chromatin condensation competitively, but not synergistically, as claimed in a recent report by Sen and Crothers (Biochemistry 25, 1495-1503, 1986). We have also estimated the extent of negative charge neutralization produced by some of the polyamines on their binding to chromatin fibers. The stoichiometry of polyamine binding at which condensation of chromatin is completed is found to be two polyamine molecules per DNA turn. The extent of neutralization of the DNA phosphates by the histones in these compact fibers is estimated to be about 55%. The model of polyamine interaction with chromatin is discussed.

THE ROLE OF HISTONE-H1 AND NON-STRUCTURED DOMAINS OF CORE HISTONES IN MAINTAINING THE ORIENTATION OF NUCLEOSOMES WITHIN THE CHROMATIN FIBER

Calf thymus chromatin was digested with trypsin and the structural alterations which occurred were followed by flow linear dichroism. After a sharp initial increase, the amplitude of the positive signal gradually decreased followed by a change of the sign of the dichroism and further increase of the negative signal up to a plateau. These changes of the dichroism were compared to the respective changes in the histone pattern. It was shown that the positive dichroism of chromatin did not depend on the condensation state of chromatin, and that the orientation of the nucleosomes along the chromatin fiber was maintained by the globular domain of H1 and the non-structured parts of core histones.

Interactions of acetylated histones with DNA as revealed by UV laser induced histone-DNA crosslinking

The interaction of acetylated histones with DNA in chromatin has been studied by UV laser-induced crosslinking histones to DNA. After irradiation of the nuclei, the covalently linked protein-DNA complexes were isolated and the presence of histones in them demonstrated immunochemically. When chromatin from irradiated nuclei was treated with clostripain, which selectively cleaved the N-terminal tails of core histones, no one of them was found covalently linked to DNA, thus showing that crosslinking proceeded solely via the N-terminal regions. However, the crosslinking ability of the laser was preserved both upon physiological acetylation of histones, known to be restricted to the N-terminal tails, and with chemically acetylated chromatin. This finding is direct evidence that the postsynthetic histone acetylation does not release the N-terminal tails from interaction with DNA.

Higher order folding of chromatin is induced in different ways by monovalent and by bivalent cations

The condensation of the 10 nm chromatin filament in the 30 nm fiber by monovalent cations, polyamines and bivalent cations was studied with light scattering at 90° and flow linear dichroism methods. It was found that monovalent cation‐ and polyamine‐induced folding was a two‐step process: a precondensation, when a rotation of nucleosomes takes place only, and a condensation step without changes in nucleosome orientation. Divalent cations affected the structure of chromatin in one step only ‐ condensation of the chromatin filament being accompanied by nucleosome reorientation.

Structure of hyperacetylated chromatin: light scattering and flow linear dichroism study

Cation‐induced folding of 10 nm chromatin filament to 30 nm fiber was studied with hyperacetylated chromatin using light scattering at 90° and flow linear dichroism. Acetylated chromatin folded in a way indistinguishable from that of the control chromatin: both the compactness of chromatin and the orientation of nucleosomes relative to the fiber axis were identical at a given salt concentration.

Chromatin superstructure: A study with an immobilized trypsin

Hen erythrocyte chromatin was treated with trypsin immobilized on collagen membranes and the unfolding of chromatin fiber was followed by light scattering at 90° and flow linear dichroism. Chromatin was found almost completely decondensed when the bulk of H1 and H5 was digested while H3 was still intact. Further digestion leading to degradation of both H3 and the rest of H1 and H5 accounted for no more than 10–15% of the total effect. When chromatin with trypsin‐cleaved H1 and H5 was titrated with increasing amounts of spermidine it folded similarly to the control sample. This finding suggests that charge neutralization appears a likely mechanism for maintaining the structure of the 30 nm chromatin fiber by the C‐terminal domain of H1 and H5.

Optical anisotropy of chromatin. Flow linear dichroism and electric dichroism studies.

The optical anisotropy of chromatin with different length of the linker DNA isolated from a variety of sources (Frend erythroleukemia cells, calf thymus, hen erythrocytes and sea urchin sperm) has been studied in a large range of mono- and bivalent cations concentrations by the use of flow linear dichroism (LD) and electric dichroism. We have found that all chromatins studied displayed negative LD values in the range of 0.25 mM EDTA - 2 mM NaCl and close positive values in the range of 2-100 mM NaCl. Mg2+ cations, in contrast to Na+ cations, induce optically isotropic chromatin fibers. All chromatin samples exhibit positive form effect amounting to 5-10% of LD amplitude observed at 260 nm. This form effect is determined by the anisotropic scattering of polarized light by single chromatin fibers. The conformational transition at 2 mM NaCl leads to the distortion of chromatin filament structure. The reversibility of this distortion depends on the length of the linker DNA - for chromatins with the linker DNA of 10-30 b.p. it is parially reversible, while for preparations with longer linker DNA it is irreversible. Relatively low electric field does not affect chromatin structure, while higher electric field (more than 7 kV/cm) distorts the structure of chromatin. Presented results explain the contradictory data obtained by electrooptical and hydrooptical methods.

The Chromatin Fiber: Structure and Conformational Transitions as Revealed by Optical Anisotropy Studies

Structure and conformational transitions of the chromatin fiber as revealed by optical anisotropy studies are reviewed. The data in the literature do not allow a definite interpretation; in fact some of them are contradictory. The major findings are reported here and an attempt is made to analyse them with respect to the internal dynamics and the various models suggested for the organization of the chromatin fiber.

Effect of ionic strength on the chromatin conformational transitions. A differential scanning calorimetry study

Abstract The thermal denaturation of hen erythrocyte chromatin at low ionic strength (0.25 m m EDTA, pH 8.0 (A), and 0.25 m m EDTA, pH 8.0, 5 m m NaCl (B)) was studied by high sensitivity differential scanning microcalorimetry and absorbance at 260 nm. The melting profiles recorded by the two methods were decomposed into sums of three (case A) and two (case B) Gauss curves and were found to be of identical shape. In case A, the three individual peaks at 61, 72, and 82°C have enthalpies 4.1, 8.4 and 12.5 kcal/mol base pairs−1, respectively. The enthalpies of pure DNA melting at these temperatures are 6.2, 6.9 and 7.6 kcal/mol base pairs−1, respectively. The smaller in comparison to pure DNA enthalpy of the peak at 61°C indicates a decreased thermal stability of the chromatin portion melting during this transition. The disappearance of this transition at increased ionic strength (case B) is accompanied by a transfer of its enthalpy to the peak at 72°C and its hyperchromicity to the peak at 82°C. During this complex transformation the overall melting enthalpy remains unchanged (9.5±0.5 kcal/mol base pairs−1).