Chromatin conformation and salt-induced compaction: three-dimensional structural information from cryoelectron microscopy.

Abstract

Cryoelectron microscopy has been used to examine the three-dimensional (3-D) conformation of small oligonucleosomes from chicken erythrocyte nuclei after vitrification in solutions of differing ionic strength. From tilt pairs of micrographs, the 3-D location and orientation of the nucleosomal disks, and the paths of segments of exposed linker can be obtained. In "low-salt" conditions (5 mM NaCl, 1 mM EDTA, pH 7.5), the average trinucleosome assumes the shape of an equilateral triangle, with nucleosomes at the vertices, and a length of exposed linker DNA between consecutive nucleosomes equivalent to approximately 46 bp. The two linker DNA segments converge at the central nucleosome. Removal of histones H1 and H5 results in a much more variable trinucleosome morphology, and the two linker DNA segments usually join the central nucleosome at different locations. Trinucleosomes vitrified in 20 mM NaCl, 1 mM EDTA, (the salt concentration producing the maximal increase in sedimentation), reveal that compaction occurs by a reduction in the included angle made by the linker DNA segments at the central nucleosome, and does not involve a reduction in the distance between consecutive nucleosomes. Frequently, there is also a change in morphology at the linker entry-exit site. At 40 mM NaCl, there is no further change in trinucleosome morphology, but polynucleosomes are appreciably more compact. Nevertheless, the 3-D zig-zag conformation observed in polynucleosomes at low salt is retained at 40 mM NaCl, and individual nucleosome disks remain separated from each other. There is no evidence for the formation of solenoidal arrangements within polynucleosomes. Comparison of the solution conformation of individual oligonucleosomes with data from physical measurements on bulk chromatin samples suggests that the latter should be reinterpreted. The new data support the concept of an irregular zig-zag chromatin conformation in solution over a range of ionic strengths, in agreement with other in situ (McDowall, A.W., J.M. Smith, and J. Dubochet. 1986, EMBO (Eur. Mol. Biol. Organ.) J.5: 1395-1402; Horowitz, R.A., D.A. Agard, J.W. Sedat, and C.L. Woodcock, 1994. J. Cell Biol. 125:1-10), and in vitro conclusions (van Holde, K., and J. Zlatanova. 1995. J. Biol. Chem. 270:8373-8376). Cryoelectron microscopy also provides a way to determine the 3-D conformation of naturally occurring chromatins in which precise nucleosome positioning plays a role in transcriptional regulation.

Nucleosome Structural Transition during Chromatin Unfolding Is Caused by Conformational Changes in Nucleosomal DNA

“…5B). This model is also consistent with the observed salt-dependent release of nucleosomal DNA termini resulting in the change in the linking number of DNA in minichromosomes (21,22) and in the altered nucleosome morphology in linker histone-depleted chromatin (21)(22)(23)(24).…”

“…In contrast, in linker histone-depleted chromatin, the distance between the points where the linker DNA enters and leaves the nucleosome core may be increased by repulsion between adjacent linker DNA segments (17,(21)(22)(23)(24) and thereby can affect the conformation of the nucleosome core DNA. To address the question of whether or not the alterations in histone-DNA contacts during chromatin unfolding, which we observed earlier (19), are caused by the changes in the nucleosome core DNA conformation, we have analyzed the histone-DNA contacts in isolated core particles under ionic conditions affecting DNA stiffness.…”

Analysis of cryo-electron microscopy images does not support the existence of 30-nm chromatin fibers in mitotic chromosomes in situ

“…4, 1 mM EDTA). This state resembles the fully open isolated chromatin fibers previously observed in the thin vitrified layer in low-salt buffer (21).…”

“…4, 0.5 mM). These filaments were reminiscent of the irregular 30-nm fibers observed in vitro (21). The peak in the 7-20 nm range persisted in the 1-DRAPS, with the maximum located at Ϸ14.3 nm.…”

“…Although 30-nm chromatin fibers can be routinely observed by cryo-EM in chromatin suspensions in vitro (21,22), cryo-EM studies of HeLa S3 cells did not reveal this structural feature in mitotic chromosomes (23). Nevertheless, because the influence of the CTF was not compensated for, it remained unclear whether the absence of 30-nm fibers in cryo-EM images of mitotic chromosomes is characteristic of the native chromatin or an artifact of the imaging properties.…”