Nucleosome repeat length and linker histone stoichiometry determine chromatin fiber structure

Abstract

To understand how nuclear processes involving DNA are regulated, knowledge of the determinants of chromatin condensation is required. From recent structural studies it has been concluded that the formation of the 30-nm chromatin fiber does not require the linker histone. Here, by comparing the linker histone-dependent compaction of long, reconstituted nucleosome arrays with different nucleosome repeat lengths (NRLs), 167 and 197 bp, we establish that the compaction behavior is both NRL-and linker histone-dependent. Only the 197-bp NRL array can form 30-nm higher-order chromatin structure. Importantly for understanding the regulation of compaction, this array shows a cooperative linker histone-dependent compaction. The 167-bp NRL array displays a limited linker histone-dependent compaction, resulting in a thinner and topologically different fiber. These observations provide an explanation for the distribution of NRLs found in nature.30-nm fiber ͉ electron microscopy ͉ heterochromatin ͉ nucleosome array reconstitution ͉ sedimentation velocity analysis D uring the past decade it has emerged that the packaging of eukaryotic DNA by histones into chromatin is a key regulator of nuclear processes involving DNA, such as transcription and replication. Although the structure of the first level of DNA folding, the nucleosome core, is known at atomic resolution (1, 2), the structure of the second level of folding, whereby a string of nucleosomes folds into a fiber with an approximate diameter of 30 nm (the 30-nm chromatin fiber) remains undetermined (3). Early evidence for the presence of a 30-nm chromatin fiber in vivo came from EM analysis of Balbiani ring genes in Chironomus tentans (4) and x-ray diffraction studies of nuclei that show spacings of 30-40 nm (5).The structure of the 30-nm chromatin fiber is controversial (reviewed in ref.3). Recent structural analyses using in vitroreconstituted model nucleosome arrays based on the strong 601 DNA nucleosome positioning sequence (6) have led to the proposal of two models for the 30-nm chromatin fiber that differ in topology, dimension, and nucleosome packing density. The first model was constructed by using the crystal structure at 9 Å of a tetra-nucleosome core array and is of a two-start helix type (7). It is based on a zigzag arrangement of nucleosome cores that stack on top of each other and has a 24-to 25-nm diameter with a packing density of five to six nucleosomes per 11 nm. The second model was derived from tight constraints obtained from measurements of the physical dimensions of long nucleosome arrays visualized by EM (8). It is of the one-start helix type in which nucleosomes from adjacent gyres are interdigitated. It has a diameter of 34 nm with a packing density of 11 nucleosomes per 11 nm.The key difference between the two 30-nm chromatin fiber models is that the interdigitated model was derived from nucleosome arrays saturated with linker histone, whereas the two-start helix model was derived from a tetra-nucleosome core array in the absence of linker histone...

A metastable structure for the compact 30‐nm chromatin fibre

“…Over this range the model algorithm gives rise to three structural classes corresponding to 177–187, 197–207 and 217–237‐bp NRLs with respectively on average 7.1, 10.9 and 15.4 nucleosomes/11 nm. These values compare with experimental determinations of ~ 6, ~ 11 and ~ 15 nucleosomes/11 nm for the packing density over the same ranges of NRL 13, 27 (Fig. S4).…”

Stable and dynamic nucleosome states during a meiotic developmental process

“…These in vivo findings contrast with one expectation from in vitro experiments that a ;165-bp nucleosome repeat length must increase by ;30 bp in order to undergo compaction (Routh et al 2008). Indeed, simple a priori expectations are that NFRs would acquire nucleosomes during compaction and become more closely spaced; nucleosomes that are not well positioned might have become more organized, but these outcomes were not observed.…”

“…Since nucleosome occupancy in NFRs has been attributed to transcriptional shutdown (Schones et al 2008;Shivaswamy et al 2008), NFRs might be expected to acquire nucleosomes during the sporulation program. In addition, the ensuing compaction might also involve nucleosome acquisition in NFRs and internucleosomal spacing changes (Routh et al 2008;Segal and Widom 2009). Compaction might also be expected to increase nucleosome positioning as the degrees of freedom for positioning become more restricted by interchromatin contacts.…”

The regulatory role of DNA supercoiling in nucleoprotein complex assembly and genetic activity

“…As the fiber compacts, γ decreases and hence the fiber becomes more supercoiled (Wu et al 2016). However, packing constraints suggest that, for fibers with short NRLs (e.g., 167 and 177 bp), the pitch angle is higher (Routh et al 2008) and, therefore, these fibers in principle constrain less negative superhelicity. Importantly, this same principle implies that, as the fiber untwists, unconstrained negative superhelicity can be released, while conversely increasing compaction results in greater constraint.…”