Illuminating nucleosome interactions

In a recent article published in Cell Research, the structure of an array of 12 nucleosomes compacted to a 30-nm fiber reveals details about nucleosome–nucleosome interactions and the binding and folding of linker histone H5.

In eukaryotes, genomic DNA is packaged into different levels of chromatin organization in the nucleus. The fundamental repeating unit of chromatin is called a nucleosome, consisting of the histone octamer and ca.150 bp of DNA. Multiple nucleosomes are connected by segments of linker DNA, forming nucleosomal arrays with a 10-nm diameter and a ‘beads-on-a-string’ arrangement. Nucleosomal arrays were proposed to be further organized into a higher-order assembly by the linker histones, which have been shown to play a fundamental role in chromatin compaction. In a recent article published in Cell Research, Li et al. ¹ provide structural information on a nucleosome array containing 12 nucleosomes, bound to linker histone H5 and folded into a 30-nm fiber, revealing insights into the structure and mechanism of higher-order compaction.²

The concept of the 30-nm fiber was initially proposed as a level of chromatin compaction and has been extensively debated over the years.^2,3 A 30-nm chromatin fiber was first seen when chromatin released from nuclei was analyzed under an electron microscope. In vitro studies of either chromatin fragments isolated from nuclei or reconstituted oligonucleosomes, have established that in the presence of cations, 10-nm chromatin fibers fold into ‘higher-order’ structures that are ~30 nm in diameter.^4,5 Based on those observations, it has long been assumed that the 30-nm chromatin fiber is an intermediate in the packaging of 10-nm fibers into chromosomes. However, recent investigations have indicated that 30-nm fibers are not prevalent within cells, where chromatin adopts a more ‘disordered’ structure.²

Putting aside the uncertainty surrounding the existence of 30-nm fibers in cells, the study by Li et al. ¹ offers invaluable insights into how nucleosomes interact and how linker histones interact with nucleosomes and promote their organization, questions that are highly pertinent to various cellular processes. The linker histone family is the most divergent group among the highly conserved histone proteins and includes 11 variants in humans. Linker histones have been shown to play a fundamental role in chromatin compaction contributing to transcriptional gene repression. They were shown to adopt a tripartite structure, composed of a structured central globular domain flanked by a short N-terminal domain (NTD) and a long C-terminal domain (CTD).⁶ The globular domain plays an important role in binding to the nucleosome, whereas the CTD is believed to be a major determinant of chromatin folding. Since the NTD and CTD of linker histones are mostly disordered in solution, there was no structural information for full-length linker histones available up to now.

Remarkably, Li et al. ¹ solved high-resolution structure (3.6 Å) of H5-chromatin fiber and observe the full-length linker histone H5 and the majority of core histone tails in their nucleosome arrays. The structure of H5 bound to the nucleosome is most compelling, which reveals the folding of the NTD and CTD bound to DNA — a feature previously suggested but not conclusively demonstrated until now. In sharp contrast to their unstructured conformation in solution, the H5 NTD adopts an α-helix structure that binds to one segment of linker DNA at the entry/exit site of the nucleosome, using several positively charged residues. The CTD folds into three α-helices, organized into an HMG box-like motif, to tightly hold on to the second entry/exit linker DNA (Fig. 1). The globular domain contacts the nucleosomal DNA and its location appears different when bound to mono- or tetra-nucleosome (on-dyad) or in the context of longer chromatin fiber (off-dyad). This behavior might be due to the constraints on the linker DNA during folding from the open nucleosome array to the higher-order, compact chromatin fiber.

Fig. 1: Histone H5 binds nucleosome off-dyad in longer fibers.

Schematic diagram showing that disordered CTD and NTD of histone H5 fold on chromatin and bind linker DNA.

Furthermore, Li et al. ¹ explore the importance of nucleosome–nucleosome interactions in yeast cells by investigating the effects of histone mutations that affect those contacts. Their analyses reveal growth defects, alterations in transcription, and changes in chromatin structure. While these mutations may impact the binding of various chromatin factors, it is intriguing that the authors can partially alleviate those defects through compensatory mutations. These observations suggest that nucleosome–nucleosome interactions, as described by Li et al. ¹ in the 30-nm fiber structure, contribute to normal chromatin structure and function in cells.

While the 30-nm fiber is not considered a fundamental structure of chromatin any longer, shorter, fiber-like structures such as tetrasomes have been observed in yeast and mammalian cells,² suggesting that longer fibers may assemble and play a functional role under specific cellular conditions. In recent years, cryo-electron tomography has emerged as an approach that can inform on the chromatin structure in cells, and we look forward to the technological developments that will allow us to analyze chromatin structure and organization in cells at much higher resolution.