Structure of the ‘30 nm’ chromatin fibre: A key role for the linker histone
Introduction
During the past decade, it became evident that, in eukaryotes, the packaging of DNA by histones into chromatin is a key regulator of transcription and other nuclear processes that involve DNA. Although the structure of the first level of DNA organisation, the nucleosome core, has been determined to high resolution (1.9 Å) [1], the next level of DNA compaction, the ‘30 nm’ fibre, lacks direct structural information and has remained controversial. In the nucleosome core particle, 147 bp of DNA is wrapped in 1.7 left-handed superhelical turns around the histone octamer, which consists of two copies each of the core histones (H2A, H2B, H3 and H4). Binding of the linker histone, H1/H5, organises an additional 20 bp of DNA to complete the nucleosome, which contains ∼167 bp of DNA [2]. Such binding stabilises the structure of the nucleosome, and determines the trajectory of the entering and exiting DNA [3]. Consequently, the linker histone is likely to direct the relative positioning of successive nucleosomes and the pattern of nucleosome–nucleosome contacts.
Adjacent nucleosomes are linked via DNA (linker DNA), which varies in length in a cell- and species-specific manner [4]. Nucleosome arrays have an inherent propensity to compact into tightly folded filamentous structures at physiological salt concentrations. Early electron micrographs of thin sections of HeLa metaphase chromosomes showed thick fibres with a diameter of ∼30 nm [5], whose integrity was dependent on raised ionic strength and the presence of the linker histone [6]. Although a variety of biophysical studies have been in agreement that the nucleosomes are oriented essentially parallel to the fibre axis [7, 8], aspects such as the connectivity between adjacent nucleosomes and the path of the linker DNA remained elusive. This has led to the proposal of a number of different models for the 30 nm chromatin fibre. These models fall into two main classes (Figure 1): the one-start helix, or solenoid, and the two-start helix. In the solenoid model, a nucleosome array coils up so that successive nucleosomes are adjacent in the compact structure and connected by linker DNA, which bends into the fibre interior to accommodate variable DNA lengths [9]. The two-start helix model is based on a zigzag arrangement of nucleosomes, with essentially straight linker DNA connecting nucleosomes on opposite sides of the fibre [10, 11]. Despite fundamental differences between the two models, the limitations of data derived from the inherently irregular native chromatin samples have prevented earlier studies from differentiating between them unequivocally.
In this review, we will focus on recent progress in elucidating the structure of the 30 nm chromatin fibre using in vitro reconstituted model nucleosome arrays in which regularity has been imposed on the structure by using a strong nucleosome-positioning DNA sequence [12]. Regularity of structure is required in order to establish its fundamental features. This approach has provided more detailed structural information than was previously possible. The two new models proposed, one derived from a crystal structure [13••] and the other from EM analysis [14••], provide strong evidence that the topology and degree of compaction of chromatin fibres are determined by the linker histone. We will argue that the formation of the 30 nm chromatin fibre requires the linker histone and will discuss whether the two models represent different functional states.
Section snippets
Model of the chromatin fibre in the absence of the linker histone
Recently, Richmond and colleagues [13••] proposed a two-start crossed-linker model for the 30 nm chromatin fibre based on the crystal structure of a four-nucleosome core array lacking the linker histone (Figure 2). The array was based on the ‘Widom 601’ strong nucleosome-positioning DNA sequence, which yields a single position for the histone octamer [12] and has a short nucleosome repeat length, 167 bp, which is uncommon in nature [4]. Crystals were obtained at very high divalent salt
Model of the chromatin fibre in the presence of the linker histone
Robinson et al. [14••, 18•] have taken a different approach to investigating the structure of the 30 nm chromatin fibre. Our model was derived from tight constraints obtained from measurements of the physical dimensions and compaction ratios of long tightly folded nucleosome arrays visualised by EM. These in vitro reconstituted nucleosome arrays were also based on the Widom 601 nucleosome-positioning DNA sequence [12], but, in contrast to those studied by Richmond and colleagues [13••], contain
The H4 N-terminal tail plays a major role in nucleosome–nucleosome contacts
A variety of evidence suggests that electrostatic interactions between nucleosomes are the driving force in chromatin fibre compaction. These interactions are favoured by increasing salt concentrations (or divalent cations), which reduce the repulsive forces between linker DNA [21], and are likely to be modulated by post-transcriptional modifications that alter the charge of the very long histone tails [22]. Recent results suggest that a key interaction is made by the N-terminal tail of histone
The role of the linker histone in 30 nm fibre formation
It has long been accepted that the linker histone (H1/H5) influences the degree of chromatin compaction [6] and that its removal leads to decondensation [26••]. It is also known that transcriptionally active chromatin regions have a more open structure. This view has recently been contradicted by analysis of the compaction of short model nucleosome arrays in the presence and absence of bound linker histone. From these results, it was concluded that the linker histone does not affect the degree
Conclusions
The proposal of two strikingly different models for the structure of the 30 nm chromatin fibre, based on more reliable and detailed structural information, appears at first sight to continue the controversy in the field. However, this controversy can be resolved if we accept the existence of at least two different levels of higher order chromatin structure: one highly compact structure containing the linker histone, which represents the 30 nm fibre, and a looser structure formed in the absence of
References and recommended reading
Papers of particular interest, published within the annual period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
We apologize to those authors whose work we could not quote due to space restrictions. We thank our colleagues for useful discussions and the British Medical Research Council for funding our research.
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