Higher-Order Chromatin Structure

Levels of chromatin folding beyond the linear array of nucleosomes are defined as higher-order chromatin structures (Woodcock, 2006). A hierarchical system has been proposed for classifying chromatin structure. The term higher-order chromatin structure encompasses a wide range of hierarchical levels of chromatin folding from positioned nucleosomes up to the large-scale organization of interphase chromosomes (Woodcock and Dimitrov, 2001).

The formation of higher-order chromatin structures is based upon the systematic build-up of the characteristic hierarchical structure of chromatin starting from the primary 10 nm chromatin fibre, a nucleosomal array. The secondary structure, termed 30 nm chromatin fibre, is formed by a folding of the primary structure and involves internucleosomal contacts and interactions with linker histones as well as non-histone chromosomal proteins.

The tertiary structure of chromatin that can reach diameters of 300-400 nm and ultimately concludes the chromosome assembly is formed by interactions between secondary structure elements and sustained by intranucleosomal contacts or looped chromatin domains (Woodcock, 2006; Woodcock and Dimitrov, 2001) (Figure 1.5).

Reconstitution of nucleosomal arrays using recombinant histones revealed that chromatin fibres can still fully condense when any of the histone tails is deleted, with the exception of histone H4 (Dorigo et al., 2003). Still, chromatin structure is dynamic and regulated by a plethora of factors, including PTMs, histone variants, nucleosome repeat length, and the presence of linker histones and non-histone chromosomal proteins. Thereby, a high degree of heterogeneity and complexity in structure is being created within all levels of chromatin folding.

Figure 1.5: Formation of higher-order chromatin structures.

Schematic illustration of the packaging of DNA into higher-order chromatin structures. Taken from Weier, 2001.

Investigation of dynamic properties of chromatin organization became possible by advancements in imaging technology, fluorescent protein assays and monitoring of the mobility of many chromosomal proteins including histones in living cells of higher eukaryotes (Gasser, 2002; Kimura, 2005; Phair et al., 2004). Fluorescence Recovery after Photobleaching (FRAP) experiments using GFP-tagged histones in HeLa cells allowed monitoring of histone turnover, revealing different turnover rates for H2A-H2B and (H3-H4)2 tetramers (Kimura and Cook, 2001). Other photobleaching studies reported rapid turnover of proteins from chromatin involved in transcription and DNA repair (Bustin et al., 2005; Hager et al., 2004; Mone et al., 2004; Phair et al., 2004).

Still, all these approaches face limitations in regard to studying chromatin structure in vivo (Horowitz-Scherer and Woodcock, 2006). Although they all create a highly detailed description of the local and global primary structure of chromatin, the results remain essentially one-dimensional (Horowitz-Scherer and Woodcock, 2006; Woodcock, 2006).

Therefore, experimental studies of chromatin secondary structure were conducted using structural biological methods. Built upon observations from transmission microscopy, electron cryo-microscopy, atomic force microscopy and X-ray diffraction of chromatin in various ionic strength environments, distinct structural

Most models have an open zig-zag arrangement of nucleosome arrays in common that results in a helical structure, 30 nm in diameter. These models propose either a one-start or a two-start helix. In a one-start helix, consecutive nucleosomes of an array coil up and follow each other immediately along the same helical path (Kruithof et al., 2009; Robinson et al., 2006; Robinson and Rhodes, 2006). The linker DNA between adjacent nucleosomes continues the curvature established in the nucleosome and thus is bent. In a two-start helix, consecutive nucleosomes of an array arrange in a zig-zag course into a helical structure, which is interconnected by straight linker DNA segments (Dorigo et al., 2004). A pronounced example for a one-start helix is the solenoid model in which nucleosome arrays coil around a central cylinder with linker DNA in the interior of the fibre and with six to eight nucleosomes per turn (Finch and Klug, 1976; McGhee et al., 1983;

Thoma et al., 1979).

Until recently, the one-start solenoid model and the two-start zig-zag model were dominating the organization of this secondary chromatin structure in vitro (Chen and Li, 2010; Robinson and Rhodes, 2006; Schalch et al., 2005). However, inclusion of the existence of one structure must not exclude the other. It was proposed that each structure can form depending on the linker DNA length (Routh et al., 2008). This would lead to the assumption that both structures can co-exist within a 30 nm chromatin fibre under certain conditions (Grigoryev et al., 2009). Therefore, the controversy of how nucleosomes are organized in condensed 30 nm chromatin fibres continues.

Different models have been described for the formation of large-scaled higher-order chromatin structures. The radial loop model suggests an arrangement of loops made from 30 nm chromatin fibres, forming smaller, thicker loops, which are distributed radially around the axis of the chromatid. Stabilization of the structure is supposed to be maintained by non-histone structural proteins at the base of each loop (Paulson and Laemmli, 1977; Laemmli et al., 1978). The hierarchical helical model proposes a progressive folding of a 30 nm chromatin fibre into larger fibres, including ~100 nm and then ~200 nm fibres, up to a final ~400 nm chromatin fibre (Sedat and Manuelidis, 1978; Belmont et al., 1989; Horn and Peterson, 2002).

The common dogma of all models is the actual existence of the elusive 30 nm fibre in vivo. In 1986, pioneering cryo-EM work allowed the first imaging of native mammalian chromosomes (Dubochet et al., 1986). The observed mitotic chromosomes revealed a homogeneous and grainy texture with ~11 nm spacing instead of higher-order chromatin structures including 30 nm chromatin fibres (Dubochet et al., 1986; Dubochet et al., 1988). This observation aligns with a number of publications that have questioned whether mammalian chromosomes contain regular 30 nm chromatin fibres (Dekker, 2008; Fussner et al., 2011a, b; Fussner et al., 2012; Maeshima et al., 2010; Nishino et al., 2012; Woodcock, 1994).

Cryo-EM studies by Woodcock et al. (1994) were able to show that 30 nm chromatin fibres are only present in a minor group of cells defined by highly condensed, transcriptionally inactive chromatin and rather long (~210 bp) nucleosomal repeat lengths. In yeast, investigation of transcriptionally active loci using Chromosome-conformation-capture (3C) exposed a loose arrangement of 10 nm chromatin fibres instead of compact 30 nm fibres (Dekker, 2008). Further visual proof was produced by electron spectroscopic imaging (ESI) that allows assessing the folding of genomic DNA by mapping its phosphorus and nitrogen. It revealed

a highly diffuse mesh of 10 nm fibres in pluripotent mammalian cells (Fussner et al., 2011a, b) and condensed chromatin structures of 10 nm fibres in differentiated mammalian cells (Fussner et al., 2012).

A combinational effort in investigating the structure of mitotic chromosomes in HeLa cells by cryo-EM, SAXS and ultra-SAXS revealed an arrangement of nucleosome fibres in an irregular manner lacking any kind of higher-order chromatin structures above a diameter of 11 nm (Nishino et al., 2012). Maeshima et al. postulate that the folding of nucleosome arrays into irregularly arranged fibres is regulated by nucleosome array concentrations and exhibits a “polymer melt” behavior (2010). Their model states that low nucleosome array concentrations allow the formation of 30 nm chromatin fibres by selective intra-array interactions. An increase in nucleosome array concentrations to a level resembling in vivo conditions would lead to a disruption of these interactions and formation of inter-array interactions. Therefore, cellular intermolecular crowding of nucleosome arrays would interfere with the formation of a 30 nm chromatin fibre in vivo (Maeshima et al., 2010).

Conclusively, these observations challenge the traditional view of chromatin from possessing static regular to dynamic irregular structural properties.