How is a long strand of human genome organized in the cell??

Research Summary
Since Flemming described a nuclear substance in the 19th century and named it “chromatin”, this substance has fascinated biologists. What is the structure of chromatin? Human genome DNA is wrapped around core histones, forming a nucleosome fiber (10-nm fiber). This fiber has long been assumed to fold into a 30-nm chromatin fiber, and subsequently into helically folded larger fibers, or radial loops. However, our cryo-EM (1, 2) and X-ray scattering analyses (3,4) demonstrated that chromatin is composed of irregularly folded 10-nm fibers, without 30-nm chromatin fibers, in interphase chromatin and mitotic chromosomes. This irregular folding implies a chromatin state that is physically less constrained, which could be more dynamic compared with classical regular helical folding structures. Consistent with this, recently using single nucleosome imaging, we uncovered by single nucleosome imaging large nucleosome fluctuations in living mammalian cells (~50 nm/30 ms) (5,6). Subsequent computational modeling suggested that nucleosome fluctuation increases chromatin accessibility, which is advantageous for many “target searching” biological processes such as transcriptional regulation. Therefore, we have provided a novel view on chromatin structure, in which chromatin consists of dynamic and disordered 10-nm fibers (polymer-melt)(2,7).

1. Eltsov et al. (2008) Proc Natl Acad Sci U S A 105, 19732-19737.
2. Maeshima et al. (2010) Curr Opin Cell Biol 22, 291-297.
3. Nishino et al. (2012) EMBO J. 31, 1644-53.
4. Joti et al. (2012) Nucleus 3, 404-410.
5. Hihara et al. (2012) Cell Reports 2, 1645-56.
6. Nozaki et al. (2013) Nucleus 4, 3349-356.
7. Maeshima et al. (2014) Chromosoma 123, 225-237

More detailed description

Introductory movie by Virginia Hughes

The following research description was modified from Maeshima et al., 2014.
For more comprehensive description in the chromatin field, see Maeshima et al., 2016.

There are 60 trillion cells in the human body. Each cell contains 2 m of genomic DNA in a small nucleus with an approximately 10-mm diameter (a volume of only ~100 fL to 1 pL), and yet it is able to search and read the information in its genomic DNA to execute diverse cellular functions. Therefore, it is important to understand how this long genomic DNA is organized in the nucleus. In the 19th century, W. Flemming described a nuclear substance that was clearly visible after staining with a basic dye using primitive light microscopes, and named it “chromatin.” This is now thought to be the basic unit of genomic DNA organization (Olins and Olins 2003). Since then, even before the discovery of the structure of DNA (Watson and Crick 1953), chromatin has attracted significant interest from biologists.

DNA and nucleosomes
Deoxyribonucleic acid (DNA) is a negatively charged polymer that produces electrostatic repulsion between adjacent DNA regions. Therefore, it would be difficult for a long DNA molecule alone to fold into a small space like the nucleus (Bloomfield 1996). To overcome this problem, the long, negatively charged polymer is wrapped around a basic protein complex known as a core histone octamer, which consists of the histone proteins H2A, H2B, H3, and H4, to form a nucleosome (Fig. 1)(Olins and Olins 1974; Kornberg 1974; Woodcock et al. 1976). This nucleosome fiber is also known as the 10-nm fiber (Fig. 1). A single histone octamer in the nucleosome has ~220 positively charged lysine and arginine residues, and ~74 negatively charged aspartic acid and glutamic acid residues. There are also 400 negative charges in the phosphate backbone of 200 bp of DNA. Because only about half of the negative charges in the DNA are neutralized, the remaining charge must be neutralized by other factors (e.g., linker histone H1, cations, and other positively charged molecules) for further folding.

Figure 1.Old and novel views of chromatin structure.
A long DNA molecule with a diameter of ~2 nm is wrapped around a core histone octamer, and forms a nucleosome with a diameter of 11 nm. The nucleosome has long been assumed to fold into 30-nm chromatin fibers (left), and subsequently into the higher-order organization of interphase nuclei or mitotic chromosomes. The right panel shows the novel hypothesis of irregularly folded nucleosome fibers.

30-nm chromatin fibers and further helical holding structures
In 1976, Finch and Klug first found, under transmission electron microscopy (EM), that purified nucleosome fibers (10-nm fibers) with linker histone H1 or Mg2+ ions were folded into fibers with a diameter of 30 nm. They named these fibers ‘30-nm chromatin fibers’ (Figs. 1, 2A, and 2B) (Finch and Klug 1976). In their structural model of the 30-nm fibers called “solenoids”, consecutive nucleosomes are located adjacent to one another in the fiber, and fold into a simple “one-start helix” (Fig. 2A). Subsequently, a second model of the “two-start helix” was proposed based on microscopic observations of isolated nucleosomes (Fig. 2B) (Woodcock et al. 1984). The second model assumed that nucleosomes were arranged in a zigzag manner, where a nucleosome in the fiber was bound to the second neighbor (Bassett et al. 2009) (Fig. 2B). In addition to these two famous structural models, many other structural variations of 30-nm chromatin fibers have been proposed (van Holde and Zlatanova 2007).

It has long been assumed that the 10-nm nucleosome fibers form a 30-nm chromatin fiber, and subsequently the higher-order chromatin structures of interphase nuclei and mitotic chromosomes. Several models have been proposed to describe the structure of higher-order chromatin. The “hierarchical helical folding model” suggests that a 30-nm chromatin fiber is folded progressively into larger fibers, including ~100-nm and then ~200-nm fibers, to form large interphase chromatin fibers (chromonema fibers) or mitotic chromosomes (Fig. 2C) (Sedat and Manuelidis 1978; Belmont et al. 1989; Belmont and Bruce 1994). In contrast, the “radial loop model” assumes that a 30-nm chromatin fiber folds into radially oriented loops to form mitotic chromosomes (Fig. 2C) (Paulson and Laemmli 1977; Laemmli et al. 1978; Marsden and Laemmli 1979).

Figure 2.Two classical models of 30-nm chromatin fibers and higher-order chromatin structures.
(A) One-start helix (solenoid); (B) two-start helix (zigzag). (Top) A scheme of the two different topologies of chromatin fibers is shown Robinson and Rhodes (2006). Positions from the first (N1) to the eighth (N8) nucleosome are labeled. (C) Two classical higher-order chromatin structure models: the radial loop model (Laemmli et al., 1978), and the hierarchical helical folding model (Sedat and Manuelidis, 1978). In the radial loop model, many loop structures of the 30-nm fiber (red) wrap around the scaffold structure (gray) (Laemmli et al., 1978), which consists of condensin and topoisomerase IIα (Maeshima and Laemmli, 2003).

Does the 30-nm chromatin fiber exist in vivo? The cryo-electron microscopy (EM) study
In 1986, the Dubochet group performed a pioneering study to visualize native cellular structures using cryo-EM (McDowall et al. 1986). Mammalian mitotic cells were frozen rapidly, sectioned, and observed directly under a cryo-EM with no chemical fixation or staining (cryo-EM of vitreous sections; CEMOVIS). Surprisingly, the chromosomes had a homogeneous, grainy texture with ~11-nm spacing. No higher-order or periodic structures, including 30-nm fibers, were observed.

Interphase chromatin has also been visualized using cryo-EM. It was suggested that interphase nuclei in most higher eukaryote cells might not contain 30-nm chromatin fibers (Dubochet et al. 2001; Bouchet-Marquis et al. 2006; Fakan and van Driel 2007).

On the other hand, it is unclear whether the absence of 30-nm structures in cryo-EM images truly demonstrates a lack of 30-nm chromatin fibers because when researchers capture cryo-EM images they use a technique called “defocusing” to produce high-contrast images. This process results in artificial amplification or suppression of the signal intensity, which affects different structural features depending on the defocus value (contrast transfer function [CTF] effect; for a review, see Frank 2006). It is thus possible that the degree of defocusing needed to image chromosomes or chromatin with high contrast prevents the visualization of 30-nm chromatin fibers.

To solve this problem, our laboratory collaborated with Eltsov, Frangakis, and Dubochet to compensate for the CTF effect by merging several images taken at different levels of defocus into a single image (Conway and Steven 1999). Even after this correction, we were unable to detect 30-nm structures in the chromosomal areas. In addition, the detection of periodic structures in the chromosomal region by power spectral (Fourier transform) analysis revealed a prominent peak at 11 nm, but not at 30 nm. This cryo-EM study suggested that 30-nm chromatin fibers were essentially absent from mitotic chromosomes; therefore, we proposed that 10-nm nucleosome fibers exist in a highly disordered, interdigitated state similar to a “polymer melt” (Figs. 1 and 4) (Eltsov et al. 2008; Maeshima et al. 2010).

Small angle X-ray scattering analyses revealed no 30-nm chromatin structures in interphase nuclei and mitotic chromosomes
Although our cryo-EM study did not detect any 30-nm structures in mitotic chromosomes, it might have been challenging to observe potential hierarchical regular structures because only a small number of 50-nm sections were examined (Eltsov et al. 2008). Langmore and Paulson (Langmore and Paulson 1983; Paulson and Langmore 1983) detected a 30-nm structure in interphase nuclei and mitotic chromosomes using small angle X-ray scattering (SAXS) analysis, which can detect bulky periodic structures in non-crystal materials in solution without chemical fixation or staining (Fig. 3A and B). Therefore, this study provided evidence for the existence of 30-nm chromatin fibers in interphase chromatin and mitotic chromosomes (Langmore and Paulson 1983; Paulson and Langmore 1983). Because these findings were inconsistent with the cryo-EM findings described above, we performed a comprehensive investigation of the structure of interphase nuclei and mitotic chromosomes using SAXS and cryo-EM (Nishino et al. 2012; Joti et al. 2012; for review, see Hansen 2012). Isolated human interphase nuclei and mitotic chromosomes were exposed to synchrotron X-ray beams (Fig. 3A). A typical scattering pattern of interphase nuclei and mitotic chromosomes exhibited three peaks at 30-, weakly at 11-, and 6-nm (Fig. 3C, left) (Nishino et al. 2012; Joti et al. 2012). This was consistent with the previous findings of Langmore and Paulson (1983), who suggested that the 6- and 11-nm peaks were derived from the face-to-face and edge-to-edge positioning of nucleosomes, respectively. They concluded that the 30-nm peak represented the side-by-side packaging of 30-nm chromatin fibers. However, this fails to explain why the 30-nm structures were not observed in interphase chromatin and mitotic chromosomes using cryo-EM.

To understand the nature of the 30-nm peak observed using SAXS, isolated chromosomes were examined using cryo-EM (Nishino et al. 2012; Joti et al. 2012). Again, no 30-nm chromatin fibers were observed in chromosomes. However, the cryo-EM images revealed that the surface of the chromosome was coated with electron-dense granules the size of ribosomes. Subsequent immunostaining and western blotting confirmed that the chromosome surface was contaminated with ribosomes. The ribosomes were stacked regularly at ~30-nm intervals, which could explain the ~30-nm peak observed using SAXS. To test this hypothesis, we removed ribosomes from the surface of the chromosome by washing with an isotonic buffer containing polyamine and EDTA while maintaining the size and shape of the chromosomes, and then analyzed mitotic chromosomes using SAXS. Importantly, no 30-nm peaks were detected (Fig 3C, right), but the 11- and 6-nm peaks resulting from the internal structure of the nucleosomes remained (Fig. 3C, right). Similarly, when we examined nuclei after ribosome removal, the 30-nm peak in the SAXS pattern disappeared (Joti et al. 2012). These results suggested the absence of a 30-nm chromatin fiber in interphase chromatin and mitotic chromosomes.

Next, we investigated the larger-scale chromatin structure of interphase nuclei and mitotic chromosomes using a newly developed apparatus for ultra-small-angle X-ray scattering (USAXS) (Nishino et al. 2009). Consistent with our previous observations, there were no regular periodic structures, between ~30- and 1000- nm in interphase nuclei and mitotic chromosomes. This contradicts the hierarchical helical folding model (Figure 2C)(Nishino et al. 2012; Joti et al. 2012). The scattering properties also suggested the existence of a scale-free structure or fractal nature up to ~275-nm in interphase chromatin, and ~1000-nm in mitotic chromosomes. This suggests that interphase and mitotic chromatin share the common structural features of up to ~275 nm of condensed and irregularly folded 10-nm nucleosome fibers, without 30-nm structures (discussed below). Taken together, the cryo-EM, SAXS, and USAXS data suggest that irregularly folded 10-nm nucleosome fibers form the bulk structure of human interphase chromatin and mitotic chromosomes (Nishino et al. 2012; Joti et al. 2012).

Other evidence supporting the absence of 30-nm chromatin fibers
Dekker (2008) used the chromosome-conformation-capture (3C) technique to investigate the folding of a specific genomic DNA region within yeast cells. He measured the average distance between two loci in the genome by confocal microscopy and the flexibility of the intervening chromatin fiber by the 3C technique. In combination with polymer modeling, the mass density of the chromatin fiber was determined. His conclusion was that yeast chromatin in a transcriptionally active domain did not form a compact 30-nm chromatin fiber but, rather, was extended with a loose arrangement of 10-nm nucleosome fibers.

More recently, Bazett-Jones et al. (2008) used electron spectroscopic imaging (ESI), a process that involves electron microscopy with an energy filter. ESI makes it possible to perform phosphorus and nitrogen mapping in cells with high contrast and resolution (Ahmed et al. 2009; Fussner et al. 2011). The signals from phosphorus and nitrogen, which are the main components of DNA, may be used to assess the folding of genomic DNA, and can distinguish 10- from 30-nm fibers. They observed that pluripotent cells were characterized by a highly dispersed mesh of 10-nm, but not 30-nm, fibers (Fussner et al. 2011; Fussner et al. 2012). In contrast, differentiated cells form compact chromatin domains that leave a large space in the nucleus that is devoid of DNA. Surprisingly, ESI combined with tomography methods revealed that condensed heterochromatin domains such as chromocenters consisted of 10-nm, rather than 30-nm, chromatin fibers (Fussner et al. 2012), consistent with the observations using cryo-EM. Furthermore, Gan et al. investigated the picoplankton Ostreococcus tauri, the smallest known free-living eukaryote, using cryo-EM tomography of ice sections and subsequent computational analysis (Gan et al. 2013). They demonstrated that O. tauri chromatin resembles a disordered assembly of nucleosomes without the 30-nm chromatin structure, compatible with the polymer melt model. Therefore, several lines of evidence suggest the absence of regular 30-nm chromatin fibers in eukaryotic cells.

Figure 3.Small angle X-ray scattering (SAXS) analysis of chromatin structure. (A) Experimental design. The chromosome pellet in a quartz capillary tube was exposed to synchrotron X-ray beams, and the scattering patterns were recorded using the imaging plate (Nishino et al., 2012). (B) When non-crystal materials were irradiated with X-rays, scattering at small-angles generally reflected periodic structures. Images (A) and (B) were reproduced from (Joti et al., 2012), with some modifications. (C) (Upper left) Typical SAXS patterns of purified mitotic HeLa chromosome fractions. Three peaks at ~6, ~11 (weak), and ~30 nm were detected (arrows). (Upper right) After the removal of ribosome aggregates, the 30-nm peak disappeared, whereas the other peaks remained. (Bottom) A model whereby the 30-nm peak in SAXS results from regularly spaced ribosome aggregates, and not from the chromosomes. Image (C) was reproduced from (Nishino et al., 2012), with some modification.

Why can 30-nm chromatin fibers be observed in vitro?
Although the near absence of 30-nm chromatin fibers in eukaryotic cells was suggested, these structures are shown in EM images in molecular biology textbooks. We propose that most 30 nm chromatin fibers in EM images are in vitro artifacts caused by the low salt buffer conditions. The formation of 30-nm chromatin fibers requires the selective binding of nucleosomes, which are close neighbors on the DNA strand, via intra-fiber nucleosomal association (Fig. 4A). In low salt buffer conditions of <1 mM MgCl2 or <100 mM NaCl, nucleosomal fibers gently repel each other due to their negative charges. This “isolation of nucleosome fibers” facilitates the intra-fiber nucleosomal association and the subsequent formation of stable 30-nm chromatin fibers (Figs. 4A and B). In conventional EM imaging studies, these 30-nm fibers might be stabilized through chemical cross-linking (such as glutaraldehyde fixation) and then shrunk further after alcohol dehydration during sample preparation (Maeshima et al. 2010).


Polymer melt
It is important to assess chromatin structure under more physiological salt conditions. Under these conditions, inter-fiber nucleosome interactions become increasingly dominant (Fig. 4A and B) (Maeshima et al. 2010). Nucleosome fibers (10 nm) are forced to interdigitate, which interferes with the formation and maintenance of 30-nm chromatin fibers. This leads to the “polymer melt” (Maeshima et al. 2010) or “self-oligomer” state (for review, see Hansen 2002; Hansen 2012)) (Fig. 4A and B). In addition, inter-fiber nucleosome interactions increase significantly in the presence of >2 mM Mg2+ ions (Zheng et al. 2005; Kan et al. 2009). However, it is important to note that the tail domain of histone H4 mediates both 30 nm fiber formation (Dorigo et al. 2003) and inter-fiber nucleosome association (Kan et al. 2009). Consequently, inter-fiber nucleosome association can prevent the formation of 30-nm fibers by sequestering the H4 tail domain (Hansen 2012).

Figure 4.Polymer-melt model.
(A) Under low-salt conditions, nucleosome fibers could form 30-nm chromatin fibers via intra-fiber nucleosome associations. An increase in salt (cation) concentration results in inter-fiber nucleosomal contacts that interfere with intra-fiber nucleosomal associations, leading to a polymer melt scenario. Note that in these illustrations, we show a highly simplified two-dimensional nucleosome model. Arrows and dotted lines show repulsion forces and interactions, respectively. (B) During the melting process, the 30-nm chromatin fibers become irregularly folded nucleosome fibers.

Dynamic 10-nm fibers in living mammalian cells
The original liquid chromatin model proposed by Dubochet (McDowall et al., 1986; Dubochet et al. 1988) and our polymer melt model (Eltsov et al., 2008; Maeshima et al., 2010) both imply a less physically constrained chromatin state, and a more dynamic state locally; the 10-nm nucleosome fibers fluctuate locally. Therefore, we attempted to visualize local nucleosome fluctuation. Previous studies of chromatin dynamics employed very large chromatin regions such as the LacO array that encompasses 20-50 nucleosomes (Straight et al., 1996; Belmont et l., 1999; Heun et al., 2001; Vazquezet al., 2001; Chubb et al., 2002; Levi et al., 2005; Hajjoul et al., 2013). The motion of these large regions in living mammalian cells was measured by monitoring the movement of the GFP-LacI signal bound to the LacO array at specific chromatin regions.

To observe and analyze more local nucleosome dynamics, we performed single nucleosome imaging in living mammalian cells (Fig. 5) (Hihara et al., 2012; Nozaki et al., 2013). We fused histone H4 with photoactivatable (PA)-GFP, and expressed the fusion protein in mammalian cells at a very low level (Fig. 5A). We then used an oblique illumination microscope to illuminate a limited thin area within the cell for single nucleosome imaging (Hihara et al., 2012; Nozaki et al., 2013; for principle, see Tokunaga et al., 2008). Generally, PA-GFP shows green fluorescence only after activation with a 405-nm laser (Lippincott-Schwartz et al., 2009). Surprisingly, we observed that a small fraction of H4-PA-GFP and PA-GFP-H4 in the cells was activated spontaneously without laser stimulation (Fig. 5A). Fig. 5B shows a typical single nucleosome image of a living mammalian cell. Each bright dot in the nucleus represents a single H4-PA-GFP (PA-GFP-H4) within the single nucleosome. Strikingly, we observed significant nucleosome fluctuation (~50 nm movement/30 ms) in both interphase chromatin and mitotic chromosomes (Fig. 5C), likely caused by Brownian motion (Hihara et al., 2012; Nozaki et al., 2013). Mean square displacement (MSD) plots, measuring the spatial extent of the random motion, and fitting to an anomalous diffusion curve suggested a restricted nucleosome movement. The McNally group also published single nucleosome tracking data using H2B-EGFP (Mazza et al., 2012), which appear to be consistent with our single nucleosome tracking results using PA-GFP-H4.

Local fluctuation of nucleosomes as a basis for scanning genome information
Some computational modeling studies, including our own, have suggested that nucleosome fluctuations facilitate the mobility of diffusing proteins in the chromatin environment (Fig. 5D) (Wedemeier et al., 2009; Fritsch and Langowski 2011; Hihara et al., 2012). Such nucleosome fluctuations may also contribute to the frequent exposure of genomic DNA sequences. Because both facilitating protein mobility and DNA exposure increase chromatin accessibility, these local dynamics may be advantageous in template-directed biological processes such as transcriptional regulation, DNA replication, and DNA repair/recombination. Therefore, we propose that the local fluctuation of nucleosomes forms the basis for scanning genome information (Fig. 5D).

Figure 5. Single nucleosome imaging.
(A) A small portion of PA-GFP-H4 was activated spontaneously without laser activation, and was used for single nucleosome imaging. (B) Single nucleosome image of a DM cell (Indian Muntjac cell) nucleus that expresses PA-GFP-H4. PA-GFP-H4 is observed as a bright dot using oblique illumination microscopy. The dots were fitted to an assumed Gaussian point spread function to determine the precise center of signals with higher resolution. Bar = 5 µm. (C) Representative three trajectories of fluorescently tagged single nucleosomes. (D) Chromatin fluctuations as a basis for scanning genome information. In cells, nucleosome fibers (red spheres and lines) are folded irregularly. The nucleosomes fluctuate, and these nucleosome dynamics facilitate chromatin accessibility. The images were reproduced from (Hihara et al. 2012; Nozaki et al., 2013)

The traditional view of chromatin is changing from one of static regular structures including 30-nm chromatin fibers to a dynamic irregular folding structure of 10-nm nucleosome fibers. Although the term “irregular” or “disordered” might give the impression that the organization is functionally irrelevant, the irregular folding results in less physical constraint and increased dynamism, increasing the accessibility of the DNA (Fig. 5D). This dynamic state may be essential for various genome functions, including transcription, replication, and DNA repair/recombination.

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