approximately 20° relative to the dyad axis, this would result in a pattern in which nucleosomes would alternate on either side of a central region of linker DNA bound by histone HI (Figure 7-29).

Nucleosome Arrays Can Form More Complex Structures: the 30-nm Fiber

Binding of 111 stabilizes higher-order chromatin structures. In the test tube, as salt concentrations are increased, the addition of histone Hi results in the nucleosomal DNA forming a 30-nm fiber. This structure, which can also be observed in vivo, represents the next level of DNA compaction, More importantly, the incorporation of DNA into this fiber makes the DNA less accessible to many DNA-dependent enzymes (such as RNA polymerases)-

There are two models for the structure of the 30-nm fiber. In the solenoid model, the nucleosomal DNA forms a superhelix containing approximately six nucleosomes per turn (see Figure 7-lBa). This structure is supported by both EM and X-ray diffraction studies, which indicate that the 30-nm fiber has a helical pitch of approximately 11 nm. This is also the approximate diameter of the nucleosome disc, suggesting that the 30-nm fiber is composed of nucleosome discs stacked on edge in the form of a helix (Figure 7-30a). In this model, the flat surfaces on either face of ihe histone octamer disc are adjacent to each other and the DNA surface of the nucleosomes forms the outside accessible surface of the superhelix. The linker DNA is buried in the center of the superhelix, but it never passes through the axis of the fiber. Rather, the linker DNA circles around the central axis as the DNA moves from one nucleosome to the next.

FIGURE 7-29 Histone HI induces tighter DNA wrapping around the nucleosome. The two illustrations show a comparison of the wrapping of DNA around the nudeosonc in the presence and absence of histone H1. One histor.e H1 can associate with each nucleosome. Histone H 3 binds to both linker DNA and the DNA helix located in the middle of the nUcieosome-hound DNA.

b ztgzag

FIGURE 7-30 Two models for the 30-nm chromatin fiber, (a) The solenoid model, tote that the linker DNA does not pass throu^i the central axis ot the superheiix and that the sides and entry and exit points of the nudcosomes are relatively inaccessible, (b) The "zigzag" model. In this model, the linker DNA frequently passes through the central axis of the fiber and the sides and even the entry and exit points are more accessible. (Source: Pollard T and Earns haw w 2002. Cell biology, 1st edition, p. 202, f 13-6. Copyright © 2002. Reproduced by permission uf WB. Sounders Inc.)

a solenoid b ztgzag

An alternative model for the 30-nm fiber is the "zigzag" model (Figure 7-3Ob). This model is based on the zigzag pattern of nucleosomes formed upon Hi addition. In this case, the 30-nm fiber is g compacted form of these zigzag nucleosome arrays. Analysis of the spring-like nature of isolated 30-nm fibers supports this zigzag model. Unlike the solenoid model, the zigzag conformation requires the linker DNA to pass through the central axis of the fiber in a relatively straight form (see Figure 7-30b). Thus, longer linker DNA favors this conformation. Because the average linker DNA varies between different species (see Table 7-4), the form of the 30-nm fiber may not always be the same.

The Histone N-Terminal Tails Are Required for the Formation of the 30-nm Fiber

Clore histones lacking their N-terminal tails are incapable of forming the 30-nm fiber. The most likely role of the tails is to stabilize the 30-nm fiber by interacting with adjacent nucleosomes. This model is supported by the three-dimensional structure of the nucleosome. which shows that the amino terminal tails of H2A, H3, and H4 each interact with adjacent nucleosomes in the crystal lattice (Figure 7-31). For example, the histone H4 N-terminus makes multiple hydrogen bonds with H2A and H2B on the surface of an adjacent nucleosome in the crystal. The residues of H2A and H2B that interact with the 114 tail are conserved across many eukaryotic organisms but are not involved in DNA binding or formation of the histone octamer. One possibility is that these regions of H2A and H2B are conserved to mediate ixiter-nucleosomal interactions with the H4 tail. As we shall see below, the histone tails are frequent targets for modification in the cell. It is likely that these modifications influence the ability to form the 30-nm fiber and other higher-order nucleosome structures.

Further Compaction of DNA Involves Large Loops of Nucleosomal DNA

Together, the packaging of DNA into nucleosomes and the 30-nm fiber results in the compaction of the linear length of DNA by approxb

nucleosome histone core DNA

linker DNA

30-nm fiber

30-nm fiber

FIGURE 7-31 A speculative model for the stabilization of the 30-nm fiber by histone N-terminal tails. In this model the iO-nm fiber is illustrated using the "zigzag" mode!. Several different tait-histone core interactions ate possible. Here the interactions are shown as between every alternate histone but they could also be with adjacent or more distant histones.

mately 40-fold. This is still insufficient to fit 1-2 meters of DNA into a nucleus approximately ltr5 meters across. Additional folding of the 30-nm fiber is required to compact the DNA further. Although the exact nature of this folded structure remains unclear, one popular model proposes that the 30-nin fiber forms loops of 40-90 kb that are held together a! their bases by a proteinacious structure referred to as the nuclear scaffold fFigure 7-32). A variety of methods have been developed to identify proteins that are part of this structure although the true nature of the nuclear scaffold remains mysterious.

Two classes of proteins that contribute to the nuclear scaffold have been identified. One of these is topoisomerase 11 (Topo II). which is abundant in both scaffold preparations and purified mitotic chromosomes. Treating cells with drugs that result in DNA breaks at the sites of Topo fl DNA binding generates DNA fragments that are ahout 50 kb in size. This is similar to l he size range observed for limited nuclease digestion of chromosomes and suggests that Topo II may be part of the mechanism that holds the DNA at the base of these loops,

The SMC proteins aré also abundant components of the nuclear scaffold. As we discussed earlier (see section on Chromosome Duplication and Segregation), these proteins are key components of the machinery that condenses arid holds daughter chromosomes together after chromosome duplication. The associations of these proteins with the nuclear scaffold may serve to enhance their functions by providing an underlying foundation for their interactions with chromosomal DNA.

Histone Variants Alter Nucleosome Function

The core histones are among the most conserved eukaryotic proteins; therefore, the microsomes formed by these proteins are very similar in all eukaryotes (Figure 7-33a). But there are several histone variants found in eukaryotic cells. Such unorthodox histones can replace one of the four standard histones to form alternate nucleosomes. Such nucleosomes may serve to demarcate particular regions oí chromosomes or confer specialized functions to the nucleosomes into which I hey are incorporated. For example, H2A.z is a variant of H2A that is widely distributed in eukaryotic nucleosomes and is generally associ-

b chromatin fiber

FIGURE 7-32 The higher-order structure of chromatin, (a) A transmission electron micrograph shows chromatin emerging iron a centra) structure of a chromosome. The electron-dense regions are the nuclear scaffold that acts to organize the large amounts of DNA found in eukaryotic chromosomes The bar represents 2DO nm. (b) A mode! for the structure of a eukaryotic chromosome shows that the majority of the DMA is packaged into large bops of 50-nm fiber that are tethered to the nude at scaffold at their base Sites of active DNA manipulation (for example, sites of transcription or DNA replication) are in the form of io-nm fiber or even naked DMA. {Source: (a) Courtesy of J.R Paulson and U K. laemrnli.)

ated with transcribed regions of DNA, There is little change in the overall structure of a nucleosome containing this variant histone. instead, the presence of the H2A.2 histone inhibits nucleosomes from forming repressive chromatin structures, creating regions of easily accessible chromatin that are more compatible with transcription.


DNA loop

10 nm

30 nm

chromosome scaffold naked

10 nm

30 nm

DNA loop chromosome scaffold a normal (nonvariant) histoncs

30-nm fiber b with CENP-A

kinetochore binding protern interaction with kinetochore

FIGURE 7-33 Alteration of chromatin by incorporation of histone variants, (a) Transition be tween 10-nm and 30-nm fibers for standard histories, (b) Incorporation of CENP-A in place of histone Hi is proposed to act as a binding site for one or more components of the kinetochore

30-nm fiber

FIGURE 7-33 Alteration of chromatin by incorporation of histone variants, (a) Transition be tween 10-nm and 30-nm fibers for standard histories, (b) Incorporation of CENP-A in place of histone Hi is proposed to act as a binding site for one or more components of the kinetochore a normal (nonvariant) histoncs b with CENP-A

kinetochore binding protern interaction with kinetochore

A second histone variant, CENP-A, is associated with nucleosomes that include centromeric DNA. In this chromosomal region, CENP-A replaces the histone H3 subumts in nucleosomes. These nucleosotnes are incorporated into the kinetochore which mediates attachment of the chromosome to the mitotic spindte (see Figure 7-12}. Compared to H3, CENP-A includes a substantial extension of the N-terminal tail region. Thus, like nucleosotnes with H2A,z, it is unlikely that incorporation of CENP-A changes the core structure of the nucleosome. Instead, the extended tail of CENP-A may generate novel binding sites for other protein components of the kinetochore (Figure 7-33b}. Civen the critical role of the histone N-iermini in the formation of higher-order chromatin structures, these changes may alter the interactions between nncleosomes at the centrnmere/kinetochore as well,

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