Histones, the most abundant proteins in chromatin, constitute a family of small, basic proteins. The five major types of histone proteins—termed H1, H2A, H2B, H3, and H4—are rich in positively charged basic amino acids, which interact with the negatively charged phosphate groups in DNA.

When chromatin is extracted from nuclei and examined in the electron microscope, its appearance depends on the salt concentration to which it is exposed. At low salt concentrations and in the absence of divalent cations such as Mg²⁺, isolated chromatin resembles beads on a string (Figure 8-23a). In this extended form, the “string” is composed of free DNA, called “linker” DNA, connecting beadlike structures termed nucleosomes. Composed of DNA and histones, nucleosomes are about 10 nm in diameter and are the primary structural units of chromatin. If chromatin is isolated at a physiological salt concentration, it assumes a more condensed fiberlike form that is about 30 nm in diameter (Figure 8-23b).

Structure of Nucleosomes The DNA component of nucleosomes is much less susceptible to nuclease digestion than is the linker DNA between them. If nuclease treatment is carefully controlled, all the linker DNA can be digested, releasing individual nucleosomes with their DNA component. A nucleosome consists of a protein core with DNA wound around its surface like thread around a spool. The core is an octamer containing two copies each of histones H2A, H2B, H3, and H4. X-ray crystallography has shown that the octameric histone core is a roughly disk-shaped structure made of interlocking histone subunits (Figure 8-24). Histones H3 and H4 fold into a tetramer in the absence of histones H2A and H2B. Two heterodimers of H2A and H2B then associate with the H3-H4 tetramer. Positive charges on the surface of the histone octamer in the region interacting with DNA hold the negatively charged DNA against the surface of the histone octamer at the center of the nucleosome. Nucleosomes from all eukaryotes contain about 147 bp of DNA wrapped one and two-thirds turns around the globular histone core. The length of the linker DNA is more variable among species, and even between different cells of the same organism, ranging from about 10 to 90 base pairs. During cell replication, DNA is assembled into nucleosomes shortly after the replication fork passes (see Figure 5-32). This process depends on specific chaperone molecules that bind to histones and assemble them, together with newly replicated DNA, into nucleosomes.

Structure of the 30-nm Fiber When extracted from cells in isotonic buffers (i.e., buffers with the same salt concentration found in cells, about 0.15 M KCl, 0.004 M MgCl₂), most chromatin appears as fibers about 30 nm in diameter (see Figure 8-23b). Despite many years of study, the arrangement of nucleosomes in the 30-nm chromatin fiber remains controversial. In one widely accepted model with considerable experimental support, nucleosomes are arranged in a helical conformation forming a solenoid with six or more nucleosomes per turn of the helix (Figure 8-25a). In a second strongly supported model, the two-start helix, nucleosomes alternately stack like coins into two “strands,” and those strands wind into a left-handed double helix (Figure 8-25b). The 30-nm fibers also include H1, the fifth major histone. H1 is bound to the DNA as it enters and exits the nucleosome core, but its structure in the 30-nm fiber is not known at atomic resolution.

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The chromatin in chromosomal regions that are not being transcribed or replicated exists predominantly in the condensed, 30-nm fiber form and in higher-order folded structures whose detailed conformation is not currently understood. The regions of chromatin actively being transcribed and replicated are thought to assume the extended beads-on-a-string form.

Conservation of Chromatin Structure The general structure of chromatin is remarkably similar in the cells of all eukaryotes, including fungi, plants, and animals, indicating that the structure of chromatin was optimized early in the evolution of eukaryotic cells. The amino acid sequences for the four core histones (H2A, H2B, H3, and H4) are highly conserved between distantly related species. For example, the sequences of histone H3 from sea urchin tissue and calf thymus differ by only a single amino acid, and H3 from the garden pea and calf thymus differ by only four amino acids. Apparently, significant deviations from the histone amino acid sequences were selected against strongly during evolution. The amino acid sequence of H1, however, varies more from organism to organism than do the sequences of the other major histones. The similarity in sequence among histones from all eukaryotes indicates that they fold into very similar three-dimensional conformations, which were optimized for histone function early in evolution in a common ancestor of all modern eukaryotes.

Minor histone variants encoded by genes that differ from the highly conserved major types also exist, particularly in vertebrates. For example, a special form of H2A, designated H2AX, is incorporated into nucleosomes in place of H2A in a small fraction of nucleosomes in all regions of chromatin. At sites of double-stranded breaks in chromosomal DNA, H2AX becomes phosphorylated and participates in the chromosome-repair process, probably by functioning as a binding site for repair proteins. In the nucleosomes at centromeres, H3 is replaced by another variant histone called CENP-A, which participates in the binding of spindle microtubules during mitosis. A variant of histone H3, known as H3.3, replaces the major histone H3 in transcribed regions of DNA, probably when the histone octamer must be moved out of the way by histone chaperones as RNA polymerase transcribes the DNA in chromatin. Most minor histone variants differ only slightly in sequence from the major histones. These slight changes in histone sequence may influence the stability of the nucleosome as well as its tendency to fold into the 30-nm fiber and other higher-order structures.